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28569221
|
10.1038/embor.2008.246
|
DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy
|
2008
|
Figure 1
|
Functional interaction between DAPK and beclin 1. (<b>A</b>) HEK293 cells were transfected with DAPK ΔCaM or with a control vector (pcDNA3‐luciferase, LUC), together with shRNAs targeting beclin 1 or HcRed, and with GFP–LC3 plasmid. After 72 h, cells were counted and lysates were prepared. (<b>A</b>) The percentage of cells with punctate GFP–LC3 fluorescence per total GFP–LC3‐positive cells was quantified. Data presented are the mean±s.d. from a triplicate of 100 transfected cells. The asterisks denote a significance level of <i>P</i>=0.001. (<b>B</b>) Western blot analysis was performed using the indicated antibodies. (<b>C</b>) Representative GFP–LC3 staining of cells transfected with the control shRNA (HcRed) together with pcDNA3‐luciferase or DAPKΔCaM. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; HEK, human embryonic kidney; shRNA, short hairpin RNA.
|
https://api.sourcedata.io/file.php?figure_id=1629
|
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|
28569221
|
10.1038/embor.2008.246
|
DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy
|
2008
|
Figure 2
|
Beclin 1 is a new substrate of DAPK. (<b>A</b>) Flag‐tagged DAPK (100 ng) was incubated with GST (900 ng) or GST–beclin‐1 (750 ng) in the presence of Ca<sup>2+</sup>, calmodulin and [γ‐<sup>33</sup>P]ATP for 30 min or 60 min. Phosphorylated proteins were visualized by X‐ray film exposure, and GST/GST–beclin‐1 levels were visualized by Ponceau S staining. The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples. (<b>B</b>) Flag‐tagged DAPK (60 ng) was incubated with Flag‐tagged beclin 1 (250 ng), which was purified from HEK293T cells, and a kinase assay was performed for 60 min. Where indicated (+LiCl), beclin‐1‐bound beads were first washed stringently in 0.5 M LiCl and 0.5 M KCl. Phosphorylated proteins were visualized by X‐ray film exposure, and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies. DAPK, death‐associated protein kinase; GST, glutathione <i>S</i>‐transferase; HEK, human embryonic kidney.
|
https://api.sourcedata.io/file.php?figure_id=1630
|
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|
28569221
|
10.1038/embor.2008.246
|
DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy
|
2008
|
Figure 3
|
Physical interaction between DAPK and beclin 1. (<b>Aa</b>) Protein extracts from COS7 cells, HEK293T cells, or no extracts were added to bacterially produced GST or GST–beclin‐1. The pulled‐down proteins, as well as the total cell extracts, were blotted with the indicated antibodies. (<b>Ab</b>) Ponceau S staining of GST and GST–beclin‐1 to which the extracts were added. (<b>B</b>) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 or with Flag‐tagged beclin 1 lacking the Bcl‐2‐binding domain (ΔBD) together with Bcl‐X<sub>L</sub> and HA‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted with DAPK, Bcl‐X<sub>L</sub> and beclin 1 antibodies. (<b>C</b>) HEK293 cells were transfected with Flag–beclin‐1 or Flag–GFP, and the Flag‐tagged proteins were immunoprecipitated using Flag antibodies, and eluted with an excess of Flag peptides. The blot was reacted with DAPK antibodies or with Flag antibodies at different time exposures. DAPK, death‐associated protein kinase; GFP, green fluorescent protein; GST, glutathione <i>S</i>‐transferase; HA, haemagglutinin; HEK, human embryonic kidney.
|
https://api.sourcedata.io/file.php?figure_id=1631
|
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|
28569221
|
10.1038/embor.2008.246
|
DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy
|
2008
|
Figure 4
|
DAPK phosphorylates beclin 1 on Thr 119 located within its BH3 domain. (<b>A</b>) A bacterially purified catalytic domain of DAPK was incubated for 15 min at 30°C with increasing concentrations (5–10 nmol) of a peptide corresponding to the BH3 domain of beclin 1 (aa 108–127) and with the same peptide in which Thr 119 was substituted by alanine. An <i>in vitro</i> kinase assay was performed, and the reactions were applied to Whatman filters. Total levels of TCA‐insoluble counts were measured and plotted against substrate concentration. Data are the mean±s.d. of three experiments. (<b>B</b>) A model of the interaction between the BH3 domain of beclin 1, and the hydrophobic pocket of Bcl‐X<sub>L</sub>. The cyan arrow points to the groove in which phosphorylated Ser 113 can be accommodated. The black arrow points to the site that might be disrupted by phosphorylation of Thr 119. The magenta arrow points to the positively charged groove in which phosphorylated Ser 127 can be accommodated. (<b>C</b>) Flag‐tagged DAPK (60 ng) was incubated with GST–WT beclin 1 or with GST–T119A beclin 1 (1000 ng) in the presence of Ca<sup>2+</sup>, calmodulin and ATP for 30 min. GST‐beclin 1 levels were visualized by Ponceau S staining, and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody (Western blot). (<b>D</b>) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 with or without ΔCaM DAPK. After 24 h, beclin 1 was immunoprecipitated from cells, using Flag antibodies, and the immunoprecipitates were reacted with phosphoThr 119 antibodies. The Ponceau staining shows equal amounts of immunoprecipitated beclin 1. The cell extract blots were reacted with haemagglutinin antibodies or with beclin 1 antibodies. CaM, calmodulin; DAPK, death‐associated protein kinase; GST, glutathione <i>S</i>‐transferase; HEK, human embryonic kidney; TCA, trichloro‐acetic acid.
|
https://api.sourcedata.io/file.php?figure_id=1632
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"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "beclin 1",
"type": "geneprod",
"uniprot_ids": [
"Q14457"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q14457",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "beclin 1",
"type": "geneprod",
"uniprot_ids": [
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}
] |
|
28569221
|
10.1038/embor.2008.246
|
DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy
|
2008
|
Figure 5
|
DAPK promotes the dissociation of beclin 1 from Bcl‐X<sub>L</sub>. (<b>A</b>) HEK293 cells were transfected with Flag‐tagged beclin 1 and Bcl‐X<sub>L</sub> with or without haemagglutinin‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. (<b>B</b>) HEK293 cells were co‐transfected with Flag‐tagged T119A or T119E beclin 1 mutants and Bcl‐X<sub>L</sub>. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. (<b>C</b>) HEK293 cells were transfected with 5 or 10 μg T119A or T119E beclin 1 mutants together with GFP–LC3 plasmid. After 24 h, cells were counted and lysates were prepared. (<b>a</b>) Representative GFP–LC3 staining. (<b>b</b>) The percentage of cells with punctate GFP–LC3 fluorescence per total GFP–LC3‐positive cells was quantified. Data presented are the mean±s.d. from a triplicate experiment with 100 transfected cells. The asterisks denote significance level: <sup>*</sup><i>P</i>=0.01; <sup>**</sup><i>P</i>=0.005. (<b>c</b>) Western blot analysis was performed using the indicated antibodies. (<b>D</b>) HEK293 cells were transfected with Bcl‐X<sub>L</sub>, Flag‐tagged beclin 1 (WT) or Flag‐tagged T119A beclin 1 mutant with or without haemagglutinin‐tagged activated DAPK (ΔCaM). Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. Levels of Bcl‐X<sub>L</sub> were quantified using NIH image software, and the ratio between immunoprecipitated and expressed Bcl‐X<sub>L</sub> was calculated. (<b>E</b>) Transmission electron micrographs of HEK293 cells 24 h after they were transfected with Bcl‐X<sub>L</sub>, Flag‐tagged beclin 1 and ΔCaM or pcDNA3‐luciferase as a control. The images in (<b>c</b>) and (<b>d</b>) were taken at higher magnifications of the ΔCaM treatment (see the scale bars). ‘AV’ indicates autophagic vacuoles. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; GST, glutathione <i>S</i>‐transferase; HEK, human embryonic kidney; NIH, National Institutes of Health.
|
https://api.sourcedata.io/file.php?figure_id=1633
|
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}
] |
|
28569221
|
10.1038/embor.2009.242
|
The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells
|
2009
|
Figure 1
|
DOR moves out of the nucleus in response to the activation of autophagy. (<b>A</b>) The intracellular distribution of DOR is modified by starvation. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal), HBSS (starvation), 50 μM chloroquine or 10 mM 3MA. The intracellular localization of DOR was analysed by immunofluorescence and is shown in red. The nuclei are shown in blue (DAPI staining). Scale bars, 10 μm. (<b>B</b>) Endogenous DOR leaves the nucleus under starvation. C2C12 myoblasts were incubated for 30 min at 37°C with DMEM (basal) or HBSS (starvation). DOR is shown in red and nuclei are in blue (DAPI staining). Scale bars, 5 μm. (<b>C</b>,<b>D</b>) De‐activation of autophagy returns DOR to the nucleus. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal) or HBSS (starvation). Cells were then incubated with normal DMEM for an additional hour (starvation+GM). The intracellular distribution of DOR was determined by immunofluorescence. DOR is shown in red and the nuclei are in blue. Scale bars, 5 μm. Cell lysates were also obtained and western blot assays were performed with specific antibodies. 3MA, 3‐methyladenine; DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; HBSS, Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.
|
https://api.sourcedata.io/file.php?figure_id=1634
|
[
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"ncbi_gene_id": null,
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"text": "DOR",
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"uniprot_ids": [
"Q8IXH6"
]
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"Q8IXH6"
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"ext_ids": "Q8CFU8",
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"ext_tax_names": "Mus musculus",
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{
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},
{
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]
},
{
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"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
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]
},
{
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"ext_ids": "Q8IXH6",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
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"role": "assayed",
"text": "DOR",
"type": "geneprod",
"uniprot_ids": [
"Q8IXH6"
]
}
] |
|
28569221
|
10.1038/embor.2009.242
|
The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells
|
2009
|
Figure 2
|
DOR localizes to autophagosomes when autophagy is activated. (<b>A</b>) Confocal images of HeLa cells transiently transfected with DOR and GFP‐LC3, and incubated for 1h with DMEM, HBSS (starvation), 2 μM rapamycin or HBSS containing 50 μM chloroquine. Nuclei were labelled with DAPI. Scale bars, 10 μm. (<b>B</b>,<b>C</b>) Confocal images of HeLa cells transiently transfected with DOR or GFP‐LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS. Scale bars, 10 μm. (<b>D</b>) Confocal images of HeLa cells transiently transfected with DOR and LAMP1‐GFP, or with YFP‐LC3 and LAMP1‐GFP. Scale bars, 10 μm. Contrast‐corrected merge RGB pictures and the <i>Z</i>‐projection of Product of the differences from the mean (PDM) images are shown in all panels. Colour scales of PDM images have different maximal values, so the different conditions are not comparable from these projections. Product of the differences from the mean (PDM) values closer to 1 show reliable colocalized pixels. DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; LAMP1, lysosomal‐associated membrane protein 1; LC3, microtubule‐associated protein 1A/1B‐light chain 3.
|
https://api.sourcedata.io/file.php?figure_id=1635
|
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] |
|
28569221
|
10.1038/embor.2009.242
|
The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells
|
2009
|
Figure 3
|
DOR stimulates autophagy. (<b>A</b>) DOR gain‐of‐function accelerates the degradation of proteins of middle–long half‐life. Untreated HeLa cells and those transiently transfected with DOR were incubated in DMEM or HBSS. <sup>*</sup>Significant effects of starvation, <i>P</i>0.01; <sup>τ</sup>significant effects caused by DOR overexpression, <i>P</i>0.01. (<b>B</b>) DOR overexpression enhances autophagosomal formation. LC3‐GFP‐positive vacuoles were counted in 100 transfected HeLa cells (with GFP‐LC3 and TRα1, or GFP‐LC3 and DOR). <sup>*</sup>Significant effects of starvation, <i>P</i>0.001; <sup>τ</sup>significant effects of DOR overexpression, <i>P</i>0.001. (<b>C</b>,<b>D</b>) HeLa cells transiently transfected with empty pCDNA3 or DOR were incubated in DMEM (basal) or in HBSS (starvation). Cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes (diameter, 407±63 nm). Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen transfected cells. <sup>*</sup>Significant effects of starvation, <i>P</i>0.05; <sup>τ</sup>significant effects of DOR overexpression, <i>P</i>0.05. DMEM; Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS; Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.
|
https://api.sourcedata.io/file.php?figure_id=1636
|
[
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"ext_dbs": "Uniprot///Uniprot///Uniprot",
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},
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"uniprot_ids": [
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{
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"ext_tax_names": "Homo sapiens",
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"text": "DOR",
"type": "geneprod",
"uniprot_ids": [
"Q8IXH6"
]
}
] |
|
28569221
|
10.1038/embor.2009.242
|
The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells
|
2009
|
Figure 4
|
DOR loss‐of‐function reduces autophagy. (<b>A</b>,<b>B</b>) C2C12 myoblasts were previously infected with lentiviruses encoding scrambled RNA, DOR siRNA1 or DOR siRNA2. Endogenous LC3 was detected by immunofluorescence. LC3‐positive puncta were counted in 100 cells per group under basal conditions or starved for 20 min. Scale bars, 10 μm. <sup>*</sup>Significant effects of starvation, <i>P</i>0.001; τsignificant effects caused by DOR knockdown, <i>P</i>0.001. (<b>C</b>,<b>D</b>) Scramble or DOR siRNA1 C2C12 cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes. Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen cells. <sup>*</sup>Significant effects of DOR repression, <i>P</i>0.001. (<b>E</b>) Scramble or DOR siRNA1 C2C12 cells were incubated in DMEM with and without 10 mM 3MA. <sup>*</sup>Significant effects of DOR knockdown, <i>P</i>0.01; τsignificant effects caused by 3MA, <i>P</i>0.01. 3MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; LC3, microtubule‐associated protein 1A/1B‐light chain 3.
|
https://api.sourcedata.io/file.php?figure_id=1637
|
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]
},
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},
{
"ext_dbs": "Uniprot///Uniprot",
"ext_ids": "Q9CQV6///Q91VR7",
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},
{
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]
},
{
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"ext_tax_names": "Mus musculus",
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"ncbi_gene_id": "68728",
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"text": "DOR",
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"uniprot_ids": [
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]
},
{
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"ext_ids": "68728",
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"role": "intervention",
"text": "DOR",
"type": "geneprod",
"uniprot_ids": [
"Q8CFU8"
]
}
] |
|
28569221
|
10.1038/embor.2009.242
|
The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells
|
2009
|
Figure 5
|
CG11347 is required for autophagy activation in the fat body of <i>Drosophila</i>. (<b>A</b>) Decrease of mRNA levels in TubG4, UAS‐CG11347‐RNAi animals compared with TubG4 controls. All measurements were normalized to rp49. <sup>*</sup>Significant change, <i>P</i>0.05. (<b>B</b>) Lysotracker staining of fat bodies. No Lysotracker staining is observed in feeding early third‐instar larvae, whereas Lysotracker‐positive granules (red) accumulate in fat body cells of wandering late third‐instar controls (upper panels, TubG4/+). dDOR knockdown animals (TubG4, UAS‐CG11347‐RNAi T4, lower panels) show decreased staining of Lysotracker‐positive granules in wandering late third‐instar larvae. Nuclei are shown in blue. Scale bars, 50 μm. (<b>C</b>) The number of Lysotracker‐positive puncta per unit area are shown for each genotype (control and RNAi), normalized to the value for control cells processed in parallel. <sup>*</sup>Significant change, <i>P</i>0.05. (<b>D</b>) Transmission electron microscopy images of fat body cells from wandering late third‐instar larvae. Developmental autophagy results in the accumulation of large autolysosomes (AL) in control (TubG4/+) larvae, whereas only a few autolysosomes are seen in CG11347 RNAi fat body cells of the same age. Control and RNAi fat bodies present lipid droplets (LP) with a ‘striped’ appearance, which is an electron microscopy artefact. Scale bars, 5 nm. dDor, CG11347, a homologue of DOR; DOR, diabetes‐ and obesity‐regulated gene; mRNA, messenger RNA; wt, wild type.
|
https://api.sourcedata.io/file.php?figure_id=1638
|
[
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"text": "CG11347",
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{
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"uniprot_ids": [
"A8JNK7",
"M9NFM7",
"Q8IRB4",
"Q9VZC7"
]
}
] |
|
28569221
|
10.1038/embor.2013.111
|
Autophagy in Myf5+ progenitors regulates energy and glucose homeostasis through control of brown fat and skeletal muscle development
|
2013
|
Figure 1
|
<sd-panel>Deleting <sd-pretag parent-gene-tag-id='9' parent-protein-tag-id='17' id='sdPretag593924121sd'><i>Atg7</i></sd-pretag> in <sd-pretag parent-tag-id='99' id='sdPretag1035017798sd'>Myf5</sd-pretag>+ progenitors disrupts macroautophagy (MA) in <sd-pretag role='component' id='sdPretag1833636228sme' type='tissue' >brown adipose tissue</sd-pretag> (<sd-pretag parent-tag-id='1' id='sdPretag1238641529sd'>BAT</sd-pretag>) and <sd-pretag parent-tag-id='271' id='sdPretag1625487632sd'>skeletal muscle</sd-pretag> (<sd-pretag parent-tag-id='22' id='sdPretag1063145095sd'>SKM</sd-pretag>). (<b>A</b>-<b>C</b>) <sd-pretag parent-tag-id='8' id='sdPretag486635496sd'>Immunoblots</sd-pretag> for indicated proteins in <sd-pretag parent-tag-id='1' id='sdPretag633998489sd'>BAT</sd-pretag>, <sd-pretag parent-tag-id='2' id='sdPretag951414607sd'>extensor digitorum longus</sd-pretag> (<sd-pretag parent-tag-id='3' id='sdPretag427524538sd'>EDL</sd-pretag>), <sd-pretag parent-tag-id='4' id='sdPretag508416920sd'>epididymal white adipose tissue</sd-pretag> (<sd-pretag parent-tag-id='5' id='sdPretag1956897059sd'>eWAT</sd-pretag>) and <sd-pretag parent-tag-id='6' id='sdPretag1422317987sd'>heart</sd-pretag> from 10‐ to 12‐month (mo)‐old control (Con) and knock out (KO) <sd-pretag parent-tag-id='16' id='sdPretag222659653sd'>mice</sd-pretag>. Arrows depict <sd-pretag parent-tag-id='41' id='sdPretag1322299508sd'>LC3</sd-pretag>‐I and II. (<b>D</b>) <sd-pretag parent-gene-tag-id='18' parent-protein-tag-id='39' id='sdPretag1197365711sd'>ATG5</sd-pretag> and <sd-pretag parent-gene-tag-id='9' parent-protein-tag-id='17' id='sdPretag241177768sd'>ATG7</sd-pretag> mRNA levels in indicated tissues (<i>n</i>=4), and (<b>E</b>) <sd-pretag parent-tag-id='8' id='sdPretag264732163sd'>immunoblots</sd-pretag> for indicated proteins in <sd-pretag parent-tag-id='329' id='sdPretag752151019sd'>spleen</sd-pretag>, <sd-pretag parent-tag-id='27' id='sdPretag1538915143sd'>liver</sd-pretag>, <sd-pretag parent-tag-id='28' id='sdPretag429638906sd'>lung</sd-pretag>, <sd-pretag role='component' id='sdPretag451329955sme' type='tissue' >kidney</sd-pretag>, <sd-pretag parent-tag-id='30' id='sdPretag38460992sd'>mediobasal hypothalamus</sd-pretag> (<sd-pretag parent-tag-id='31' id='sdPretag1011766746sd'>MBH</sd-pretag>), <sd-pretag parent-tag-id='32' id='sdPretag753703900sd'>inguinal white adipose tissue</sd-pretag> (<sd-pretag parent-tag-id='33' id='sdPretag1166453570sd'>ingWAT</sd-pretag>) and <sd-pretag parent-tag-id='34' id='sdPretag1754973989sd'>perinephric fat</sd-pretag> (<sd-pretag parent-tag-id='35' id='sdPretag29763030sd'>pnWAT</sd-pretag>) from 10‐ to 12‐mo‐old Con and KO <sd-pretag parent-tag-id='16' id='sdPretag285400110sd'>mice</sd-pretag>. (<b>F</b>) Body weights (wt) of chow diet (RD)‐fed (<i>n</i>=6-29), and (<b>G</b>) high‐<sd-pretag parent-tag-id='346' id='sdPretag600001817sd'>fat</sd-pretag> diet (HFD)‐fed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag2055355227sd'>mice</sd-pretag> at indicated ages (<i>n</i>=4-17). (<b>H</b>) Total body <sd-pretag parent-tag-id='346' id='sdPretag642613873sd'>fat</sd-pretag> and lean mass of 4-7 mo RD‐fed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag2024153699sd'>mice</sd-pretag> (<i>n</i>=8-12). (<b>I</b>) <sd-pretag parent-tag-id='1' id='sdPretag1115920137sd'>BAT</sd-pretag> wt (<i>n</i>=4-7), (<b>J</b>) <sd-pretag parent-tag-id='51' id='sdPretag2119356351sd'>gastrocnemius</sd-pretag> (GA) wt (<i>n</i>=5-7), (<b>K</b>) <sd-pretag parent-tag-id='52' id='sdPretag752006033sd'>soleus</sd-pretag> wt (<i>n</i>=4-7), (<b>L</b>) <sd-pretag parent-tag-id='5' id='sdPretag991489143sd'>eWAT</sd-pretag> wt (<i>n</i>=5-7) and (<b>M</b>) visceral (Visc) and subcutaneous (Subc) body <sd-pretag parent-tag-id='346' id='sdPretag111730488sd'>fat</sd-pretag> distribution in 10‐mo‐old Con and KO <sd-pretag parent-tag-id='16' id='sdPretag59110958sd'>mice</sd-pretag> (<i>n</i>=4). Values are mean±s.e., *<i>P</i>0.05, **<i>P</i>0.01, ***<i>P</i>0.001.</sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=1700
|
[
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"ext_tax_names": "Mus musculus///Mus musculus",
"mapping_source": "unknown",
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}
] |
|
28569221
|
10.1038/embor.2013.111
|
Autophagy in Myf5+ progenitors regulates energy and glucose homeostasis through control of brown fat and skeletal muscle development
|
2013
|
Figure 3
|
<sd-panel>Loss of <sd-pretag parent-gene-tag-id='9' parent-protein-tag-id='17' id='sdPretag939711638sd'><i>Atg7</i></sd-pretag> in <sd-pretag parent-tag-id='99' id='sdPretag1960412435sd'>Myf5</sd-pretag>+ progenitors increases energy expenditure. (<b>A</b>) <sd-pretag parent-tag-id='375' id='sdPretag1255405343sd'>Body temperature</sd-pretag> before (Basal) and during <sd-pretag parent-tag-id='371' id='sdPretag1835594132sd'>cold exposure</sd-pretag> (<i>n</i>=7-8). (<b>B</b>) <sd-pretag parent-tag-id='132' id='sdPretag440479403sd'>Oxygen consumption</sd-pretag> (VO<sub>2</sub>), (<b>C</b>) carbon <sd-pretag role='assayed' id='sdPretag2075542126sme' type='molecule' >dioxide</sd-pretag> production (<sd-pretag role='assayed' id='sdPretag1069616353sme' type='geneprod' >VCO<sub>2</sub></sd-pretag>) and (<b>D</b>) energy expenditure in <sd-pretag parent-tag-id='441' id='sdPretag566941606sd'>10‐month (mo)‐old</sd-pretag> chow diet (RD)‐fed control (Con) and knock out (KO) <sd-pretag parent-tag-id='16' id='sdPretag627400570sd'>mice</sd-pretag> (<i>n</i>=4). (<b>E</b>) VO<sub>2</sub>, (<b>F</b>) <sd-pretag role='intervention' id='sdPretag44429163sme' type='geneprod' >VCO<sub>2</sub></sd-pretag> and (<b>G</b>) energy expenditure in 10‐mo‐old high‐<sd-pretag parent-tag-id='346' id='sdPretag805189336sd'>fat</sd-pretag> diet (HFD)‐fed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1125547888sd'>mice</sd-pretag> (<i>n</i>=3-5). (<b>H</b>) Ambulation (AMB) in 10‐mo‐old RD‐fed (<i>n</i>=4) and HFD‐fed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag463695785sd'>mice</sd-pretag> (<i>n</i>=3-5). (<b>I</b>) <sd-pretag parent-tag-id='494' id='sdPretag547954211sd'>Hematoxylin and eosin</sd-pretag> (HE)‐stained <sd-pretag parent-tag-id='4' id='sdPretag1181136243sd'>epididymal white adipose tissue</sd-pretag> (<sd-pretag parent-tag-id='5' id='sdPretag493956624sd'>eWAT</sd-pretag>) from 6‐mo‐old RD‐fed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1856855714sd'>mice</sd-pretag>. Quantification for cell size is shown (<i>n</i>=3). Scale bar, 50 μm. (<b>J</b>) <sd-pretag parent-tag-id='159' id='sdPretag1218005388sd'>Adβ3</sd-pretag> mRNA in <sd-pretag parent-tag-id='5' id='sdPretag1515526027sd'>eWAT</sd-pretag> from 10‐mo‐old RD‐fed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag301911598sd'>mice</sd-pretag> (<i>n</i>=4), and in (<b>K</b>) <sd-pretag role='component' id='sdPretag859018651sme' type='tissue' >inguinal</sd-pretag> (<sd-pretag parent-tag-id='33' id='sdPretag1115856413sd'>ingWAT</sd-pretag>) from <sd-pretag parent-tag-id='114' id='sdPretag476430176sd'>4‐mo‐old</sd-pretag> <sd-pretag parent-tag-id='396' id='sdPretag680245194sd'>cold</sd-pretag>‐challenged RD‐fed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1138558745sd'>mice</sd-pretag> (<i>n</i>=3). (<b>L</b>) <sd-pretag parent-tag-id='400' id='sdPretag986735196sd'>Serum</sd-pretag>‐<sd-pretag parent-tag-id='401' id='sdPretag53951552sd'>free fatty acid</sd-pretag> (FFA) and <sd-pretag parent-tag-id='171' id='sdPretag1991896790sd'>glycerol</sd-pretag> (Gly) from 4‐ to 6‐mo‐old Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1953760904sd'>mice</sd-pretag> untreated (basal) or treated with <sd-pretag parent-tag-id='173' id='sdPretag1926714641sd'>isoproterenol</sd-pretag> (<sd-pretag role='intervention' id='sdPretag78095795sme' type='molecule' >Iso</sd-pretag>) i.p. for 20 min (<i>n</i>=4-6), and (<b>M</b>) from <sd-pretag parent-tag-id='114' id='sdPretag405368150sd'>4‐mo‐old</sd-pretag> RD‐fed <sd-pretag parent-tag-id='396' id='sdPretag884549702sd'>cold</sd-pretag>‐challenged (<sd-pretag parent-tag-id='116' id='sdPretag625511589sd'>75 min</sd-pretag>) <sd-pretag parent-tag-id='16' id='sdPretag1889125153sd'>mice</sd-pretag> (<i>n</i>=3-4). Values are mean±s.e. *<i>P</i>0.05, **<i>P</i>0.01, ***<i>P</i>0.001.</sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=1702
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "11556",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "11556",
"original_type": "gene",
"role": "assayed",
"text": "Adβ3",
"type": "geneprod",
"uniprot_ids": [
"P25962",
"Q3UP63"
]
}
] |
|
28569221
|
10.1038/embor.2013.111
|
Autophagy in Myf5+ progenitors regulates energy and glucose homeostasis through control of brown fat and skeletal muscle development
|
2013
|
Figure 4
|
<sd-panel>Beige cells in <sd-pretag parent-tag-id='32' id='sdPretag393982345sd'>inguinal white adipose tissue</sd-pretag> (<sd-pretag parent-tag-id='33' id='sdPretag947210568sd'>ingWAT</sd-pretag>) and <sd-pretag role='component' id='sdPretag1999827059sme' type='tissue' >brown adipose tissue</sd-pretag> (<sd-pretag parent-tag-id='1' id='sdPretag466967213sd'>BAT</sd-pretag>) contribute to increased energy expenditure in knock out (KO) <sd-pretag parent-tag-id='16' id='sdPretag1022460760sd'>mice</sd-pretag>. (<b>A</b>,<b>B</b>) <sd-pretag parent-tag-id='180' id='sdPretag686301121sd'><i>Tmem26</i></sd-pretag> and <sd-pretag parent-tag-id='181' id='sdPretag825758807sd'><i>tbx1</i></sd-pretag> mRNA levels in <sd-pretag role='component' id='sdPretag1415135837sme' type='tissue' >epididymal eWAT</sd-pretag>, and (<b>C</b>,<b>D</b>) <sd-pretag parent-tag-id='33' id='sdPretag842111934sd'>ingWAT</sd-pretag>, and (<b>E</b>,<b>F</b>) <sd-pretag parent-tag-id='494' id='sdPretag1397093667sd'>hematoxylin and eosin</sd-pretag> (HE)‐stained <sd-pretag parent-tag-id='33' id='sdPretag1354808470sd'>ingWAT</sd-pretag> section from <i>Atg7</i><sup>Flox/Flox</sup> <sd-pretag parent-tag-id='16' id='sdPretag1340974644sd'>mice</sd-pretag> (4 months (mo)) injected in <sd-pretag parent-tag-id='1' id='sdPretag1380696043sd'>BAT</sd-pretag> with empty (Con AdV) or <sd-pretag parent-tag-id='184' id='sdPretag2127397793sd'>Cre</sd-pretag>‐expressing (<sd-pretag parent-tag-id='184' id='sdPretag1229465864sd'>Cre</sd-pretag> AdV) <sd-pretag parent-tag-id='185' id='sdPretag799011557sd'>adenoviruses</sd-pretag> and <sd-pretag parent-tag-id='396' id='sdPretag2042708868sd'>cold</sd-pretag>‐exposed or not (basal) for <sd-pretag parent-tag-id='116' id='sdPretag339742570sd'>75 min</sd-pretag> (<i>n</i>=4-5). (<b>G</b>) mRNA for indicated genes in <sd-pretag parent-tag-id='33' id='sdPretag747038691sd'>ingWAT</sd-pretag> from <sd-pretag parent-tag-id='114' id='sdPretag373294969sd'>4‐mo‐old</sd-pretag> <sd-pretag parent-tag-id='396' id='sdPretag1181407343sd'>cold</sd-pretag>‐exposed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1512404743sd'>mice</sd-pretag> (<i>n</i>=4). (<b>H,I</b>) mRNA levels in <sd-pretag parent-tag-id='5' id='sdPretag123183225sd'>eWAT</sd-pretag> from 10‐mo‐old Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1782781754sd'>mice</sd-pretag> (<i>n</i>=4). (<b>J</b>) β‐oxidation in <sd-pretag parent-tag-id='5' id='sdPretag483015202sd'>eWAT</sd-pretag> and <sd-pretag parent-tag-id='33' id='sdPretag368624477sd'>ingWAT</sd-pretag> from <sd-pretag parent-tag-id='114' id='sdPretag2068489502sd'>4‐mo‐old</sd-pretag> Con and KO <sd-pretag parent-tag-id='16' id='sdPretag323923862sd'>mice</sd-pretag> (<i>n</i>=4). (<b>K</b>) mRNA levels in <sd-pretag parent-tag-id='52' id='sdPretag876761229sd'>soleus</sd-pretag> from <sd-pretag parent-tag-id='114' id='sdPretag2036174312sd'>4‐mo‐old</sd-pretag> <sd-pretag parent-tag-id='396' id='sdPretag1126950339sd'>cold</sd-pretag>‐exposed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1887159127sd'>mice</sd-pretag> (<i>n</i>=3-6). (<b>L</b>) β‐oxidation in <sd-pretag parent-tag-id='52' id='sdPretag1519005233sd'>soleus</sd-pretag> and (<b>M</b>) <sd-pretag parent-tag-id='1' id='sdPretag260909447sd'>BAT</sd-pretag> from <sd-pretag parent-tag-id='114' id='sdPretag1788692054sd'>4‐mo‐old</sd-pretag> Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1562483771sd'>mice</sd-pretag> (<i>n</i>=4). Values are mean±s.e. *<i>P</i>0.05, **<i>P</i>0.01, ***<i>P</i>0.001.</sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=1703
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "74244",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "74244",
"original_type": "gene",
"role": "intervention",
"text": "Atg7",
"type": "geneprod",
"uniprot_ids": [
"Q9D906",
"A0A0A0MQN4",
"Q3TM87"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "21380",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "21380",
"original_type": "gene",
"role": "assayed",
"text": "tbx1",
"type": "geneprod",
"uniprot_ids": [
"P70323",
"B9EII4",
"F6ZP09"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "327766",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "327766",
"original_type": "gene",
"role": "assayed",
"text": "Tmem26",
"type": "geneprod",
"uniprot_ids": [
"Q3UP23",
"Q149T4"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "74244",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "74244",
"original_type": "gene",
"role": "intervention",
"text": "Atg7",
"type": "geneprod",
"uniprot_ids": [
"Q9D906",
"A0A0A0MQN4",
"Q3TM87"
]
}
] |
|
28569221
|
10.1038/embor.2013.111
|
Autophagy in Myf5+ progenitors regulates energy and glucose homeostasis through control of brown fat and skeletal muscle development
|
2013
|
Figure 5
|
<sd-panel>Knock out (KO) <sd-pretag parent-tag-id='16' id='sdPretag217004713sd'>mice</sd-pretag> show reduced <sd-pretag parent-tag-id='286' id='sdPretag367977178sd'>myofiber</sd-pretag> size and impaired <sd-pretag parent-tag-id='259' id='sdPretag822692641sd'>glucose</sd-pretag> clearance. (<b>A</b>) <sd-pretag parent-tag-id='426' id='sdPretag1908801182sd'>Blood</sd-pretag> <sd-pretag parent-tag-id='259' id='sdPretag82669766sd'>glucose</sd-pretag> in 6‐ to <sd-pretag parent-tag-id='441' id='sdPretag1198218055sd'>10‐month (mo)‐old</sd-pretag> <sd-pretag parent-tag-id='16' id='sdPretag1429795418sd'>mice</sd-pretag> (<i>n</i>=12-15), and (<b>B</b>) <sd-pretag parent-tag-id='400' id='sdPretag775708972sd'>serum</sd-pretag> <sd-pretag parent-tag-id='314' id='sdPretag865490755sd'>insulin</sd-pretag> levels in 10‐mo‐old fed control (Con) and KO <sd-pretag parent-tag-id='16' id='sdPretag389375040sd'>mice</sd-pretag> (<i>n</i>=6). (<b>C</b>) <sd-pretag parent-tag-id='259' id='sdPretag886356207sd'>Glucose</sd-pretag> tolerance tests in 10‐mo‐old chow diet (RD)‐fed (<i>n</i>=5), and (<b>D</b>) in 10‐ to 12‐mo‐old high‐<sd-pretag parent-tag-id='346' id='sdPretag2066013069sd'>fat</sd-pretag> diet (HFD)‐fed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1043256877sd'>mice</sd-pretag> (<i>n</i>=4-9). (<b>E</b>) <sd-pretag parent-tag-id='314' id='sdPretag522881469sd'>Insulin</sd-pretag> tolerance test in 10‐mo‐old RD‐fed Con and KO <sd-pretag parent-tag-id='16' id='sdPretag405336381sd'>mice</sd-pretag> (<i>n</i>=5). (<b>F</b>) <sd-pretag parent-tag-id='8' id='sdPretag737695157sd'>Immunoblots</sd-pretag> for indicated proteins in <sd-pretag parent-tag-id='271' id='sdPretag831664401sd'>skeletal muscle</sd-pretag> from 10‐mo‐old Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1377955224sd'>mice</sd-pretag>. (<b>G</b>) <sd-pretag parent-tag-id='494' id='sdPretag404884184sd'>Hematoxylin and eosin</sd-pretag> (<sd-pretag category='assay' id='sdPretag1257827052sme'>HE</sd-pretag>)‐stained <sd-pretag parent-tag-id='51' id='sdPretag55872059sd'>gastrocnemius</sd-pretag> (GA) sections from 6‐mo‐old Con and KO <sd-pretag parent-tag-id='16' id='sdPretag1014855467sd'>mice</sd-pretag>. <sd-pretag parent-tag-id='286' id='sdPretag687506860sd'>Myofiber</sd-pretag> cross‐sectional area (μm<sup>2</sup>) is shown (<i>n</i>=3). (<b>H</b>) Expression of <sd-pretag parent-tag-id='287' id='sdPretag1503919314sd'>MuRF‐1</sd-pretag> and <sd-pretag parent-tag-id='288' id='sdPretag1388028132sd'>Atrogin‐1</sd-pretag> in GA and TA (<i>n</i>=4) and (<b>I</b>) myogenic genes in GA from 10‐mo‐old Con and KO <sd-pretag parent-tag-id='16' id='sdPretag968506772sd'>mice</sd-pretag> (<i>n</i>=6). (<b>J</b>) <sd-pretag parent-tag-id='8' id='sdPretag139166631sd'>Immunoblots</sd-pretag> for indicated proteins in tissues from <sd-pretag parent-tag-id='114' id='sdPretag658367629sd'>4‐mo‐old</sd-pretag> Con and KO <sd-pretag parent-tag-id='16' id='sdPretag946864165sd'>mice</sd-pretag> (<i>n</i>=4). (<b>K</b>) mRNA for <sd-pretag parent-tag-id='319' id='sdPretag1350757261sd'>IRS1</sd-pretag>, (<b>L</b>) <sd-pretag parent-tag-id='320' id='sdPretag1165052301sd'>IRS2</sd-pretag> in <sd-pretag parent-tag-id='4' id='sdPretag1529324101sd'>epididymal white adipose tissue</sd-pretag> (<sd-pretag parent-tag-id='5' id='sdPretag455687179sd'>eWAT</sd-pretag>) and GA from 10‐mo‐old Con and KO <sd-pretag parent-tag-id='16' id='sdPretag285307412sd'>mice</sd-pretag> (<i>n</i>=6). Values are mean±s.e. *<i>P</i>0.05, **<i>P</i>0.01, ***<i>P</i>0.001.</sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=1704
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "67731",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "67731",
"original_type": "gene",
"role": "assayed",
"text": "Atrogin‐1",
"type": "geneprod",
"uniprot_ids": [
"Q9CPU7"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "433766",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "433766",
"original_type": "gene",
"role": "assayed",
"text": "MuRF‐1",
"type": "geneprod",
"uniprot_ids": [
"B9EJZ0",
"F8VPZ1",
"Q8CFN7"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "16367",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "16367",
"original_type": "gene",
"role": "assayed",
"text": "IRS1",
"type": "geneprod",
"uniprot_ids": [
"P35569",
"Q543V3"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "16368",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "unmapped",
"mapping_status": "unmapped",
"ncbi_gene_id": "16368",
"original_type": "gene",
"role": "assayed",
"text": "IRS2",
"type": "geneprod",
"uniprot_ids": []
}
] |
|
28569221
|
10.1038/embor.2012.208
|
Structure of the Atg12–Atg5 conjugate reveals a platform for stimulating Atg8–PE conjugation
|
2012
|
Figure 1
|
Crystal structure of the Atg12–Atg5 conjugate bound to Atg16N. (<b>A</b>) Ribbon representation of the Atg12–Atg5 conjugate bound to Atg16N. Atg12, Atg5 and Atg6 are coloured salmon pink, green and orange, respectively. Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 are shown with a stick model. The disordered C‐terminal region of Atg12 (residues 182–184) is indicated with a broken line. The left figure was obtained by a 90° rotation of the right figure along the vertical axis. The amino‐ and carboxy‐termini are denoted as N and C, respectively. All of the figures representing the molecular structures were generated with PyMOL [<link href="#embr2012208-bib-0026">26</link>]. (<b>B</b>) Stereo view of the detailed interactions between Atg12 and Atg5. The side chains of the residues involved in the Atg12–Atg5 interaction are shown with a stick model, in which nitrogen and oxygen atoms are coloured red and blue, respectively. (<b>C</b>) Electron density map of the isopeptide linkage between Atg12 Gly 186 and Atg5 Lys 149. The simulated annealing <i>F</i>o–<i>F</i>c difference Fourier map was calculated by omitting Atg12 Phe 185 and Gly 186 and Atg5 Lys 149, and is shown with black meshes at 5.0σ. (<b>D</b>) Superimposition of the Atg5‐Atg16N complex structure (PDB ID 2DYM) on that of the Atg12–Atg5‐Atg16N complex. The Atg12–Atg5‐Atg16N complex is coloured red, while the Atg5‐Atg16N complex is coloured grey. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model. (<b>E</b>) <i>In vivo</i> analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non‐covalent Atg12‐Atg5 interactions. Yeast cells with or without starvation were lysed and subjected to urea SDS–polyacrylamide gel electrophoresis (PAGE) followed by western blotting. Ape1, aminopeptidase I; mApe1, mature form of Ape1; PE, phosphatidylethanolamine; prApe1, preform of Ape1; WT, wild‐type.
|
https://api.sourcedata.io/file.php?figure_id=1666
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P38316",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Atg12",
"type": "geneprod",
"uniprot_ids": [
"P38316"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38316",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Atg12",
"type": "geneprod",
"uniprot_ids": [
"P38316"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38316",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Atg12",
"type": "geneprod",
"uniprot_ids": [
"P38316"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38316",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Atg12",
"type": "geneprod",
"uniprot_ids": [
"P38316"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q03818",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Atg16",
"type": "geneprod",
"uniprot_ids": [
"Q03818"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q12380",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Atg5",
"type": "geneprod",
"uniprot_ids": [
"Q12380"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q12380",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Atg5",
"type": "geneprod",
"uniprot_ids": [
"Q12380"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q12380",
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] |
|
28569221
|
10.1038/embor.2012.208
|
Structure of the Atg12–Atg5 conjugate reveals a platform for stimulating Atg8–PE conjugation
|
2012
|
Figure 2
|
Atg12 functions as a binding module for Atg3. (<b>A</b>) <i>In vitro</i> pull‐down assay using recombinant plant Atg homologues. (<b>B</b>) Mapping of the mutated residues on the Atg12–Atg5‐Atg16N complex structure. Atg5 and Atg16N are shown with a ribbon model, whereas Atg12 is shown with both surface and ribbon models. The side chains of Tyr 147, Tyr 149 and Phe 154 of Atg12 are shown with a stick model and coloured grey (Tyr 147) or blue (Tyr 149 and Phe 154). Corresponding AtAtg12b residues are indicated in parentheses. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model. (<b>C</b>) Proposed model of Atg8–PE conjugate formation mediated by the Atg12–Atg5‐Atg16 complex. CBB, Coomassie Brilliant Blue staining; GST, glutathione <i>S</i>‐transferase; HA, haemagglutinin; PE, phosphatidylethanolamine; WT, wild‐type.
|
https://api.sourcedata.io/file.php?figure_id=1667
|
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] |
|
28569221
|
10.1038/embor.2013.151
|
Structural coupling of the EF hand and C‐terminal GTPase domains in the mitochondrial protein Miro
|
2013
|
Figure 1
|
<sd-panel>Domain architecture and structure of <sd-pretag parent-tag-id='1' id='sdPretag988607498sd'>Miro</sd-pretag>. (<b>A</b>) Bar diagram of <sd-pretag parent-tag-id='1' id='sdPretag633392624sd'>Miro</sd-pretag> domain structure. Numbering corresponds to dMiro isoform D, domain names are described in the text (<b>B</b>) Ribbon diagram of the <sd-pretag parent-tag-id='4' id='sdPretag1386951386sd'>MiroS</sd-pretag> structure in the "side view". Note three distinct domains: <sd-pretag role='assayed' id='sdPretag1367907262sme' type='geneprod' >ELM1</sd-pretag> (blue), <sd-pretag role='assayed' id='sdPretag1947540215sme' type='geneprod' >ELM2</sd-pretag> (green), <sd-pretag role='assayed' id='sdPretag2118993452sme' type='geneprod' >cGTPase</sd-pretag> (grey/yellow), joined by two linkers: <sd-pretag role='assayed' id='sdPretag577350837sme' type='geneprod' >Lnk1</sd-pretag> (orange), and <sd-pretag role='assayed' id='sdPretag705554579sme' type='geneprod' >Lnk2</sd-pretag> (red). The structure shown is <sd-pretag role='reporter' id='sdPretag1829268717sme' type='geneprod' >MgGDP</sd-pretag>‐<sd-pretag parent-tag-id='4' id='sdPretag757175606sd'>MiroS</sd-pretag>, with the <sd-pretag parent-tag-id='5' id='sdPretag1829578572sd'>Ca<sup>2+</sup></sd-pretag> ion from the Ca‐<sd-pretag parent-tag-id='4' id='sdPretag1675764457sd'>MiroS</sd-pretag> structure overlaid for orientation (<sd-pretag parent-tag-id='5' id='sdPretag2036586055sd'>Ca<sup>2+</sup></sd-pretag>/<sd-pretag role='assayed' id='sdPretag98325171sme' type='geneprod' >Mg<sup>2</sup></sd-pretag><sup>+</sup>/<sd-pretag parent-tag-id='3' id='sdPretag1331082657sd'>GDP</sd-pretag> as yellow/purple/multicoloured spheres). <sd-pretag role='assayed' id='sdPretag747951136sme' type='geneprod' >cGTPase</sd-pretag>, C‐terminal <sd-pretag role='assayed' id='sdPretag33635121sme' type='geneprod' >GTPase</sd-pretag>; dMiro, <sd-pretag role='component' id='sdPretag79007978sme' type='organism' ><i>Drosophila</i></sd-pretag> <sd-pretag parent-tag-id='1' id='sdPretag1031084064sd'>Miro</sd-pretag>; ELM, EF hand pair with ligand mimic.</sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=1705
|
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"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "MiroS",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "MiroS",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
}
] |
|
28569221
|
10.1038/embor.2013.151
|
Structural coupling of the EF hand and C‐terminal GTPase domains in the mitochondrial protein Miro
|
2013
|
Figure 2
|
<sd-panel>Miro ELM domains. (<b>A</b>) Top, <sd-pretag parent-tag-id='4' id='sdPretag2032693057sd'>MiroS</sd-pretag> shown in the "top view". <sd-pretag role='assayed' id='sdPretag1043588948sme' type='geneprod' >ELM1</sd-pretag> and <sd-pretag role='assayed' id='sdPretag567300341sme' type='geneprod' >ELM2</sd-pretag> are related by pseudo twofold symmetry, represented by the dyad between them. Bottom, overlay of the two ELM domains (rmsd of 1.8 Å over 84 Cα positions). <sd-pretag role='assayed' id='sdPretag116199936sme' type='geneprod' >ELM1</sd-pretag> (blue), <sd-pretag role='assayed' id='sdPretag113003511sme' type='geneprod' >ELM2</sd-pretag> (green) and identical or similar conserved residues in identical positions are shown as sticks. (<b>B</b>) Comparison of the ELM domain hydrophobic cores. The <sd-pretag role='assayed' id='sdPretag1426609899sme' type='geneprod' >ELM1</sd-pretag> core contains the UNL, modelled here as <sd-pretag role='component' id='sdPretag208845994sme' type='molecule' >homoserine</sd-pretag> shown in <sd-pretag role='component' id='sdPretag1222516069sme' type='tissue' >spheres</sd-pretag>, occupying a ∼442 Å<sup>3</sup> cavity highlighted with mesh. The <sd-pretag role='intervention' id='sdPretag2141962453sme' type='geneprod' >ELM2</sd-pretag> core includes three tryptophans in close proximity (W367, W391, W395 in orange). Hydrophobic residues in the LM/EF hand pair interfaces are also shown in sticks. LM, ligand mimic; UNL, unidentified ligand.</sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=1706
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "MiroS",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
}
] |
|
28569221
|
10.1038/embor.2013.151
|
Structural coupling of the EF hand and C‐terminal GTPase domains in the mitochondrial protein Miro
|
2013
|
Figure 4
|
<sd-panel>The <sd-pretag role='assayed' id='sdPretag2130992242sme' type='geneprod' >cGTPase</sd-pretag> and its interface. (<b>A</b>) The <sd-pretag role='assayed' id='sdPretag744679274sme' type='geneprod' >ELM2</sd-pretag>/<sd-pretag role='assayed' id='sdPretag912857899sme' type='geneprod' >cGTPase</sd-pretag> interface can be divided into three regions (insets 1-3). Select residues are shown as sticks, polar contacts as dashed lines, and <sd-pretag role='assayed' id='sdPretag182672200sme' type='geneprod' >Mg<sup>2</sup></sd-pretag><sup>+</sup> and <sd-pretag role='assayed' id='sdPretag1423573873sme' type='geneprod' >MgGDP</sd-pretag> as spheres. Region 1: αC2 and the <sd-pretag role='component' id='sdPretag922960357sme' type='cell' >hEF2</sd-pretag> loop form both backbone and side‐chain contacts with αG1 and Switch I in the <sd-pretag role='assayed' id='sdPretag794973179sme' type='geneprod' >cGTPase</sd-pretag>, including the Switch I threonine T481. Region 2: αD2 in <sd-pretag role='component' id='sdPretag139679056sme' type='geneprod' >hEF2</sd-pretag> packs against the twisting <sd-pretag role='intervention' id='sdPretag347623950sme' type='geneprod' >cGTPase</sd-pretag> β‐strands <sd-pretag role='component' id='sdPretag929900982sme' type='geneprod' >Gβ1‐3</sd-pretag>. Region 3: the candidate <sd-pretag parent-tag-id='25' id='sdPretag2055380008sd'>Pink1</sd-pretag> phosphorylation site residue S324 in <sd-pretag role='component' id='sdPretag200242093sme' type='geneprod' >Lnk1</sd-pretag> <sd-pretag role='component' id='sdPretag1531119946sme' type='molecule' >hydrogen</sd-pretag> bonds with R444 in <sd-pretag role='intervention' id='sdPretag456957749sme' type='geneprod' >Lnk2</sd-pretag>, which also forms a salt bridge with D521, at the end of the Switch II loop. (<b>B</b>) <sd-pretag role='assayed' id='sdPretag1474886482sme' type='geneprod' >cGTPase</sd-pretag> rearrangements in <sd-pretag role='component' id='sdPretag888778013sme' type='undefined' >MgGDP</sd-pretag> binding. Displayed is the <sd-pretag parent-tag-id='4' id='sdPretag2135258588sd'>MiroS</sd-pretag>‐<sd-pretag role='reporter' id='sdPretag751806636sme' type='geneprod' >MgGDP</sd-pretag> structure, with select residues from the aligned apo‐<sd-pretag parent-tag-id='4' id='sdPretag1865249839sd'>MiroS</sd-pretag> structure shown in orange. <sd-pretag parent-tag-id='3' id='sdPretag137629455sd'>GDP</sd-pretag> is shown as sticks (2Fo‐Fc map, 1.2σ), and <sd-pretag role='reporter' id='sdPretag670129530sme' type='geneprod' >Mg<sup>2</sup></sd-pretag><sup>+</sup> as a purple sphere. In the apo state, D505 in the Switch II <sup>503</sup>DIDV<sub>506</sub> motif is far from the nucleotide‐binding pocket and is <sd-pretag role='assayed' id='sdPretag710335047sme' type='molecule' >hydrogen</sd-pretag> bonded to <sd-pretag role='component' id='sdPretag2059667693sme' type='cell' >R507</sd-pretag>, which is also stabilized by interactions with P512. In the <sd-pretag role='reporter' id='sdPretag556131407sme' type='geneprod' >MgGDP</sd-pretag>‐bound state, the D505 side‐chain rotates ∼7 Å to interact with the β‐phosphate (arrow), locking the Switch II loop in a <sd-pretag role='intervention' id='sdPretag1635252406sme' type='geneprod' >β‐strand</sd-pretag> conformation and extending <sd-pretag role='assayed' id='sdPretag784535533sme' type='geneprod' >Gβ3</sd-pretag>. V506 rotates away from the nucleotide‐binding pocket, and both <sd-pretag role='component' id='sdPretag784982063sme' type='cell' >R507</sd-pretag> and <sd-pretag role='component' id='sdPretag607446106sme' type='cell' >Y537</sd-pretag> become solvent exposed. <sd-pretag role='assayed' id='sdPretag505324018sme' type='geneprod' >cGTPase</sd-pretag>, C‐terminal <sd-pretag role='assayed' id='sdPretag597204396sme' type='geneprod' >GTPase</sd-pretag>; Sw I/II, Switch I/II; P loop, phosphate‐binding loop.</sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=1708
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "MiroS",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "MiroS",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
}
] |
|
28569221
|
10.1038/embor.2013.151
|
Structural coupling of the EF hand and C‐terminal GTPase domains in the mitochondrial protein Miro
|
2013
|
Figure 5
|
<sd-panel></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=1709
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Miro",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Miro",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Miro",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Miro",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Miro1",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Miro1",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "MiroS",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q8IMX7",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "MiroS",
"type": "geneprod",
"uniprot_ids": [
"Q8IMX7"
]
},
{
"ext_dbs": "Uniprot///Uniprot",
"ext_ids": "Q86LE7///Q86CZ3",
"ext_tax_ids": "7227///7227",
"ext_tax_names": "Drosophila melanogaster///Drosophila melanogaster",
"mapping_source": "unknown",
"mapping_status": "unmapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Parkin",
"type": "geneprod",
"uniprot_ids": []
},
{
"ext_dbs": "Uniprot///Uniprot",
"ext_ids": "Q86LE7///Q86CZ3",
"ext_tax_ids": "7227///7227",
"ext_tax_names": "Drosophila melanogaster///Drosophila melanogaster",
"mapping_source": "unknown",
"mapping_status": "unmapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Parkin",
"type": "geneprod",
"uniprot_ids": []
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q961J3",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Pink1",
"type": "geneprod",
"uniprot_ids": [
"Q961J3"
]
}
] |
|
25533845
|
10.1083/jcb.147.2.435
|
Formation process of autophagosome is traced with Apg8/Aut7p in yeast.
|
1999
|
Figure 1
|
<p>ALP assay. The cells expressing Pho8Δ60p were cultured in SD+CA medium (open bars) and shifted to SD(−N) medium for three hours (filled bars). The ALP activity of the lysates was measured to estimate autophagic activity. Wild-type cells, TN124; Δ<italic>apg8</italic> mutant harboring vector, TK303; Δ<italic>apg8</italic> mutant harboring <italic>APG8</italic> on a centromeric vector, TK301; Δ<italic>apg8</italic> mutant harboring 3 × HA-tagged APG8 plasmid, TK307.</p>
|
https://api.sourcedata.io/file.php?figure_id=4375
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "apg8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "APG8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "apg8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "apg8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "APG8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P11491",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "ALP",
"type": "geneprod",
"uniprot_ids": [
"P11491"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P11491",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "ALP",
"type": "geneprod",
"uniprot_ids": [
"P11491"
]
}
] |
|
25533845
|
10.1083/jcb.147.2.435
|
Formation process of autophagosome is traced with Apg8/Aut7p in yeast.
|
1999
|
Figure 3
|
<p>Expression of <italic>APG8</italic>. A, The lysates prepared from the growing cells were subjected to immunoblotting with the anti-Apg8p antibody. Lane 1, wild-type cells (YW5-1B); lane 2, Δ<italic>apg8</italic> cells harboring <italic>APG8</italic> on a multicopy vector (TK201); lane 3, Δ<italic>apg8</italic> cells (TK404). B, The wild-type (YW5-1B) cells were cultured in YEPD medium until 1–2 × 10<sup>7</sup> cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, 3, 4.5, and 6 h at 30°C. The amount of Apg8p was estimated by immunoblotting with anti-Apg8p antibody. Each lane has 10 μg of total protein. C, YW5-1B cells were cultured in YEPD medium until 1–2 × 10<sup>7</sup> cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, and 6 h at 30°C, and mRNA was prepared from each culture as described in Materials and Methods. <italic>APG8</italic> mRNA and <italic>ACT1</italic> mRNA were detected by Northern blotting with each specific probe. Each lane has 4 μg of total mRNA. D, YW5-1B cells were cultured in YEPD medium until 1 × 10<sup>7</sup> cells/ml at 30°C. Rapamycin was added to the culture at a final concentration of 0.2 μg/ml and the cells were further incubated in YEPD medium for two hours at 30°C. Western blotting was performed with the anti-Apg8p antibody. Each lane has 20 μg of total protein.</p>
|
https://api.sourcedata.io/file.php?figure_id=4377
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "apg8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "APG8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "apg8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "APG8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "assayed",
"text": "APG8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
}
] |
|
25533845
|
10.1083/jcb.147.2.435
|
Formation process of autophagosome is traced with Apg8/Aut7p in yeast.
|
1999
|
Figure 4
|
<p>Subcellular fractionation and solubilization of Apg8p. The wild-type cells (YW5-1B) growing logarithmically in YEPD medium or starved in SD(−N) medium for three hours were lysed. A, The lysates (T) were centrifuged at 13,000 <italic>g</italic> for 15 min to generate pellet (LSP) and supernatant (LSS). LSS fraction was further centrifuged at 100,000 <italic>g</italic> for one hour and separated to pellet (HSP) and supernatant (HSS). LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates. Equal volume of each sample was applied to each well, and immunoblotting was performed with anti-Apg8p antibody. B, The lysates (T) were sonicated and centrifuged at 100,000 <italic>g</italic> for one hour to separate pellet (P) and supernatant (S). The distribution of CPY and Apg8p was examined by immunoblotting with each specific antibody, respectively. C, The sonicated lysates were centrifuged at 100,000 <italic>g</italic> for one hour to generate pellet fraction. The pellet fraction was treated with 2% Triton X-100 (TX-100) for 30 min on ice. The sample was centrifuged at 100,000 <italic>g</italic> for one hour and separated to supernatant (S) and pellet (P) fractions. The distribution of Apg8p was examined by immunoblotting with the anti-Apg8p antibody.</p>
|
https://api.sourcedata.io/file.php?figure_id=4378
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P32319",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "CPY",
"type": "geneprod",
"uniprot_ids": [
"P32319"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
}
] |
|
25533845
|
10.1083/jcb.147.2.435
|
Formation process of autophagosome is traced with Apg8/Aut7p in yeast.
|
1999
|
Figure 5
|
<p>Immunofluores-cent staining of Δ<italic>apg8</italic> cells expressing 3 × HA–Apg8p. A, The Δ<italic>apg8</italic> cells harboring 3 × HA-tagged APG8 plasmid, TK114 cells, were grown until logarithmic phase. The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts. The spheroplasts were permeabilized by 0.5% Triton X-100, and incubated with the anti-HA antibody, 16B12, followed with the FITC-conjugated anti–mouse IgG. Left, Fluorescence image of 3 × HA–Apg8p; right, Nomarski image of the cells. Arrows show punctate signals that are a little larger than tiny dot signals. Bars, 10 μm. B, Immunofluorescent staining of YW5-1B cells before (0 h) and after (3 h) shift to starvation (negative control). Left, Fluorescence image; right, Nomarski image. Bars, 10 μm. C, The cells were shifted to starvation for 0, 0.5, and 3 h. Upper panels, Fluorescence images of 3 × HA–Apg8p; lower panels, Nomarski images of the cell. Bars, 5 μm.</p>
|
https://api.sourcedata.io/file.php?figure_id=4379
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
}
] |
|
25533845
|
10.1083/jcb.147.2.435
|
Formation process of autophagosome is traced with Apg8/Aut7p in yeast.
|
1999
|
Figure 6
|
<p>Change of Apg8p localization in Δ<italic>pep4</italic> cells after shift to starvation. The Δ<italic>apg8</italic>Δ<italic>pep4</italic> cells harboring 3 × HA-tagged APG8 plasmid, TK116 cells, were shifted to starvation for 0, 0.5, 1, 3, and 6 h. Immunofluorescence microscopy was performed as described in <xref ref-type="fig" rid="F5">Fig. 5</xref>. A–E, FITC staining of 3 × HA–Apg8p. F–J, Nomarski images of the cells. Arrows show an autophagic body. Bars, 5 μm.</p>
|
https://api.sourcedata.io/file.php?figure_id=4380
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
}
] |
|
25533845
|
10.1083/jcb.147.2.435
|
Formation process of autophagosome is traced with Apg8/Aut7p in yeast.
|
1999
|
Figure 7
|
<p>Immunostaining image of a cell containing autophagic bodies in the vacuole. The Δ<italic>apg8</italic>Δ<italic>pep4</italic> cells expressing 3 × HA–Apg8p, TK116 cells, were shifted to SD(−N) medium for two hours. Cells were immunolabeled with anti-HA mAb, 16B12, followed by 10-nm gold-conjugated goat anti–mouse IgG. AB, autophagic body; N, nucleus; V, vacuole.</p>
|
https://api.sourcedata.io/file.php?figure_id=4381
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
}
] |
|
25533845
|
10.1083/jcb.147.2.435
|
Formation process of autophagosome is traced with Apg8/Aut7p in yeast.
|
1999
|
Figure 8
|
<p>Immuno-EM of autophagosomes. A and B, Mature autophagosome. C and D, Premature autophagosome. Arrows show expanded regions of the intramembrane space of the premature autophagosomes. TK116 cells were incubated in SD(−N) medium for one hour (D), two hours (B and C), or three hours (A). The localization of 3 × HA–Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A and D) or 10-nm gold- (B and C) conjugated goat anti–mouse IgG. AP, autophagosome; N, nucleus; V, vacuole.</p>
|
https://api.sourcedata.io/file.php?figure_id=4382
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
}
] |
|
25533845
|
10.1083/jcb.147.2.435
|
Formation process of autophagosome is traced with Apg8/Aut7p in yeast.
|
1999
|
Figure 9
|
<p>Possible intermediate structures of autophagosome. A, A membrane sac under construction. Arrows show gold particles. B and C, Isolation membranes. B, Isolation membrane is detected along the arrows. C, The small arrow shows a semicircular isolation membrane and a large arrow marks its open area. D, Apg8p-residing structures gathered in the area close to the vacuole. TK116 cells were incubated in SD(−N) medium for one hour (A and C), or two hours (B and D). The localization of 3 × HA–Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A) or 10-nm gold- (B–D) conjugated goat anti–mouse IgG. V, vacuole.</p>
|
https://api.sourcedata.io/file.php?figure_id=4383
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P38182",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Apg8p",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
}
] |
|
25533845
|
10.1083/jcb.147.2.435
|
Formation process of autophagosome is traced with Apg8/Aut7p in yeast.
|
1999
|
Figure 10
|
<p>Fine morphology of Δ<italic>ypt7</italic> and Δ<italic>apg8</italic> cells and proteinase-K accession to proAPI in these mutants. A, EM image of Δ<italic>ypt7</italic> cell starved in SD(−N) medium for four hours. Autophagosomes (AP, arrows) were accumulated. B, EM image of the starved Δ<italic>apg8</italic> cell. C–E, The representatives of the membrane structures detected in the starved Δ<italic>apg8</italic> cells. C, Autophagosome-like structure indistinguishable from autophagosome. D and E, Aberrant multivesicular structures. N, nucleus; V, vacuole. F, Proteinase K-sensitivity of proAPI. Cell lysates were prepared from Δ<italic>ypt7</italic> and Δ<italic>apg8</italic> cells starved in SD(−N) medium for 4.5 h. The lysates were treated with 100 μg/ml proteinase K in the presence or absence of Triton X-100. Asterisk shows mature API and digestion product of proAPI by proteinase K.</p>
|
https://api.sourcedata.io/file.php?figure_id=4384
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "apg8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "apg8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "apg8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "852200",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "852200",
"original_type": "gene",
"role": "intervention",
"text": "apg8",
"type": "geneprod",
"uniprot_ids": [
"P38182"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "855012",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "855012",
"original_type": "gene",
"role": "intervention",
"text": "ypt7",
"type": "geneprod",
"uniprot_ids": [
"P32939"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "855012",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "855012",
"original_type": "gene",
"role": "intervention",
"text": "ypt7",
"type": "geneprod",
"uniprot_ids": [
"P32939"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "855012",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "855012",
"original_type": "gene",
"role": "intervention",
"text": "ypt7",
"type": "geneprod",
"uniprot_ids": [
"P32939"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P14904",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "proAPI",
"type": "geneprod",
"uniprot_ids": [
"P14904"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P14904",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "proAPI",
"type": "geneprod",
"uniprot_ids": [
"P14904"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P14904",
"ext_tax_ids": "559292",
"ext_tax_names": "Saccharomyces cerevisiae S288C",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "proAPI",
"type": "geneprod",
"uniprot_ids": [
"P14904"
]
}
] |
|
10.15252/embj.2019103476
|
Multilineage stem cell-derived organoids recapitulate murine distal lung development and disease
|
2020
|
Figure 1
|
<sd-panel> <p><strong>Figure 1. 3D co-culture of BASC and rMC results in the formation of BALO</strong></p> <ol type="a"> <li> <p><strong>Gating strategy for sorting of EpCAM<sup>high</sup>CD24<sup>low</sup>Sca‑1<sup>+</sup> cells and rMC from lung homogenate of adult mice.</strong></p> </li> <li> <p><strong>Time-course of epithelial stem/progenitor cell proliferation and differentiation within BALO at days 8, 11 and 21 of co-culture with rMC.</strong></p> </li> <li> <p><strong>Clonal expansion of EpCAM<sup>high</sup>CD24<sup>low</sup>Sca‑1<sup>+</sup> cells derived from either GFP-expressing mice or tdTomato-expressing ROSA<sup>mT/mG</sup> mice at day 21 of co‑culture.</strong></p> </li> <li> <p><strong>Percentages of SCGB1A1<sup>+</sup>SFTPC<sup>+</sup> (BALO), SCGB1A1<sup>+</sup>SFTPC<sup>-</sup> (bronchiolospheres) and SCGB1A1<sup>-</sup>SFTPC<sup>+</sup> (alveolospheres) organoids per well at day 21 of culture derived from EpCAM<sup>high</sup>CD24<sup>low</sup>Sca-1<sup>+</sup> cells isolated from <em>Scgb1a1<sup>mCherry</sup>Sftpc<sup>YFP</sup></em> reporter mice (n=4 biological replicates).</strong></p> </li> <li> <p><strong>Representative confocal images of days 8 to 21 of culture showing endogenous SCGB1A1 and SFTPC expression during BALO formation derived from EpCAM<sup>high</sup>CD24<sup>low</sup>Sca-1<sup>+</sup>SCGB1A1<sup>+</sup>SFTPC<sup>+</sup> cells isolated from <em>Scgb1a1<sup>mCherry</sup>Sftpc<sup>YFP</sup></em> reporter mice.</strong></p> </li> </ol> <p><strong>F-H. Heat map (left) and tSNE plot (right) (F) of digested day 21 BALO cultures depicting four distinct clusters (C1, airway, blue; C2, alveolar, purple; C3, MYO, orange, and C4, LIF, green). Violin plots of selected genes representing airway associated-genes (G) (<em>Itgb4, Trp63, Krt7,</em> and <em>Sox2</em>) and alveoli associated‑genes (H) (Cxcl15, <em>Lyz2, Sftpc,</em> and <em>Hopx</em>). Each violin plot shows the frequency distribution of the mean transcript level (log<sub>2</sub>).</strong></p> <p><strong>Data information: Scale bars = 100 μm (B, C right, E) or 500 μm (C left). Bar charts presented as the mean ± S.E.M.</strong></p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35366
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P54254",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Sca‑1",
"type": "geneprod",
"uniprot_ids": [
"P54254"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P24807",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "CD24",
"type": "geneprod",
"uniprot_ids": [
"P24807"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q99JW5",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "EpCAM",
"type": "geneprod",
"uniprot_ids": [
"Q99JW5"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P54254",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Sca‑1",
"type": "geneprod",
"uniprot_ids": [
"P54254"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P24807",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "CD24",
"type": "geneprod",
"uniprot_ids": [
"P24807"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q99JW5",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "EpCAM",
"type": "geneprod",
"uniprot_ids": [
"Q99JW5"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q06318",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "SCGB1A1",
"type": "geneprod",
"uniprot_ids": [
"Q06318"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q06318",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "SCGB1A1",
"type": "geneprod",
"uniprot_ids": [
"Q06318"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q06318",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
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||
10.15252/embj.2019103476
|
Multilineage stem cell-derived organoids recapitulate murine distal lung development and disease
|
2020
|
Figure 2
|
<sd-panel> <p><strong>Figure 2. The BALO model mimics the 3D morphology and cellular composition of the bronchioalveolar compartment with proximo-distal patterning and pulmonary surfactant secretion</strong></p> <ol type="a"> <li> <p><strong>mRNA expression analysis of epithelial cell differentiation markers <em>Sftpc</em> (AEC II), <em>Hopx</em> (AEC I), <em>Foxj1</em> (ciliated cells), <em>Muc5ac</em> (secretory cells) and <em>p63</em> (basal cells) in BALO at days 0, 10, and 21 of culture (n=3 biological replicates with pooled cells from 4 cultures per replicate)</strong></p> </li> <li> <p><strong>Electron microscopy of BALO alveoli showing two cuboidal epithelial cells (AEC II) connected by tight junctions (red square) with numerous mitochondria (M) and lamellar bodies (LB). A LIF with numerous lipid droplets (*) and a MYO with cisterns of rough ER (red arrow) are located at the basal side (left). The alveolar lumen is filled with lamellar surfactant including tubular myelin (white arrow) (lower right). Exocytosis of a lamellar body (*) from an AEC II (upper right). Scale bars indicate 500 nm.</strong></p> </li> <li> <p><strong>Electron microscopy of bronchiolar-like airway depicting columnar ciliated cells (red arrowhead) with basal bodies (black arrowhead) at the left side of the longitudinally sectioned lumen. The boxed area indicates an apical junctional complex between two ciliated cells. 1 = tight junction, 2 = adherens junction. Scale bar indicates 500 nm (in insert: 100 nm).</strong></p> </li> <li> <p><strong>Staining of lamellar bodies in BALO with RFP LysoTracker. Scale bars represent 100 μm.</strong></p> </li> <li> <p><strong>Western blot analysis of the surfactant proteins: pro-SPC, mature SPC, pro-SPB, mature SPB, and SPA in lung homogenate (LH), AEC and day 21 BALO (n=3 biological replicates).</strong></p> </li> </ol> <p><strong>F-G. Alveolar diameter (F) and number of alveoli (G) in BALO at days 15, 21, 30 and 40 of culture was measured from n=5 BALO in n=3 biological replicates with tdTomato<sup>+</sup> rMC.</strong></p> <p><strong>H-I. Representative scheme and images of day 40 BALO alveoli (H) and airway (I). BALO alveolar-like structures (H) are shown (h') (red arrowheads) in semithin section (0.5 µm) stained with Toluidine blue. Scale bar indicates 100 μm (left). Electron microscopy showing AEC I (h'') ultrastructure within BALO. Scale bars indicate 2500 nm (center) and 1000 nm (right). An airway-like structure (i') is shown with secretory and ciliated cells (red arrowheads) in semithin sections (0.5 µm) longitudinally cut and stained with Toluidine blue. Scale bar indicates 50 μm (far left). Electron microscopy of a bronchiolar-like airway (I) depicting pseudostratified epithelium (i'') with a basal-like cell (BC), not reaching the lumen in which cilia (C) are seen, located between a secretory (SC) and a ciliated cell (Ci). Scale bars indicate 1000 nm (left and right). Mature cilia (i''') in BALO at higher magnification depicting the 9x2+2 structure with central doubled microtubules (insert, red arrowhead), a characteristic for motile cilia. Scale bar indicates 100 nm (far right).</strong></p> <p><strong>Data information: Bar and dot charts presented as the mean ± S.E.M. and probability determined using one‐way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).</strong></p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35368
|
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||
10.15252/embj.2019103476
|
Multilineage stem cell-derived organoids recapitulate murine distal lung development and disease
|
2020
|
Figure 3
|
<sd-panel> <p><strong>Figure 3. Distinct subsets of EpCAM<sup>-</sup>Sca-1<sup>+</sup> rMC-derived fibroblasts are indispensable for BALO growth, cell differentiation, and branching morphogenesis, and model the mesenchymal niche of the lung</strong></p> <ol type="a"> <li> <p>Representative images of day 21 BALO and tdTomato<sup>+</sup> rMC stained with LipidTOX (green). Scale bar represents 100 μm.</p> </li> <li> <p>Fluorescence images of αSMA (green) and neutral lipids (LipidTOX red) staining in WT‑derived rMC and BALO (dotted lines indicate single BALO or insert) at day 21 of culture. Scale bars represent 50 μm (left) and 25 μm (right).</p> </li> <li> <p>Representative flow cytometric dot plots and histograms of PDGFRα expression in rMC (EpCAM<sup>-</sup>CD45<sup>-</sup>CD31<sup>-</sup>Sca-1<sup>+</sup>) isolated from the lung homogenate of <em>Pdgfra<sup>GFP</sup></em> reporter mice.</p> </li> <li> <p>Representative images of BALO formation at day 8, 15 and 21 of co-culture. WT BASC were co‑cultivated with sorted rMC (total PDGFRα+ population) or rMC expressing either low or high levels of PDGFRα‑GFP. Scale bars represent 100 μm.</p> </li> <li> <p>tSNE plots and violin plots depicting selected genes representing MYO (<em>Pdgfrα, Tagln, Acta2, Eln,</em> and <em>Axin2</em>) (top panels) and LIF associated-genes (<em>Fgf10, Apoe, Serpina3n, Gsn,</em> and <em>Gas6</em>) (bottom panels). Each violin plot shows the frequency distribution of the mean transcript level (log<sub>2</sub>). C3 (MYO, orange) and C4 (LIF, green) refer to the scRNA-Seq experiment in Fig. 1G.</p> </li> <li> <p>Fluorescence image of αSMA<sup>+</sup>PDGFRα<sup>high</sup> MYO (yellow arrows) and LipidTOX<sup>+</sup>PDGFRα<sup>low</sup> LIF (red arrows) from sorted PDGFRα-GFP rMC after 21 days of BALO culture. Scale bars represent 50 μm (left) and 25 μm (right).</p> </li> <li> <p>Fluorescence image containing PDGFRα<sup>high</sup> MYO from sorted PDGFRα-GFP rMC within BALO derived from tdTomato<sup>+</sup> mice at day 21 of culture. Scale bars represent 100 μm (left) and 50 μm (right).</p> </li> </ol> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35370
|
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"P26618"
]
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"P26618"
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"role": "assayed",
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"P26618"
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}
] |
||
10.15252/embj.2019103476
|
Multilineage stem cell-derived organoids recapitulate murine distal lung development and disease
|
2020
|
Figure 4
|
<sd-panel> <p><strong><br> Figure 4. Tissue-resident macrophages engraft into BALO alveolar-like regions, maintain their identity and drive epithelial cell differentiation</strong></p> <ol type="a"> <li> <p><strong>Gating strategy to define TR‑Mac from BAL of adult tdTomato<sup>+</sup> mice.</strong></p> </li> <li> <p><strong>Representative images of BALO after microinjection of tdTomato<sup>+</sup> TR-Mac at day 14. TR-Mac are preferentially found in alveoli (right). Scale bar represent 100 μm (left) and 50 μm (right).</strong></p> </li> <li> <p><strong>Fluorescence confocal images of CD206, Siglec-F, and isotype control staining 14 days after tdTomato<sup>+</sup> TR‑Mac microinjection in a day 28 BALO. Scale bars represent 100 μm (left) and 25 μm (right).</strong></p> </li> <li> <p><strong>Electron microscopy depicting filopodium of TR‑Mac (white dashed lines) with a characteristic actin filament bundle (yellow background) (left panel) in contact with AEC I within BALO alveolar-like structures. Uptake of surfactant by TR-mac is depicted by a (*). Scale bar indicates 1000nm (left, in insert: 500 nm) and 500 nm (right, in insert: 250 nm).</strong></p> </li> <li> <p><strong>Representative confocal images of Cx43 staining in tdTomato<sup>+</sup> TR-Mac monoculture in Matrigel and tdTomato<sup>+</sup> TR‑Mac microinjected at day 14 and analyzed in a mature day 21 BALO. Scale bars represent 50 μm (overview) and 5 μm (close up).</strong></p> </li> <li> <p><strong>Heat map (left) and tSNE plot (right) of the comparative analysis of digested day 23 BALO cultures with (+) and without (-) microinjected TR‑Mac depicting six distinct clusters (C1, club/secretory cells, red; C2, basal cells, yellow; C3, rMC, green; C4, AEC II, blue; C5, AEC I, purple; and C6, ciliated cells, pink).</strong></p> </li> <li> <p><strong>Expression data dot plots of genes found differentially regulated in cluster C1 between day 23 BALO with (C1+) and without (C1-) microinjected TR-Mac. The circle size illustrates the number of cells expressing a specific gene.</strong></p> </li> <li> <p><strong>Percentage of terminally differentiated cells (AEC I and ciliated cells) in day 23 BALO cultures with (+) and without (-) microinjected TR‑Mac.</strong></p> </li> </ol> <p><strong>Data information: Bar and dot charts presented as the mean ± S.E.M. and probability determined using t-test (**P < 0.01).</strong></p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35371
|
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"ext_ids": "Q61830",
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"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "CD206",
"type": "geneprod",
"uniprot_ids": [
"Q61830"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q80ZE2",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Siglec-F",
"type": "geneprod",
"uniprot_ids": [
"Q80ZE2"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P23242",
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"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Cx43",
"type": "geneprod",
"uniprot_ids": [
"P23242"
]
}
] |
||
10.15252/embj.2019103476
|
Multilineage stem cell-derived organoids recapitulate murine distal lung development and disease
|
2020
|
Figure 5
|
<sd-panel> <p><strong>Figure 5. Manipulation of WNT signaling pathway during BALO morphogenesis by knock-down of miR142-3p gene expression recapitulates developmental defects observed in embryonic lung explants.</strong></p> <ol type="a"> <li> <p><strong>Representative images of E11.5 lung explants after treatment for 48 h with 100 μM Scra or mo142-3p reveals reduced size and branching morphogenesis after miR142-3p knockdown.</strong></p> </li> <li> <p><strong>Organoid diameter (n=30-50 per group) in µm before addition of 4 μM Scra or mo142-3p at day 6 (control) and 5 days after treatment (at day 11 BALO culture) in n=3 biological replicates</strong></p> </li> </ol> <p><strong>C-D. Representative images of β-galactosidase staining in <em>TOPGAL</em> epithelium (C) and <em>TOPGAL</em> rMC (D) BALO cultures before (day 6, control) or 5 days after treatment with either 4 μM Scra or mo142-3p (day 11 of co-culture). BASC and rMC were isolated form the lung homogenate of TOPGAL mice. β-galactosidase<sup>+</sup> rMC at day 11 of culture are indicated with arrows.</strong></p> <ol start="5" type="A"> <li> <p><strong>Representative transmission and confocal images after LysoTracker staining indicating branching and number of branching points in n=4 BALO 15 days after treatment with 4 μM Scra or mo142-3p (day 21 of co-culture) in n=3 biological replicates.</strong></p> </li> <li> <p><strong>mRNA levels of epithelial and mesenchymal <em>miR142-3p</em> expression in Scra and mo142-3p-treated organoids 5 days after treatment (n=3-4 biological replicates with pooled cells from 4 cultures per replicate).</strong></p> </li> </ol> <p><strong>Data information: Scale bars=100 μm. Bar charts presented as the mean ± S.E.M. and probability determined using t-test ((*P < 0.05, **P < 0.01).</strong></p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35372
|
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"mapping_source": "unmapped",
"mapping_status": "unmapped",
"ncbi_gene_id": "387160",
"original_type": "gene",
"role": "intervention",
"text": "miR142-3p",
"type": "geneprod",
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},
{
"ext_dbs": "Uniprot",
"ext_ids": "P23780",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
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"mapping_status": "mapped",
"ncbi_gene_id": null,
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"text": "β-galactosidase",
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"P23780"
]
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{
"ext_dbs": "Uniprot",
"ext_ids": "P23780",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
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"role": "assayed",
"text": "β-galactosidase",
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"P23780"
]
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{
"ext_dbs": "Uniprot",
"ext_ids": "P23780",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "β-galactosidase",
"type": "geneprod",
"uniprot_ids": [
"P23780"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "387160",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "unmapped",
"mapping_status": "unmapped",
"ncbi_gene_id": "387160",
"original_type": "gene",
"role": "assayed",
"text": "miR142-3p",
"type": "geneprod",
"uniprot_ids": []
}
] |
||
10.15252/embj.2019103476
|
Multilineage stem cell-derived organoids recapitulate murine distal lung development and disease
|
2020
|
Figure 6
|
<sd-panel> <p><strong><br> Figure 6. BALO support influenza virus infection and allow modeling of lung infection and injury</strong></p> <ol type="a"> <li> <p><strong>Representative images of day 21 BALO after microinjection of SC35M-GFP IAV (green) into the central airway-like (*) after 0 and 12 h pi visualizes IVA spread to the alveolar-like regions. Dotted lines illustrate BALO-borders. Scale bars represent 100 μm.</strong></p> </li> <li> <p><strong>Quantification of plaque forming units in supernatants from mock or SC35M IAV infected BALO 48 h after infection (n=5 biological replicates). N.D.: not-detectable.</strong></p> </li> <li> <p><strong>Relative <em>NP</em> expression in PR8 IAV infected (HA<sup>+</sup>) or non-infected (HA<sup>-</sup>) epithelial cells isolated from BALO 48 h pi, FACS-sorted according to EpCAM and HA expression (n=3 biological replicates with pooled cells from 4 cultures per replicate).</strong></p> </li> <li> <p><strong>Representative flow cytometric data showing the percentage of EpCAM<sup>+</sup>NP<sup>+</sup> cells in mock- and SC35M IAV‑infected BALO at 48 h pi in n=3 biological replicates with pooled cells from 4 cultures per replicate.</strong></p> </li> <li> <p><strong>Representative fluorescence images of a day 21 distal region in BALO generated from mTmG reporter mouse and infected by SC35M-Cre IAV after 0, 10, 14 and 25 h. Arrows indicate cell death. Scale bars represent 25 μm and 10 μm in the insert.</strong></p> </li> <li> <p><strong>mRNA expression of <em>Ifnb</em> in mock and PR8 IAV-infected BALO at 48 h pi (n=3 biological replicates with pooled cells from 4 cultures per replicate).</strong></p> </li> <li> <p><strong>Release of TNFα (6 h pi), IL-6 (48 h pi), IL-1β (48 h pi) detected by Bio-Plex® Multiplex Immunoassay in the supernatant of mock and IAV infected BALO cultures with and without TR-Mac (n=3 biological replicates).</strong></p> </li> </ol> <p><strong>Data information: Bar charts presented as the mean ± S.E.M. and probability determined using t-test (F) and one‐way ANOVA (G) (*P < 0.05, **P < 0.01).</strong></p> <p><strong>Table 1. Adjusted p-value of genes in LIF and MYO clusters.</strong> Adjusted p-value of genes associated with MYO (C3) and LIF (C4) phenotypes including <em>Pdgfrα, Tagln, Acta2, Eln</em>, <em>Axin2</em> and <em>Apoe, Serpina3n, Gsn Gas6,</em> and <em>Fgf10,</em> respectively.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35373
|
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"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "956531",
"original_type": "gene",
"role": "assayed",
"text": "NP",
"type": "geneprod",
"uniprot_ids": [
"P03466"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "A0A482E1P7",
"ext_tax_ids": "11320",
"ext_tax_names": "Influenza A virus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "intervention",
"text": "HA",
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"uniprot_ids": [
"A0A482E1P7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "A0A482E1P7",
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"ext_tax_names": "Influenza A virus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "intervention",
"text": "HA",
"type": "geneprod",
"uniprot_ids": [
"A0A482E1P7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "A0A482E1P7",
"ext_tax_ids": "11320",
"ext_tax_names": "Influenza A virus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "intervention",
"text": "HA",
"type": "geneprod",
"uniprot_ids": [
"A0A482E1P7"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q99JW5",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "EpCAM",
"type": "geneprod",
"uniprot_ids": [
"Q99JW5"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "A0A2P1E2Q4",
"ext_tax_ids": "11320",
"ext_tax_names": "Influenza A virus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "NP",
"type": "geneprod",
"uniprot_ids": [
"A0A2P1E2Q4"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "15977",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "15977",
"original_type": "gene",
"role": "assayed",
"text": "Ifnb",
"type": "geneprod",
"uniprot_ids": [
"P01575",
"Q0VE17"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P10749",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "IL-1β",
"type": "geneprod",
"uniprot_ids": [
"P10749"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P08505",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "IL-6",
"type": "geneprod",
"uniprot_ids": [
"P08505"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P06804",
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"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
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"role": "assayed",
"text": "TNFα",
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"P06804"
]
}
] |
||
10.15252/emmm.202114123
|
Mitogen-activated protein kinase activity drives cell trajectories in colorectal cancer
|
2021
|
Figure 2
|
<p><strong>Figure 2. CRC cell clusters are distinguished by signaling pathway activities</strong>.</p><p><strong>A</strong> Transcriptional activity associated with key oncogenic traits and signals, by tumor-specific cell type and patient, as indicated. Red: High activity, blue: low activity. Significance was assessed by Kruskal Wallis Test (FDR corrected p<0.05), followed by a post-hoc analysis using a Wilcox-test of each group against all other groups, FDR corrected significance levels are shown (*=p<0.05; **=p<0.01; ***p<0.001).</p><p><strong>B</strong> Visualization of signatures corresponding to oncogenic traits and signals in the tumor cell transcriptome UMAP.</p><p><strong>C</strong> Cell cycle distribution of TC1-4 epithelial tumor cells, as inferred from single cell transcriptomes.</p><p><strong>D</strong> Immunofluorescence of DNA-damage-associated nuclear protein PARP. Images show adjacent normal and tumor crypts of tissue P009T, marked by N and T, respectively, and tumor tissue of patients P008 and P009, as indicated. Scale bar 100µm. Significance was assessed by an unpaired t-test, after blinded analysis of 10 random images per tumor.</p><p><strong><br></strong></p>
|
https://api.sourcedata.io/file.php?figure_id=42551
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P09874",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "PARP",
"type": "geneprod",
"uniprot_ids": [
"P09874"
]
}
] |
||
10.15252/emmm.202114123
|
Mitogen-activated protein kinase activity drives cell trajectories in colorectal cancer
|
2021
|
Figure 3
|
<p><strong>Figure 3: RNA metabolic labeling defines tumor cell trajectories in patient-derived organoids</strong>.</p><p><strong>A</strong> Phenotypes of patient-derived organoid lines P009T and P013T.</p><p><strong>B</strong> UMAPs of organoid single cell transcriptomes. Organoid lines and medium conditions as indicated. LGR5-ISC stem cell, enterocyte and Goblet cell signatures are visualized.</p><p><strong>C</strong> Schematic representation of SLAM-Seq workflow to infer RNA dynamics ("RNA velocity"). In short, organoids were passaged and assigned to different medium conditions. After three days, nascent RNA was metabolically labelled for 2 h using 4sU. Organoids were harvested, dissociated, and fixed. RNA in single cells was alkylated, and cells were subjected to single cell sequencing. Reads were assigned to nascent or old RNA status, depending on diagnostic T-C mutational status.</p><p><strong>D</strong> Developmental trajectories inferred from RNA metabolic labelling. Bold and thin arrows indicate high versus low directionality of RNA velocity. Colors below RNA velocity show latent time (yellow: early latent time; blue: late latent time).</p><p><strong>E</strong> Activities of Wnt/β-catenin and MAPK target genes in organoid single cell transcriptomes, ordered along latent time.</p><p><strong>F</strong> Activities of <em>TFF3</em>, <em>FABP1</em>, <em>MKI67</em> and <em>MMP7</em> in organoid single cell transcriptomes, ordered along latent time.</p><p>Data information: Color code for panels E and F: Red: high activity; blue: low activity.</p>
|
https://api.sourcedata.io/file.php?figure_id=42553
|
[
{
"ext_dbs": "NCBI gene",
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"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "2168",
"original_type": "gene",
"role": "assayed",
"text": "FABP1",
"type": "geneprod",
"uniprot_ids": [
"P07148",
"Q05CP7",
"Q6FGL7"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "4288",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "4288",
"original_type": "gene",
"role": "assayed",
"text": "MKI67",
"type": "geneprod",
"uniprot_ids": [
"P46013"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "4316",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "4316",
"original_type": "gene",
"role": "assayed",
"text": "MMP7",
"type": "geneprod",
"uniprot_ids": [
"P09237"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "7033",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "7033",
"original_type": "gene",
"role": "assayed",
"text": "TFF3",
"type": "geneprod",
"uniprot_ids": [
"Q07654"
]
}
] |
||
10.15252/emmm.202114123
|
Mitogen-activated protein kinase activity drives cell trajectories in colorectal cancer
|
2021
|
Figure 4
|
<p><strong>Figure 4: MAPK activity is linked to CRC cell differentiation states</strong>.</p><p><strong>A</strong> Gene expression of <em>LGR5</em> and <em>EPHB2</em>, along activity gradients of LGR5-ISC or MAPK target gene signatures.</p><p><strong>B</strong> Cell state distribution of SCN-aberrant CRC cells along gradients of LGR5-ISC or MAPK transcriptional signatures, as in A.</p><p><strong>C</strong> Cell state distribution of SCN-aberrant CRC tumor cells along MAPK signature activity, as in B, per tumor. Correlation between cell state distributions and MAPK target gene was calculated using Pearson´s r. For correlations and significances, see Table EV6. Color code as in Fig. 4B.</p><p><strong>D</strong> UMAP representations of single cell transcriptomes derived from P009T or P013T organoids, after MAPK blockade using MEK or combined MEK and EGFR inhibition. Color codes are treatment conditions or expression strength of signature, as indicated. Dashed line in P013T UMAP roughly separates control (DMSO) and MEK/EGFR inhibitor treated cells.</p><p><strong><br></strong></p>
|
https://api.sourcedata.io/file.php?figure_id=42555
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "2048",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "2048",
"original_type": "gene",
"role": "assayed",
"text": "EPHB2",
"type": "geneprod",
"uniprot_ids": [
"P29323",
"B4DSE0",
"Q6NVW1"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "8549",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "8549",
"original_type": "gene",
"role": "assayed",
"text": "LGR5",
"type": "geneprod",
"uniprot_ids": [
"O75473",
"A0A0A8K8C7"
]
}
] |
||
10.15252/embj.2020105479
|
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium
|
2020
|
Figure 1
|
<sd-panel> <p><strong>Figure 1. Massive progenitor cell apoptosis without global loss of proliferation in rostral telencephalon of <em>Rx-Dicer</em> mutants.</strong></p> <p><strong>A</strong> - Distribution of Casp3+ cells in rostral and ventral domains of an E11.5 <em>Rx-Dicer</em> mutant embryo. NCx, neocortex; OB, olfactory bulb; Spt, septum.</p> <p><strong>B,C</strong> - Marker analysis of Casp3+ cells in the rostral telencephalon of E12.5 <em>Rx-Dicer</em> mutant embryos. Most apoptotic cells are Pax6+ RGCs (solid arrowheads) and not Tbr1+ neurons (open arrowheads). <em>N</em> = 3 replicates per marker.</p> <p><strong>D,E</strong> - Distribution and abundance of apoptotic cells (Casp3+) in the rostral telencephalic primordium of control and <em>Rx-Dicer</em> mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. <em>N</em> = 3 replicates per genotype and age.</p> <p><strong>F,G</strong> - Distribution and abundance of mitoses (PH3+) and neurons (Tuj1) in the rostral telencephalic primordium of control and <em>Rx-Dicer</em> mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. <em>N</em> = 3-4 replicates per genotype and age. No significant differences were found between control and mutant embryos in apical nor basal mitoses.</p> <p>Data information: Histograms represent mean ± SEM; symbols in plots indicate values for individual embryos; <em>X<sup>2</sup></em>-test (C), t-test (E,G); *<em>p</em><0.05, ** <em>p</em> <0.01, *** <em>p</em> <0.001.</p> <p>Scale bars, 200 µm (A), 40 µm (B), 25 µm (D,F).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35428
|
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||
10.15252/embj.2020105479
|
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium
|
2020
|
Figure 2
|
<sd-panel> <p><strong>Figure 2. Formation of rosettes in the rostral telencephalon of <em>Rx-Dicer</em> mutants.</strong></p> <p><strong>A</strong> - DAPI stain of sagittal sections through the rostral telencephalon of E17.5 control and <em>Rx-Dicer</em> mutant littermates showing the neocortex (NCx) and olfactory bulb (OB; dashed line).</p> <p><strong>B</strong> - Expression pattern of <em>Grm1</em> mRNA in OB (dotted line) of control and <em>Rx-Dicer</em> mutants.</p> <p><strong>C</strong> - Quantification of OB perimeter (mean ± SEM; symbols indicate values for individual embryos); t-test, ***<em>P</em> < 0.001. <em>N</em> = 14 replicates per genotype.</p> <p><strong>D</strong> - Immunostains of E17.5 control and <em>Rx-Dicer</em> mutant brains showing the distribution of progenitor cells (Ki67, red) and neurons (Tuj1, green). Arrowheads indicate rosettes. Sp, septum.</p> <p><strong>E</strong> - Detail of a rosette from an E17.5 <em>Rx-Dicer</em> mutant embryo displaying typical features: closed apical surface (dotted line) with PH3+ apical mitoses (white arrowhead) and basal mitoses, surrounded by Tuj1+ neurons (green). Dashed line indicates the basal border of the rosette.</p> <p><strong>F</strong> - Rostral half of an <em>Rx-Dicer</em> mutant E17.5 brain immunostained for Pax6, clarified and segmented to reveal rosettes (yellow). C, caudal; R, rostral.</p> <p><strong>G,H</strong> - BrdU incorporation and cell cycle re-entry analysis with the progenitor cell marker Ki67 at E17.5, in the rostral cortex of control embryos compared to rostral rosettes of <em>Rx-Dicer</em> mutants. Dotted lines indicate apical surface, dashed lines indicate basal border of VZ. Arrowheads indicate double-positive cells. Data in histograms are mean ± SEM, symbols indicate values for individual embryos; <em>n</em> ≥ 3 embryos; <em>X<sup>2</sup></em>-test, *<em>P</em> < 0.05, ***<em>P</em> < 0.001.</p> <p><strong>I</strong> - Apical lumen of rosettes immunostained against apical complex proteins (Par3, β-Catenin), primary cilia (Arl13b), apical mitoses (PhVim) and neurons (Tuj1).</p> <p><strong>J</strong> - Coronal section through the rostral telencephalon of an E14.5 <em>Rx-Dicer</em> mutant embryo illustrating the high abundance and location of rosettes (arrows), as revealed by the distribution of mitoses (PH3) and neurons (Tuj1).</p> <p><strong>K-S</strong> - Analysis of the regional identity of rosettes in E14.5 <em>Rx-Dicer</em> mutants. (K) Schema of normal transcription factor expression patterns defining telencephalic regional identity. Expression of <em>Ngn2, Tbr2</em> and <em>Pax6</em> identifies rosettes in the rostro-dorsal telencephalon as having dorsal identity (L-N,P); <em>Gsx2</em>, <em>Dlx2</em> and <em>Dlx5</em> identify rosettes in the ventral telencephalon as having ventral identity (O,Q); <em>Nkx2.1</em> identifies MGE rosettes as being normotopic (R); absence of <em>Gsx2</em> in LGE rosette cells identifies them as ectopic (S). Tuj1 labels neurons. In (L,M), arrowheads indicate the border between dorsal and ventral territories, and dotted line indicates the outer border of the telencephalon. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; P, pallium; SP, subpallium.</p> <p>Data information: Scale bars, 500 µm (A,B,D,F), 25 µm (E,G,I,P-S), 200 µm (J-O).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35429
|
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||
10.15252/embj.2020105479
|
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium
|
2020
|
Figure 3
|
<sd-panel> <p><strong>Figure 3. Disturbance of ventricular integrity precedes the formation of rosettes.</strong></p> <p><strong>A</strong> - Coronal section through the rostral telencephalon of an E14.5 <em>Rx-Dicer</em> mutant embryo displaying the three types of rosettes.</p> <p><strong>B</strong> - High magnification of rosettes in <em>Rx-Dicer</em> mutants at E12.5 (Type 1), E14.5 (Type 2) and E17.5 (Type 3), immunostained for PH3 and Tuj1. Dashed line indicates ventricular surface; arrowheads indicate basal mitoses; arrows indicate a stream of apical mitoses connecting the lumen of the rosette with the lumen of the telencephalic ventricle.</p> <p><strong>C,D</strong> - Abundance of rosette types at the indicated ages (C), and rostro-caudal distribution of total rosette abundance per age, independent of type (D). <em>n</em> ≥ 2 brains per age.</p> <p><strong>E</strong> - Schematic of the progression of rosette formation in the rostral telencephalon of <em>Rx-Dicer</em> mutant embryos.</p> <p><strong>F,G</strong> - Distribution of neurons (Tuj1+ cells) and apical adherens junction protein Par3 in the OB primordium of control and <em>Rx-Dicer</em> mutant embryos at the indicated ages. Dotted lines indicate basal border, dashed lines indicate apical border. White arrowheads indicate ectopic neurons and/or absence of Par3. Arrow in (G) indicates accumulation of Par3 at the lumen of a nascent rosette.</p> <p>Data information: Scale bars, 100 µm (A), 40 µm (B), 50 µm (F,G).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35431
|
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10.15252/embj.2020105479
|
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium
|
2020
|
Figure 4
|
<sd-panel> <p><strong>Figure 4. Changes in expression of miRNAs and mRNAs upon early loss of Dicer.</strong></p> <p><strong>A,B</strong> - Changes in expression levels of miRNAs between control and <em>Rx-Dicer</em> mutants at E11.5 in the rostral telencephalon. Red dots indicate statistically differentially expressed miRNAs according to FDR correction of <em>P</em> values based on the Wald statistic (DESeq2 analysis) (B). Dashes lines indicate stringency limits in evaluation of results.</p> <p><strong>C</strong> - Gene ontology terms (top) and signalling pathways (bottom) associated with target genes of the miRNAs differentially-expressed between control and <em>Rx-Dicer</em> mutants.</p> <p><strong>D,E</strong> - Changes in mRNA expression levels between control and <em>Rx-Dicer</em> mutants at E11.5 in the rostral telencephalon. Statistically differentially expressed mRNAs according to FDR correction of <em>P</em> values based on the Wald statistic (DESeq2 analysis; Adj. <em>P</em> < 0.1 and FC > 1.25) are highlighted in red, and the three with the highest Adj. <em>P</em> value are identified by name. Blue dot indicates <em>Irs2</em>: FC=1.748, Adj. <em>P</em> = 5.95e-08.</p> <p><strong>F</strong> - Gene ontology terms (top) and signalling pathways (bottom) associated with mRNAs differentially-expressed between control and <em>Rx-Dicer</em> mutants.</p> <p><strong>G</strong> - Heatmaps of relative expression levels of DEGs related to apoptosis (top) and proliferation (bottom). Rows correspond to independent biological replicates. Indicated are some example genes.</p> <p><strong>H</strong> - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis (NES = 2.98; p = 0; Adj. <em>P</em> = 0.00045), Regulation of cell proliferation (NES = 2.40; p=0; Adj. <em>P</em> = 0.0033), Positive regulation of cell adhesion (NES = 2.06; p = 0.0058; Adj. <em>P</em> = 0.013), Regulation of response to stress (NES = 2.50; p = 0; Adj. <em>P</em> = 0.0023), Positive regulation of cell death (NES = 1.84; p = 0.0179; Adj. <em>P</em> = 0.0326), Apoptotic signaling pathway (NES = 1.59; p= 0.047; Adj. <em>P</em> = 0.084).</p> <p><strong>I,J</strong> - ISH stains of <em>Irs2</em> mRNA, and qPCR for <em>Irs2</em> mRNA and <em>let-7-5p</em> miRNA, in the rostral telencephalon of control and <em>Rx-Dicer</em> mutant embryos. Dashed line indicates basal border, dotted line indicates apical surface. Arrowheads indicate area with the greatest increase in <em>Irs2</em> expression. BG, basal ganglia; H, hippocampus; NCx, neocortex; OB, olfactory bulb; Spt, septum. Histograms represent mean ± SEM (logarithmic scale); symbols in plots indicate values for individual embryos; t-test, *<em>P</em> <0.05, **<em>P</em> <0.01. <em>N</em> = 6-9 replicates per group. Scale bar, 100 µm.</p> <p><strong>K</strong> - Working model of two potential genetic mechanisms leading to rosette formation in <em>Dicer</em> mutants: red, direct effect of increased p53 pathway activity upon loss of <em>let-7</em>; green, <em>Irs2</em> expression is massively increased by the combined effect of <em>let-7</em> loss and increased p53-related cell stress signals, which enhances proliferation and decreases cell adhesion, leading to rosettes.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35433
|
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||
10.15252/embj.2020105479
|
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium
|
2020
|
Figure 5
|
<sd-panel> <p><strong>Figure 5. Loss of <em>p53</em> in <em>Dicer</em> mutants blocks the formation of rosettes.</strong></p> <p><strong>A-C</strong> - Immunostains for activated p53 (red) in the rostral telencephalon of control and <em>Rx-Dicer</em> mutant embryos at E12.5, and quantification of positive cells (C). Dashed lines indicate apical side, dotted lines indicate basal side. <em>N</em> = 4 replicates per genotype. Scale bar, 50µm.</p> <p><strong>D-L</strong> - Rostral telencephalon of E12.5 embryos of the indicated genotypes immunostained for the detection of apoptotic cells (Casp3), neurons (Tuj1) and the apical complex protein Par3, and quantification of cells positive for Casp3 (H). <em>N</em> = 4-7 replicates per genotype. Scale bar, 50 µm.</p> <p><strong>M-O</strong> - Sagittal sections through the rostral telencephalon of E17.5 embryos of the indicated genotypes. Note the rosettes and general disorganization in <em>Rx-Dicer</em> single mutants, and complete rescue of this phenotype in <em>Rx-Dicer-p53</em> double mutants. Images in panels M,N are re-used from Fig 2A. NCx, neocortex; OB, olfactory bulb. Scale bar, 1 mm.</p> <p>Data information: Data in histograms are mean ± SEM, symbols indicate values for individual embryos; t-test; ns, not significant; *<em>P</em> < 0.05, ***<em>P</em> < 0.001, ****<em>P</em> < 0.0001.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35435
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10.15252/embj.2020105479
|
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium
|
2020
|
Figure 6
|
<sd-panel> <p><strong>Figure 6. Rosettes form by upregulation of <em>Irs2</em> expression.</strong></p> <p><strong>A</strong> - Relative <em>Irs2</em> mRNA levels in HEK cells upon transfection with <em>Gfp</em>- or <em>Irs2</em>-encoding plasmids. Mean ± SEM; t-test, ****<em>P</em> < 0.0001. <em>N</em> = 6 replicates per group.</p> <p><strong>B-D</strong> - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 or E17.5 mouse embryos, as indicated, electroporated at E12.5 with the indicated plasmid combinations. Dashed line indicates the apical surface. Insets show the same field of view, with location and extent of electroporations. Note the overlap of rosettes (arrows) with ectopic neurons at the apical surface (arrowheads) and increased basal mitoses, upon overexpression of <em>Irs2</em> (C). Asterisk indicates absence of malformation in nearby cortex. At E17.5 (D) GFP+ rosettes remain in deep cortical layers (arrowheads).</p> <p><strong>E-H</strong> - Details of rosettes (large arrows in E; dashed lines in F-H) from <em>Irs2+Gfp</em>-overexpressing E14.5 embryos immunostained as indicated. Small arrows indicate apical mitoses, arrowheads indicate basal mitoses (F,G) and Par3+ apical surface at the centre of rosettes (H), dotted lines indicate apical surface, dashed lines indicate basal side of rosettes. Inset in (H) is at the same scale.</p> <p><strong>I-M</strong> - Distribution of neurons (Tuj1, red), GFP (green) and mitotic (PH3, white) or apoptotic (Casp3, white) cells in the rostral telencephalon of wild-type E13.5 embryos electroporated at E12.5 with the indicated plasmids, and quantification of Casp3+ cells (M). Dashed line indicates apical surface, dotted lines indicate borders of the cortical plate (CP). Nascent rosettes (arrows) are next to ectopic ventricular neurons (arrowheads in K') and basal mitoses, without significant apoptosis in VZ (L',M). Mean ± SEM; t-test; ns, not significant, *<em>P</em> < 0.05. <em>N</em> = 3-6 replicates per group.</p> <p><strong>N</strong> - Distribution of neurons (Tuj1, red) in the rostral telencephalon of wild-type E14.5 embryos electroporated at E12.5 with a combination of plasmids encoding <em>Irs2, let-7a</em>, <em>let-7b</em> and <em>let-7c</em>, and <em>Gfp</em>. Dashed line indicates apical surface. Formation of rosettes in <em>Irs2</em>-expressing embryos is rescued by overexpression of <em>let-7</em>. Inset shows the same field of view, with location and extent of electroporation.</p> <p>Data information: Scale bars, 150 µm (B-D,N), 50 µm (E-H), 100 µm (I-L).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35437
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"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Tuj1",
"type": "geneprod",
"uniprot_ids": [
"Q587J6"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q587J6",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Tuj1",
"type": "geneprod",
"uniprot_ids": [
"Q587J6"
]
}
] |
||
10.15252/embj.2020105479
|
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium
|
2020
|
Figure 7
|
<sd-panel> <p><strong>Figure 7. Rosettes form by loss of <em>let-7</em> and upregulation of <em>Irs2</em> expression.</strong></p> <p><strong>A</strong> - Frequency of rosette formation in the experimental conditions indicated. <em>n</em>, number of embryos per condition.</p> <p><strong>B-G</strong> - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 mouse embryos electroporated at E12.5 with the indicated plasmid combinations. Inset shows the same field of view, with location and extent of electroporations. In (C) and (E), arrows indicate rosettes and arrowheads indicate neuronal ventricular ectopias. (D) and (F) are high magnification details of individual rosettes (dashed lines), where arrowheads indicate apical and basal mitoses. Loss of endogenous <em>let-7</em> drives the formation of rosettes and ventricular neuronal ectopias (C), which is rescued with the additional loss of <em>Irs2</em> (G).</p> <p>Data information: Scale bars, 150 µm (B,C,E,G), 50 µm (D,F).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35438
|
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{
"ext_dbs": "NCBI gene",
"ext_ids": "384783",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "384783",
"original_type": "gene",
"role": "intervention",
"text": "Irs2",
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"uniprot_ids": [
"P81122"
]
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{
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"ext_ids": "723966///387246///387245///723965///387244",
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"ext_tax_names": "Mus musculus///Mus musculus///Mus musculus///Mus musculus///Mus musculus",
"mapping_source": "unknown",
"mapping_status": "unmapped",
"ncbi_gene_id": null,
"original_type": "gene",
"role": "intervention",
"text": "let-7",
"type": "geneprod",
"uniprot_ids": []
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P68433",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "PH3",
"type": "geneprod",
"uniprot_ids": [
"P68433"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q587J6",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
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"role": "assayed",
"text": "Tuj1",
"type": "geneprod",
"uniprot_ids": [
"Q587J6"
]
}
] |
||
10.15252/embj.2020105479
|
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium
|
2020
|
Figure 8
|
<sd-panel> <p><strong>Figure 8. <em>Irs2</em> and <em>let-7</em> regulate progenitor cell proliferation in human cerebral organoids.</strong></p> <p><strong>A</strong> - Schema of experimental design. At 40 days in culture, human cerebral organoids were electroporated with one of the indicated DNA plasmid combinations, followed by 7 additional days in culture prior to analysis.</p> <p><strong>B-G</strong> - Stains and quantifications of human cerebral organoids electroporated with the indicated construct combinations, to reveal the frequency of electroporated cells remaining as progenitors (%GFP+Ki67+/GFP+). Histograms represent mean ± SEM; symbols in plots indicate values for individual organoids; <em>X<sup>2</sup></em>-test, *<em>P</em> < 0.05. <em>N</em> = 8-11 replicates per group. Dashed lines indicate ventricular surface, solid arrowheads indicate Ki67+/GFP+ cells, open arrowheads indicate Ki67-/GFP+ cells. Scale bars, 50 µm.</p> <p><strong>H</strong> - Schematic drawing of conclusions from this study. <em>Let-7</em> expressed in the early telencephalic neuroepithelium represses the expression of p53 and Irs2, which maintains the cellular homeostasis and epithelial integrity. Loss of <em>let-7</em>, as in <em>Dicer1</em> mutants, de-represses expression of <em>Irs2</em> and of p53 pathway components, which further increase <em>Irs2</em> levels. High Irs2 enhances progenitor cell proliferation and loss of apical adhesion, altering neuroepithelial homeostasis and integrity, and leading to the formation of hyperproliferative rosettes. <strong><br></strong></p> <p><strong>Expanded View Figure legends</strong></p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=35439
|
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"ext_ids": "P46013",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
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"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Ki67",
"type": "geneprod",
"uniprot_ids": [
"P46013"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P46013",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Ki67",
"type": "geneprod",
"uniprot_ids": [
"P46013"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P46013",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Ki67",
"type": "geneprod",
"uniprot_ids": [
"P46013"
]
}
] |
||
19339967
|
10.1038/nature07976
|
Autophagy regulates lipid metabolism
|
2009
|
figf1
|
<p><b>a</b>, TG levels in hepatocytes untreated (None) or treated with 3-methyladenine (3MA) and cultured in regular medium (RM) or oleate (OL) (*<i>P</i> 0.02, **<i>P</i> 0.002, <i>n</i> = 3). <b>b</b>, TG levels in vector-infected (VEC) and si<i>Atg5</i> cells in RM, OL or in MCDM (*<i>P</i> 0.001, <i>n</i> = 5). OL values are in mM. <b>c</b>, TG levels in wild-type (WT) or <i>Atg5</i> knockout mice embryonic fibroblasts (<i>Atg5<super>-/-</super></i>) (*<i>P</i> 0.01 or **<i>P</i> 0.0001, <i>n</i> = 5). <b>d</b><b>f</b>, Cells from <b>a</b> and <b>b</b> stained with BODIPY 493/503 (<b>d</b>), oil red O (<b>e</b>) or visualized by electron microscopy (<b>f</b>). Right: quantifications of LD number and size (*<i>P</i> 0.01, **<i>P</i> 0.001 with untreated wild-type cells; #<i>P</i> 0.01, # #<i>P</i> 0.001 with cells in RM; <i>n</i> = 46). Nuclei are highlighted with 4,6-diamidino-2-phenylindole (DAPI; <b>d</b>). Error bars, s.e.m.</p>
|
https://api.sourcedata.io/file.php?figure_id=2684
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "365601",
"ext_tax_ids": "10116",
"ext_tax_names": "Rattus norvegicus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "365601",
"original_type": "gene",
"role": "intervention",
"text": "Atg5",
"type": "geneprod",
"uniprot_ids": [
"Q3MQ06",
"A6K6V9",
"Q5XIS0"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "11793",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "11793",
"original_type": "gene",
"role": "intervention",
"text": "Atg5",
"type": "geneprod",
"uniprot_ids": [
"Q99J83"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "11793",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "11793",
"original_type": "gene",
"role": "intervention",
"text": "Atg5",
"type": "geneprod",
"uniprot_ids": [
"Q99J83"
]
}
] |
|
19339967
|
10.1038/nature07976
|
Autophagy regulates lipid metabolism
|
2009
|
figf2
|
<p><b>a</b>, <b>b</b>, VEC and si<i>Atg5</i> cells cultured with oleate (OL) or in MCDM were examined for their rates of TG synthesis (<b>a</b>) and &bgr;-oxidation (<b>b</b>) as compared to cells in regular medium alone (*<i>P</i> 0.03, **<i>P</i> 0.004 with VEC cells in the same medium, <i>n</i> = 34). <b>c</b>, <b>d</b>, Rates of TG decay in OL (<b>c</b>) and MCDM (<b>d</b>) (*<i>P</i> 0.05, **<i>P</i> 0.01, ***<i>P</i> 0.001 as compared to VEC cells, <i>n</i> = 37). <b>e</b>, TG levels in wild-type cells treated with dimethyl sulphoxide vehicle (DMSO), 3-methyladenine (3MA) or diethylumbelliferyl phosphate (DEUP) (*<i>P</i> 0.00001 with DMSO-treated cells, #<i>P</i> 0.003 with 3MA-treated cells, <i>n</i> = 6). Error bars, s.e.m. <b>f</b>, Co-localization of BODIPY 493/503 (green) with LAMP1 (red) or LC3 (red) in hepatocytes in MCDM. <b>g</b>, High-magnification regions of hepatocytes in MCDM alone (none) or treated with vinblastine and stained as labelled. Arrows indicate co-localization.</p>
|
https://api.sourcedata.io/file.php?figure_id=2685
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "365601",
"ext_tax_ids": "10116",
"ext_tax_names": "Rattus norvegicus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "365601",
"original_type": "gene",
"role": "intervention",
"text": "Atg5",
"type": "geneprod",
"uniprot_ids": [
"Q3MQ06",
"A6K6V9",
"Q5XIS0"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P14562",
"ext_tax_ids": "10116",
"ext_tax_names": "Rattus norvegicus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "LAMP1",
"type": "geneprod",
"uniprot_ids": [
"P14562"
]
},
{
"ext_dbs": "Uniprot///Uniprot",
"ext_ids": "Q62625///Q6XVN8",
"ext_tax_ids": "10116///10116",
"ext_tax_names": "Rattus norvegicus///Rattus norvegicus",
"mapping_source": "unknown",
"mapping_status": "unmapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "LC3",
"type": "geneprod",
"uniprot_ids": []
}
] |
|
19339967
|
10.1038/nature07976
|
Autophagy regulates lipid metabolism
|
2009
|
figf3
|
<p><b>a</b>, Electron micrographs of cultured hepatocytes. dm, double-membrane cytosolic vesicles. Arrows indicate membranes in LDs. Insets show the double membrane in nucleus (N), lipid-containing vesicles (L) and mitochondria (mit), but not in LDs. <b>b</b>, Mouse liver LC3 immunogold. Insets show higher magnification. Arrowheads indicate gold particles (black) and LC3-labelled bilayer membranes (white). <b>c</b>, Percentages of autophagic vacuoles (AVs) containing only lipid (Lipids, *), other cargo (Other) or mixed cargo (Mixed) in cells treated as in <b>b</b> (*<i>P</i> 0.01, **<i>P</i> 0.001 with cells in RM, <i>n</i> = 46). Left: representative examples. Error bars, s.e.m.</p>
|
https://api.sourcedata.io/file.php?figure_id=2686
|
[
{
"ext_dbs": "Uniprot///Uniprot",
"ext_ids": "Q91VR7///Q9CQV6",
"ext_tax_ids": "10090///10090",
"ext_tax_names": "Mus musculus///Mus musculus",
"mapping_source": "unknown",
"mapping_status": "unmapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "LC3",
"type": "geneprod",
"uniprot_ids": []
},
{
"ext_dbs": "Uniprot///Uniprot",
"ext_ids": "Q91VR7///Q9CQV6",
"ext_tax_ids": "10090///10090",
"ext_tax_names": "Mus musculus///Mus musculus",
"mapping_source": "unknown",
"mapping_status": "unmapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "LC3",
"type": "geneprod",
"uniprot_ids": []
}
] |
|
19339967
|
10.1038/nature07976
|
Autophagy regulates lipid metabolism
|
2009
|
figf4
|
<p><b>a</b>, Immunoblots of cellular homogenates (Hom) and LDs from fed (F) or 24-h starved (S) mice. I&kgr;B, inhibitor of the nuclear factor of kappa light polypeptide gene enhancer; GPDH, glyceraldehyde-3-phosphate dehydrogenase. <b>b</b>, Percentages of autophagic vacuoles (AVs) containing only lipid, other cargo or mixed cargo calculated from samples processed as in <sir rid="s1">Supplementary Fig. 16b</sir> (*<i>P</i> 0.001, **<i>P</i> 0.0001, <i>n</i> = 46). <b>c</b>, Immunoblots of Hom, AV and lysosomes (Lys). L-1, LAMP1. <b>d</b>, Immunoblots of liver Hom and LDs from HFD-fed mice. <b>e</b>, Liver homogenate immunoblots. PDI, protein disulphide isomerase. <b>f</b>, Total hepatic TG and cholesterol content (*<i>P</i> 0.01, **<i>P</i> 0.00001, <i>n</i> = 817). <b>g</b>, Hepatic TG concentration (*<i>P</i> 0.05, <i>n</i> = 3). <b>h</b>, Percentage of cellular cholesterol in lysosomes (*<i>P</i> 0.02, <i>n</i> = 4). <b>i</b>, Immunoblots of homogenates and isolated LDs. Error bars, s.e.m.</p>
|
https://api.sourcedata.io/file.php?figure_id=2687
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P13707",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "GPDH",
"type": "geneprod",
"uniprot_ids": [
"P13707"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q60778",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "IκB",
"type": "geneprod",
"uniprot_ids": [
"Q60778"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P11438",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "LAMP1",
"type": "geneprod",
"uniprot_ids": [
"P11438"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P09103",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "PDI",
"type": "geneprod",
"uniprot_ids": [
"P09103"
]
}
] |
|
26657708
|
10.15252/embj.201592593
|
A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory
|
2015
|
Figure 1
|
<p><strong>Figure 1: HS induces sustained H3K4</strong><strong>me3 and H3K4me2</strong><strong> methylation at </strong><strong><em>HSP22.0</em></strong><strong>,</strong><strong> but not at </strong><strong><em>HSP70</em></strong><strong>. </strong></p><p>Dynamics of histone modifications after an acclimatizing HS (ACC) or no HS (NHS) in Col-0 wild type at <em>HSP22.0</em> (A) and <em>HSP70</em> (B). Seedlings were subjected to ACC or a control treatment (NHS) 4 d after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac, H3K4me3, H3K4me2 and H3. Schematics show positions of regions analyzed. Amplicon positions relative to TSS are <em>HSP22.0</em>: 1, -2570 bp; 2, +235 bp. HSP70: 1, 4192 bp downstream of the 3’UTR; 2, +47 bp. Data shown are averages over three biological replicates. Amplification values were normalized to input, H3 and 4 h NHS region 2. The bottom panel shows the H3 signal normalized to input and 4 h NHS region 2. Squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively, Student´s t-test) between ACC and NHS samples of the same time point. Error bars indicate SE.</p>
|
https://api.sourcedata.io/file.php?figure_id=5382
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "826616",
"ext_tax_ids": "3702",
"ext_tax_names": "Arabidopsis thaliana",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "826616",
"original_type": "gene",
"role": "assayed",
"text": "HSP22.0",
"type": "geneprod",
"uniprot_ids": [
"Q38806"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "826616",
"ext_tax_ids": "3702",
"ext_tax_names": "Arabidopsis thaliana",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "826616",
"original_type": "gene",
"role": "assayed",
"text": "HSP22.0",
"type": "geneprod",
"uniprot_ids": [
"Q38806"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "820438",
"ext_tax_ids": "3702",
"ext_tax_names": "Arabidopsis thaliana",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "820438",
"original_type": "gene",
"role": "assayed",
"text": "HSP70",
"type": "geneprod",
"uniprot_ids": [
"Q9LHA8"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "820438",
"ext_tax_ids": "3702",
"ext_tax_names": "Arabidopsis thaliana",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "820438",
"original_type": "gene",
"role": "assayed",
"text": "HSP70",
"type": "geneprod",
"uniprot_ids": [
"Q9LHA8"
]
}
] |
|
26657708
|
10.15252/embj.201592593
|
A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory
|
2015
|
Figure 2
|
<p><strong>Figure 2: </strong><strong>S</strong><strong>ustained </strong><strong>induction </strong><strong>of HS memory-related gene</strong><strong> expression</strong><strong> after </strong><strong>an acclimatizing HS</strong><strong> depends on HSFA2</strong><strong><em>.</em></strong></p>
<p>A. Transcript levels of the indicated HS-inducible genes after ACC in Col-0 (blue bars) and <em>hsfa2</em> (orange bars) as determined by quantitative Reverse Transcription-PCR (qRT-PCR). Transcript levels were normalized to <em>TUB6</em> and the respective NHS harvested at the same time point ([GENE OF INTEREST<sub>ACC x h</sub>/ TUB6<sub>ACC x h</sub>]/ [GENE OF INTEREST<sub>NHS</sub><sub> x h</sub>/ TUB6<sub>NHS</sub><sub> x h</sub>]). Note that the y-axis is on a log<sub>10</sub> scale. Error bars show SE of at least three biological replicates. Asterisks show significant differences between the genotypes at the same time point based on Student´s t-test (*: p<0.05; **: p<0.01).</p>
<p>B. Immunoblotting of HSP21 and HSP101 in Col-0 (C) and <em>hsfa2 </em>(<em>h</em>) at the indicated time points after ACC or NHS. TUBULIN was used as a loading control. M, marker.</p>
|
https://api.sourcedata.io/file.php?figure_id=5383
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "817155",
"ext_tax_ids": "3702",
"ext_tax_names": "Arabidopsis thaliana",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "817155",
"original_type": "gene",
"role": "intervention",
"text": "hsfa2",
"type": "geneprod",
"uniprot_ids": [
"O80982",
"B3H5P6"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "817155",
"ext_tax_ids": "3702",
"ext_tax_names": "Arabidopsis thaliana",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "817155",
"original_type": "gene",
"role": "intervention",
"text": "hsfa2",
"type": "geneprod",
"uniprot_ids": [
"O80982",
"B3H5P6"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P42730",
"ext_tax_ids": "3702",
"ext_tax_names": "Arabidopsis thaliana",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "HSP101",
"type": "geneprod",
"uniprot_ids": [
"P42730"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q9FIT9",
"ext_tax_ids": "3702",
"ext_tax_names": "Arabidopsis thaliana",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "HSP21",
"type": "geneprod",
"uniprot_ids": [
"Q9FIT9"
]
}
] |
|
26657708
|
10.15252/embj.201592593
|
A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory
|
2015
|
Figure 3
|
<p><strong>Figure </strong><strong>3</strong><strong>: Sustained H3K4</strong><strong>me3 and H3K4me2 at</strong><strong> HS memory-related </strong><strong>loci depends</strong><strong> on HSFA2. </strong></p><p>H3K4me3 (A) or H3K4me2 (B) levels after an acclimatizing HS (ACC) in Col-0 and <em>hsfa2</em> at <em>HSP18.2</em>, <em>HSP22.0</em>,<em> HSP70 </em>and <em>APX2 </em>as detected by ChIP-qPCR. Col-0 (blue bars) and <em>hsfa2</em> (orange bars) seedlings were subjected to ACC or no treatment (NHS) 4 d after germination. At the indicated time points after the treatment, ChIP-qPCR was performed with antibodies against H3K4me3 (A), H3K4me2 (B) and H3 (for normalization). Schematics show positions of regions analyzed (grey bars, UTR; black bar, exons). Intergenic control region 1 is 3123 bp (<em>APX2</em>) or 2570 bp (<em>HSP22.0</em>) upstream of the TSS, or 5311 bp (<em>HSP18.2</em>) or 6725 bp (<em>HSP70</em>) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4h NHS region 2 (<em>HSP18.2, HSP22.0 </em>and<em> HSP70</em>) or region 3 (for <em>APX2</em>). *, p<0.05; **, p<0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student´s t-test. Error bars indicate SE.</p>
|
https://api.sourcedata.io/file.php?figure_id=5384
|
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] |
|
26657708
|
10.15252/embj.201592593
|
A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory
|
2015
|
Figure 4
|
<p><strong>Figure </strong><strong>4</strong><strong>: </strong><strong>H</strong><strong>istone H3K9ac</strong><strong> profiles</strong><strong> after </strong><strong>ACC</strong><strong> in Col-0 and </strong><strong><em>hsfa2</em></strong><strong> at </strong><strong><em>HSP18.2</em></strong><strong>, </strong><strong><em>HSP22.0</em></strong><strong>,</strong><strong><em> HSP70 </em></strong><strong>and </strong><strong><em>APX2 </em></strong><strong>as detected by </strong><strong>ChIP-qPCR</strong><strong>.</strong></p><p>Col-0 (blue bars) and <em>hsfa2</em> (orange bars) seedlings were subjected to ACC or control treatment (NHS) 4 d after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac or H3. Schematics (grey bars, UTR; black bar, exons) show positions of regions analyzed. Intergenic control region 1 is 3123 bp (<em>APX2</em>) or 2570 bp (<em>HSP22.0</em>) upstream of the TSS, or 5311 bp (<em>HSP18.2</em>) or 6725 bp (<em>HSP70</em>) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4h NHS region 2 (<em>HSP18.2, HSP22.0 </em>and<em> HSP70</em>) or 3 (for <em>APX2</em>). *, p<0.05; **, p<0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (p<0.01 and p<0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student´s t-test. Error bars indicate SE.</p>
|
https://api.sourcedata.io/file.php?figure_id=5385
|
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] |
|
26657708
|
10.15252/embj.201592593
|
A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory
|
2015
|
Figure 5
|
<p><strong>Figure </strong><strong>5</strong><strong>: HSFA2 associates with HS memory-related genes transiently after </strong><strong>an acclimatizing HS</strong><strong>. </strong></p>
<p>Binding of HSFA2-YFP to the promoter regions of <em>HSP18.2</em> (A), <em>HSP22.0 </em>(B), <em>APX2</em> (C), HSP70 (D) and <em>ACTIN7</em> (E) was determined by ChIP-qPCR. Quantifications represent the enrichment relative to input in %. Red-orange-yellow bars represent time points (0.5 h-4 h-28 h) after a ACC and grey bars represent the respective NHS control samples. Schematics show the promoter region of the respective gene with regions analyzed (arrow, TSS; grey bar, 5’UTR; black bar, exon; black boxes, heat shock elements (5’-nnGnAnnTnCtn-3’). Regions are drawn to scale. Intergenic control region 1 is 4610 bp (<em>HSP18.2</em>) and 4192 bp (<em>HSP70</em>) downstream of the 3’UTR and 2570 bp (<em>HSP22.0</em>) or 3123 bp (<em>APX2</em>) upstream of the TSS, respectively. One representative experiment out of at least four biological replicates is shown. Error bars are SD of three replicates.</p>
|
https://api.sourcedata.io/file.php?figure_id=5386
|
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] |
|
26657708
|
10.15252/embj.201592593
|
A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory
|
2015
|
Figure 6
|
<p><strong>Figure </strong><strong>6</strong><strong>: </strong><strong>Expression p</strong><strong>rofiles of HS memory-related gene</strong><strong>s</strong><strong> after a recurring HS show HSFA2-dependent transcriptional memory</strong></p>
<p>Response of selected HS-inducible loci upon recurring HS as determined by qRT-PCR. Transcript levels of <em>APX</em><em>2</em> (A),<em> HSP</em><em>18.2</em> (B),<em> HSP</em><em>22.0</em> (C), and <em>HSP70</em> (D) in Col-0 (blue bars) and <em>hsfa2</em> (orange bars) at the indicated time points after a single HS or two HS separated by 2 d. Plants were subjected to 37°C for 60 min on either d 4, d 6 or on d 4 + d 6 after germination. NHS samples were harvested at the same time points as HS d 4 samples. Transcript levels of four biological replicates normalized to <em>TUB6</em> are shown. Small brackets compare the two genotypes at the same condition, large brackets compare the same genotype across two treatments (at 1 h after treatment) with Col-0 indicated first (Col-0/ <em>hsfa2</em>). Error bars show SE over four biological replicates. *, p<0.05; **, p<0.01; Student´s t-test.</p>
|
https://api.sourcedata.io/file.php?figure_id=5387
|
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|
10.15252/embr.202154350
|
Distinct biogenesis pathways may have led to functional divergence of the human and Drosophila Arglu1 sisRNA
|
2022
|
Figure 1
|
<p><strong>Figure 1 - Characterization and biogenesis of Arglu1 sisRNA.</strong></p><p>A Schematics showing the Arglu1 gene model and the different splicing isoforms.</p><p>B Top: Arglu1 gene locus indicating the primers' location (black arrow) and smFISH probes designed for Arglu1 sisRNA (green line) and Arglu1 pre-mRNA (magenta line). Bottom: RT-PCR showing the presence of Arglu1 sisRNA (P1+P2) detected from the different breast cancer cell lines.</p><p>C Experimental scheme for transcription inhibition treatment using alpha-amanitin and the indicated time points for cell harvesting.</p><p>D qPCR of Arglu1 sisRNA, pre-mRNA and mRNA expression levels normalized to <em>gapdh</em> from alpha-amanitin treatment at indicated time points. (n=3 biological replicates). Data are presented as mean ± SEM. **P≤0.01 (Student's t-test). See also Appendix Figure S1.</p><p>E RT-PCR indicating presence of Arglu1 sisRNA in the nuclear (N) and but not in the cytoplasmic (C) fraction. U85 gene was used as nuclear marker and 28S and 18S rRNA as cytoplasmic marker.</p><p>F smFISH of Arglu1 pre-mRNA (magenta) and Arglu1 sisRNA (green). Merged dots (white, arrowheads) represent Arglu1 pre-mRNA transcript. Scale bar: 20 μm.</p><p>G Quantification of Arglu1 pre-mRNA and Arglu1 sisRNA copy number from (F). (n = 50 cells) Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.</p><p>H Diagram indicating the locations of antisense morpholino oligonucleotides (AMOs) targeting the UCE region of Arglu1 intron 2 and primers used in (I).</p><p>I RT-PCR of Arglu1 sisRNA, spliced mRNA levels and <em>actin</em> as control after AMOs treatment.</p>
|
https://api.sourcedata.io/file.php?figure_id=51344
|
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||
10.15252/embr.202154350
|
Distinct biogenesis pathways may have led to functional divergence of the human and Drosophila Arglu1 sisRNA
|
2022
|
Figure 2
|
<p><strong>Figure 2 - Arglu1 sisRNA is involved in autoregulation of its host gene in a UCE-dependent manner.</strong></p><p>A Arglu1 gene locus indicating the locations of antisense oligonucleotides (ASOs) (red lines) designed for Arglu1 sisRNA KD and primers used in (B-D).</p><p>B, C qPCR of Arglu1 sisRNA and Arglu1 pre-mRNA expression levels in Arglu1 sisRNA ASO1 and ASO2 cells vs control cells. (n = 3 biological replicates).</p><p>D Chart showing the splicing indices (spliced/unspliced ratio) in Arglu1 sisRNA ASO1 and ASO2 cells vs control cells as measured by qPCR. (n = 3 biological replicates).</p><p>E Arglu1 gene locus indicating the location of antisense oligonucleotide (ASO) (red line) designed for Arglu1 sisRNA KD and primers used in (F).</p><p>F qPCR of Arglu1 mRNA expression levels at different exon junctions in Arglu1 sisRNA KD cells vs control cells. (n = 3 biological replicates).</p><p>G Left: Western blot analysis of ARGLU1 protein and GAPDH as loading control in control cells and Arglu1 sisRNA KD cells. Right: Quantification of relative ARGLU1 protein levels normalized to GADPH in Arglu1 sisRNA KD cells vs control cells from western blot analysis (left). (n = 3 biological replicates).</p><p>H Top: Schematic showing minigene expressing Arglu1 intron 2 wild type (WT) or intron 2 with UCE deletion (Δ) (location of UCE as indicated by orange bar). Bottom: Arglu1 gene locus indicating the primers' location (black arrows) for (I and J).</p><p>I Chart showing the splicing indices (spliced/unspliced ratio) in Arglu1 intron 2 WT OE cells and Arglu1 Intron 2 ΔUCE OE cells as measured by qPCR. (n = 3 biological replicates).</p><p>J qPCR of Arglu1 sisRNA levels in Arglu1 intron 2 WT OE cells and Arglu1 Intron 2 ΔUCE OE cells vs control cells (n = 3 biological replicates).</p><p>K, L qPCR of c-Myc and pS2 expression levels in 3 days Arglu1 sisRNA KD cells vs control cells under 100 nM estrogen treatment at indicated time points. (n = 3 biological replicates).</p><p>M Left: RT-PCR of AXIN1a and AXIN1b spliced levels and <em>actin</em> as loading control in 3 days Arglu1 sisRNA KD cells vs control cells under 100 nM estrogen treatment for 2 hrs. Right: Quantification of relative spliced AXIN1a/AXIN1b expression ratio normalized to <em>actin</em> from RT-PCR analysis (left). (n = 3 biological replicates).</p><p>N Schematics of Arglu1 gene autoregulatory loop model indicating involvement of Arglu1 sisRNA in promoting splicing of host gene (red arrow).</p><p>Data information: In (B-D, F, G, I-M), data are presented as mean ± SEM. ns=not significant, *P≤0.05, **P≤0.01 and ***P≤0.001 (Student's t-test). See also Fig EV1 and EV2.</p>
|
https://api.sourcedata.io/file.php?figure_id=51345
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10.15252/embr.202154350
|
Distinct biogenesis pathways may have led to functional divergence of the human and Drosophila Arglu1 sisRNA
|
2022
|
Figure 3
|
<p><strong>Figure 3 - Arglu1 sisRNA binds ARGLU1 protein and inhibits its interaction with Arglu1 pre-mRNA.</strong></p><p>A Top: Arglu1 gene locus indicating the primers' location (black arrows) for (A-C). Bottom: RIP-qPCR of Arglu1 pre-mRNA (P3+P4) and Arglu1 sisRNA (P1+P2) levels from ARGLU1 protein immunoprecipitation. Samples with beads and no antibodies were used as control. (n = 3 biological replicates).</p><p>B Left: Western blot analysis of ARGLU1 protein and GAPDH as loading control in 1 day Arglu1 sisRNA KD cells vs control cells. Right: Quantification of relative ARGLU1 protein expression level normalized to GADPH in 1 day Arglu1 sisRNA KD cells vs control cells from western blot analysis (left). (n = 3 biological replicates).</p><p>C RIP-qPCR of Arglu1 pre-mRNA and Arglu1 sisRNA levels normalized to <em>gapdh</em> in 1 day Arglu1 sisRNA KD cells vs control cells from ARGLU1 protein immunoprecipitation. (n = 3 biological replicates).</p><p>D Left: smFISH of Arglu1 sisRNA (green) coupled with immunostaining of ARGLU1 protein (red) shown in z-stack. Right: Zoom in z-slice on the boxed area showing co-localization (arrowheads) of Arglu1 sisRNA with ARGLU1 protein. The intensity plot shows the signal quantification and co-localization of Arglu1 sisRNA and ARGLU1 protein in the direction of the white arrow. Scale bar: 20 μm.</p><p>E Left: smFISH of Arglu1 pre-mRNA (green) coupled with ARGLU1 protein (red) immunostaining shown in z-stack. Right: Zoom in z-slice on the boxed area showing co-localization (arrowhead) of Arglu1 pre-mRNA with ARGLU1 protein. The intensity plot shows the signal quantification and co-localization of Arglu1 pre-mRNA and ARGLU1 protein in the direction of the white arrow.</p><p>F smFISH of Arglu1 sisRNA (green) and Arglu1 pre-mRNA (magenta) in 1 day Arglu1 sisRNA KD cells vs control cells. Scale bar: 20 μm.</p><p>G, H Quantification of Arglu1 sisRNA and Arglu1 pre-mRNA copy number from (F). (n = 90 cells). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.</p><p>I Quantification of Arglu1 pre-mRNA and ARGLU1 protein co-localization from Arglu1 pre-mRNA smFISH coupled with ARGLU1 immunostaining in 1 day Arglu1 sisRNA KD cells vs control cells. (n = 5 biological replicates).</p><p>Data information: In (A-C, and G-I), data are presented as mean ± SEM. ns=not significant, *P≤0.05 and ***P≤0.001 (Student's t-test). See also Appendix Figure S2.</p>
|
https://api.sourcedata.io/file.php?figure_id=51347
|
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] |
||
10.15252/embr.202154350
|
Distinct biogenesis pathways may have led to functional divergence of the human and Drosophila Arglu1 sisRNA
|
2022
|
Figure 4
|
<p><strong>Figure 4 - Arglu1 sisRNA promotes robust ARGLU1 protein phase separation and localization to nuclear speckles.</strong></p><p>A Immunostaining of MED1 (magenta) and SC35 (red) with transiently expressed mCherry-ARGLU1 protein (green) in MCF-7 cells. Zoom in of boxed area and the intensity plot shows the co-localization and signal quantification of mCherry-ARGLU1 protein, MED1 and SC35 in the direction of the white arrow. Scale bar: 20 μm.</p><p>B Confocal images of GFP-SRSF2 (green, control) and mCherry-ARGLU1 protein (red) at the pre-bleach, bleach, and post-bleach time points in FRAP analysis. The boxed area showing the speckle that was being bleached. Scale bar: 5 μm.</p><p>C Fluorescence intensities of the nuclear speckle over time in GFP-SRSF2 (green, control) and mCherry-ARGLU1 protein (red) FRAP analysis. (n = 3 separate nuclear speckles).</p><p>D Immunostaining of ARGLU1 protein (green) and SC35 (red) in 1 day Arglu1 sisRNA KD cells vs control cells. Zoom in of boxed area and the intensity plot shows the co-localization and signal quantification of ARGLU1 protein and SC35 in the direction of the white arrow. Scale bar: 20 μm.</p><p>E Confocal images of 125 nM ARGLU1-GFP protein (green) only and ARGLU1-GFP protein added with 50 nM IVT RNA (magenta), of either Arglu1 intron 2 with UCE (Intron 2 WT) or IVT Arglu1 intron without UCE (Intron 2 ΔUCE). White arrowhead indicates ARGLU1-GFP droplet incorporated with IVT Intron 2 WT and blue arrowhead indicates free ARGLU1-GFP droplet not incorporated with IVT Intron 2 WT. Scale bar: 10 μm.</p><p>F Quantification of total ARGLU1-GFP droplets from (E). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.</p><p>G Quantification of average ARGLU1-GFP droplets size with/without incorporation of IVT intron 2 WT from (E, ARGLU1-GFP + Intron 2 WT). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.</p><p>Data information: In (C), data are presented as mean ± SEM. ns=not significant and *P≤0.05 (Student's t-test). See also Figure EV3 and Appendix Figure S3.</p>
|
https://api.sourcedata.io/file.php?figure_id=51349
|
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||
10.15252/embr.202154350
|
Distinct biogenesis pathways may have led to functional divergence of the human and Drosophila Arglu1 sisRNA
|
2022
|
Figure 6
|
<p><strong>Figure 6. dArglu1 sisRNA interacts with U1 snNRP, Arglu1 pre-mRNA and dARGLU1 protein in forming nuclear bodies and promotes dARGLU1 protein phase separation.</strong></p><p>A Western blot analysis of dARGLU1 protein level from dARGLU1 protein immunoprecipitation in S2 cells. Samples with rabbit-IgG antibodies were used as control. ARGLU1 antibody #1, but not #2, worked for IP.</p><p>B RIP-qPCR of dArglu1 sisRNA, dArglu1 pre-mRNA, U1 snRNA and U85 snoRNA levels from dARGLU1 protein immunoprecipitation using ovary lysates. Samples with rabbit-IgG antibodies were used as control. (n = 3 biological replicates).</p><p>C-E RIP-qPCR of dArglu1 sisRNA, pre-mRNA and U1 snRNA levels normalized to U85 from dARGLU1 protein immunoprecipitation using dArglu1 sisRNA KD 1 and 2 ovaries vs control (sibling control, <em>MTD-GAL4/CyO</em>) ovaries. (n = 3 biological replicates).</p><p>F Left: Western blot analysis of dARGLU1 protein levels and ACTIN5C as loading control in dArglu1 sisRNA KD ovaries vs control (sibling control, <em>MTD-GAL4/CyO</em>) ovaries. Right: Quantification of relative dARGLU1 protein levels in dArglu1 sisRNA KD ovaries vs control ovaries from the western blot analysis. (n = 3 biological replicates)</p><p>G Left: Immunostaining of dARGLU1 protein (green) and VASA (red) in dArglu1 sisRNA KD germaria vs control (sibling control, <em>MTD-GAL4/TM3</em>) germaria. The asterisk (*) marks the anterior of the germarium. Right: Zoom in of the boxed area and the intensity plot shows the signal quantification of dARGLU1 protein and VASA in the direction of the white arrow. Scale bar: 10 μm.</p><p>H Quantification of germaria with and without nuclear speckle formation from dARGLU1 protein staining in dArglu1 sisRNA KD germaria vs control (sibling control, <em>MTD-GAL4/TM3</em>) germaria from (G). (n = 20 germaria)</p><p>I Confocal images of 125 nM dARGLU1-GFP protein (green) only and dARGLU1-GFP protein added with 50 nM IVT RNA (magenta), of either dArglu1 intron 2 until the 3' end of sisRNA (dArglu1 intron 2) or IVT dArglu1 intron 2 starting after 3' end of sisRNA (dArglu1 pre-mRNA). White arrowheads indicate dARGLU1-GFP droplets incorporated with IVT dArglu1 intron 2 and blue arrowheads indicate free dARGLU1-GFP droplets not incorporated with IVT dArglu1 intron 2. Scale bar: 10 μm.</p><p>J Quantification of total dARGLU1-GFP droplets from (I). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.</p><p>K Quantification of average dARGLU1-GFP droplets size with/without incorporation of IVT dArglu1 intron 2 from (I, dARGLU1-GFP + dArglu1 intron 2). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.</p><p>Data information: In (B-F), data are presented as mean ± SEM. ns=not significant, *P≤0.05, **P≤0.01 and ***P≤0.001 (Student's t-test). See also Figure EV5.</p>
|
https://api.sourcedata.io/file.php?figure_id=51353
|
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]
},
{
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"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
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]
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] |
||
10.15252/embr.202154350
|
Distinct biogenesis pathways may have led to functional divergence of the human and Drosophila Arglu1 sisRNA
|
2022
|
Figure 7
|
<p><strong>Figure 7. Cross-species binding of Arglu1 intron 2 to ARGLU1 protein and processing into sisRNAs.</strong></p><p>A Confocal images of 125 nM d/hARGLU1-GFP protein added with 50 nM IVT RNA of either hArglu1 intron 2 WT (magenta) or dArglu1 intron 2 (red). White arrowheads indicate free IVT RNA dots that are not co-localized with the d/hARGLU1-GFP droplets. Scale bar: 10 μm.</p><p>B Quantification of non co-localized RNA dots from (A, white arrowheads). (n = 16 biological replicates).</p><p>C, D Quantification of total hARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates).</p><p>E, F Quantification of total dARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates).</p><p>G Top: Schematic showing exon2-intron2-exon3 (E2i2E3) of dArglu1 used for exogenous expression in MCF-7 cells and locations of primers used for RT-PCR (bottom). Bottom: RT-PCR of processed dArglu1 spliced isoforms and <em>actin</em> as control in dArglu1 E2i2E3 OE MCF-7 cells vs untransfected (WT) MCF-7 cells.</p><p>H hArglu1 gene locus showing location of primers used in (I-K).</p><p>I-K qPCR of hArglu1 sisRNA, mRNA and pre-mRNA levels in dArglu1 E2i2E3 OE MCF-7 cells vs untransfected (WT) MCF-7 cells. (n = 3 biological replicates).</p><p>L Top: Schematic showing exon2-intron2-exon3 (E2i2E3) of hArglu1 used for exogenous expression in S2 cells and locations of primers used for RT-PCR (bottom). Bottom: RT-PCR of processed hArglu1 spliced isoforms and <em>actin5C</em> as control in hArglu1 E2i2E3 OE S2 cells vs untransfected (WT) S2 cells.</p><p>M dArglu1 gene locus showing location of primers used in (N-P).</p><p>N-P qPCR of dArglu1 sisRNA, mRNA and pre-mRNA levels in hArglu1 E2i2E3 OE S2 cells vs untransfected (WT) S2 cells (n = 3 biological replicates).</p><p>Data information: In (B-F), Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. In (I-K, N-P), data are presented as mean ± SEM. ns=not significant, *P≤0.05, **P≤0.01 and ***P≤0.001 (Student's t-test).</p>
|
https://api.sourcedata.io/file.php?figure_id=51354
|
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] |
||
28011581
|
10.15252/embj.201695734
|
Hippo kinases maintain polarity during directional cell migration in C. elegans
|
2016
|
Figure 1
|
<p><strong>Figure 1. </strong><strong>Hippo kinases </strong><strong>promote </strong><strong>AP </strong><strong>migration</strong> <strong>via prevention of</strong><strong> DV polarization</strong><strong>.</strong></p>
<p><strong>(A)</strong> Model of Q cell position and migration direction in <em>C. </em><em>elegans</em> L1 Larvae. Green arrow indicates that Q cells migrate along the AP body axis, whereas the dotted red arrows indicate that Q cells are potent to move towards the DV direction. <strong>(B)</strong> Schematics of the <em>cst-1</em> and <em>cst-2</em> gene models and DNA sequences of the <em>cst-1 </em>and/or <em>cst-2</em> mutants that were generated via CRISPR-Cas9. Purple bar, Serine/Threonine kinase domain; blue bar, SARAH (Salvador-Rassf-Hippo homolog) domain. Arrow, sgRNA target site; green, target sequence; red, PAM sequence; blue, inserted nucleotide; dashed lines, deleted nucleotides. <strong>(C)</strong> Quantification of AQR position in adult WT, <em>cst-1</em> and/or <em>cst-2</em> mutant animals. The full length between the URXs and the end of the nematode tail is divided into 10 blocks, and the percent of AQR that stopped within each block is indicated. <em>***, P < 0.001</em> by Fisher’s exact test. N = 50 - 109. <strong>(D)</strong> Quantification scheme of the angle (α) between the longitudinal axis of a migrating cell and the AP body axis. <strong>(</strong><strong>E</strong> <strong>-</strong> <strong>F</strong><strong>)</strong> Fluorescence time-lapse images of F-actin, plasma membrane and histone during AQR/QR.ap migration in WT (<strong>E</strong>) or <em>cst-1; cst-2</em> double mutant (<strong>F</strong>) animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrows, migration distance. F-actin is labeled with GFP-tagged with the actin-binding domain of Moesin (GFP::Moesin ABD), and the plasma membrane and Histone are labeled with mCherry-tagged with a myristoylation signal and a histone. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bars, 5 μm. <strong>(</strong><strong>G </strong><strong>- </strong><strong>J</strong><strong>) </strong>Quantification of the QR.ap migration angle <strong>(G & H)</strong>, distance <strong>(I) </strong>and speed <strong>(J)</strong>. Each line in <strong>(G & I) </strong>represents the measurement from one time-lapse movie. Error bars indicate SD. N = 10 - 17. Statistical significance is based on Student’s <em>t-</em>tests, <em>**, P < 0.01</em>; <em>***, P < 0.001</em>.</p>
|
https://api.sourcedata.io/file.php?figure_id=11497
|
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}
] |
|
28011581
|
10.15252/embj.201695734
|
Hippo kinases maintain polarity during directional cell migration in C. elegans
|
2016
|
Figure 2
|
<p><strong>Figure 2. </strong><strong>E</strong><strong>ctopic DV polarization in </strong><strong><em>mig-2</em></strong><strong> gain-of-function mutants and the phosphorylation of </strong><strong>MIG-2</strong><strong><sup>S139</sup></strong> <strong>by Hippo kinases</strong><strong>.</strong></p>
<p><strong>(A)</strong> Fluorescence time-lapse images of F-actin, plasma membrane and histone during QR.ap migration in <em>mig-</em><em>2</em><em>(</em><em>rh17)</em> gain-of-function mutant animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrow, migration distance. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bar, 5 μm. <strong>(B - </strong><strong>C</strong><strong>) </strong>Quantification of the QR.ap migration angle <strong>(B)</strong> and distance <strong>(C)</strong>. N = 10. <strong>(</strong><strong>D</strong><strong>)</strong> Mass spectrum of a MIG-2 peptide that was phosphorylated at Serine 139 in <em>C. </em><em>elegans</em>. The peptide was identified from the GFP::MIG-2 immunoprecipitate. <strong>(</strong><strong>E</strong><strong>)</strong> In vitro phosphorylation assay of CST-1/2 to MIG-2. Bacterially expressed His-tagged MIG-2 or His-tagged MIG-2<sup>S139A</sup> were incubated with the GST-tagged kinase domain of GST-CST-1/2 (1-318 aa). The kinase may use ATP-γ-S as a phosphodonor to thiophosphorylate its substrate; thus, the phosphorylation reaction was performed with ATP-γ-S, and anti-thiophosphate ester antibody was used to detect substrate phosphorylation (Allen et al, 2005; Allen et al, 2007). The final reaction was probed via immunoblotting with antibodies as indicated. Thio, thiophosphate ester. <strong>(</strong><strong>F</strong><strong>) </strong>Sequence alignment of MIG-2. Serine 139 (red) is conserved among the Rac1 homologs across species. The accession version is as follows: <em>C. </em><em>elegans</em>, CAB01691.1; <em>Drosophila</em>, CAC88352.1, AAA67041.1; Human, CAB53579.5; Rat, NP_599193.1; Mouse, AAH51053.1; Chicken, NP_990348.1, NP_001188381.1; Zebrafish, AAV85902.1; <em>X</em><em>enopus</em>, NP_001089332.1. <strong>(G & H)</strong> Three-dimensional ribbon (PDB 3TH5) representations of the human Rac1 polypeptide which indicate the position of the T135 (S139 in MIG-2) located between the last two α-helixes (Krauthammer et al, 2012).</p>
|
https://api.sourcedata.io/file.php?figure_id=11498
|
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] |
|
28011581
|
10.15252/embj.201695734
|
Hippo kinases maintain polarity during directional cell migration in C. elegans
|
2016
|
Figure 3
|
<p><strong>Figure 3.</strong> <strong>MIG-2</strong><strong><sup>S139</sup></strong> <strong>phosphorylation regulates actin assembly during cell migration</strong><strong>.</strong></p>
<p><strong>(A) </strong>Quantification of AQR position in <em>mig-</em><em>2</em><em>(</em><em>mu28)</em> null mutant animals with different rescue transgenes. N = 108 - 114. *, <em>P < 0.05</em>; ##, <em>P < 0.0</em><em>1</em>, phenotype enhanced. P values are based on Fisher’s exact tests. <strong>(B) </strong>Quantification of AQR position in WT and knock-in animals that harbor <em>mig-2</em><em><sup>S139</sup></em> point mutations. N = 100 - 111. ***, <em>P < 0.001</em> by Fisher’s exact test. <strong>(C - F) </strong>Quantification of the QR.ap migration angle <strong>(C & D)</strong>, distance <strong>(E) </strong>and speed <strong>(F)</strong> in <em>mig-2</em><em><sup>S139A/E</sup></em> mutant animals. Each line in <strong>(</strong><strong>C</strong><strong> & </strong><strong>E</strong><strong>) </strong>represents the measurement from one movie of QR.ap migration. Error bars indicate SD. N = 12 -17. <em>**, P < 0.01</em>; <em>***, P < 0.001</em> by Student’s <em>t</em> tests. <strong>(</strong><strong>G</strong><strong>)</strong> Inverted fluorescence images (up) and schematics (down) of QR.ap in WT, <em>cst-1</em><em>; cst-2</em> double or <em>mig-2</em> mutant animals. The plasma membrane and histones were labeled with mCherry. Scale bar, 5 μm. <strong>(</strong><strong>H</strong><strong>) </strong>Area ratio between the cell body and the nucleus in WT and mutant animals. Error bars indicate SD. N = 17 - 22. *, <em>P < 0.05</em>; **, <em>P < 0.01</em>; ***, <em>P < 0.001</em> by Student’s <em>t</em> tests. <strong>(</strong><strong>I</strong> <strong>-</strong> <strong>K</strong><strong>)</strong> Representative frames (upper) and quantification (lower) of F-actin enrichment at the QR.ap periphery in WT <strong>(</strong><strong>I</strong><strong>)</strong> and <em>mig-2</em><em><sup>S139A/E</sup></em> mutant animals <strong>(</strong><strong>J</strong><strong> & </strong><strong>K</strong><strong>)</strong>. The trace starts from the rear of the QR.ap and moves along the cell periphery to the leading edge (L) and back to the rear (R). F-actin was labeled with GFP::Moesin ABD; the plasma membrane and histone were marked with mCherry fluorescence. Green lines, F-actin fluorescence intensity; red lines, plasma membrane fluorescence intensity; dotted lines, cell periphery. Double-headed arrows indicate the F-actin enrichment region, in which the fluorescence intensity is 2-fold higher than the background. Scale bars, 5 μm. <strong>(</strong><strong>L</strong><strong>)</strong> Quantification of F-actin enrichment ratio in WT and <em>mig-2</em> mutants. Error bars indicate SD. N = 28 - 68. **, <em>P < 0.01</em>; ***, <em>P < 0.001</em> by Student’s <em>t</em><em>-</em>tests.</p>
|
https://api.sourcedata.io/file.php?figure_id=11499
|
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] |
|
28011581
|
10.15252/embj.201695734
|
Hippo kinases maintain polarity during directional cell migration in C. elegans
|
2016
|
Figure 4
|
<p><strong>Figure 4.</strong> <strong>Hippo kinases </strong><strong>are </strong><strong>restricted</strong> <strong>in</strong> <strong>the </strong><strong>cell body by MIG-2. </strong></p>
<p><strong>(A</strong> <strong>-</strong> <strong>C)</strong> Fluorescence images (upper) and quantifications (lower) of GFP-tagged CST-1 <strong>(A</strong><strong> & </strong><strong>B)</strong> or GFP-labeled F-actin <strong>(</strong><strong>C) </strong>and mCherry tagged plasma membrane and histone in WT and <em>mig-2</em> null mutant animals. The membrane protrusions in the white boxes are enlarged 3 times at the bottom. Yellow dashed lines indicate the cell periphery. White lines indicate the line to measure the fluorescence intensity. Green lines denote the CST-1 fluorescence intensity; red lines denote the plasma membrane fluorescence intensity; double-headed arrows denote the distance between the GFP signal and cell periphery. Scale bars, 5 μm. <strong>(D)</strong> Distance between GFP::CST-1 or GFP::Moesin ABD and the plasma membrane at the leading edge in WT and<em> mig-2</em> null mutants. Error bars indicate SD. N = 9 - 14. ***, <em>P < 0.001</em> by Student’s <em>t </em>test. <strong>(E</strong> <strong>-</strong> <strong>F) </strong>Fluorescence images (left) and quantifications (right) of GFP-tagged CST-1 and mCherry tagged plasma membrane and histone in WT and <em>mig-2</em> null mutant animals. Blue lines, sites of measurements; dotted lines, cell periphery; Green lines; CST-1 fluorescence intensity; red lines, mCherry fluorescence intensity. Scale bars, 5 μm. <strong>(G)</strong> Quantification of the fluorescence intensity ratio of GFP::CST-1 and mCherry-membrane between the leading edge and the lagging edge of AQR in WT and <em>mig-2 </em>null mutant animals. Error bars indicate SD. N = 20. ***, <em>P < 0.001</em> by Student’s <em>t</em> test. <strong>(H)</strong> Proposed model of Hippo-RhoG signaling negative feedback loop in the prevention of secondary fronts during directional cell migration.</p>
|
https://api.sourcedata.io/file.php?figure_id=11500
|
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] |
|
10.15252/embj.2020107403
|
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6
|
2021
|
Figure 1
|
<sd-panel> <p><strong>Figure 1. CRTH2 is located in the endoplasmic reticulum (ER) and cell membrane in fibroblasts.</strong></p> <p><strong>A,</strong> CRTH2 subcellular localization in GFP-fused CRTH2 expressing plasmid-transfected cells. <strong>Red arrows indicate CRTH2 localized in the</strong> ER and white arrows indicate CRTH2 localized in the plasma membrane<strong>. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.</strong></p> <p><strong>B,</strong> Western blot analysis of CRTH2 expression in cell membrane and ER in NIH 3T3 and HEK293T cells. Myc-tagged CRTH2 expressing plasmid was transfected into NIH 3T3 and HEK293T cells. Whole cell lysate, plasma, and ER fractions were obtained 48 h after transfection. Calnexin and ATP1B were used as markers for ER and cell membrane, respectively.</p> <p><strong>C,</strong> The effect of TGF-β1 (10 ng/mL) on the expression of caveolin-1 in NIH 3T3 cells.</p> <p><strong>D,</strong> Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF-β1 (10 ng/mL) treatment. <strong>Red arrows indicate CRTH2 localized in the</strong> ER and white arrows indicate CRTH2 localized in the plasma membrane<strong>. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.</strong></p> <p><strong>E, Quantification of CRTH2 colocalization with endoplasmic reticulum after</strong> TGF-β1 or vehicle treatment<strong>.</strong> *<em>P</em> < 0.05 vs vehicle (Mann-Whitney U test); <em>n</em> = 4 for all groups. Data are expressed as the mean ± standard error of the mean.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41481
|
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"text": "Calnexin",
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] |
||
10.15252/embj.2020107403
|
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6
|
2021
|
Figure 2
|
<sd-panel> <p><strong>Figure 2. CRTH2 interacts with LARP6 and GRP78 in the ER.</strong></p> <p><strong>A, Immunopurification and mass spectrometric analysis of CRTH2-containing protein complex in NIH 3T3 cells. Cellular extracts from NIH 3T3 cells stably expressing myc-CRTH2 protein, were immunopurified with anti-myc affinity beads and eluted with myc peptide. The eluates were used for SDS-PAGE and the gels were silver stained and analyzed using mass spectrometry.</strong></p> <p><strong>B-D, Representative mass spectrogram of CRTH2 (B), LARP6 (C) and GRP78 (D) from CRTH2-containing protein complex in NIH 3T3 cells.</strong></p> <p><strong>E-F,</strong> Co-<strong>immunoprecipitation analysis of association of CRTH2 with LARP6. HEK293T cells were co-transfected with Myc-CRTH2 and Flag-LARP6 expressing plasmids. Whole cell lysates from HEK293T cells were immunoprecipitated and then immunoblotted with the indicated antibodies</strong>.</p> <p><strong>G, Subcellular localization of CRTH2 and LARP6 in NIH 3T3 cells. NIH 3T3 cells were transfected with GFP-fused CRTH2 expressing plasmid, and fixed and immunostained with antibodies against the indicated proteins. Red arrow indicates colocalization of CRTH2 and LARP6 in the ER. Scale bar, 20 μm.</strong></p> <p><strong>H-I,</strong> Co-<strong>IP analysis of association of CRTH2 with GPR78. HEK293T cells were co-transfected with Flag-CRTH2 and Myc-GPR78 expressing plasmids.</strong></p> <p><strong>J, Colocalization of CRTH2, LARP6 and GRP78 in NIH 3T3 cells. White arrow indicates colocalization of CRTH2, LARP6 and GRP78. Scale bar, 20 μm.</strong></p> <p><strong>K,</strong> Schematic diagram depicting different domains of CRTH2 involved in the interaction with LARP6 and GRP78 in the endoplasmic reticulum membrane.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41483
|
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"text": "CRTH2",
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"P20029",
"Q3U7T8"
]
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"role": "assayed",
"text": "CRTH2",
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"uniprot_ids": [
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]
}
] |
||
10.15252/embj.2020107403
|
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6
|
2021
|
Figure 3
|
<sd-panel> <p><strong>Figure 3. CRTH2 deficiency increases collagen synthesis in fibroblasts through LARP6.</strong></p> <p><strong>A,</strong> RNA immunoprecipitation and qRT-PCR (RIP-qPCR) analysis of collagen type 1, α1 (Col1a1), collagen type 1, α2 (Col1a2), and collagen type 3, α1 (Col3a1) mRNA in CRTH2<sup>-/-</sup> and wild type (WT) fibroblasts using LARP6 antibody. *<em>P</em> < 0.05 vs WT (two-tailed <strong>Student's t test</strong>); <em>n</em> = 6 for all groups.</p> <p><strong>B,</strong> Effect of the CRTH2 deficiency on the stability of Col1a1, Col1a2, and Col3a1 mRNA in primary mouse fibroblasts. Fibroblasts were treated with 5 μg/mL Actinomycin D (Act D). Total RNA was extracted immediately after addition of Act D at 0, 2, 4, 6, 8, 12, and 24 h. Col1a1, Col1a2, and Col3a1 mRNA expression was quantitated by qRT-PCR. Dotted lines indicate mRNA half-lives; *<em>P</em> < 0.05 vs WT (<strong>two-way ANOVA</strong>); <em>n</em> = 6 for all groups.</p> <p><strong>C,</strong> Effect of CRTH2 deficiency on Col1a1, Col1a2, and Col3a1 mRNA expression in primary mouse fibroblasts. GAPDH was used as housekeeping gene. *<em>P</em> < 0.05 vs WT (two-tailed <strong>Student's t test</strong>); <em>n</em> = 8 for all groups.</p> <p><strong>D,</strong> Effect of CRTH2 deficiency on Collagen (Col) I and III protein expression in primary mouse fibroblasts with and without TGF-β1 (10 ng/mL) treatment.</p> <p><strong>E,</strong> Knockdown efficiency of LARP6 si-RNA (Si-LARP6) detected by qRT-PCR in primary fibroblasts. *<em>P</em> < 0.05 vs scramble, (two-tailed <strong>Student's t test</strong>); <em>n</em> = 5 for all groups.</p> <p><strong>F,</strong> Effect of LARP6 knockdown on Col1a1, Col1a2 and Col3a1 mRNA stability in WT and CRTH2<sup>-/-</sup> fibroblasts. *<em>P</em> < 0.05 vs WT, (<strong>two-way ANOVA</strong>); <em>n</em> = 8 for all groups.</p> <p><strong>G-I,</strong> Effect of LARP6 knockdown on Col1a1, Col1a2, and Col3a1 mRNA expression in WT and CRTH2<sup>-/-</sup> fibroblasts. Fibroblasts were transfected with Si-LARP6 or scramble siRNA. After 48 h transfection, RNA was prepared for qRT-PCR analysis. GAPDH was used as housekeeping gene. *<em>P</em> < 0.05 vs WT, <em><sup>#</sup>P</em> < 0.05 vs scramble (two-way ANOVA); <em>n</em> = 6 for all groups.</p> <p><strong>J,</strong> Effect of LARP6 knockdown on Collagen I and III protein expression in WT and CRTH2<sup>-/-</sup> fibroblasts.</p> <p>Data information: All data are expressed as the mean ± standard error of the mean.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41485
|
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"text": "Col3a1",
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"mapping_status": "mapped",
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"role": "assayed",
"text": "Collagen I",
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"P11087"
]
}
] |
||
10.15252/embj.2020107403
|
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6
|
2021
|
Figure 4
|
<sd-panel> <p><strong>Figure 4. CRTH2 deficiency in fibroblasts exacerbates injury-induced organ fibrosis in mice.</strong></p> <p><strong>A,</strong> Schematic diagram of the bleomycin-induced lung fibrosis model in mice.</p> <p><strong>B,</strong> Representative images of Masson′s trichrome staining of lung sections from CRTH2<sup>flox/flox</sup>Col1a2-Cre<sup>ERT</sup> <strong>(F-CRTH2 KO) and</strong> CRTH2<sup>flox/flox</sup> (Control) mice 14 days after bleomycin challenge. Blue indicates the collagen deposition regions. Scale bar, 100 μm.</p> <p><strong>C, Quantification of collagen content in mouse lung sections after</strong> bleomycin challenge<strong>.</strong> *<em>P</em> < 0.05 vs control, <em><sup>#</sup>P</em> < 0.05 vs saline group (two-way ANOVA); saline groups, <em>n</em> = 4 each; bleomycin groups, <em>n</em> = 8 each.</p> <p><strong>D,</strong> Hydroxyproline content <strong>in lungs from</strong> control and F-CRTH2 KO mice treated with saline or bleomycin. *<em>P</em> < 0.05 vs control, <em><sup>#</sup>P</em> < 0.05 vs saline group (two-way ANOVA); saline groups, <em>n</em> = 4 each, bleomycin groups, <em>n</em> = 8 each.</p> <p><strong>E,</strong> Western blot analysis of <strong>collagen (Col)</strong> Ⅰ and Ⅲ expression in lung tissues from control and F-CRTH2 KO mice 14 days after bleomycin challenge.</p> <p><strong>F,</strong> Protocol for CCl<sub>4</sub>-induced liver fibrosis in mice.</p> <p><strong>G,</strong> Representative images of Masson′s trichrome staining of liver sections from control and F-CRTH2 KO mice 4 weeks after CCl<sub>4</sub> challenge; Scale bar, 100 μm.</p> <p><strong>H, Quantification of collagen content in mouse liver sections after</strong> CCl<sub>4</sub> challenge<strong>.</strong> *<em>P</em> < 0.05 vs control, <em><sup>#</sup>P</em> < 0.05 vs mineral oil group (two-way ANOVA); mineral oil groups, <em>n</em> = 4 each; CCl<sub>4</sub> groups, <em>n</em> = 8 each.</p> <p><strong>I,</strong> Hydroxyproline content <strong>in lungs from</strong> control and F-CRTH2 KO mice treated with CCl<sub>4</sub>. *<em>P</em> < 0.05 vs control, <em><sup>#</sup>P</em> < 0.05 vs mineral oil group (two-way ANOVA); mineral oil groups, <em>n</em> = 4 each, CCl<sub>4</sub> groups, <em>n</em> = 8 each.</p> <p><strong>J,</strong> Western blot analysis of Collagen Ⅰ and Ⅲ expression in liver tissues from control and F-CRTH2 KO mice 4 weeks after CCl<sub>4</sub> challenge.</p> <p><strong>K,</strong> Schematic diagram of isoproterenol (ISO)-induced heart fibrosis in mice.</p> <p><strong>L,</strong> Representative images of Masson′s trichrome staining of heart sections from control and F-CRTH2 KO mice 21 days after ISO challenge; Scale bar, 50 μm.</p> <p><strong>M, Quantification of collagen content in mouse heart sections after</strong> ISO challenge<strong>.</strong> *<em>P</em> < 0.05 vs control, <em><sup>#</sup>P</em> < 0.05 vs saline group (two-way ANOVA); PBS groups, <em>n</em> = 3 each, ISO groups, <em>n</em> = 7-8.</p> <p><strong>N,</strong> Hydroxyproline content <strong>in lungs from</strong> control and F-CRTH2 KO mice treated with ISO. *<em>P</em> < 0.05 vs control, <em><sup>#</sup>P</em> < 0.05 vs saline group (two-way ANOVA); saline groups, <em>n</em> = 4 each; ISO groups, <em>n</em> = 8 each.</p> <p><strong>O,</strong> Western blot analysis of Collagen Ⅰ and Ⅲ expression in heart tissues from control and F-CRTH2 KO mice 21 days after ISO challenge.</p> <p>Data information: All data are expressed as the mean ± standard error of the mean.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41487
|
[
{
"ext_dbs": "NCBI gene",
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"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "14764",
"original_type": "gene",
"role": "intervention",
"text": "CRTH2",
"type": "geneprod",
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]
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{
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"ext_tax_names": "Mus musculus",
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{
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{
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{
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"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
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"text": "CRTH2",
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}
] |
||
10.15252/embj.2020107403
|
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6
|
2021
|
Figure 5
|
<sd-panel> <p><strong>Figure 5. LARP6 is required for deteriorated organ fibrosis in CRTH2<sup>-/-</sup> mice.</strong></p> <p><strong>A,</strong> Representative images of Masson's trichrome staining of lung sections from F-LARP6 KO and F-LARP6/F-CRTH2 double KO (F-DKO) mice 14 days after bleomycin challenge; Scale bar, 100 μm.</p> <p><strong>B, Quantification of collagen content in mouse lung sections after</strong> bleomycin challenge<strong>.</strong> <em><sup>#</sup>P</em> < 0.05 vs saline group (two-way ANOVA); saline groups, <em>n</em> = 4 each, bleomycin groups, <em>n</em> = 7-8.</p> <p><strong>C,</strong> Hydroxyproline content <strong>in lungs from</strong> F-LARP6 KO and F-DKO mice treated with bleomycin. <em><sup>#</sup>P</em> < 0.05 vs saline group (two-way ANOVA); saline groups, <em>n</em> = 4 each, bleomycin groups, <em>n</em> = 8 each.</p> <p><strong>D,</strong> Western blot analysis of Collagen (Col) Ⅰ and Ⅲ expression in lung tissues from F-LARP6 KO and F-DKO mice 14 days after bleomycin challenge.</p> <p><strong>E,</strong> Representative images of Masson's trichrome staining of liver sections from F-LARP6 KO and F-DKO mice 4 weeks after CCl<sub>4</sub> challenge; Scale bar, 100 μm.</p> <p><strong>F, Quantification of collagen content in mouse liver sections after</strong> CCl<sub>4</sub> challenge<strong>.</strong> <em><sup>#</sup>P</em> < 0.05 vs mineral oil group (two-way ANOVA); mineral oil groups, <em>n</em> = 4 each, CCl<sub>4</sub> groups, <em>n</em> = 8 each.</p> <p><strong>G,</strong> Hydroxyproline content <strong>in livers from</strong> F-LARP6 KO and F-DKO mice treated with CCl<sub>4</sub>. <em><sup>#</sup>P</em> < 0.05 vs mineral oil group (two-way ANOVA); saline groups, <em>n</em> = 4 each, CCl<sub>4</sub> groups, <em>n</em> = 8 each.</p> <p><strong>H,</strong> Western blot analysis of Collagen Ⅰ and Ⅲ expression in liver tissues from F-LARP6 KO and F-DKO mice 4 weeks after CCl<sub>4</sub> challenge.</p> <p><strong>I,</strong> Representative images of Masson's trichrome staining of heart sections from F-LARP6 KO and F-DKO mice 21 days after ISO challenge; Scale bar, 50 μm.</p> <p><strong>J, Quantification of collagen content in mouse heart sections after</strong> ISO challenge<strong>.</strong> <em><sup>#</sup>P</em> < 0.05 vs saline group (two-way ANOVA); saline groups, <em>n</em> = 3 each, ISO groups, <em>n</em> = 7 each.</p> <p><strong>K,</strong> Hydroxyproline content <strong>in hearts from</strong> F-LARP6 KO and F-DKO mice treated with ISO. <em><sup>#</sup>P</em> < 0.05 vs saline group (two-way ANOVA); saline groups, <em>n</em> = 4 each, ISO groups, <em>n</em> = 8 each.</p> <p><strong>L,</strong> Western blot analysis of Collagen Ⅰ and Ⅲ expression in heart tissues from F-LARP6 KO and F-DKO mice 21 days after ISO challenge.</p> <p><strong>M,</strong> Schematic diagram of the working model of CRTH2-mediated collagen homeostasis in fibroblasts<strong>.</strong></p> <p>Data information: All data are expressed as the mean ± standard error of the mean.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41489
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "Q9Z2J6",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "intervention",
"text": "CRTH2",
"type": "geneprod",
"uniprot_ids": [
"Q9Z2J6"
]
}
] |
||
10.15252/embj.2020107403
|
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6
|
2021
|
Figure 6
|
<sd-panel> <p><strong>Figure 6. Bumetanide inhibits bleomycin-induced lung fibrosis in mice by targeting the LARP6 RRM domain.</strong></p> <p><strong>A,</strong> Structure-based flow chart for the virtual screening of small molecules interacting with the LARP<strong>6-RRM domain.</strong></p> <p><strong>B-C, Microscale thermophoresis (MST) analysis of the binding affinity of</strong> Bumetanide with MBP-LARP6 <strong>and</strong> MBP control protein<strong>.</strong></p> <p><strong>D,</strong> Binding mode of the LARP6-RRM domain with Bumetanide. The protein is shown as cartoons and is green. Bumetanide and dominant residues are shown as sticks. Bumetanide is cyan and the dominant residues are green.</p> <p><strong>E-F,</strong> Effect of Bumetanide (2 μM) on direct interaction of CRTH2 N-terminal (CRTH2-2) with LARP6 RRM domain (LARP6-4) using GST and MBP pulldown assays.</p> <p><strong>G,</strong> Effect of Bumetanide on LARP6 binding to Col1a1, Col1a2, and Col3a1 <strong>mRNA in</strong> primary mouse fibroblasts. *<em>P</em> < 0.05 vs DMSO, (<strong>unpaired two-tailed t test</strong>); <em>n</em> = 6 for all groups.</p> <p><strong>H, Protocol for administration of Bumetanide to bleomycin-challenged mice. Bumetanide</strong> (2 mg/kg) wa<strong>s subcutaneously injected in mice daily for 14 days.</strong></p> <p><strong>I, Representative</strong> Masson's trichrome staining of lung sections from <strong>Bumetanide-treated</strong> control <strong>and F-CRTH2 KO mice</strong>. Scale bars, 100 µm.</p> <p><strong>J, Quantification of collagen content in lung sections from Bumetanide-treated</strong> control (CRTH2<sup>flox/flox</sup>) <strong>and F-CRTH2 KO mice.</strong> *<em>P</em> < 0.05 vs control, <sup>#</sup><em>P</em> < 0.05 vs Vehicle (two-way ANOVA); <em>n</em> = 7 for all groups.</p> <p><strong>K, Effect of Bumetanide treatment on pulmonary hydroxyproline levels in</strong> control <strong>and F-CRTH2 KO mice. *P < 0.05</strong> vs control; <sup>#</sup><em>P</em> < 0.05 vs Vehicle (two-way ANOVA); <strong>n = 7</strong> for all groups<strong>.</strong></p> <p><strong>L, Effect of Bumetanide treatment on Collagen (Col) I and III expression in lungs from</strong> control <strong>and F-CRTH2 KO mice.</strong></p> <p>Data information: All data are expressed as the mean ± standard error of the mean.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41491
|
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"original_type": "protein",
"role": "assayed",
"text": "LARP6",
"type": "geneprod",
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"Q8BN59"
]
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{
"ext_dbs": "NCBI gene",
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"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
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"text": "CRTH2",
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{
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"original_type": "gene",
"role": "intervention",
"text": "CRTH2",
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"Q9Z2J6"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "14764",
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"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "14764",
"original_type": "gene",
"role": "intervention",
"text": "CRTH2",
"type": "geneprod",
"uniprot_ids": [
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}
] |
||
10.15252/embj.2020107403
|
ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6
|
2021
|
Figure 7
|
<sd-panel> <p><strong>Figure 7. Bumetanide reduces TGF-β1-induced collagen biosynthesis in human fibroblasts.</strong></p> <p><strong>A-B,</strong> Bumetanide interferes with the interaction of CRTH2 with LARP6 in MRC-5 <strong>human lung fibroblasts MRC-5</strong>. <strong>MRC-5</strong> cells were co-transfected with myc-LARP6 and flag-CRTH2 expressing plasmids for 24 h, and then treated with Bumetanide (0.1, 2 μM) for an additional 24 h. Whole cell lysates from <strong>MRC-5</strong> cells were immunoprecipitated and immunoblotted with the indicated antibodies.</p> <p><strong>C-E,</strong> Effect of Bumetanide on LARP6 binding to Col1a1, Col1a2, and Col3a1 <strong>mRNA in</strong> cultured <strong>MRC-5 cells. MRC-5</strong> cells were treated with TGF-β1 and Bumetanide (2 μM) for 24 h. The cells were ultraviolet-cross-linked and RNA-bound proteins were immunoprecipitated with LARP6 antibody followed by RNA extraction for qRT-PCR<strong>.</strong> *<em>P</em> < 0.05 vs Vehicle, <sup>#</sup><em>P</em> < 0.05 vs Control (two-way ANOVA); <em>n</em> = 6 for all groups.</p> <p><strong>F-H,</strong> Effect of Bumetanide on Col1a1, Col1a2 and Col3a1 <strong>mRNA expression in MRC-5 cells</strong>. GAPDH was used as housekeeping gene. *<em>P</em> < 0.05 vs vehicle, <sup>#</sup><em>P</em> < 0.05 vs Control (two-way ANOVA); <em>n</em> = 6 for all groups.</p> <p><strong>I,</strong> Effect of Bumetanide on Collagen (Col) I and III protein <strong>expression in MRC-5 cells</strong>.</p> <p>Data information: All data are expressed as the mean ± standard error of the mean.</p> <p><strong>Expanded View</strong></p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41492
|
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"text": "(Col) I",
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||
10.15252/embj.2019101704
|
The TRAPP complex mediates secretion arrest induced by stress granule assembly
|
2019
|
Figure 1
|
<p>Figure 1.</p><p>The TRAPP complex is recruited to stress granules</p><p>A. Localization of TRAPP complex components after sodium arsenite (SA) treatment in HeLa cells treated with 300 µM SA for 30 min. Fluorescence microscopy of fixed cells using antibodies against TRAPPC2 and eIF3 (to label SGs) at steady state and after SA treatment (top two rows). Other panels show localization of GFP-TRAPPC3 and endogenous TRAPPC1 to eIF3-labeled SGs and of endogenous TRAPPC9 and TRAPPC8 to G3BP-labeled SGs. DAPI (blue). Scale bar, 10 µm.</p><p>B. Representative images of a time course analysis of TRAPPC2 redistribution to SGs. Cells were treated as in (A). The graph (B') shows quantification of TRAPPC2 localization at SGs over time as the ratio between TRAPPC2 (mean fluorescence intensity) in SG puncta and cytosolic TRAPPC2. Mean ± s.e.m. n = 50 cells per experiment, N = 3. Scale bar, 10 µm.</p><p>C. Analysis of TRAPPC2 (green), TRAPPC8 (blue) and TRAPPC9 (orange) localization at SGs upon TRAPPC2, TRAPPC4 TRAPPC8 and TRAPPC9 depletion. Mean ± s.e.m. n>100. *p<0.05; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.</p><p>D. Schematic representation of MS/MS analysis of TRAPPC2 interactors. In addition to the TRAPP components and known TRAPPC2 interactors, 53 RNA binding proteins known to associate with SGs were co-IP with TRAPPC2. Proteins that associate with TRAPPC2 only upon SA-treatment are highlighted in red.</p><p>E. Analysis of TRAPPC2 localization at SGs upon CLIC1, CRYAB and ENO1 depletion. Mean ± s.e.m. n>100. ****p<0.0001; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.</p>
|
https://api.sourcedata.io/file.php?figure_id=27520
|
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||
10.15252/embj.2019101704
|
The TRAPP complex mediates secretion arrest induced by stress granule assembly
|
2019
|
Figure 2
|
<p>Figure 2.</p><p>TRAPPC2 is required for Sec23/24 relocalization to SGs</p><p>A. HeLa cells, untreated or treated with SA, were fixed and visualized by fluorescence microscopy using anti-Sec24C Ab, anti-eIF3 Ab, and DAPI (blue).</p><p>B. Quantification of Sec24C redistribution to SGs over time after SA treatment (the ratio between Sec24C (mean fluorescence intensity) in SG puncta and cytosolic Sec24C). Mean ± s.e.m. n = 50 cells per experiment, N = 3.</p><p>C. Quantification of residual Sec24C in ERES after SA treatment. The data are expressed as percentage of steady state values (CTRL). Mean ± s.e.m. n = 100 cells per experiment, N = 3.</p><p>D. Representative images of TRAPPC2 localization in Sec23AB-KD and Sec24ABCD-KD cells treated with SA. Cells were fixed and visualized by fluorescence microscopy using anti-TRAPPC2 Ab, anti-G3BP Ab (to label SGs) and DAPI (blue).</p><p>E. Quantification of TRAPPC2 redistribution to SGs after KD of the indicated Sec23 and Sec24 combinations, calculated as the ratio between TRAPPC2 (mean intensity) in SG puncta and cytosolic TRAPPC2 and expressed as % of CTRL. Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.</p><p>F. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC3-KD and TRAPPC2-KD cells treated with SA. Depletion of the entire TRAPP complex (via TRAPPC3 depletion) or of only TRAPPC2 reduces Sec24C recruitment. Graph, quantification of Sec24C at SGs, calculated as in (E). Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ****p< 0.0001, One-way ANOVA with Dunnett's multiple comparison test.</p><p>G. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC2-KO cells treated with SA.</p><p>Data information: (A, D, F, G) Scale bars, 10 µm.</p>
|
https://api.sourcedata.io/file.php?figure_id=27522
|
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] |
||
10.15252/embj.2019101704
|
The TRAPP complex mediates secretion arrest induced by stress granule assembly
|
2019
|
Figure 3
|
<p>Figure 3.</p><p>Comparison and relationships between the ERES-associated and the SG-associated pools of TRAPP and COPII</p><p>A. TRAPP and COPII associate more stably with SGs than with ERES. The membrane association of Sec24C was evaluated in non-permeabilized or permeabilized cells with or without SA treatment, as indicated. G3BP was used as an SG marker. Left panel insets, G3BP staining in non-permeabilized cells. Dashed white lines show the outline of permeabilized SA-untreated cells. Blue, DAPI.</p><p>B. Stabilizing TRAPP and COPII at the ERES prevents their relocalization to SGs. HeLa cells overexpressing GFP-Sar1H79G were treated with SA and immunostained for Sec24C, TRAPPC2, and eIF3, as indicated. Blue, DAPI. Graphs show quantification of Sec24C or TRAPPC2 at SGs in GFP-Sar1H79G-expressing cells. Data are the ratio between Sec24C or TRAPPC2 mean intensity in SG puncta and Sec24C or TRAPPC2 mean intensity at ERES, expressed as a percentage of the non-transfected (NT) cells. Mean ± s.e.m. n = 60-70, three independent experiments. ****p<0.0001, Student's unpaired two-tailed <em>t</em>-test.</p><p>C. Effect of Sec31A depletion on TRAPPC2 recruitment to SGs. Cells were mock-treated or KD for Sec31, treated with SA, and then immunostained for TRAPPC2 and eIF3 as indicated. Graphs, quantification of TRAPPC2 at ERES and SGs after SA treatment. Mean ± s.e.m., one representative experiment; n=60-80. ** p<0.001, ****p<0.0001, Student's unpaired two-tailed <em>t</em>-test.</p><p>D. Effect of Sec31A depletion on Sec24C recruitment to SGs. Cells were mock-treated or KD for Sec31 and then immunostained for Sec24C and cTAGE5 (to stain ERES, top panels) and for eIF3 (bottom panels). Insets show eIF3. Graphs, quantification of Sec24C at ERES and in SGs after SA treatment. n=80. ****p<0.0001, Student's unpaired two-tailed <em>t</em>-test.</p><p>Data information; (A-D) Scale bars, 10 µm.</p>
|
https://api.sourcedata.io/file.php?figure_id=27524
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||
10.15252/embj.2019101704
|
The TRAPP complex mediates secretion arrest induced by stress granule assembly
|
2019
|
Figure 4
|
<p>Figure 4.</p><p>The association of TRAPP with SGs is under control of CDK1/2</p><p>A. Cells were treated with 273 kinase inhibitors (10 µM) from the Sellekchem library and with SA, and Sec24C localization in G3BP puncta (% of total G3BP area) was analyzed by automated microscopy as described in Methods under High content screening and is reported as a percentage of the control (cells treated with SA alone). Gray dashed line, mean of control; gray box, control standard deviation (± 18.2%); red line, threshold of positive hits; red box: positive hits.</p><p>B. Enrichment Analysis of the positive hits; for each class of inhibitor, the enrichment (in percentage) of compounds falling into positive hits and the enrichment (in percentage) in the total number of compounds was calculated and expressed as a ratio.</p><p>C. Evaluation of CDK kinase activity upon SA treatment. Western blot analysis of phosphoSer-CDK substrates in control, SA (300µM, 30 min), Dinaciclib (10 µM, 180 min) and SA+Dinaciclib (150 min Dinaciclib and 30 min SA). CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition. Left, Western blot, right: Ponceau was used as loading control. Image, one representative experiment out of 3 independent replicates.</p><p>D. Drug specificity from high (++++) to low (+) of the different CDKs as described by Selleckchem, and Sec24C recruitment to SGs as a percentage of control.</p><p>E. Analysis of Sec24C recruitment to SGs in control and CDK1+2-KD HeLa cells. Different levels of CDK1+2 KD were achieved by transfecting cells with different amounts of siRNA for different times (2 nM 24 h; 5 nM 24 h; 5 nM 48 h; 20 nM 48 h) to give 50, 30, 20 and 10%, respectively, of the CDK1+2 levels in the control. CDK1+2 levels measured by qRT-PCR and Sec24C recruitment to SGs were evaluated from parallel cultures. Sec24C recruitment to SGs was performed by automated microscopy as described in (A) and is expressed as a percentage of mock (CTRL, set at 100%) ± s.d. N=8.</p><p>F. Representative images of HeLa cells treated with the three best hits (Flavopiridol, SNS-032, Dinaciclib; 1µM 150 min) and then exposed to SA (300 µM, 30 min), followed by immunostaining for Sec24C and G3BP. Scale bar, 10 µm.</p><p>G. HeLa cells were treated with Flavopiridol, SNS-032, or Dinaciclib for 150 min at the indicated concentrations, subsequently treated with SA (300 µM, 30 min), and then immunostained for Sec24C and G3BP and imaged by OPERA. BS-181, a specific CDK7 inhibitor, was used as negative control. Sec24C localization to SGs is expressed as a percentage of the control (cells treated with SA alone). Dashed red line, mean of control; yellow box, standard deviation of control (± 9.8%), N=12.</p><p>H. Cells were treated as in (G) and immunostained for TRAPPC2 and eIF3. Scale bar, 10 µm. The graph shows the quantification of TRAPPC2 localization in SG puncta (mean intensity). Data are mean ± s.e.m. expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol, SNS-032 or Dinaciclib treatment compared to the control (cells treated with SA alone). N=3, three independent experiments, n=60-80 cells per experiment. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.</p>
|
https://api.sourcedata.io/file.php?figure_id=27526
|
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] |
||
10.15252/embj.2019101704
|
The TRAPP complex mediates secretion arrest induced by stress granule assembly
|
2019
|
Figure 5
|
<p>Figure 5.</p><p>CDK1/2 modulate the COPII cycle at ERES</p><p>A. Immunofluorescence images of Sec24C and TRAPPC2 in vehicle-treated and SNS-032-treated (1 µM, 3 h) cells. Graphs show the quantification of Sec24C and TRAPPC2 at ERES (mean intensity) normalized in SNS-032-treated cells relative to vehicle-treated cells (set as 100%). Mean ± s.e.m. of three independent experiments. **p<0.05, ****p<0.0001, Student's unpaired two-tailed <em>t</em>-test.</p><p>B. HeLa cells were treated with Dinaciclib (1 µM, 3 h) and permeabilized or not with digitonin as described in Materials and Methods. A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency. Upper panels, non-permeabilized (NP) control cells; middle panels, permeabilized control cells; lower panels, permeabilized Dinaciclib-treated cells.</p><p>C. Quantification of Sec24C membrane association after CDK inhibitor treatment. Digitonin-permeabilized cells treated with vehicle (CTRL) or the indicated CDK inhibitor (1 µM, 3 h) were immunostained for Sec24C. The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug-treated cells is expressed as fold change compared to the control; n= 60-80; mean ± s.e.m. of three independent experiments ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.</p><p>D. HeLa cells transfected with HA-hnRNPK were left untreated (Steady state) or treated with SA (300µM, 30 min) or SA+Dinaciclib (10 µM, 150 min) and then immunostained with anti-HA and anti-G3BP antibodies.</p><p>E. TRAPPC2 interacts with hnRNPK. (Top) SDS-PAGE and Western blot analysis of endogenous hnRNPK after immunoprecipitation with an anti-TRAPPC2 Ab or IgG, and (bottom) after immunoprecipitation with an anti-HA Ab from cells transfected (+) or not (-) with hnRNPK-HA detected with the indicated antibodies.</p><p>F. Mock or hnRNPK KD cells treated with SA were immunostained for TRAPPC2 and eIF3.</p><p>G. Mock or hnRNPK KD cells treated with SA were immunostained for Sec24C and G3BP.</p><p>H. Graphs show quantification of TRAPPC2 and Sec24C redistribution to SGs in mock treated (CTRL) and hnRNPK KD cells. n>100, N=3 mean ± s.d. ***p<0.001.****p<0.0001, Student's unpaired two-tailed <em>t</em>-test.</p><p>Data information: (A, B, D, F, G) Scale bars, 10 µm.</p>
|
https://api.sourcedata.io/file.php?figure_id=27528
|
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||
10.15252/embj.2019101704
|
The TRAPP complex mediates secretion arrest induced by stress granule assembly
|
2019
|
Figure 6
|
<p>Figure 6.</p><p>TRAPP and Sec23/24 migration to SGs depends on the proliferation state of cells</p><p>A. HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA (30 min, 300 µM), and immunostained for Sec24C or TRAPPC2 and eIF3. Scale bar 10 µm.</p><p>B. Analysis of the proliferation status of the cells after 8 h starvation in HBSS. Top, representative images of starved and non-starved (CTRL) cells using EdU incorporation (see Materials and Methods). Scale bar 100 µm. Bottom, quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells, N=3, mean ± s.d.</p><p>C. HeLa cells were seeded at different confluency, treated with SA and stained for Sec24C and G3BP as an SG marker. Scale bar, 10 µm. Flow cytometry (FACS) analysis (right panels) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency. The graph shows quantification of Sec24C mean intensity in SG puncta (normalized for the cytosolic Sec24C) at the indicated cell confluency. Mean ± s.e.m. of a representative experiment out of 5 biological replicates. n= 50-80. ns: not significant; ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.</p><p>D. Differentiation of podocytes. Western blot of growing and differentiated podocytes. Synaptopodin (SYNPO) was used as a differentiation marker, GAPDH as a loading control.</p><p>E. Growing and differentiated podocytes were treated with SA (300 µM, 30 min) and stained for TRAPPC2 or Sec24C and eIF3. Scale bar, 10 µm. The graphs show quantification of TRAPPC2 and Sec24C (mean intensity) in SGs. Mean ± s.e.m. of three independents experiments. ****p<0.0001, Student's unpaired two-tailed <em>t</em>-test.</p><p>F. Western blot analysis of total (RB) and phosphorylated (p-RB) retinoblastoma in growing (non-differentiated) versus differentiated podocytes. ß-actin was used as loading control.</p><p>G. CDK kinase activity in growing and differentiated podocytes. Western blot using a specific antibody recognizing phosphoSer-CDK substrates was used on total cell lysates.</p>
|
https://api.sourcedata.io/file.php?figure_id=27530
|
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||
10.15252/embj.2019101704
|
The TRAPP complex mediates secretion arrest induced by stress granule assembly
|
2019
|
Figure 7
|
<p>Figure 7.</p><p>The TRAPP complex controls SG composition and function</p><p>A. SG area (see Materials and Methods) in mock, TRAPPC2 TRAPPC3, TRAPPC8, and TRAPPC9-depleted cells treated with SA. Data are shown in box-and-whisker plots: Box-plot central line, median; box limits, upper and lower quartiles; whiskers show the minimum to maximum. N=3. ***p<0.001, ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.</p><p>B. HeLa cells microinjected with control IgG or a TRAPPC3-specific antibody (right panels in green) were treated with SA. The TRAPPC3 Ab disrupts the Golgi (Yu <em>et al,</em> 2006), monitored using an anti-TGN46 Ab. Anti-G3BP was used to stain SGs. The graph shows quantification of the SG area. Mean ± s.e.m. three independent experiments; n>80. ****p<0.0001, Student's unpaired two-tailed <em>t</em>-test. Scale bar, 10 µm.</p><p>C. Structured Illumination Microscopy (SIM)-Super resolution (SR) images of endogenous TRAPPC2 and GFP-Sec23 localizing at SGs, stained for G3BP. Scale bar 5 µm. Right, magnification of boxed area. Scale bar, 0.5 µm.</p><p>D,E. Localization of Raptor (D) and RACK1 (E) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 µm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock (TRL). Mean ± S.D. three independent replicates. *p<0.02; **p<0.009 in (D), *p<0.05; ****p<0.0001 in (E), One-way ANOVA with Dunnett's multiple comparison test.</p><p>F,G. Immunoprecipitation (IP) with an anti-TRAPPC2 Ab or IgG from cells with or without SA treatment, as indicated, followed by SDS-PAGE and Western blot with ant-Raptor (F) or anti-RACK1 (G) Abs. Input: cell lysate (40 µg for TRAPPC2, 10 µg for RACK1 and Raptor).</p><p>H,I. Localization of Raptor (H) and RACK1 (I) in untreated cells or cells pre-treated with the indicated CDK inhibitor (1 µM, 150 min) and then with SA (300 µM, 30 min). G3BP was used to stain SGs. Scale bar, 10 µm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.</p><p>J. Analysis of cell death after overnight recovery of HeLa cells treated or untreated with CDKi (SNS-032; Flavopiridol or Dinaciclib, 1 µM, 150 min) and then treated or not with SA (500 µM, 3 h). Images were acquired automatically by OPERETTA microscope. Values indicate the percentage of the total number of nuclei (stained with DAPI) positive for BoBo-3 staining. Mean ± s.d. of one representative experiment out of three independent replicates. ns: not significant, ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.</p><p>K. Analysis of cell death, as described in (J), in HeLa cells that were left untreated, treated with non-targeting siRNAs or siRNAs against TRAPPC2, and in two cell clones knocked out for TRAPPC2, with and without SA treatment. Mean ± s.d. of three independent replicates.</p>
|
https://api.sourcedata.io/file.php?figure_id=27531
|
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||
10.15252/embj.2019101704
|
The TRAPP complex mediates secretion arrest induced by stress granule assembly
|
2019
|
Figure 8
|
<p>Figure 8.</p><p>The sequestering of TRAPP and COPII in SGs slows down ER export</p><p>A. Left, control or SA-treated (500 µM, 120 min) human fibroblasts (HFs) were incubated at 40°C for 180 min and stained for eIF3, PCI, and Sec24C. Right, the cells were imaged 10 min after shifting the temperature from 40°C to 32°C and stained for eIF3, PCI, and Giantin (to label the Golgi).</p><p>B. HeLa cells were exposed to SA (200 µM) for the indicated time, alone or in combination with ISRIB and SGs were analyzed. Quantification of SG area (µm) of three independent experiments. Mean ± s.e.m. n = 60-80 cells per experiment, N = 3.</p><p>C. Quantification of Sec24C localization at SGs over time (the ratio between Sec24C (mean fluorescence intensity) in SG puncta and cytosolic Sec24C). Mean ± SD. n = 60-80 cells per experiment, N = 3.</p><p>D. HeLa cells expressing VSV-G-UVR8 mEOS were left untreated or treated with SA alone or in combination with ISRIB (1 µM) (120 min ISRIB pre-treatment and 30 min SA+ISRIB) and then pulsed with blue light. Images were taken 10 min after the UV pulse and processed for staining with the indicated markers.</p><p>E. Cells were treated with SA for 30 min, the SA was washed out (WO), and cells were left to recover for 120 min in the presence of cycloheximide, followed by a blue light pulse and processing for staining as described in (D).</p><p>F. Quantification of VSV-G (mean intensity) in the Golgi area to the total VSV-G per cell under the conditions described in (D,E). Data are expressed as percentage of the control. Mean ± s.e.m. of three independent experiment. ****p<0.0001; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.</p><p>Data information: (A, D, E) Scale bars, 10 µm.</p>
|
https://api.sourcedata.io/file.php?figure_id=27532
|
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"ext_ids": "836506",
"ext_tax_ids": "3702",
"ext_tax_names": "Arabidopsis thaliana",
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"mapping_status": "mapped",
"ncbi_gene_id": "836506",
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"role": "intervention",
"text": "UVR8",
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"uniprot_ids": [
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]
},
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"ext_dbs": "Uniprot",
"ext_ids": "P04882",
"ext_tax_ids": "11283",
"ext_tax_names": "Vesicular stomatitis virus (serotype New Jersey / strain Ogden)",
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] |
||
10.15252/embj.2019101704
|
The TRAPP complex mediates secretion arrest induced by stress granule assembly
|
2019
|
Figure 9
|
<p>Figure 9.</p><p>The sequestering of TRAPP in SGs induces the fragmentation of the GC in a Rab1-dependent manner</p><p>A. AiryScan images of HeLa cells treated with SA for the indicated times and stained with GM130 (gray). Scale bar, 10 µm. The graph shows quantification of Golgi objects in SA-treated cells. Mean ± s.e.m. of three independent experiments. n=90-100. ****p<0.0001; ns: not significant, Student's unpaired two-tailed <em>t</em>-test.</p><p>B. Control cells and cells treated with Dinaciclib (CDKi) were treated with SA and stained for GM130 (in gray). Insets, eIF3. Blue, nuclear DAPI staining. The graph shows quantification of Golgi objects in HeLa cells untreated or treated with SNS-032, Flavopiridol, or Dinaciclib for 150 min and then treated with SA (300 µM, 30 min). Mean ± s.e.m. of three independent experiments. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.</p><p>C. Quantification of Golgi objects in cells plated at different percentage of confluency and treated or untreated with SA. Mean ± s.e.m. of three independent experiments. **p<0.001; ***p<0.0002; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.</p><p>D. Quantification of Golgi objects in cells pre-treated with ISRIB (1 µM) for 3 h and then exposed to SA (30 min) in the presence of ISRIB. ***p<0.002, Student's unpaired two-tailed <em>t</em>-test.</p><p>E. Electron microscopy images of cells treated (300 µM, 30 min) or untreated with SA. The arrow indicates the Golgi complex. Scale bar, 200 nm.</p><p>F. AiryScan images of HeLa cells at steady state or exposed to SA for the indicated times. A Rab1-GTP specific antibody was used to monitor the pool of active Rab1, and GM130 to stain the Golgi complex. Insets, eIF3. Scale bar, 10 µm. The graph shows quantification of Rab1-GTP at the Golgi complex expressed as a percentage of the control. Mean ± s.e.m. of three independent experiments. *p<0.011, **p<0.002, Student's unpaired two-tailed <em>t</em>-test.</p><p>G. HeLa cells transfected or not with WT GFP-Rab1B were left untreated or treated with SA and stained for GM130 and G3BP to monitor SGs. Dashed white line, WT GFP-Rab1B transfected cells. Scale bar, 10 µm. The graph shows quantification of Golgi objects in the different conditions. NT: non-transfected. Mean ± s.e.m. of three independent experiments. ****p<0.0001, Student's unpaired two-tailed <em>t</em>-test.</p><p>Expanded View Figure Legends</p>
|
https://api.sourcedata.io/file.php?figure_id=27533
|
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||
10.15252/embj.2021109202
|
Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding
|
2022
|
Figure 1
|
<sd-panel><p><strong>Fig 1. Loss of <sd-pretag id="sdPretag996063586sm" type="geneprod" role="assayed"><em>UPF3B</em></sd-pretag> in <sd-pretag id="sdPretag1786084315sm" type="organism" role="component">human</sd-pretag> cells affects EJC-dependent NMD.</strong></p> <p>A. Schematic of <sd-pretag id="sdPretag1459231795sm" type="geneprod" role="intervention"><em>UPF3B</em></sd-pretag> mutagenesis strategies using CRISPR-<sd-pretag id="sdPretag890602876sm" type="geneprod" role="assayed">Cas9</sd-pretag>. <sd-pretag id="sdPretag1126514150sm" type="geneprod" role="assayed"><em>UPF3B</em></sd-pretag> locus is in black where rectangles represent exons and horizontal line denotes introns; coding region is shown as wider rectangles. Red arrowheads represent guide RNA targeting sites. Top: two guide RNAs delete the <sd-pretag id="sdPretag2127488581sm" type="geneprod" role="assayed">UPF2</sd-pretag> binding domain (2BD) of <sd-pretag id="sdPretag1936813313sm" type="geneprod" role="assayed">UPF3B</sd-pretag> protein coding region as shown to create 3B<sup>Δ2BD</sup> cells. Bottom: a donor template is used to insert <sd-pretag id="sdPretag1941564264sm" type="small" role="intervention">puromycin</sd-pretag> resistant gene (PuroR) and bovine growth hormone (BGH) polyadenylation signal at the cut site to generate <sd-pretag id="sdPretag932262025sm" type="geneprod" role="intervention">UPF3B</sd-pretag> knockout (3B<sup>KO</sup>) cells.</p> <p>B. <sd-pretag id="sdPretag1886414003sm" category="assay">Immunoblot</sd-pretag> of parental wild-type (WT) and <sd-pretag id="sdPretag681914697sm" type="geneprod" role="intervention">UPF3B</sd-pretag> mutant cell lines showing levels of proteins on the right. In 3B<sup>Δ2BD</sup>, a smaller <sd-pretag id="sdPretag954915602sm" type="geneprod" role="assayed">UPF3B</sd-pretag> protein with deletion of amino acids 20-155 (UPF3BΔ20-155) is expressed. Relative Expression (Rel Exp) of this deletion protein as compared to the full-length <sd-pretag id="sdPretag1322896364sm" type="geneprod" role="assayed">UPF3B</sd-pretag> along with standard error of mean (SEM) are indicated below lane 2. <sd-pretag id="sdPretag20953721sm" type="geneprod" role="assayed">UPF3B</sd-pretag> antibody recognizes antigen outside the deleted region in 3B<sup>Δ2BD</sup>. <sd-pretag id="sdPretag1881588213sm" type="geneprod" role="assayed">HNRNPA1</sd-pretag> is used as a loading control. ND = <sd-pretag id="sdPretag725300843sm" type="geneprod" role="assayed">UPF3B</sd-pretag> levels not determined.</p> <p>C. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from the same set of genes. X-axis represents fold change in 3B<sup>Δ2BD</sup> versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.</p> <p>D. Bar plots from isoform specific <sd-pretag id="sdPretag2035342155sm" category="assay">RT-qPCR</sd-pretag> analysis showing average fold change (y-axis) of PTC+ and PTC- isoforms from genes indicated on the bottom in WT and the two <sd-pretag id="sdPretag1681677466sm" type="geneprod" role="intervention">UPF3B</sd-pretag> mutant cells identified in the legend on the top right. For each isoform, levels in mutant cells are compared to the levels in WT cells (set to 1). Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means. The asterisk (*) represents p<0.05 in t-test with null hypothesis of true mean being 1 (n=3 biological replicates).</p> <p>E. Cumulative Distribution Function (CDF) plots of <sd-pretag id="sdPretag677594744sm" category="disease">NMD+</sd-pretag> isoforms and <sd-pretag id="sdPretag1579788430sm" category="disease">NMD</sd-pretag>- isoforms. X-axis represents fold change in 3B<sup>Δ2BD</sup> versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.</p> <p>F. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from same set of genes. X-axis represents fold change in <sd-pretag id="sdPretag788377784sm" type="geneprod" role="intervention">UPF1</sd-pretag> knockdown (siUPF1) versus control knockdown (siNC) in 3B<sup>Δ2BD</sup> cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.</p> <p>G. Cumulative Distribution Function (CDF) plots of <sd-pretag id="sdPretag575819054sm" category="disease">NMD+</sd-pretag> isoforms and NMD- isoforms. X-axis represents fold change in <sd-pretag id="sdPretag2014790102sm" type="geneprod" role="intervention">UPF1</sd-pretag> knockdown (siUPF1) versus control knockdown (siNC) in 3B<sup>Δ2BD</sup> cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=47039
|
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] |
||
10.15252/embj.2021109202
|
Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding
|
2022
|
Figure 2
|
<sd-panel><p><strong>Fig 2. <sd-pretag id="sdPretag728680379sm" type="geneprod" role="assayed">UPF3A</sd-pretag> activates NMD in the absence of <sd-pretag id="sdPretag1324304135sm" type="geneprod" role="intervention">UPF3B</sd-pretag>.</strong></p> <p>A. <sd-pretag id="sdPretag144020437sm" category="assay">Immunoblots</sd-pretag> showing levels of proteins on the right in input or <sd-pretag id="sdPretag642598755sm" type="geneprod" role="reporter">FLAG</sd-pretag> <sd-pretag id="sdPretag1714656043sm" category="assay">immunoprecipitates</sd-pretag> (IP) from WT and <sd-pretag id="sdPretag2001989322sm" type="geneprod" role="intervention">UPF3B</sd-pretag> mutant cells expressing endogenously <sd-pretag id="sdPretag463454120sm" type="geneprod" role="reporter">FLAG</sd-pretag>-tagged <sd-pretag id="sdPretag676822748sm" type="geneprod" role="assayed">UPF1</sd-pretag> protein as indicated above each lane. The presence of <sd-pretag id="sdPretag1666705214sm" type="geneprod" role="assayed">RNase A</sd-pretag> during <sd-pretag id="sdPretag85889243sm" type="geneprod" role="reporter">FLAG</sd-pretag>-<sd-pretag id="sdPretag1005472154sm" category="assay">IP</sd-pretag> is indicated above each lane. The asterisk (*) represents p<0.05 in t-test with null hypothesis of true mean being 1.</p> <p>B. <sd-pretag id="sdPretag179973747sm" category="assay">Immunoblots</sd-pretag> showing levels of proteins (right) in input and <sd-pretag id="sdPretag25706078sm" category="disease">IP</sd-pretag> with normal rabbit IgG (IgG-IP) or antibody targeting <sd-pretag id="sdPretag1700122377sm" type="geneprod" role="assayed">EIF4A3</sd-pretag> (<sd-pretag id="sdPretag1924097896sm" type="geneprod" role="assayed">EIF4A3</sd-pretag>-IP) from WT and <sd-pretag id="sdPretag704233968sm" type="geneprod" role="intervention">UPF3B</sd-pretag> mutant cells. The asterisk (*) represents p<0.05 in t-test with null hypothesis of true mean being 1.</p> <p>C, D. CDF plots of (C) PTC+ and PTC-isoforms, (D) NMD+ and NMD- isoforms. X-axis represents fold change upon <sd-pretag id="sdPretag181351459sm" type="geneprod" role="intervention">UPF3A</sd-pretag> knockdown (siUPF3A) versus negative control knockdown (siNC) in WT cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.</p> <p>E, F. CDF plots of (E) PTC+ and PTC-isoforms, (F) NMD+ and NMD- isoforms. X-axis represents fold change upon <sd-pretag id="sdPretag99150858sm" type="geneprod" role="intervention">UPF3A</sd-pretag> knockdown (siUPF3A) versus negative control knockdown (siNC) in 3B<sup>Δ2BD</sup> cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.</p> <p>G, H. CDF plots of <sd-pretag id="sdPretag684359552sm" type="geneprod" role="intervention">UPF3B</sd-pretag>-dependent (G) and -independent (H) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon <sd-pretag id="sdPretag1717048774sm" type="geneprod" role="intervention">UPF3A</sd-pretag> knockdown (siUPF3A) versus negative control knockdown (siNC) in 3B<sup>Δ2BD</sup> cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.</p> <p>I. CDF plots of <sd-pretag id="sdPretag2106909866sm" type="geneprod" role="intervention">UPF3B</sd-pretag>-dependent and UPF3B-independent NMD+ isoforms as compared to NMD- isoforms. X-axis represents fold change upon <sd-pretag id="sdPretag940106746sm" type="geneprod" role="intervention">UPF3A</sd-pretag> knockdown (siUPF3A) versus negative control knockdown (siNC) in 3B<sup>Δ2BD</sup> cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.</p> <p>J. Bar plot showing average fold change as measured by isoform specific <sd-pretag id="sdPretag999622293sm" category="assay">RT-qPCR</sd-pretag> of PTC+ and PTC- isoform from genes indicated on the bottom in WT and two independent clones of 3A<sup>KO</sup>, 3B<sup>Δ2BD</sup>, 3B<sup>KO</sup>, and 3<sup>DKO</sup> cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means. The asterisk (*) represents p<0.05 in t-test with null hypothesis of true mean being 1 (n=3 biological replicates).</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=47041
|
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] |
||
10.15252/embj.2021109202
|
Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding
|
2022
|
Figure 3
|
<sd-panel><p><strong>Fig 3. EJC binding is dispensable for <sd-pretag id="sdPretag249205076sm" category="disease">NMD</sd-pretag> activity of <sd-pretag id="sdPretag1452235739sm" type="geneprod" role="assayed">UPF3</sd-pretag> paralogs.</strong></p> <p>A. Protein sequence alignment of C-terminal regions of <sd-pretag id="sdPretag1385982967sm" type="geneprod" role="assayed">UPF3</sd-pretag> proteins from different mammalian species (labeled on the left).</p> <p>B. <sd-pretag id="sdPretag1429476271sm" category="assay">Immunoblot</sd-pretag> showing levels of <sd-pretag id="sdPretag1192766737sm" type="geneprod" role="assayed">EJC</sd-pretag> and <sd-pretag id="sdPretag146378960sm" category="disease">UPF</sd-pretag> proteins (on the right) in input or <sd-pretag id="sdPretag797771286sm" type="geneprod" role="reporter">FLAG</sd-pretag>-<sd-pretag id="sdPretag791492836sm" category="assay">IP</sd-pretag> samples from 3<sup>DKO#2</sup> cells stably expressing different <sd-pretag id="sdPretag1596252818sm" type="geneprod" role="reporter">FLAG</sd-pretag>-tagged proteins indicated above each lane.</p> <p>C. Bar plots showing isoform specific <sd-pretag id="sdPretag443262514sm" category="assay">RT-qPCR</sd-pretag>-based measurement of relative levels of PTC+ and PTC- isoforms of genes indicated below from wild-type (WT) or 3<sup>DKO#2</sup> cells stably expressing the specified proteins. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means. The asterisk (*) represents statistically significant difference (p<0.05, t-test) of indicated samples from <sd-pretag id="sdPretag1482954655sm" type="geneprod" role="reporter">EGFP</sd-pretag>-transfected control cells, which has a value of 1 (n=3 biological replicates).</p> <p>D. <sd-pretag id="sdPretag1393713867sm" category="assay">Immunoblot</sd-pretag> showing EJC/UPF proteins (on the right) in input or <sd-pretag id="sdPretag1987436413sm" type="geneprod" role="reporter">FLAG</sd-pretag>-<sd-pretag id="sdPretag1720233022sm" category="assay">IP</sd-pretag> samples from 3<sup>DKO#2</sup> cells stably expressing <sd-pretag id="sdPretag1024545478sm" type="geneprod" role="reporter">FLAG-</sd-pretag>tagged proteins given above each lane (hUPF3B = <sd-pretag id="sdPretag1788721327sm" type="organism" role="component">human</sd-pretag> <sd-pretag id="sdPretag1944535884sm" type="geneprod" role="assayed">UPF3B</sd-pretag>; <sd-pretag id="sdPretag897326822sm" type="geneprod" role="assayed">hUPF3B</sd-pretag>-h3AC = <sd-pretag id="sdPretag1160940791sm" type="organism" role="component">human</sd-pretag> <sd-pretag id="sdPretag1251742841sm" type="geneprod" role="assayed">UPF3B</sd-pretag> protein with C-terminal domain from <sd-pretag id="sdPretag545585033sm" type="organism" role="component">human</sd-pretag> <sd-pretag id="sdPretag1386764969sm" type="geneprod" role="intervention">UPF3A</sd-pretag>; <sd-pretag id="sdPretag271143735sm" type="geneprod" role="intervention">hUPF3B</sd-pretag>-m3AC = <sd-pretag id="sdPretag1391509981sm" type="organism" role="component">human</sd-pretag> <sd-pretag id="sdPretag1990755719sm" type="geneprod" role="assayed">UPF3B</sd-pretag> protein with C-terminal domain from <sd-pretag id="sdPretag1613188796sm" type="organism" role="component">mouse UPF3A</sd-pretag>).</p> <p>E. Bar plots of isoform specific <sd-pretag id="sdPretag2110456049sm" category="assay">RT-qPCR</sd-pretag>-based analysis of <sd-pretag id="sdPretag111345488sm" type="geneprod" role="assayed">PTC+</sd-pretag> and <sd-pretag id="sdPretag1644356305sm" type="geneprod" role="assayed">PTC</sd-pretag>- transcript levels from wild-type (WT) or 3<sup>DKO#2</sup> cells stably expressing the proteins specified in the legend. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means. The asterisk (*) represents statistically significant difference (p<0.05, t-test) of indicated samples from <sd-pretag id="sdPretag964290759sm" type="geneprod" role="reporter">EGFP</sd-pretag>-transfected control cells, which has a value of 1 (n=3 biological replicates).</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=47043
|
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]
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"ext_ids": "65109",
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] |
||
10.15252/embj.2021109202
|
Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding
|
2022
|
Figure 4
|
<sd-panel><p><strong>Fig 4. <sd-pretag id="sdPretag1511202273sm" type="organism" role="component">Human</sd-pretag> <sd-pretag id="sdPretag687557869sm" type="geneprod" role="assayed">UPF3</sd-pretag> paralogs differ in <sd-pretag id="sdPretag1912878291sm" category="disease">NMD</sd-pretag> activity</strong>.</p> <p>A. <sd-pretag id="sdPretag150324822sm" category="assay">Northern blots</sd-pretag> showing levels of <sd-pretag id="sdPretag1748767497sm" type="geneprod" role="reporter">β-globin</sd-pretag> reporter mRNAs in wild-type <sd-pretag id="sdPretag951975341sm" type="cell" role="component">HeLa</sd-pretag> Tet-off cells and <sd-pretag id="sdPretag445219537sm" type="geneprod" role="intervention">UPF3B</sd-pretag> knockout <sd-pretag id="sdPretag2125721394sm" type="cell" role="component">HeLa</sd-pretag> Tet-off cells. <sd-pretag id="sdPretag1007273336sm" type="geneprod" role="assayed">β39</sd-pretag> is a <sd-pretag id="sdPretag248036963sm" type="small" role="intervention">tetracycline</sd-pretag> (Tet)-inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut-off (chase) as indicated above each lane. <sd-pretag id="sdPretag376696320sm" type="geneprod" role="assayed">β-GAP</sd-pretag> is a stable, constitutively-expressed, longer <sd-pretag id="sdPretag501770354sm" type="geneprod" role="assayed">β-globin</sd-pretag> mRNA used as transfection control. Proteins overexpressed (OE) in each condition are indicated on top and reporter mRNA half-lives (t<sub>1/2</sub>) along with standard errors of means are on the bottom.</p> <p>B. Schematic of <sd-pretag id="sdPretag160214696sm" type="organism" role="component">human</sd-pretag> <sd-pretag id="sdPretag1813816596sm" type="geneprod" role="assayed">UPF3A</sd-pretag>, <sd-pretag id="sdPretag54695485sm" type="geneprod" role="assayed">UPF3B</sd-pretag> and the UPF3A chimeric proteins where UPF3A domains are replaced by the corresponding domains from <sd-pretag id="sdPretag106071159sm" type="geneprod" role="assayed">UPF3B</sd-pretag> (see material and methods for detailed domain definition). Previously characterized <sd-pretag id="sdPretag150620891sm" type="geneprod" role="assayed">UPF2</sd-pretag> binding domain (<sd-pretag id="sdPretag673368261sm" type="geneprod" role="assayed">UPF2</sd-pretag>-BD) and EJC-binding motif (EBM) are shown.</p> <p>C. <sd-pretag id="sdPretag26967170sm" category="assay">Northern blot</sd-pretag> showing steady-state levels of <sd-pretag id="sdPretag1636057436sm" type="geneprod" role="assayed">β39</sd-pretag> NMD reporter and <sd-pretag id="sdPretag1148306131sm" type="geneprod" role="assayed">β-GAP</sd-pretag> control in <sd-pretag id="sdPretag354309201sm" type="cell" role="component">HeLa</sd-pretag> Tet-off <sd-pretag id="sdPretag94783842sm" type="geneprod" role="intervention">UPF3B</sd-pretag> knockout cells upon transient overexpression of wild-type <sd-pretag id="sdPretag1165952529sm" type="geneprod" role="intervention">UPF3</sd-pretag> proteins or different <sd-pretag id="sdPretag1052790280sm" type="geneprod" role="assayed">UPF3A</sd-pretag> chimeric proteins indicated above each lane. Below each lane, relative fold-change (Rel FC) indicates β39 reporter levels (normalized to <sd-pretag id="sdPretag676278989sm" type="geneprod" role="assayed">β-GAP</sd-pretag> control) as compared to the normalized β39 reporter levels in <sd-pretag id="sdPretag1082377853sm" type="geneprod" role="intervention">UPF3B</sd-pretag> expressing cells.</p> <p>D. <sd-pretag id="sdPretag280403277sm" category="assay">Immunoblot</sd-pretag> showing levels of EJC proteins or <sd-pretag id="sdPretag1108576643sm" type="geneprod" role="assayed">HNRNPA1</sd-pretag> in input or <sd-pretag id="sdPretag417855121sm" type="geneprod" role="assayed">EIF4A3</sd-pretag>-IP from 3<sup>DKO#2</sup> cells stably expressing different <sd-pretag id="sdPretag1745311211sm" type="geneprod" role="assayed">UPF3</sd-pretag> proteins or <sd-pretag id="sdPretag81007356sm" type="geneprod" role="reporter">EGFP</sd-pretag> as a control as indicated above each lane. Relative IP of <sd-pretag id="sdPretag2045938717sm" type="geneprod" role="reporter">FLAG</sd-pretag>-tagged proteins are quantified against <sd-pretag id="sdPretag531900497sm" type="geneprod" role="intervention">EIF4A3</sd-pretag>.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=47045
|
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"type": "geneprod",
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{
"ext_dbs": "Uniprot",
"ext_ids": "P38919",
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"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
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"text": "EIF4A3",
"type": "geneprod",
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{
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"ext_ids": "P38919",
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"ncbi_gene_id": null,
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"ext_tax_names": "Homo sapiens///Homo sapiens",
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"mapping_status": "unmapped",
"ncbi_gene_id": null,
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] |
||
10.15252/embj.2021109202
|
Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding
|
2022
|
Figure 5
|
<sd-panel><p><strong>Fig 5. <sd-pretag id="sdPretag46092878sm" category="disease">NMD</sd-pretag> activity in <sd-pretag id="sdPretag1456413772sm" type="organism" role="component">human</sd-pretag> cells in the absence of both <sd-pretag id="sdPretag802966980sm" type="geneprod" role="intervention">UPF3</sd-pretag> paralogs.</strong></p> <p>A, B. CDF plots of (A) PTC+ and PTC-isoforms, (B) NMD+ and NMD- isoforms. X-axis represents fold change upon <sd-pretag id="sdPretag1517064670sm" type="geneprod" role="intervention">UPF1</sd-pretag> knockdown (siUPF1) versus negative control knockdown (siNC) in 3<sup>DKO#2</sup> cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.</p> <p>C. Two possible models for <sd-pretag id="sdPretag1916805731sm" type="geneprod" role="intervention">UPF3A/3B</sd-pretag>-dependent and -independent NMD. Model 1 predicts that mRNAs (represented by wavy lines) from one set of genes (mRNA 1) undergoes <sd-pretag id="sdPretag3806228sm" type="geneprod" role="intervention">UPF3A/3B</sd-pretag>-dependent NMD and those from another set of genes (mRNA 2) are targeted by <sd-pretag id="sdPretag1841602766sm" type="geneprod" role="intervention">UPF3A/3B</sd-pretag>-independent NMD. Model 2 posits that same mRNAs are targeted to both <sd-pretag id="sdPretag545034081sm" type="geneprod" role="intervention">UPF3A/3B</sd-pretag>-dependent and -independent NMD. The number of arrows signify the probability/rate of <sd-pretag id="sdPretag1274083969sm" category="disease">NMD</sd-pretag> commitment via <sd-pretag id="sdPretag298233908sm" category="disease">each</sd-pretag> branch. In Model 2, commitment to <sd-pretag id="sdPretag1078689625sm" category="disease">NMD</sd-pretag> is higher in the presence of <sd-pretag id="sdPretag1297035021sm" type="geneprod" role="intervention">UPF3</sd-pretag> paralogs and lower in their absence.</p> <p>D, E. CDF plots of <sd-pretag id="sdPretag1617350390sm" type="geneprod" role="intervention">UPF3A/3B</sd-pretag>-independent (UPF3A/3B-indep., D) and -dependent (UPF3A/3B-dep., E) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon <sd-pretag id="sdPretag56365337sm" type="geneprod" role="intervention">UPF1</sd-pretag> knockdown (siUPF1) versus negative control knockdown (siNC) in 3<sup>DKO#2</sup> cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.</p> <p>F. CDF plots of UPF3A/3B-dependent (UPF3A/3B-dep.), UPF3A/3B-independent (UPF3A/3B-indep.) NMD+ isoforms and <sd-pretag id="sdPretag1944304793sm" category="disease">NMD</sd-pretag>- isoforms. X-axis represents fold change upon <sd-pretag id="sdPretag1639372941sm" type="geneprod" role="intervention">UPF1</sd-pretag> knockdown (siUPF1) versus negative control knockdown (siNC) in 3<sup>DKO#2</sup> cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=47047
|
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"mapping_status": "mapped",
"ncbi_gene_id": "5976",
"original_type": "gene",
"role": "intervention",
"text": "UPF1",
"type": "geneprod",
"uniprot_ids": [
"Q92900",
"A0A024R7L5",
"B3KY55"
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{
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] |
||
10.15252/embj.2021109202
|
Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding
|
2022
|
Figure 6
|
<sd-panel><p><strong>Fig 6. <sd-pretag id="sdPretag1651276803sm" type="geneprod" role="assayed">CASC3</sd-pretag> promote <sd-pretag id="sdPretag1028749219sm" type="geneprod" role="assayed">EJC</sd-pretag> association of <sd-pretag id="sdPretag1655178951sm" type="geneprod" role="assayed">UPF3</sd-pretag> paralogs to enhance NMD.</strong></p> <p>A, B. <sd-pretag id="sdPretag601618762sm" category="assay">Western blots</sd-pretag> showing levels of EJC proteins or <sd-pretag id="sdPretag1268894394sm" type="geneprod" role="assayed">HNRNPA1</sd-pretag> (control) in input, IgG IP or <sd-pretag id="sdPretag779518330sm" type="geneprod" role="intervention">EIF4A3</sd-pretag> IP following overexpression (OE) of <sd-pretag id="sdPretag2052185940sm" type="geneprod" role="intervention">CASC3</sd-pretag> wild-type (WT) and EJC binding deficient (<sd-pretag id="sdPretag1691752613sm" category="disease">HDAA</sd-pretag>) mutant proteins in (A) <sd-pretag id="sdPretag1760200356sm" type="cell" role="component">HeLa</sd-pretag> <sd-pretag id="sdPretag1261771436sm" type="small" role="assayed">Tet</sd-pretag>-off cells, and (B) 3B<sup>KO#3</sup> <sd-pretag id="sdPretag428627929sm" type="cell" role="component">HeLa</sd-pretag> Tet-off cells. Ramps above lanes indicate expression levels of the <sd-pretag id="sdPretag1745596161sm" type="geneprod" role="assayed">CASC3</sd-pretag> proteins. The asterisk (*) represents p<0.05 in t-test with null hypothesis of true mean less or equal than 1.</p> <p>C. <sd-pretag id="sdPretag2088175302sm" category="assay">Western blots</sd-pretag> showing levels of <sd-pretag id="sdPretag1447913420sm" type="geneprod" role="assayed">EJC/UPF</sd-pretag> proteins and <sd-pretag id="sdPretag186432931sm" type="geneprod" role="assayed">HNRNPA1</sd-pretag> in input and <sd-pretag id="sdPretag1536659216sm" type="geneprod" role="reporter">FLAG</sd-pretag> followed by <sd-pretag id="sdPretag421501566sm" type="geneprod" role="assayed">EIF4A3</sd-pretag> tandem <sd-pretag id="sdPretag230229883sm" category="assay">IP</sd-pretag> from <sd-pretag id="sdPretag1310920172sm" type="cell" role="component">HCT116</sd-pretag> cells expressing the <sd-pretag id="sdPretag2059414002sm" type="geneprod" role="reporter">FLAG</sd-pretag>-tagged endogenous protein indicated above each lane. Quantifications of <sd-pretag id="sdPretag637180624sm" type="geneprod" role="assayed">UPF3B</sd-pretag> and <sd-pretag id="sdPretag1807893959sm" type="geneprod" role="assayed">CASC3</sd-pretag> protein enrichment from two replicates are shown at the bottom.</p> <p>D. Meta-exon plot showing read distributions within the 100 nucleotide (nt) window from the exon 3′ end in <sd-pretag id="sdPretag1074744201sm" category="assay">RIPiT-Seq</sd-pretag> replicates of <sd-pretag id="sdPretag1152998973sm" type="geneprod" role="assayed">MAGOH</sd-pretag>:<sd-pretag id="sdPretag770770573sm" type="geneprod" role="assayed">EIF4A3</sd-pretag>, <sd-pretag id="sdPretag999004043sm" type="geneprod" role="assayed">UPF3B</sd-pretag>:<sd-pretag id="sdPretag413676835sm" type="geneprod" role="assayed">EIF4A3</sd-pretag>, and <sd-pretag id="sdPretag779681865sm" type="geneprod" role="assayed">CASC3</sd-pretag>:<sd-pretag id="sdPretag1455717310sm" type="geneprod" role="assayed">EIF4A3</sd-pretag>. The black vertical line indicates the -24 nt position.</p> <p>E. Venn diagram showing the degree of overlap between genes significantly enriched in <sd-pretag id="sdPretag1491130343sm" type="geneprod" role="intervention">CASC3:EIF4A3</sd-pretag> EJC and <sd-pretag id="sdPretag319839172sm" type="geneprod" role="intervention">UPF3B</sd-pretag>:<sd-pretag id="sdPretag1529862628sm" type="geneprod" role="intervention">EIF4A3</sd-pretag> EJC occupancy as compared to <sd-pretag id="sdPretag1257995295sm" type="geneprod" role="intervention">MAGOH</sd-pretag>:<sd-pretag id="sdPretag1997199187sm" type="geneprod" role="intervention">EIF4A3</sd-pretag> EJC occupancy.</p> <p>F. Scatter plot comparing log<sub>2</sub>-transformed fold change in occupancy of <sd-pretag id="sdPretag863226114sm" type="geneprod" role="intervention">CASC3:EIF4A3</sd-pretag> EJC as compared to <sd-pretag id="sdPretag1347656607sm" type="geneprod" role="intervention">MAGOH</sd-pretag>:<sd-pretag id="sdPretag1497063465sm" type="geneprod" role="intervention">EIF4A3</sd-pretag> EJC (x-axis) and <sd-pretag id="sdPretag617562345sm" type="geneprod" role="intervention">UPF3B</sd-pretag>:<sd-pretag id="sdPretag115508038sm" type="geneprod" role="intervention">EIF4A3</sd-pretag> EJC compared to <sd-pretag id="sdPretag1123386867sm" type="geneprod" role="intervention">MAGOH</sd-pretag>:<sd-pretag id="sdPretag400273350sm" type="geneprod" role="intervention">EIF4A3</sd-pretag> EJC. Each dot represents a gene where gene-level occupancy of each EJC composition was quantified at the canonical position for EJC footprints. Pearson correlation coefficient is shown on the top left. The black dotted line indicates the x=y diagonal and the red dashed line indicates the total least square regression fit for the data points.</p> <p>G. Box plots comparing occupancy of a specific EJC composition on mRNA (indicated on y-axis; normalized by gene expression from <sd-pretag id="sdPretag2083458809sm" category="assay">RNA-Seq</sd-pretag>) between genes of different <sd-pretag id="sdPretag1021651050sm" category="disease">NMD efficiency</sd-pretag>. Up, no, and down stands for upregulation, no obvious change or downregulation of a representative PTC+ isoform for each gene (see material and methods) that is used to reflect overall <sd-pretag id="sdPretag1983430576sm" category="disease">NMD</sd-pretag> efficiency for each gene. In the box plots, central band represents the median, boxes represent the interquartile range (IQR), whiskers indicate maximum (top) and minimum (bottom) values (1.5-times the highest or lowest IQR values, respectively), and circles represent the outliers. p-values are from Wilcoxon signed-rank test comparing the two indicated distributions.</p> <p>H. Box plots as in G comparing specific EJC composition occupancy relative to EJC core occupancy (normalized by <sd-pretag id="sdPretag1913688977sm" type="geneprod" role="intervention">MAGOH</sd-pretag>:<sd-pretag id="sdPretag460101079sm" type="geneprod" role="intervention">EIF4A3</sd-pretag> <sd-pretag id="sdPretag1846102109sm" type="geneprod" role="assayed">RIPiT</sd-pretag>-<sd-pretag id="sdPretag22739519sm" category="assay">Seq</sd-pretag>) between genes of different <sd-pretag id="sdPretag662851307sm" category="disease">NMD efficiency</sd-pretag>. Up, no, down, central band, boxes, and whiskers are as in G.</p> <p>I. Bar plots showing fold changes measured by isoform specific <sd-pretag id="sdPretag1810389206sm" category="assay">RT-qPCR</sd-pretag> of PTC+ and <sd-pretag id="sdPretag834189925sm" type="geneprod" role="assayed">PTC</sd-pretag>- isoform from genes indicated on the bottom in WT and <sd-pretag id="sdPretag98077362sm" type="geneprod" role="intervention">CASC3</sd-pretag>-KO <sd-pretag id="sdPretag132938694sm" type="cell" role="component">HCT116</sd-pretag> cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard error of means. The asterisk (*) represents p<0.05 in t-test with null hypothesis of true mean being 1 (n=3 biological replicates).</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=47049
|
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] |
||
30389665
|
10.15252/embj.201899277
|
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH
|
2018
|
Figure 1
|
<sd-panel><p><strong>Figure 1. Liver-specific <sd-pretag id="sdPretag218192730sm" type="geneprod" role="intervention">Sel1L</sd-pretag> deficiency in <sd-pretag id="sdPretag474237445sm" type="organism" role="component">mice</sd-pretag> (<em>Sel1L<sup>Alb</sup></em>) causes growth retardation.</strong></p><p> (A) <sd-pretag id="sdPretag37137754sm" category="assay">Western blot</sd-pretag> analysis and quantitation of <sd-pretag id="sdPretag1853482430sm" type="geneprod" role="assayed">Sel1L</sd-pretag>-<sd-pretag id="sdPretag313106566sm" type="geneprod" role="assayed">Hrd1 ERAD</sd-pretag> proteins in the <sd-pretag id="sdPretag102059239sm" type="tissue" role="component">livers</sd-pretag> of WT <sd-pretag id="sdPretag961628481sm" type="organism" role="component">mice</sd-pretag> at 3, 9 and 24 weeks of age (n=3-6 per group, 2 independent repeats).</p><p> (B) <sd-pretag id="sdPretag1377953687sm" category="assay">Western blot</sd-pretag> analysis and quantitation of <sd-pretag id="sdPretag1074124157sm" type="geneprod" role="assayed">Sel1L</sd-pretag>-<sd-pretag id="sdPretag2009553478sm" type="geneprod" role="assayed">Hrd1</sd-pretag> ERAD proteins in the <sd-pretag id="sdPretag2030352219sm" type="tissue" role="component">livers</sd-pretag> of 10-week-old WT <sd-pretag id="sdPretag546460656sm" type="organism" role="component">mice</sd-pretag> under fasted (overnight) or ad libitum fed conditions (n=3-6 per group, 2 independent repeats).</p><p> (C) <sd-pretag id="sdPretag1636018254sm" category="assay">Western blot</sd-pretag> analysis and quantitation of <sd-pretag id="sdPretag1386079007sm" type="geneprod" role="assayed">ERAD</sd-pretag> proteins in <em><sd-pretag id="sdPretag694539717sm" type="geneprod" role="intervention">Sel1L<sup>f</sup></sd-pretag><sup>/f</sup></em> and <em><sd-pretag id="sdPretag1445787898sm" type="geneprod" role="intervention">Sel1L<sup>Alb</sup></sd-pretag></em> <sd-pretag id="sdPretag948811763sm" type="tissue" role="component">livers</sd-pretag> (n=4 per group, 3 independent repeats).</p><p> (D) Growth curves of male (n=10 each) and female (n=7 each) <sd-pretag id="sdPretag1834142110sm" type="organism" role="component">mice</sd-pretag>.</p><p> (E-F) Representative images (E) and nose-to-anus length (F) of male <sd-pretag id="sdPretag1865875022sm" type="organism" role="component">mice</sd-pretag> at 6 weeks of age (n=6-10 per group).</p><p> (G) Organ-to-body-weight ratios of <sd-pretag id="sdPretag876491633sm" type="tissue" role="component">liver</sd-pretag> and kidney in 6-week-old male <sd-pretag id="sdPretag846855241sm" type="organism" role="component">mice</sd-pretag> (n=6 per group, 3 independent repeats).</p><p> (H) Daily food intake (g/d) normalized to gram of body weight (gbw) (n=3 per group, measured over 3 days).</p><p> (I) Representative estrus cycle mapping in 2-4-month old females (n=6 per group).</p><p> (J) H&E images of paraffin-embedded <sd-pretag id="sdPretag1322808497sm" type="tissue" role="component">liver</sd-pretag> sections from 9-week-old <sd-pretag id="sdPretag1474174655sm" type="organism" role="component">mice</sd-pretag> (n=4 per group, 3 independent repeats).</p><p> (K) <sd-pretag id="sdPretag400333686sm" category="assay">Western blot</sd-pretag> analysis of cell death (cleaved and pro-<sd-pretag id="sdPretag98991045sm" type="geneprod" role="assayed">caspase-3</sd-pretag>) in <sd-pretag id="sdPretag1800957023sm" type="tissue" role="component">livers</sd-pretag> of 9-week-old <sd-pretag id="sdPretag1209695850sm" type="organism" role="component">mice</sd-pretag>, with WT ileum as positive control (n=3 per group, 3 independent repeats).</p><p> (L) Representative <sd-pretag id="sdPretag306376718sm" category="assay">transmission electron microscope</sd-pretag> (TEM) images obtained from 9-week-old female <sd-pretag id="sdPretag1439264611sm" type="organism" role="component">mice</sd-pretag> <sd-pretag id="sdPretag1348235307sm" type="tissue" role="component">livers</sd-pretag> (n=10-12 cells from one <sd-pretag id="sdPretag1743172993sm" type="organism" role="component">mouse</sd-pretag> each). N, <sd-pretag id="sdPretag512193947sm" type="subcellular" role="component">nucleus</sd-pretag>; mito, <sd-pretag id="sdPretag161130878sm" type="subcellular" role="component">mitochondria</sd-pretag>; ER, <sd-pretag id="sdPretag2082562162sm" type="subcellular" role="component">endoplasmic reticulum</sd-pretag>.</p><p> Data information: <sd-pretag id="sdPretag891853949sm" type="geneprod" role="component">Hsp90</sd-pretag> and <sd-pretag id="sdPretag1082636527sm" type="geneprod" role="intervention">α-tubulin</sd-pretag>, loading controls for <sd-pretag id="sdPretag1175418730sm" category="assay">Western blot</sd-pretag> analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001, n.s., non-significant by Student's t test.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=23312
|
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] |
|
30389665
|
10.15252/embj.201899277
|
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH
|
2018
|
Figure 2
|
<sd-panel><p><strong>Figure 2. Elevated <sd-pretag id="sdPretag1099558650sm" type="geneprod" role="assayed"><em>Fgf21</em></sd-pretag> expression in the <sd-pretag id="sdPretag1495085116sm" type="tissue" role="component">liver</sd-pretag> as well as circulating <sd-pretag id="sdPretag527663501sm" type="geneprod" role="component">Fgf21</sd-pretag> in the absence of <sd-pretag id="sdPretag373535224sm" type="geneprod" role="intervention">Sel1L</sd-pretag>.</strong></p><p> (A) Volcano plot depicting transcriptomics data from the <sd-pretag id="sdPretag644273211sm" type="tissue" role="component">livers</sd-pretag> of 9-week-old <em><sd-pretag id="sdPretag1999404586sm" type="geneprod" role="intervention">Sel1L<sup>f</sup></sd-pretag><sup>/f</sup></em> and <em>Sel1L<sup>Alb</sup></em> <sd-pretag id="sdPretag190230356sm" type="organism" role="component">mice</sd-pretag> (n=3 per group); dotted line marks p=0.05; black dots represent fold change>2.</p><p> (B-C) <sd-pretag id="sdPretag437267244sm" category="assay">qPCR</sd-pretag> (B) and <sd-pretag id="sdPretag1007881255sm" category="assay">Western blot</sd-pretag> (C) analyses of <sd-pretag id="sdPretag1727396523sm" type="geneprod" role="assayed"><em>Fgf21</em></sd-pretag> expression in 9-week-old <sd-pretag id="sdPretag72320130sm" type="tissue" role="component">livers</sd-pretag> (n=3-6 per group, 3 independent repeats).</p><p> (D) <sd-pretag id="sdPretag987286869sm" category="assay">ELISA</sd-pretag> analysis of <sd-pretag id="sdPretag543066525sm" type="geneprod" role="assayed">Fgf21</sd-pretag> in <sd-pretag id="sdPretag1549800626sm" type="tissue" role="component">serum</sd-pretag> from 8-9-week-old <sd-pretag id="sdPretag446521555sm" type="organism" role="component">mice</sd-pretag> (n=6-7 per group).</p><p> (E) Protein levels of <sd-pretag id="sdPretag1223817452sm" type="geneprod" role="assayed">Sel1L</sd-pretag> (left panel) and mRNA levels of <sd-pretag id="sdPretag1820701279sm" type="geneprod" role="assayed">Fgf21</sd-pretag> (right panel) in primary <sd-pretag id="sdPretag1338121484sm" type="organism" role="component">mouse</sd-pretag> <sd-pretag id="sdPretag1940097975sm" type="cell" role="component">hepatocytes</sd-pretag> isolated from the <sd-pretag id="sdPretag361692412sm" type="molecule" role="component">tamoxifen</sd-pretag>-inducible <sd-pretag id="sdPretag2008944191sm" type="geneprod" role="intervention">Sel1L</sd-pretag>-knockout <em>Sel1L<sup>ERCre</sup></em> <sd-pretag id="sdPretag710687373sm" type="organism" role="component">mice</sd-pretag> (2 independent repeats).</p><p> (F-G) Acute loss-of-function model where 8-week-old <em><sd-pretag id="sdPretag927070663sm" type="geneprod" role="intervention">Sel1L<sup>f</sup></sd-pretag><sup>/f</sup></em> <sd-pretag id="sdPretag1187104428sm" type="organism" role="component">mice</sd-pretag> were injected i.v. with either <sd-pretag id="sdPretag1808094478sm" type="organism" role="component">AAV8</sd-pretag>-Cre or control <sd-pretag id="sdPretag1767823646sm" type="geneprod_organism" role="intervention">AAV8</sd-pretag>-<sd-pretag id="sdPretag1225977999sm" type="geneprod" role="reporter">GFP</sd-pretag>: (F) <sd-pretag id="sdPretag907143432sm" category="assay">Western blot</sd-pretag> analysis of <sd-pretag id="sdPretag1939778947sm" type="tissue" role="component">hepatic</sd-pretag> and control adipose <sd-pretag id="sdPretag1735035059sm" type="geneprod" role="component">Sel1L</sd-pretag> protein (n=3 per group); and (G) <sd-pretag id="sdPretag647755229sm" category="assay">qPCR</sd-pretag> analysis of hepatic <sd-pretag id="sdPretag726023426sm" type="geneprod" role="assayed"><em>Fgf21</em></sd-pretag> expression and <sd-pretag id="sdPretag850811904sm" category="assay">ELISA</sd-pretag> analysis of <sd-pretag id="sdPretag675742562sm" type="tissue" role="component">serum</sd-pretag> <sd-pretag id="sdPretag183304868sm" type="geneprod" role="assayed">Fgf21</sd-pretag> (n=3 per group, 2 independent repeats).</p><p> (H) Heatmaps of top 15 significantly upregulated and downregulated genes in <em><sd-pretag id="sdPretag207005967sm" type="geneprod" role="intervention">Fgf21</sd-pretag> Tg</em> <sd-pretag id="sdPretag991523331sm" type="tissue" role="component">livers</sd-pretag> and their expression levels in <em><sd-pretag id="sdPretag610317480sm" type="geneprod" role="assayed">Sel1L<sup>Alb</sup></sd-pretag></em> <sd-pretag id="sdPretag364947113sm" type="tissue" role="component">livers</sd-pretag> (n=3 per group).</p><p> (I) Scatter plot depicting the logarithmic fold-change (FC) for 16,402 genes in <em><sd-pretag id="sdPretag20594386sm" type="geneprod" role="intervention">Sel1L<sup>Alb</sup></sd-pretag></em> and <sd-pretag id="sdPretag1184329998sm" type="geneprod" role="intervention"><em>Fgf21</em></sd-pretag> transgenic <em>(Tg)</em> <sd-pretag id="sdPretag670003152sm" type="tissue" role="component">livers</sd-pretag> (n=3 per group); genes that are highly upregulated or downregulated in both datasets are marked in red and blue, respectively; genes that are upregulated unique to each data set (e.g. <sd-pretag id="sdPretag540765314sm" type="geneprod" role="intervention"><em>Derl3</em></sd-pretag> for <sd-pretag id="sdPretag1466732720sm" type="geneprod" role="intervention">Sel1L</sd-pretag>-<sd-pretag id="sdPretag1876582994sm" type="geneprod" role="component">Hrd1</sd-pretag> ERAD-deficient <sd-pretag id="sdPretag1255883718sm" type="tissue" role="component">liver</sd-pretag>) are marked in <sd-pretag id="sdPretag1431203125sm" type="tissue" role="component">green</sd-pretag>.</p><p> (J-L) Data from rescue experiments where 5-week-old <em><sd-pretag id="sdPretag1459008867sm" type="geneprod" role="intervention">Sel1L<sup>f</sup></sd-pretag><sup>/f</sup></em> and <em>Sel1L<sup>Alb</sup></em> <sd-pretag id="sdPretag1476660941sm" type="organism" role="component">mice</sd-pretag> were injected i.v. with <sd-pretag id="sdPretag1229410602sm" type="organism" role="component">AAV8</sd-pretag>-<sd-pretag id="sdPretag1582105397sm" type="geneprod" role="intervention"><em>shFgf21</em></sd-pretag> or control AAV8-<em>shLuc</em>: (J-K) <sd-pretag id="sdPretag821839762sm" category="assay">qPCR</sd-pretag> analysis of <sd-pretag id="sdPretag1050076542sm" type="geneprod" role="assayed"><em>Fgf21</em></sd-pretag> mRNA (J) and <sd-pretag id="sdPretag1306730665sm" category="assay">ELISA</sd-pretag> analysis of <sd-pretag id="sdPretag2108610077sm" type="geneprod" role="assayed">Fgf21</sd-pretag> in <sd-pretag id="sdPretag900951046sm" type="tissue" role="component">serum</sd-pretag> (K) 3 weeks after injection (n=4 per group). (L) Weight gain curve after injection (n=7 per group).</p><p> Data information: <sd-pretag id="sdPretag1975528347sm" type="geneprod" role="intervention">Hsp90</sd-pretag> and <sd-pretag id="sdPretag1638426587sm" type="geneprod" role="intervention">α-tubulin</sd-pretag>, loading control for <sd-pretag id="sdPretag259388202sm" category="assay">Western blot</sd-pretag> analysis. <sd-pretag id="sdPretag1213755521sm" type="subcellular" role="component">Ribosomal</sd-pretag> <sd-pretag id="sdPretag884223155sm" type="geneprod" role="intervention"><em>L32</em></sd-pretag>, loading control for <sd-pretag id="sdPretag2119496315sm" category="assay">qPCR</sd-pretag> analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001, n.s., non-significant by Student's t test (B-G) or 2-way ANOVA analysis (J-L).</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=23313
|
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"type": "geneprod",
"uniprot_ids": [
"Q9JJN1"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "56636",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "56636",
"original_type": "gene",
"role": "intervention",
"text": "Fgf21",
"type": "geneprod",
"uniprot_ids": [
"Q9JJN1",
"A0A7U3L6A3"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20338",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20338",
"original_type": "gene",
"role": "intervention",
"text": "Sel1L",
"type": "geneprod",
"uniprot_ids": [
"Q9Z2G6",
"Q5DTL8",
"Q6GTY4"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20338",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20338",
"original_type": "gene",
"role": "intervention",
"text": "Sel1L",
"type": "geneprod",
"uniprot_ids": [
"Q9Z2G6",
"Q5DTL8",
"Q6GTY4"
]
}
] |
|
30389665
|
10.15252/embj.201899277
|
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH
|
2018
|
Figure 3
|
<sd-panel><p><strong>Figure 3. <em>Sel1L<sup>Alb</sup></em> <sd-pretag id="sdPretag201792523sm" type="organism" role="component">mice</sd-pretag> phenocopy <sd-pretag id="sdPretag2024387118sm" type="geneprod" role="intervention">Fgf21</sd-pretag>-<sd-pretag id="sdPretag654821179sm" type="geneprod" role="intervention">gain</sd-pretag>-of-function <sd-pretag id="sdPretag179334059sm" type="organism" role="component">mice</sd-pretag>.</strong></p><p> (A) <sd-pretag id="sdPretag275374731sm" category="assay">Western blot</sd-pretag> analysis of p-<sd-pretag id="sdPretag860713333sm" type="geneprod" role="assayed">Stat5</sd-pretag> in <sd-pretag id="sdPretag149125496sm" type="tissue" role="component">livers</sd-pretag> of 9-week-old <sd-pretag id="sdPretag1478685966sm" type="organism" role="component">mice</sd-pretag> (n=3 per group, 3 independent repeats), with quantitation of the ratio of p- to total <sd-pretag id="sdPretag470183490sm" type="geneprod" role="assayed">Stat5</sd-pretag> shown below the blot.</p><p> (B) <sd-pretag id="sdPretag641562844sm" category="assay">qPCR</sd-pretag> analysis of p-<sd-pretag id="sdPretag761772826sm" type="geneprod" role="assayed">Stat5</sd-pretag>-associated growth genes in the <sd-pretag id="sdPretag754203432sm" type="tissue" role="component">livers</sd-pretag> of 9-week-old <sd-pretag id="sdPretag287375535sm" type="organism" role="component">mice</sd-pretag> (n=6 per group, 2 independent repeats).</p><p> (C) Z ambulatory activity of 9-week-old male <sd-pretag id="sdPretag671036963sm" type="organism" role="component">mice</sd-pretag> as measured over 24 hr (n=4 males per group).</p><p> (D-G) <sd-pretag id="sdPretag240417218sm" type="tissue" role="component">Blood</sd-pretag> <sd-pretag id="sdPretag287793436sm" type="molecule" role="assayed">glucose</sd-pretag> (D), <sd-pretag id="sdPretag969731169sm" type="molecule_tissue" role="assayed">serum</sd-pretag><sd-pretag id="sdPretag801747083sm" role=""> insulin</sd-pretag> (E), <sd-pretag id="sdPretag860773933sm" type="tissue" role="component">serum</sd-pretag> <sd-pretag id="sdPretag146999906sm" type="molecule" role="assayed">triglyceride</sd-pretag> (TG) (F), <sd-pretag id="sdPretag147114893sm" type="tissue" role="component">serum</sd-pretag> <sd-pretag id="sdPretag1491099873sm" type="molecule" role="assayed">cholesterol</sd-pretag> (CHOL) (G) levels in 9-week-old <sd-pretag id="sdPretag1855873852sm" type="organism" role="component">mice</sd-pretag> after 6 hr fasting (n=6-10 per group).</p><p> (H) H&E images of inguinal <sd-pretag id="sdPretag901523803sm" type="tissue" role="component">white adipose tissue</sd-pretag> (iWAT) from 8-week-old <sd-pretag id="sdPretag194947476sm" type="organism" role="component">mice</sd-pretag> (n=3 per group, 2 independent repeats).</p><p> (I) <sd-pretag id="sdPretag1829551331sm" category="assay">qPCR</sd-pretag> analysis of browning-related genes in iWAT (n=3-6 per group).</p><p> (J) <sd-pretag id="sdPretag523329678sm" category="assay">Western blot</sd-pretag> analysis of <sd-pretag id="sdPretag1653142466sm" type="geneprod" role="assayed">Ucp1</sd-pretag> in iWAT of 8-week-old <sd-pretag id="sdPretag1981220719sm" type="organism" role="component">mice</sd-pretag> (n=3-4 per group, 3 independent repeats).</p><p> (K) Weight gain curve of male <sd-pretag id="sdPretag540670657sm" type="organism" role="component">mice</sd-pretag> after 60% high fat diet (HFD) starting at 5 weeks of age (n=4 per group, 2 independent repeats).</p><p> (L) <sd-pretag id="sdPretag1271885246sm" type="tissue" role="component">Adipose</sd-pretag> tissue weight normalized to body weight in male <sd-pretag id="sdPretag22458464sm" type="organism" role="component">mice</sd-pretag> following 9 weeks of HFD feeding (n=4 per group).</p><p> Data information: <sd-pretag id="sdPretag1749012386sm" type="geneprod" role="intervention">Hsp90</sd-pretag>, loading control for <sd-pretag id="sdPretag1941591494sm" category="assay">Western blot</sd-pretag> analysis. <sd-pretag id="sdPretag30208563sm" type="subcellular" role="component">Ribosomal</sd-pretag> <sd-pretag id="sdPretag944172413sm" type="geneprod" role="intervention"><em>L32</em></sd-pretag>, loading control for <sd-pretag id="sdPretag390529843sm" category="assay">qPCR</sd-pretag> analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001 by Student's t test or 2-way ANOVA, as needed.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=23315
|
[
{
"ext_dbs": "Uniprot///Uniprot",
"ext_ids": "P42230///P42232",
"ext_tax_ids": "10090///10090",
"ext_tax_names": "Mus musculus///Mus musculus",
"mapping_source": "unknown",
"mapping_status": "unmapped",
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"text": "Stat5",
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},
{
"ext_dbs": "Uniprot///Uniprot",
"ext_ids": "P42230///P42232",
"ext_tax_ids": "10090///10090",
"ext_tax_names": "Mus musculus///Mus musculus",
"mapping_source": "unknown",
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"ncbi_gene_id": null,
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},
{
"ext_dbs": "Uniprot",
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"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Ucp1",
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}
] |
|
30389665
|
10.15252/embj.201899277
|
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH
|
2018
|
Figure 4
|
<sd-panel><p><strong>Figure 4. <sd-pretag id="sdPretag1300858991sm" type="geneprod" role="intervention">Sel1L</sd-pretag> deficiency leads to the accumulation of Crebh in the <sd-pretag id="sdPretag626177278sm" type="tissue" role="component">liver</sd-pretag>.</strong></p><p> (A-C) <sd-pretag id="sdPretag1349677491sm" category="assay">Western blot</sd-pretag> analysis of <sd-pretag id="sdPretag354605209sm" type="geneprod" role="assayed">Crebh</sd-pretag>, <sd-pretag id="sdPretag389430068sm" type="geneprod" role="assayed">Crebh</sd-pretag>-N, <sd-pretag id="sdPretag1075116079sm" type="geneprod" role="assayed">Pparα</sd-pretag> and <sd-pretag id="sdPretag572562597sm" type="geneprod" role="assayed">Xbp1s</sd-pretag> in whole cell lysates (A) and <sd-pretag id="sdPretag1026058178sm" type="subcellular" role="component">nuclear</sd-pretag> extracts (B) in WT and <em><sd-pretag id="sdPretag931521593sm" type="geneprod" role="intervention">Sel1L<sup>Alb</sup></sd-pretag></em> <sd-pretag id="sdPretag57345442sm" type="tissue" role="component">livers</sd-pretag> (n=3 per group, 3 independent repeats) with quantitation shown in (C).</p><p> (D) <sd-pretag id="sdPretag384121561sm" category="assay">qPCR</sd-pretag> analysis of <sd-pretag id="sdPretag1409041100sm" type="geneprod" role="assayed"><em>Crebh</em></sd-pretag>, <sd-pretag id="sdPretag221005311sm" type="geneprod" role="assayed"><em>Ppara</em></sd-pretag> and <sd-pretag id="sdPretag412287181sm" type="geneprod" role="assayed"><em>Xbp1s</em></sd-pretag> in WT and <em><sd-pretag id="sdPretag1014880613sm" type="geneprod" role="intervention">Sel1L<sup>Alb</sup></sd-pretag></em> <sd-pretag id="sdPretag606863739sm" type="tissue" role="component">livers</sd-pretag> (n=4-6 per group, 2 independent repeats).</p><p> (E) <sd-pretag id="sdPretag177991909sm" category="assay">Western blot</sd-pretag> analysis of <sd-pretag id="sdPretag395535103sm" type="geneprod" role="assayed">Crebh</sd-pretag> in acute <sd-pretag id="sdPretag308031437sm" type="geneprod" role="intervention">Sel1L</sd-pretag> loss of function model as described in Figure 2F-G (n=3 per group).</p><p> (F) <sd-pretag id="sdPretag37052004sm" category="assay">Western blot</sd-pretag> analysis of Crebh protein in 9-week-old <sd-pretag id="sdPretag1191000750sm" type="organism" role="component">mice</sd-pretag> (n=3 per group, 2 independent repeats). WT <sd-pretag id="sdPretag1050963468sm" type="organism" role="component">mice</sd-pretag> injected i.p. with <sd-pretag id="sdPretag1966512355sm" type="molecule" role="component">tunicamycin</sd-pretag> (Tm, 1.5 µg/g body weight) for 72 hours were included as a control.</p><p> (G) <sd-pretag id="sdPretag134301945sm" category="assay">Western blot</sd-pretag> analysis of Crebh in the <sd-pretag id="sdPretag2074199397sm" type="tissue" role="component">livers</sd-pretag> of 9-week-old <sd-pretag id="sdPretag1617087637sm" type="organism" role="component">mice</sd-pretag> after NP40-detergent fractionation into <sd-pretag id="sdPretag577580701sm" type="geneprod" role="intervention">NP40</sd-pretag> soluble (NP40S) and pellet (NP40P) (n=3 per group, 2 independent repeats).</p><p> (H) Representative <sd-pretag id="sdPretag876511865sm" category="assay">confocal</sd-pretag> images of Crebh in the <sd-pretag id="sdPretag1369270568sm" type="tissue" role="component">liver</sd-pretag> cryosections of 8-week-old <sd-pretag id="sdPretag1748565642sm" type="organism" role="component">mice</sd-pretag> (zoomed out versions in Figure EV3F). Note that a fraction of <sd-pretag id="sdPretag1472355942sm" type="cell" role="component">hepatocytes</sd-pretag> is binucleated. Yellow arrows represent <sd-pretag id="sdPretag946253782sm" type="geneprod" role="assayed">Crebh</sd-pretag> staining inside in the <sd-pretag id="sdPretag236747209sm" type="cell" role="component">hepatocyte</sd-pretag> <sd-pretag id="sdPretag274561411sm" type="subcellular" role="component">nucleus</sd-pretag>.</p><p> (I) <sd-pretag id="sdPretag1929526260sm" category="assay">ChIP</sd-pretag> analysis of Crebh binding onto the <sd-pretag id="sdPretag1510855538sm" type="geneprod" role="assayed"><em>Fgf21</em></sd-pretag> promoter in the <sd-pretag id="sdPretag143594486sm" type="tissue" role="component">livers</sd-pretag> of 9-week-old <sd-pretag id="sdPretag515217154sm" type="organism" role="component">mice</sd-pretag>, normalized first to 5% input group and then to no-antibody <sd-pretag id="sdPretag641936617sm" category="assay">ChIP</sd-pretag> samples (n=3 pooled per group, 2 independent repeats).</p><p> (J-K) <sd-pretag id="sdPretag1669964141sm" category="assay">Western blot</sd-pretag> analysis of <sd-pretag id="sdPretag1983823779sm" type="geneprod" role="assayed">SEL1L</sd-pretag>, <sd-pretag id="sdPretag2119318028sm" type="geneprod" role="assayed">HRD1</sd-pretag> and <sd-pretag id="sdPretag394160486sm" type="geneprod" role="assayed">CREBH</sd-pretag> proteins (J) and <sd-pretag id="sdPretag1761625220sm" category="assay">qPCR</sd-pretag> analysis of <sd-pretag id="sdPretag234295271sm" type="geneprod" role="assayed"><em>SEL1L</em></sd-pretag>, <sd-pretag id="sdPretag619345510sm" type="geneprod" role="assayed"><em>HRD1</em></sd-pretag> and <sd-pretag id="sdPretag298832301sm" type="geneprod" role="assayed"><em>FGF21</em></sd-pretag> (K) in <sd-pretag id="sdPretag140939905sm" type="organism" role="component">human</sd-pretag> <sd-pretag id="sdPretag1352018001sm" type="geneprod" role="assayed">Hep3B</sd-pretag> hepatocytes upon CRISPR deletion of <sd-pretag id="sdPretag1404551617sm" type="geneprod" role="assayed">SEL1L</sd-pretag> with two different guides.</p><p> Data information: <sd-pretag id="sdPretag218023297sm" type="geneprod" role="assayed">Hsp90</sd-pretag>, H2A, <sd-pretag id="sdPretag1056698804sm" type="geneprod" role="component">β-actin</sd-pretag> and <sd-pretag id="sdPretag1124247719sm" type="geneprod" role="intervention">lamin</sd-pretag>, loading controls for <sd-pretag id="sdPretag349424718sm" category="assay">Western blot</sd-pretag> analysis. <sd-pretag id="sdPretag1704204892sm" type="subcellular" role="component">Ribosomal</sd-pretag> <em>L32</em>, loading control for <sd-pretag id="sdPretag1197687531sm" category="assay">qPCR</sd-pretag> analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001; n.s., non-significant by Student's t test.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=23317
|
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{
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{
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},
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"text": "CREBH",
"type": "geneprod",
"uniprot_ids": [
"Q68CJ9"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q9UBV2",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "SEL1L",
"type": "geneprod",
"uniprot_ids": [
"Q9UBV2"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q86TM6",
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"ext_tax_names": "Homo sapiens",
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"mapping_status": "mapped",
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"text": "HRD1",
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"Q86TM6"
]
},
{
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"ext_ids": "26291",
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"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "26291",
"original_type": "gene",
"role": "assayed",
"text": "FGF21",
"type": "geneprod",
"uniprot_ids": [
"Q9NSA1"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "6400",
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"ext_tax_names": "Homo sapiens",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "6400",
"original_type": "gene",
"role": "assayed",
"text": "SEL1L",
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"uniprot_ids": [
"Q9UBV2",
"Q3ZCU6"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "6400",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "6400",
"original_type": "gene",
"role": "intervention",
"text": "SEL1L",
"type": "geneprod",
"uniprot_ids": [
"Q9UBV2",
"Q3ZCU6"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "84447",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "84447",
"original_type": "gene",
"role": "assayed",
"text": "HRD1",
"type": "geneprod",
"uniprot_ids": [
"Q86TM6"
]
}
] |
|
30389665
|
10.15252/embj.201899277
|
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH
|
2018
|
Figure 5
|
<sd-panel><p><strong>Figure 5. Crebh is an <sd-pretag id="sdPretag1612163921sm" type="geneprod" role="assayed">Sel1L</sd-pretag>-<sd-pretag id="sdPretag353845040sm" type="geneprod" role="assayed">Hrd1</sd-pretag> ERAD substrate</strong>.</p><p> (A) <sd-pretag id="sdPretag1406289263sm" category="assay">Western blot</sd-pretag> analysis of Crebh protein half-life in transfected WT and <sd-pretag id="sdPretag638443113sm" type="geneprod" role="intervention">HRD1</sd-pretag>-/- <sd-pretag id="sdPretag1066827606sm" type="cell" role="component">HEK293T</sd-pretag> cells treated with <sd-pretag id="sdPretag1897385713sm" type="molecule" role="intervention">cycloheximide</sd-pretag> (<sd-pretag id="sdPretag4456398sm" type="molecule" role="intervention">CHX</sd-pretag>) for indicated times. The decay of protein from one experiment is shown below.</p><p> (B-C) <sd-pretag id="sdPretag1657336579sm" category="assay">Western blot</sd-pretag> analysis and quantitation of Crebh in Crebh-transfected WT (B) and <em><sd-pretag id="sdPretag735432082sm" type="geneprod" role="intervention">HRD1</sd-pretag>-/-</em> (C) <sd-pretag id="sdPretag198816684sm" type="cell" role="component">HEK293T</sd-pretag> cells pre-treated with the <sd-pretag id="sdPretag1492793987sm" type="subcellular" role="component">proteasomal</sd-pretag> inhibitor <sd-pretag id="sdPretag456678463sm" type="molecule" role="intervention">bortezomib</sd-pretag> (<sd-pretag id="sdPretag932563127sm" type="molecule" role="intervention">BTZ</sd-pretag>) or <sd-pretag id="sdPretag1500988678sm" type="subcellular" role="component">lysosomal</sd-pretag> inhibitor <sd-pretag id="sdPretag906889289sm" type="molecule" role="intervention">chloroquine</sd-pretag> (<sd-pretag id="sdPretag1791667391sm" type="molecule" role="intervention">CHQ</sd-pretag>) for 2 hours and then with <sd-pretag id="sdPretag507487468sm" type="molecule" role="intervention">CHX</sd-pretag> for additional 1 hour (n=2 per group, 2 independent repeats).</p><p> (D) <sd-pretag id="sdPretag421028586sm" category="assay">Western blot</sd-pretag> analysis of Crebh ubiquitination following <sd-pretag id="sdPretag170438757sm" category="assay">immunoprecipitation</sd-pretag> (IP) of <sd-pretag id="sdPretag1119491959sm" type="geneprod" role="assayed">Crebh</sd-pretag>-<sd-pretag id="sdPretag761460435sm" type="geneprod" role="reporter">Flag</sd-pretag> and <sd-pretag id="sdPretag54318029sm" type="geneprod" role="assayed">Crebh</sd-pretag>-N-<sd-pretag id="sdPretag942825285sm" type="geneprod" role="reporter">Flag</sd-pretag> in <sd-pretag id="sdPretag353743709sm" type="cell" role="component">HEK293T</sd-pretag> cells transfected with indicated plasmids. Samples were boiled with SDS before IP for denaturing IP and not so for native IP. These cells were treated with proteasomal inhibitor <sd-pretag id="sdPretag1914474578sm" type="molecule" role="intervention">BTZ</sd-pretag> for the last 6 hr prior to <sd-pretag id="sdPretag772271374sm" category="assay">immunoprecipitation</sd-pretag>.</p><p> (E) <sd-pretag id="sdPretag280512227sm" category="assay">Western blot</sd-pretag> analysis of <sd-pretag id="sdPretag276807365sm" type="geneprod" role="assayed">endoglycosidase H</sd-pretag> (<sd-pretag id="sdPretag1263025912sm" type="geneprod" role="assayed">endoH</sd-pretag>)-sensitivity of Crebh in the <sd-pretag id="sdPretag1439686568sm" type="tissue" role="component">livers</sd-pretag> of 9-week-old <sd-pretag id="sdPretag565994668sm" type="organism" role="component">mice</sd-pretag>; 'r' refers to endoH-cleavage-resistant species and 's' refers to endoH-cleavage-sensitive species (n=3 per group, 2 independent repeats).</p><p> Data information: <sd-pretag id="sdPretag973720808sm" type="geneprod" role="intervention">Hsp90</sd-pretag>, loading control for <sd-pretag id="sdPretag1434501082sm" category="assay">Western blot</sd-pretag> analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; n.s., non-significant by 2-way ANOVA analysis.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=23319
|
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"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "84447",
"original_type": "gene",
"role": "intervention",
"text": "HRD1",
"type": "geneprod",
"uniprot_ids": [
"Q86TM6"
]
},
{
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"ext_ids": "Q68CJ9",
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"ext_tax_names": "Homo sapiens",
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"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Crebh",
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]
},
{
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]
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{
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},
{
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"ext_ids": "Q68CJ9",
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"mapping_source": "direct",
"mapping_status": "mapped",
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"text": "Crebh",
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]
},
{
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"ext_ids": "Q68CJ9",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
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"mapping_status": "mapped",
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},
{
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"ext_ids": "Q68CJ9",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Crebh",
"type": "geneprod",
"uniprot_ids": [
"Q68CJ9"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q91XE9",
"ext_tax_ids": "",
"ext_tax_names": "",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Crebh",
"type": "geneprod",
"uniprot_ids": [
"Q91XE9"
]
}
] |
|
30389665
|
10.15252/embj.201899277
|
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH
|
2018
|
Figure 6
|
<sd-panel><p><strong>Figure 6. Crebh links hepatic <sd-pretag id="sdPretag5185486sm" type="geneprod" role="assayed">Sel1L</sd-pretag>-<sd-pretag id="sdPretag1057091118sm" type="geneprod" role="assayed">Hrd1</sd-pretag> ERAD to <sd-pretag id="sdPretag339734093sm" type="geneprod" role="component">Fgf21</sd-pretag></strong>.</p><p> 5-week-old <em><sd-pretag id="sdPretag2025010055sm" type="geneprod" role="intervention">Sel1L<sup>f</sup></sd-pretag><sup>/f</sup></em> and <em>Sel1L<sup>Alb</sup></em> <sd-pretag id="sdPretag494433567sm" type="organism" role="component">mice</sd-pretag> were injected i.v. once with <sd-pretag id="sdPretag828347001sm" type="organism" role="component">AAV8</sd-pretag>-<em>shCrebh</em> or control AAV8-<em>shLuc</em>.</p><p> (A) <sd-pretag id="sdPretag1392432082sm" category="assay">Western blot</sd-pretag> analysis of hepatic <sd-pretag id="sdPretag883200011sm" type="geneprod" role="assayed">Sel1L</sd-pretag> and <sd-pretag id="sdPretag539386157sm" type="geneprod" role="component">Crebh</sd-pretag> 5 weeks post injection (n=3 <sd-pretag id="sdPretag172737283sm" type="organism" role="component">mice</sd-pretag> each, 2 independent repeats).</p><p> (B-C) <sd-pretag id="sdPretag728964444sm" category="assay">qPCR</sd-pretag> analysis of <sd-pretag id="sdPretag125357820sm" type="geneprod" role="assayed"><em>Crebh</em></sd-pretag> and <sd-pretag id="sdPretag1145787747sm" type="geneprod" role="assayed"><em>Fgf21</em></sd-pretag> mRNA (B) and <sd-pretag id="sdPretag1211412119sm" category="assay">ELISA</sd-pretag> analysis of circulating <sd-pretag id="sdPretag1814634399sm" type="geneprod" role="component">Fgf21</sd-pretag> (C) 5 weeks post injection (n=3 per group, 2 independent repeats).</p><p> (D) <sd-pretag id="sdPretag1605505701sm" category="assay">qPCR</sd-pretag> analysis of <sd-pretag id="sdPretag826292552sm" type="tissue" role="component">hepatic</sd-pretag> growth-associated genes 5 weeks after injection (n=6 per group).</p><p> (E) Weight gain 6 weeks post injection (n=10 per group).</p><p> (F) Insulin tolerance test (ITT) 5-weeks after injection (n=10 per group).</p><p> (G-H) <sd-pretag id="sdPretag1505107905sm" category="assay">qPCR</sd-pretag> (G) and <sd-pretag id="sdPretag1358812482sm" category="assay">Western blot</sd-pretag> analysis (H) of <sd-pretag id="sdPretag1399335073sm" type="geneprod" role="assayed">Ucp1</sd-pretag> levels in <sd-pretag id="sdPretag1976277276sm" type="tissue" role="component">inguinal white adipose tissue</sd-pretag> (iWAT) 5 weeks post injection (n=3 per group, 2 independent repeats).</p><p> Data information: <sd-pretag id="sdPretag6112763sm" type="geneprod" role="intervention">Hsp90</sd-pretag>, loading control for <sd-pretag id="sdPretag334485243sm" category="assay">Western blot</sd-pretag> analysis. <sd-pretag id="sdPretag797143535sm" type="subcellular" role="component">Ribosomal</sd-pretag> <sd-pretag id="sdPretag661308449sm" type="geneprod" role="intervention"><em>L32</em></sd-pretag>, loading control for <sd-pretag id="sdPretag1232405823sm" category="assay">qPCR</sd-pretag> analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001; n.s., non-significant by 2-way ANOVA analysis (B-G) and Student's t test (A,H).</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=23321
|
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"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20338",
"original_type": "gene",
"role": "intervention",
"text": "Sel1L",
"type": "geneprod",
"uniprot_ids": [
"Q9Z2G6",
"Q5DTL8",
"Q6GTY4"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "208677",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "208677",
"original_type": "gene",
"role": "intervention",
"text": "Crebh",
"type": "geneprod",
"uniprot_ids": [
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},
{
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"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20338",
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"text": "Sel1L",
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"mapping_status": "mapped",
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},
{
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"ext_ids": "208677",
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"ext_tax_names": "Mus musculus",
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"mapping_status": "mapped",
"ncbi_gene_id": "208677",
"original_type": "gene",
"role": "intervention",
"text": "Crebh",
"type": "geneprod",
"uniprot_ids": [
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]
},
{
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"ext_ids": "20338",
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"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20338",
"original_type": "gene",
"role": "intervention",
"text": "Sel1L",
"type": "geneprod",
"uniprot_ids": [
"Q9Z2G6",
"Q5DTL8",
"Q6GTY4"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20338",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20338",
"original_type": "gene",
"role": "intervention",
"text": "Sel1L",
"type": "geneprod",
"uniprot_ids": [
"Q9Z2G6",
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]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "22227",
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"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "22227",
"original_type": "gene",
"role": "assayed",
"text": "Ucp1",
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"uniprot_ids": [
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]
},
{
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]
},
{
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"ext_ids": "20338",
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"ext_tax_names": "Mus musculus",
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"mapping_status": "mapped",
"ncbi_gene_id": "20338",
"original_type": "gene",
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"text": "Sel1L",
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},
{
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"mapping_status": "mapped",
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"text": "Sel1L",
"type": "geneprod",
"uniprot_ids": [
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"Q5DTL8",
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]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P12242",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Ucp1",
"type": "geneprod",
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}
] |
|
30389665
|
10.15252/embj.201899277
|
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH
|
2018
|
Figure 7
|
<sd-panel><p><strong>Figure 7. <sd-pretag id="sdPretag1926854515sm" type="geneprod" role="assayed">Sel1L</sd-pretag>-<sd-pretag id="sdPretag1367424005sm" type="geneprod" role="assayed">Hrd1</sd-pretag> ERAD represses Crebh and <sd-pretag id="sdPretag983123820sm" type="geneprod" role="assayed">Fgf21</sd-pretag> under fasting-feeding and growth.</strong> Analysis of correlation between <sd-pretag id="sdPretag801205226sm" type="geneprod" role="assayed">Sel1L</sd-pretag>-<sd-pretag id="sdPretag2097054659sm" type="geneprod" role="assayed">Hrd1 ERAD</sd-pretag> and <sd-pretag id="sdPretag127659079sm" type="geneprod" role="assayed">Crebh</sd-pretag>-<sd-pretag id="sdPretag562657551sm" type="geneprod" role="assayed">Fgf21</sd-pretag> during fasting-feeding (A-D) and growth (E-H).</p><p> (A) <sd-pretag id="sdPretag1658870740sm" category="assay">Western blot</sd-pretag> analysis of hepatic <sd-pretag id="sdPretag632577973sm" type="geneprod" role="assayed">Sel1L</sd-pretag>-<sd-pretag id="sdPretag1758717204sm" type="geneprod" role="assayed">Hrd1</sd-pretag> ERAD and <sd-pretag id="sdPretag288960844sm" type="geneprod" role="assayed">Crebh</sd-pretag> of <sd-pretag id="sdPretag1219694798sm" type="subcellular" role="component">nuclear</sd-pretag> (Nuc) and <sd-pretag id="sdPretag154235567sm" type="subcellular" role="component">cytosolic</sd-pretag> (Cyto) fractions from 10-week-old <em><sd-pretag id="sdPretag414028336sm" type="geneprod" role="intervention">Sel1L<sup>f</sup></sd-pretag><sup>/f</sup></em> and <em>Sel1L<sup>Alb</sup></em> <sd-pretag id="sdPretag1174433184sm" type="organism" role="component">mice</sd-pretag> under overnight fasted or fed states.</p><p> (B) Quantitation of levels of <sd-pretag id="sdPretag1665035973sm" type="geneprod" role="assayed">Sel1L</sd-pretag>/<sd-pretag id="sdPretag458207069sm" type="geneprod" role="assayed">Hrd1</sd-pretag>/<sd-pretag id="sdPretag1687765693sm" type="geneprod" role="assayed">Crebh</sd-pretag> proteins and <sd-pretag id="sdPretag385119567sm" type="geneprod" role="assayed"><em>Fgf21</em></sd-pretag> mRNA in the <sd-pretag id="sdPretag309526493sm" type="tissue" role="component">livers</sd-pretag> of 10-week-old <em><sd-pretag id="sdPretag246693219sm" type="geneprod" role="intervention">Sel1L<sup>f</sup></sd-pretag><sup>/f</sup></em> <sd-pretag id="sdPretag1574441126sm" type="organism" role="component">mice</sd-pretag>.</p><p> (C) Quantitation of total <sd-pretag id="sdPretag1676314277sm" type="geneprod" role="assayed">Crebh</sd-pretag> (<sd-pretag id="sdPretag1352699718sm" type="geneprod" role="assayed">Crebh</sd-pretag>+<sd-pretag id="sdPretag1064849830sm" type="geneprod" role="assayed">Crebh</sd-pretag>-N) protein levels in the <sd-pretag id="sdPretag1179133741sm" type="tissue" role="component">livers</sd-pretag> of 10-week-old <em><sd-pretag id="sdPretag1492287962sm" type="geneprod" role="intervention">Sel1L<sup>f</sup></sd-pretag><sup>/f</sup></em> and <em>Sel1L<sup>Alb</sup></em> <sd-pretag id="sdPretag1249563068sm" type="organism" role="component">mice</sd-pretag>.</p><p> (D) Serum <sd-pretag id="sdPretag1017772667sm" type="geneprod" role="assayed">Fgf21</sd-pretag> levels in 10-week-old <em><sd-pretag id="sdPretag1441368092sm" type="geneprod" role="intervention">Sel1L<sup>f</sup></sd-pretag><sup>/f</sup></em> and <em>Sel1L<sup>Alb</sup></em> <sd-pretag id="sdPretag249282002sm" type="organism" role="component">mice</sd-pretag>.</p><p> (E-H) Similar to (A-D) with the exception that these experiments were done with the <sd-pretag id="sdPretag1125649289sm" type="tissue" role="component">livers</sd-pretag> from <em><sd-pretag id="sdPretag1417852993sm" type="geneprod" role="intervention">Sel1L<sup>f</sup></sd-pretag><sup>/f</sup></em> and <em>Sel1L<sup>Alb</sup></em> <sd-pretag id="sdPretag855464741sm" type="organism" role="component">mice</sd-pretag> at 3, 9 and 24 weeks of age.</p><p> Data information: <sd-pretag id="sdPretag238547675sm" type="geneprod" role="component">Hsp90</sd-pretag> and <sd-pretag id="sdPretag438183919sm" type="geneprod" role="intervention">lamin</sd-pretag>, loading controls for <sd-pretag id="sdPretag1642564753sm" type="subcellular" role="component">cytosolic</sd-pretag> and <sd-pretag id="sdPretag951423995sm" type="subcellular" role="component">nuclear</sd-pretag> fractions. Values are mean ± SEM; **, p<0.01 by two-way ANOVA analysis. n=3-4 per group, 2 independent repeats.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=23322
|
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] |
|
10.15252/embr.202153813
|
Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain
|
2022
|
Figure 2
|
<sd-panel><p><strong>Figure 2. CG2662/Lint-O binds to and functions with L(3)mbt in gene regulation.</strong></p> <p><strong>(A, B) The mRNA levels of <em>aub</em> (A) and <em>vasa</em> (B) were quantified upon the depletion (RNAi) of L(3)mbt, Lint-1, CoRest, E2F2, Myb, Mip120, and Mip130, and were compared with those in normal OSCs (control). Data represent the means ± SE (<em>n</em> = 3 biological replicates). The <em>p</em> values were calculated with the t-test. *<em>p</em>: < 0.05, **<em>p</em>: < 0.01. All t-tests were performed against samples with⎾ symbol.</strong></p> <p><strong>(C) Proteins immunoprecipitated with the anti-L(3)mbt antibody from the OSC lysates before (control) and after L(3)mbt knockdown (KD) were silver-stained. The bands corresponding to L(3)mbt-L and L(3)mbt-S are indicated.</strong></p> <p><strong>(D) Flow chart of the identification of L(3)mbt-interactors in OSCs. All peptides obtained from LC-MS/MS are listed in Appendix Table S1.</strong></p> <p><strong>(E) Summary of the sum of the −log posterior error probability (Sum Pep Score: SPS), peptide spectra match values (PSM), and known functions of the proteins in (D). Mass spectrometry analysis was performed on biological duplicates.</strong></p> <p><strong>(F, G) The mRNA levels of <em>vasa</em> (F) and <em>aub</em> (G) were quantified upon the depletion (RNAi) of L(3)mbt, Atac3, Prod, CG2662/Lint-O, and CG2199, and were compared with those in normal OSCs (control). Data represent the means ± SE (<em>n</em> = 3 biological replicates). The <em>p</em> values were calculated with the t-test. **<em>p</em>: < 0.01. All t-tests were performed against samples with⎾ symbol.</strong></p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=49171
|
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"role": "intervention",
"text": "Prod",
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},
{
"ext_dbs": "NCBI gene",
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"text": "vasa",
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] |
||
10.15252/embr.202153813
|
Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain
|
2022
|
Figure 3
|
<sd-panel><p><strong>Figure 3. The L(3)mbt−Lint-O interaction is crucial for the repression of target genes.</strong></p> <p><strong>(A) Domain structures of L(3)mbt and Lint-O. L(3)mbt has three MBT repeats (orange), a C2H2-type zinc finger (Zf) (gray), and a SAM domain (blue). Lint-O has two PHD finger domains (green) and a SAM domain (blue).</strong></p> <p><strong>(B) Immunoprecipitation (IP)/western blotting shows that WT Lint-O, but not its ∆SAM mutant (Fig EV3A), co-immunoprecipitated with L(3)mbt from the OSC lysates. n.i.: non-immune IgG.</strong></p> <p><strong>(C) Subcellular localization of WT Lint-O and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.</strong></p> <p><strong>(D) IP/western blotting shows that WT L(3)mbt-L, but not its ∆SAM mutant</strong> (Fig EV3B)<strong>, co-immunoprecipitated with Lint-O from the OSC lysates. n.i.: non-immune IgG.</strong></p> <p><strong>(E) Subcellular localization of WT L(3)mbt-L and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.</strong></p> <p><strong>(F, G) The mRNA levels of <em>vasa</em> (F) and <em>aub</em> (G) were quantified upon ectopic expression of EGFP, as well as WT Lint-O and its ∆SAM and 8CA mutants (Fig EV3A), in Lint-O-lacking OSCs (Lint-O RNAi) and were compared with those in normal OSCs (control). Data represent the means ± SE (<em>n</em> = 3 biological replicates). The <em>p</em> values were calculated with the t-test. *<em>p</em>: < 0.05. All t-tests were performed against samples with⎾ symbol.</strong></p> <p><strong>(H) IP/western blotting shows that L(3)mbt co-immunoprecipitated with WT Lint-O and its 8CA mutant, but not with ∆PHD mutant (Fig EV3A). An asterisk shows background.</strong></p> <p><strong>(I) Subcellular localization of WT Lint-O and its 8CA and ∆PHD mutants (green). All proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.</strong></p> <p><strong>(J) ChIP-qPCR shows that L(3)mbt binding to the <em>vasa</em> promoter was weakened after the loss of Lint-O. Data represent the means ± SE (<em>n</em> = 3 biological replicates). The <em>p</em> values were calculated with the t-test. **<em>p</em>: < 0.01. All t-tests were performed against samples with⎾ symbol.</strong></p> <p><strong>(K) Western blotting showing the amounts of L(3)mbt, Lint-O, and histone H3 (H3) in normal (control), L(3)mbt-depleted (RNAi), and Lint-O-depleted (RNAi) OSCs. Asterisks show background signals as in Fig EV3E.</strong></p> <p><strong>(L) The <em>lint-O</em> mRNA levels in normal (control), L(3)mbt-depleted (RNAi), and Lint-O-depleted (RNAi) OSCs are shown by fragments per kilobase million (FPKM). Data represent the means ± SE (<em>n</em> = 3 biological replicates). The <em>p</em> values were calculated with the t-test. *<em>p</em>: < 0.05, **<em>p</em>: < 0.01. All t-tests were performed against samples with⎾ symbol.</strong></p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=49173
|
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||
10.15252/embr.202153813
|
Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain
|
2022
|
Figure 5
|
<sd-panel><p><strong>Figure 5. The lack of Lint-O in flies leads to abnormal ovarian morphology and female sterility.</strong></p> <p><strong>(A)</strong> CRISPR-mediated generation of <em>lint-O</em> knockout (KO). Genomic structure of <em>lint-O</em> is shown with gRNA targeting <em>lint-O</em> exon 1 (triangle). Sequences of <em>lint-O</em> DNA in WT (<em>y w</em>) (complementary to the gRNA sequence) and the KO mutant, <em>Lint-O<sup>KO</sup>,</em> are shown. PAM: protospacer adjacent motif (underlined). E1-3: Exons 1−3. CDS: protein-coding sequence. UTR: untranslated region.</p> <p>(B) RT-qPCR analysis shows the mRNA levels of <em>lint-O</em> in <em>y w</em> and <em>Lint-O<sup>KO</sup></em> ovaries. Data are expressed as mean and error bars represent SD. <em>n</em> = 2 biological replicates.</p> <p>(C) The numbers of progeny in <em>y w</em> and <em>Lint-O<sup>KO</sup></em> are shown. Ten independent crosses were performed. Boxplot central bands, upper edges of boxes, lower edges of boxes, upper whiskers and lower whiskers show median, third quartile, first quartile, maxima, and minima, respectively.</p> <p>(D) Confocal images of <em>y w</em> and <em>Lint-O<sup>KO</sup></em> ovaries immunostained for Vasa. Scale bar: 50 <strong>μ</strong>m.</p> <p>(E) Confocal images of <em>y w</em> and <em>Lint-O<sup>KO</sup></em> ovarioles immunostained for Vasa. Vasa was ectopically expressed in follicle cells (white arrowheads). Scale bar: 50 <strong>μ</strong>m.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=49177
|
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},
{
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] |
||
10.15252/embr.202153813
|
Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain
|
2022
|
Figure 6
|
<sd-panel><p><strong>Figure 6. L(3)mbt and Lint-O cooperatively control target genes in the brain.</strong></p> <p><strong>(A)</strong> Western blotting shows that Vasa, Piwi, and Aub were ectopically expressed in <em>Lint-O<sup>KO</sup> and L(3)mbt<sup>ts1</sup></em> larval brains at 29ºC. Anti-Lint-O, anti-L(3)mbt, anti-Vasa, andi-Piwi, anti-Aub, and anti-Tubulin antibodies were used.</p> <p>(B) Confocal images of <em>y w</em>, <em>Lint-O<sup>KO</sup>,</em> and <em>L(3)mbt<sup>ts1</sup></em> immunostained for Vasa (magenta), MIRA (green), and ELAV (green). Scale bar: 100 <strong>μ</strong>m.</p> <p>(C) Quantification of brain lobe volume for the following genotypes: <em>Lint-O<sup>KO</sup></em> -/+ (<em>n</em> = 42 biological replicates), <em>Lint-O<sup>KO</sup></em> -/- (<em>n</em> = 36 biological replicates)<em>,</em> and <em>L(3)mbt<sup>ts1</sup></em> -/- (<em>n</em> = 34 biological replicates)<em>.</em> Boxplot central bands, upper edges of boxes, lower edges of boxes, upper whiskers and lower whiskers show median, third quartile, first quartile, maxima, and minima, respectively. <em>P</em>-values were calculated by the Student's t-test. (***<em>P</em>-value < 0.001).</p> <p>(D, E) <strong>The genomic regions harboring the <em>vasa</em> (D) and <em>aub</em> (E) genes. The RNA-seq reads in <em>y w,</em></strong> <em>L(3)mbt<sup>ts1</sup>,</em> <strong>and</strong> <em>Lint-O<sup>KO</sup></em> <strong>brains are shown. The shading in gray corresponds to exons. The <em>y-</em>axis shows the number of RPM.</strong> RNA-seq samples were biological triplicates.</p> <p>(F) Venn diagram shows that 620 protein-coding genes are shared with the <em>l(3)mbt</em> and <em>lint-O</em> libraries.</p> <p>(G) High-ranking GO terms for the 620 protein-coding genes in (F).</p> <p><strong>Expanded View Figure legends</strong></p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=49179
|
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}
] |
||
10.1101/241497
|
Polarization of Myosin II refines tissue material properties to buffer mechanical stress.
|
2018
|
Figure 1
|
<sd-panel> <p><strong>Figure 1: <sd-pretag parent-tag-id='177' id='sdPretag1984929077sd'>Myosin</sd-pretag> <sd-pretag role='component' id='sdPretag1312857301sme' type='cell' >RNAi</sd-pretag> clones are softer and less elastic.</strong></p> <p>(A) <sd-pretag parent-tag-id='5' id='sdPretag2010367702sd'>Stretching</sd-pretag>/<sd-pretag parent-tag-id='160' id='sdPretag1811565316sd'>compression</sd-pretag> device; 1: clamping <sd-pretag role='component' id='sdPretag1071280087sme' type='subcellular' >mechanism</sd-pretag>, 2: arms, 3: stage insert, 4: drive <sd-pretag role='component' id='sdPretag1286484657sme' type='subcellular' >mechanism</sd-pretag>, 5: media-filled Polydimethylsiloxane (<sd-pretag parent-tag-id='1' id='sdPretag1968723082sd'>PDMS</sd-pretag>) chamber, 6: two layers of stretchable elastomer (<sd-pretag parent-tag-id='1' id='sdPretag1204999753sd'>PDMS</sd-pretag>), one of which is pre- patterned with <sd-pretag category='assay' id='sdPretag463763791sme'>microchannels</sd-pretag>.</p> <p>(B) Scheme of <sd-pretag parent-tag-id='1' id='sdPretag1288566541sd'>PDMS</sd-pretag> prepatterning; the dimension of <sd-pretag category='assay' id='sdPretag1697698235sme'>microchannels</sd-pretag> are 80- 120 μm in width and 50 μm in depth.
(C) Cross-sectional schematic view of the <sd-pretag parent-tag-id='5' id='sdPretag1513533526sd'>stretching</sd-pretag> device; <sd-pretag parent-tag-id='38' id='sdPretag1605250747sd'>wing disc</sd-pretag> (in red) is positioned over the microchannel with sides clamped by the two <sd-pretag parent-tag-id='1' id='sdPretag212231044sd'>PDMS</sd-pretag> layers. The central portion of the tissue is submerged in the microchannel, perfused with <em>ex vivo</em> culture media (Mao et al., 2013; Mao et al., 2011).</p> <p>(D) <sd-pretag parent-tag-id='5' id='sdPretag1463714423sd'>Stretching</sd-pretag> of the <sd-pretag parent-tag-id='1' id='sdPretag1007904872sd'>PDMS</sd-pretag> sandwich concomitantly <sd-pretag parent-tag-id='5' id='sdPretag1424821365sd'>stretches</sd-pretag> the <sd-pretag parent-tag-id='38' id='sdPretag773813180sd'>wing disc</sd-pretag> inside the microchannel (top-down view); <sd-pretag parent-tag-id='38' id='sdPretag1523550738sd'>wing disc</sd-pretag> resting on a <sd-pretag parent-tag-id='5' id='sdPretag439449296sd'>stretching</sd-pretag> device (anchor) and 10 min after <sd-pretag parent-tag-id='5' id='sdPretag324073295sd'>stretch</sd-pretag>; M=microchannel.
(E) <sd-pretag parent-tag-id='12' id='sdPretag90449874sd'><em>zipper</em></sd-pretag> RNAi clones (<sd-pretag parent-tag-id='13' id='sdPretag1989830697sd'><em>zip</em></sd-pretag> iR, in red) in <sd-pretag parent-tag-id='15' id='sdPretag1819062255sd'>Arm</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1876768973sd'>GFP</sd-pretag> expressing anchored and <sd-pretag parent-tag-id='5' id='sdPretag1383060144sd'>stretched</sd-pretag> third <sd-pretag role='component' id='sdPretag631965087sme' type='organism' >instar</sd-pretag> <sd-pretag parent-tag-id='38' id='sdPretag1197873629sd'>wing disc</sd-pretag>; lower panel shows color coded cell <sd-pretag parent-tag-id='353' id='sdPretag1750428992sd'>aspect ratio</sd-pretag> in anchored and <sd-pretag parent-tag-id='5' id='sdPretag1587217461sd'>stretched</sd-pretag> <sd-pretag parent-tag-id='62' id='sdPretag833827436sd'>discs</sd-pretag> (bright colors indicate high elongation); <sd-pretag parent-tag-id='13' id='sdPretag222294596sd'><em>zip</em></sd-pretag> <sd-pretag role='component' id='sdPretag1161411143sme' type='cell' >RNAi</sd-pretag> clone is outlined in white.</p> <p>(F) Box plot showing distribution of cell <sd-pretag parent-tag-id='353' id='sdPretag179672054sd'>aspect ratio</sd-pretag> for control (wild-type) and <sd-pretag parent-tag-id='12' id='sdPretag895572808sd'><em>zipper</em></sd-pretag> RNAi cells in anchored and <sd-pretag parent-tag-id='5' id='sdPretag948723709sd'>stretched</sd-pretag> (20 min) <sd-pretag parent-tag-id='38' id='sdPretag2147308706sd'>wing disc</sd-pretag>; median represented by horizontal line, 75th and 25th centiles are represented by top and bottom of the boxes respectively; n=8 <sd-pretag parent-tag-id='38' id='sdPretag583682451sd'>wing discs</sd-pretag>.</p> <p>(G) Color-coded <sd-pretag parent-tag-id='353' id='sdPretag16205995sd'>aspect ratio</sd-pretag> in control and <sd-pretag parent-tag-id='13' id='sdPretag805367949sd'><em>zip</em></sd-pretag> <sd-pretag role='component' id='sdPretag1874533295sme' type='cell' >RNAi</sd-pretag> cells (<sd-pretag parent-tag-id='13' id='sdPretag522998774sd'><em>zip</em></sd-pretag> <sd-pretag role='component' id='sdPretag821086315sme' type='cell' >RNAi</sd-pretag> clone outlined in white) subjected to anchoring, <sd-pretag parent-tag-id='5' id='sdPretag455189556sd'>stretching</sd-pretag> (20 min) and relaxation (10 min, post <sd-pretag parent-tag-id='5' id='sdPretag1747232654sd'>stretch</sd-pretag>).
(H) Box plot showing distribution of cell <sd-pretag parent-tag-id='353' id='sdPretag1076335497sd'>aspect ratio</sd-pretag> in anchored, <sd-pretag parent-tag-id='5' id='sdPretag49819103sd'>stretched</sd-pretag> (20 min) and relaxed (10 min) <sd-pretag parent-tag-id='38' id='sdPretag597345874sd'>wing disc</sd-pretag> for control (wild-type) and <sd-pretag parent-tag-id='12' id='sdPretag934893327sd'><em>zipper</em></sd-pretag> RNAi cells; median represented by horizontal line; 75th and 25th centiles are represented by top and bottom of the boxes respectively, n=3 <sd-pretag parent-tag-id='38' id='sdPretag689901243sd'>wing discs</sd-pretag>.</p> <p>(I) <sd-pretag parent-tag-id='13' id='sdPretag1631837782sd'><em>zip</em></sd-pretag>-<sd-pretag role='component' id='sdPretag1495584659sme' type='cell' >RNAi</sd-pretag> clone in <sd-pretag parent-tag-id='15' id='sdPretag985372069sd'>Arm</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag449739885sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='38' id='sdPretag1433100864sd'>wing disc</sd-pretag> at the beginning (5 min) and at the end (40 min) of <sd-pretag parent-tag-id='5' id='sdPretag597102247sd'>stretching</sd-pretag> experiment. Yellow and red arrows indicate the control and <sd-pretag parent-tag-id='13' id='sdPretag652494687sd'><em>zip</em></sd-pretag> <sd-pretag role='component' id='sdPretag1521065500sme' type='cell' >RNAi</sd-pretag> cells respectively.
(J) Change of <em><sd-pretag parent-tag-id='13' id='sdPretag361164549sd'>zip</sd-pretag>-</em><sd-pretag role='component' id='sdPretag1446754296sme' type='cell' >RNAi</sd-pretag> <sd-pretag parent-tag-id='353' id='sdPretag606770553sd'>aspect ratio</sd-pretag> (a.r.) relative to change in control cells <sd-pretag parent-tag-id='353' id='sdPretag621188626sd'>aspect ratio</sd-pretag> (a.r.) in the course of <sd-pretag parent-tag-id='5' id='sdPretag126339981sd'>stretch</sd-pretag>, blue line shows fitted regression; dotted line represents 95% confidence bounds of the best fit line; n=3 <sd-pretag parent-tag-id='38' id='sdPretag1943326330sd'>wing discs</sd-pretag>.</p> <p>(K) Schematics describing behavior of <sd-pretag parent-tag-id='13' id='sdPretag364046193sd'><em>zip</em></sd-pretag>-<sd-pretag role='component' id='sdPretag1560660300sme' type='cell' >RNAi</sd-pretag> cells; spring represents control cells which contract and pull on less elastic <em><sd-pretag parent-tag-id='13' id='sdPretag1528134395sd'>zip</sd-pretag>-</em><sd-pretag role='component' id='sdPretag759869003sme' type='cell' >RNAi</sd-pretag> clone (dashpot) during 40 min <sd-pretag parent-tag-id='5' id='sdPretag1407545407sd'>stretch</sd-pretag>.
Red horizontal arrows indicate direction of <sd-pretag parent-tag-id='5' id='sdPretag2085738276sd'>stretch</sd-pretag>. * p<0.05, ****p<0.0001 with <em>t</em>-test. n.s. = non significant. Scale bars, 5 cm (A), 50 μm (D), 10 μm (E,G), 5 μm (I).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=18689
|
[
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] |
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