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10.1101/241497
|
Polarization of Myosin II refines tissue material properties to buffer mechanical stress.
|
2018
|
Figure 2
|
<sd-panel> <p><strong>Figure 2: <sd-pretag parent-tag-id='177' id='sdPretag2129194733sd'>Myosin</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1660124977sd'>polarizes</sd-pretag> with mechanical <sd-pretag parent-tag-id='5' id='sdPretag1850641689sd'>stretch</sd-pretag>.</strong></p> <p>(A) <sd-pretag parent-tag-id='38' id='sdPretag1802381130sd'>Wing disc</sd-pretag> expressing <sd-pretag parent-tag-id='63' id='sdPretag321583603sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag37417214sd'>GFP</sd-pretag> and <sd-pretag parent-tag-id='65' id='sdPretag1063623722sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag948615106sd'>mCherry</sd-pretag> prior to <sd-pretag parent-tag-id='5' id='sdPretag215905862sd'>stretch</sd-pretag> (anchor) and after <sd-pretag parent-tag-id='5' id='sdPretag708880743sd'>stretch</sd-pretag> (15 min).
(A"), Insets of <sd-pretag parent-tag-id='62' id='sdPretag1651118391sd'>discs</sd-pretag> shown in (A).
(B) Schematics demonstrating change in <sd-pretag parent-tag-id='65' id='sdPretag932425713sd'>Sqh</sd-pretag> concentration before and after <sd-pretag parent-tag-id='5' id='sdPretag24264426sd'>stretch</sd-pretag> on <sd-pretag role='component' id='sdPretag1203182709sme' type='tissue' >vertical</sd-pretag> (V) and horizontal (H) <sd-pretag role='component' id='sdPretag1598248594sme' type='subcellular' >junctions</sd-pretag></p> <p>(C) Quantification of mean <sd-pretag parent-tag-id='302' id='sdPretag1996684757sd'>fluorescent intensity</sd-pretag> of <sd-pretag parent-tag-id='63' id='sdPretag1572842546sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag378197118sd'>GFP</sd-pretag> and <sd-pretag parent-tag-id='65' id='sdPretag651730675sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag227787171sd'>mCherry</sd-pretag> on horizontal (H) <sd-pretag role='component' id='sdPretag382328017sme' type='subcellular' >junctions</sd-pretag> relative to the intensity on <sd-pretag role='component' id='sdPretag258587571sme' type='tissue' >vertical</sd-pretag> (V) <sd-pretag role='component' id='sdPretag1244294119sme' type='subcellular' >junctions</sd-pretag> (referred to as "<sd-pretag parent-tag-id='183' id='sdPretag1503990457sd'>polarity</sd-pretag>") prior to and after 15 min <sd-pretag parent-tag-id='5' id='sdPretag994784068sd'>stretch</sd-pretag>. The detailed rationale of intensity determination is described in Fig. S3 and Experimental Procedures.</p> <p>(D-E), Quantification of <sd-pretag parent-tag-id='63' id='sdPretag86594932sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag2049846694sd'>GFP</sd-pretag> (D) and <sd-pretag parent-tag-id='65' id='sdPretag535638655sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag1311943332sd'>mCherry</sd-pretag> (E) concentration (i.e. mean intensity per junctional unit area) prior and after <sd-pretag parent-tag-id='5' id='sdPretag755734863sd'>stretch</sd-pretag> on both horizontal and <sd-pretag role='component' id='sdPretag1694841590sme' type='tissue' >vertical</sd-pretag> <sd-pretag role='component' id='sdPretag1743582137sme' type='subcellular' >junctions</sd-pretag> normalized to starting concentration on unstretched (anchored) <sd-pretag role='component' id='sdPretag1705576096sme' type='tissue' >vertical</sd-pretag> <sd-pretag role='component' id='sdPretag588796441sme' type='subcellular' >junctions</sd-pretag>.</p> <p>(F) Quantification of total amount of <sd-pretag parent-tag-id='65' id='sdPretag1561391095sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag1933362475sd'>mCherry</sd-pretag> and <sd-pretag parent-tag-id='63' id='sdPretag334955561sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag917776333sd'>GFP</sd-pretag> fluorescence on <sd-pretag role='component' id='sdPretag360316420sme' type='tissue' >vertical</sd-pretag> and horizontal <sd-pretag role='component' id='sdPretag726302108sme' type='subcellular' >junctions</sd-pretag> in anchored and <sd-pretag parent-tag-id='5' id='sdPretag41226628sd'>stretched</sd-pretag> <sd-pretag parent-tag-id='62' id='sdPretag1504095903sd'>discs</sd-pretag>.</p> <p>(G) Tissue expressing <sd-pretag parent-tag-id='63' id='sdPretag326813603sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1338541374sd'>GFP</sd-pretag> and <sd-pretag parent-tag-id='65' id='sdPretag1813224475sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag738757408sd'>mCherry</sd-pretag> subjected to increasing levels of <sd-pretag parent-tag-id='5' id='sdPretag1520452145sd'>stretch</sd-pretag>.
(H) Quantification of <sd-pretag parent-tag-id='65' id='sdPretag1968995000sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag354742979sd'>mCherry</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1444980363sd'>polarity</sd-pretag> (as in c) of experiment described in (G). The level of <sd-pretag parent-tag-id='5' id='sdPretag1430963797sd'>stretch</sd-pretag> is measured as a change in <sd-pretag role='component' id='sdPretag1988187630sme' type='tissue' >tissue</sd-pretag> strain (deformation relative to unstretched <sd-pretag role='component' id='sdPretag734691418sme' type='tissue' >tissue</sd-pretag>).</p> <p>(I-K) <sd-pretag parent-tag-id='65' id='sdPretag684616317sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag1801917281sd'>mCherry</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1507066814sd'>polarity</sd-pretag> emergence as a function of cell <sd-pretag parent-tag-id='353' id='sdPretag1793550955sd'>aspect ratio</sd-pretag> change (I), <sd-pretag parent-tag-id='115' id='sdPretag1344954082sd'>stress</sd-pretag> (J), <sd-pretag parent-tag-id='115' id='sdPretag1345055375sd'>stress</sd-pretag> applied on average cell area of 10 μm2 (K). Yellow asterisks indicate equivalent regions in the <sd-pretag role='component' id='sdPretag86163274sme' type='tissue' >tissue</sd-pretag>. N=8 <sd-pretag parent-tag-id='38' id='sdPretag1021690309sd'>wing discs</sd-pretag> (A- F), N=3 <sd-pretag parent-tag-id='38' id='sdPretag1804430255sd'>wing discs</sd-pretag> (G-K); all experiments are plotted as mean ± S.E.M., * p<0.05 with <em>t</em>-test. Scale bars, 5 μm (A), 3 μm (A"), 10 μm (G).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=18690
|
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||
10.1101/241497
|
Polarization of Myosin II refines tissue material properties to buffer mechanical stress.
|
2018
|
Figure 3
|
<sd-panel> <p><strong>Figure 3: Uncoupling of <sd-pretag role='component' id='sdPretag1428018556sme' type='tissue' >tissue</sd-pretag> shape and <sd-pretag role='assayed' id='sdPretag63076020sme' type='geneprod' >MyoII</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1943209088sd'>polarity</sd-pretag> after prolonged <sd-pretag parent-tag-id='5' id='sdPretag71476873sd'>stretch</sd-pretag>.
</strong>(A) Short (15 min) <sd-pretag parent-tag-id='5' id='sdPretag1037888477sd'>stretch</sd-pretag> and relaxation (10 min) of <sd-pretag parent-tag-id='63' id='sdPretag412696743sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1812281859sd'>GFP</sd-pretag> and <sd-pretag parent-tag-id='65' id='sdPretag1192267116sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag1608531382sd'>mCherry</sd-pretag> expressing <sd-pretag parent-tag-id='62' id='sdPretag1420782014sd'>discs</sd-pretag>.</p> <p>(B) Quantification of <sd-pretag parent-tag-id='65' id='sdPretag171827419sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag840899973sd'>mCherry</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1057976028sd'>polarity</sd-pretag>, <sd-pretag parent-tag-id='63' id='sdPretag1166924933sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag546665939sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag498525979sd'>polarity</sd-pretag> and <sd-pretag parent-tag-id='353' id='sdPretag1298977193sd'>aspect ratio</sd-pretag> change of experiment described in (A); schematics below the graph illustrates quantified changes in cell <sd-pretag parent-tag-id='353' id='sdPretag1906610953sd'>aspect ratio</sd-pretag> (yellow) and <sd-pretag parent-tag-id='65' id='sdPretag1755768951sd'>Sqh</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag14833098sd'>polarity</sd-pretag> (red). Statistical test compares <sd-pretag parent-tag-id='65' id='sdPretag596064043sd'>Sqh</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1822749178sd'>polarity</sd-pretag> between anchored and <sd-pretag parent-tag-id='5' id='sdPretag294611445sd'>stretched</sd-pretag> or <sd-pretag parent-tag-id='5' id='sdPretag606907853sd'>stretched</sd-pretag> and relaxed <sd-pretag parent-tag-id='62' id='sdPretag1968352813sd'>disc</sd-pretag>.</p> <p>(C) Long (200 min) <sd-pretag parent-tag-id='5' id='sdPretag755475691sd'>stretch</sd-pretag> and relaxation (10 min) of <sd-pretag parent-tag-id='63' id='sdPretag874216702sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag156108340sd'>GFP</sd-pretag> and <sd-pretag parent-tag-id='65' id='sdPretag1465024191sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag159231320sd'>mCherry</sd-pretag> expressing <sd-pretag parent-tag-id='62' id='sdPretag702282291sd'>discs</sd-pretag>.
(D) Quantification of <sd-pretag parent-tag-id='65' id='sdPretag2102245336sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag538459747sd'>mCherry</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1530512344sd'>polarity</sd-pretag>, <sd-pretag parent-tag-id='63' id='sdPretag406678662sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1993105009sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag951649009sd'>polarity</sd-pretag> and <sd-pretag parent-tag-id='353' id='sdPretag391499844sd'>aspect ratio</sd-pretag> change of experiment described in (C); schematics below the graph illustrates quantified changes in <sd-pretag parent-tag-id='353' id='sdPretag1020439697sd'>aspect ratio</sd-pretag> (yellow) and <sd-pretag parent-tag-id='65' id='sdPretag767631409sd'>Sqh</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag362805986sd'>polarity</sd-pretag> (red). Statistical test compares changes with respect to initial (anchored) state. Yellow asterisks indicate equivalent regions in the <sd-pretag role='component' id='sdPretag616248542sme' type='tissue' >tissue</sd-pretag>. N=3 <sd-pretag parent-tag-id='38' id='sdPretag1152386134sd'>wing discs</sd-pretag>; all experiments are plotted as mean ± S.E.M., * p<0.05 with <em>t</em>-test. Scale bars, 5 μm.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=18691
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||
10.1101/241497
|
Polarization of Myosin II refines tissue material properties to buffer mechanical stress.
|
2018
|
Figure 4
|
<sd-panel> <p><strong>Figure 4: Phosphorylated <sd-pretag role='assayed' id='sdPretag1433900380sme' type='geneprod' >MyoII</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag510433584sd'>polarizes</sd-pretag> in mechanically <sd-pretag parent-tag-id='5' id='sdPretag543828090sd'>stretched</sd-pretag> cells.
</strong>(A) Overview of the <sd-pretag parent-tag-id='63' id='sdPretag897341404sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1413309026sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='62' id='sdPretag582155616sd'>disc</sd-pretag> prior to (anchor) and after <sd-pretag parent-tag-id='160' id='sdPretag1360417034sd'>compression</sd-pretag>. (B) <sd-pretag parent-tag-id='63' id='sdPretag1308453532sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag355761965sd'>GFP</sd-pretag> and <sd-pretag parent-tag-id='65' id='sdPretag199076921sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag1262222356sd'>mCherry</sd-pretag> localization in anchored and <sd-pretag parent-tag-id='160' id='sdPretag1526660311sd'>compressed</sd-pretag> <sd-pretag parent-tag-id='38' id='sdPretag49880877sd'>wing disc</sd-pretag>.</p> <p>(C) Laser cuts (perpendicular to the line of <sd-pretag parent-tag-id='5' id='sdPretag1075919988sd'>stretch</sd-pretag>) across circa 10 cells in <sd-pretag parent-tag-id='171' id='sdPretag1673146887sd'>E- cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag141154291sd'>GFP</sd-pretag> expressing <sd-pretag parent-tag-id='38' id='sdPretag1046413539sd'>wing disc</sd-pretag> tissue subjected to anchoring, short (<20 min) <sd-pretag parent-tag-id='5' id='sdPretag1562787058sd'>stretching</sd-pretag> and short (<20 min) <sd-pretag parent-tag-id='160' id='sdPretag1432036155sd'>compression</sd-pretag>. Plot shows increase in distance between recoiling short axis [μm] of the fitted <sd-pretag role='component' id='sdPretag121011699sme' type='organism' >ellipse</sd-pretag> (see Fig. S5A for details) against time [sec.]; data is plotted as mean ± S.E.M, n= 7-10 <sd-pretag parent-tag-id='62' id='sdPretag629613153sd'>discs</sd-pretag>/condition. (D) Activated <sd-pretag parent-tag-id='177' id='sdPretag325129309sd'>Myosin</sd-pretag> (anti-phosphorylated-<sd-pretag parent-tag-id='65' id='sdPretag2028008702sd'>Sqh</sd-pretag>; in red) and anti-<sd-pretag parent-tag-id='63' id='sdPretag669442455sd'>E-cad</sd-pretag> (green) staining in <sd-pretag parent-tag-id='160' id='sdPretag1388122372sd'>compressed</sd-pretag> <sd-pretag parent-tag-id='62' id='sdPretag1770699720sd'>discs</sd-pretag>.</p> <p>(D") Close up of <sd-pretag parent-tag-id='160' id='sdPretag1913370155sd'>compression</sd-pretag> experiment described in (D); yellow arrow points towards an example of <sd-pretag parent-tag-id='183' id='sdPretag1403911600sd'>polarized</sd-pretag> phosphorylated-<sd-pretag parent-tag-id='65' id='sdPretag156222457sd'>Sqh</sd-pretag> cable in <sd-pretag parent-tag-id='160' id='sdPretag804929235sd'>compressed</sd-pretag> <sd-pretag parent-tag-id='62' id='sdPretag958189501sd'>discs</sd-pretag>.
Scale bars, 5μm (B, D"), 10μm (D), 30μm (A).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=18692
|
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]
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{
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"text": "E-cad",
"type": "geneprod",
"uniprot_ids": [
"Q24298"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P40423",
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"ext_tax_names": "Drosophila melanogaster",
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{
"ext_dbs": "Uniprot",
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{
"ext_dbs": "Uniprot",
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"text": "E-cad",
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]
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{
"ext_dbs": "Uniprot",
"ext_ids": "P40423",
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"ext_tax_names": "Drosophila melanogaster",
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"text": "Myosin",
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{
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{
"ext_dbs": "Uniprot",
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"role": "assayed",
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"type": "geneprod",
"uniprot_ids": [
"P40423"
]
}
] |
||
10.1101/241497
|
Polarization of Myosin II refines tissue material properties to buffer mechanical stress.
|
2018
|
Figure 5
|
<sd-panel> <p><strong>Figure 5: Emergent tension patterns reveal tissue stiffening upon mechanical <sd-pretag parent-tag-id='5' id='sdPretag350135336sd'>stretch</sd-pretag>.
</strong>(A) Laser cuts (perpendicular to the line of <sd-pretag parent-tag-id='5' id='sdPretag1355033480sd'>stretch</sd-pretag>) across circa 10 cells in <sd-pretag parent-tag-id='171' id='sdPretag1731909127sd'>E- cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag2049976909sd'>GFP</sd-pretag> expressing <sd-pretag parent-tag-id='38' id='sdPretag1792626177sd'>wing disc</sd-pretag> tissue subjected to anchoring, short (<20 min) and long (>120 min) <sd-pretag parent-tag-id='5' id='sdPretag816420211sd'>stretching</sd-pretag> and <sd-pretag parent-tag-id='195' id='sdPretag930148803sd'>stretch retraction</sd-pretag> (tissue forced back to the original, pre-<sd-pretag parent-tag-id='5' id='sdPretag317012069sd'>stretch</sd-pretag> shape.</p> <p>(B) Quantification of the experiment described in (A). Plot shows increase in distance between recoiling short axis [μm] of the fitted ellipse (see Fig S4A-B for details) against time [sec.]; data is plotted as mean ± S.E.M; anchor and short <sd-pretag parent-tag-id='5' id='sdPretag2091944513sd'>stretch</sd-pretag> data plotted here is the same as in Fig. 4B, n=3-11 <sd-pretag parent-tag-id='38' id='sdPretag655078782sd'>wing discs</sd-pretag> per condition.</p> <p>(C) An overlay of a <sd-pretag parent-tag-id='198' id='sdPretag1553850009sd'>junction</sd-pretag> prior to (red) and 15.44 sec. after <sd-pretag parent-tag-id='199' id='sdPretag1172598736sd'>ablation</sd-pretag> (green). (D) Plot showing relative initial recoil velocity (velocity on horizontal, <sd-pretag parent-tag-id='5' id='sdPretag1743466084sd'>stretched</sd-pretag> junctions normalized to velocity on <sd-pretag role='component' id='sdPretag222831351sme' type='tissue' >vertical</sd-pretag>, unstretched junctions) for individual junctions <sd-pretag parent-tag-id='199' id='sdPretag301286143sd'>ablated</sd-pretag> in anchored or <sd-pretag parent-tag-id='5' id='sdPretag1916814347sd'>stretched</sd-pretag> (<30 min, >100 min) <sd-pretag parent-tag-id='63' id='sdPretag1608295176sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1122708489sd'>GFP</sd-pretag> expressing <sd-pretag parent-tag-id='62' id='sdPretag861972673sd'>discs</sd-pretag>. Data is plotted as a mean ± S.E.M, n=17-24 cuts per condition.
(E) Laser cuts (perpendicular or parallel to the line of <sd-pretag parent-tag-id='5' id='sdPretag85707113sd'>stretch</sd-pretag>) across circa 10 cells in <sd-pretag parent-tag-id='63' id='sdPretag1840433667sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1553910307sd'>GFP</sd-pretag> expressing <sd-pretag parent-tag-id='38' id='sdPretag1927437349sd'>wing disc</sd-pretag> tissue subjected to anchoring or short <sd-pretag parent-tag-id='5' id='sdPretag1070479333sd'>stretching</sd-pretag> (< 30 min)..</p> <p>(F) Quantification of experiment described in (E). Data is quantified and presented as in (B). N=4-7 <sd-pretag parent-tag-id='38' id='sdPretag1958922638sd'>wing discs</sd-pretag> per condition.
(G) Image depicting injury site (yellow asterisk) and selection of cell rows used for tissue recoil propagation measurement; r: row.</p> <p>(H) An overlay of anchored and <sd-pretag parent-tag-id='5' id='sdPretag382054430sd'>stretched</sd-pretag> <sd-pretag parent-tag-id='62' id='sdPretag1000479166sd'>discs</sd-pretag> prior to <sd-pretag parent-tag-id='199' id='sdPretag1722110438sd'>ablation</sd-pretag> (red) and 15.44 seconds post <sd-pretag parent-tag-id='199' id='sdPretag997647125sd'>ablation</sd-pretag> (green). Inset zooms in on the area next to <sd-pretag parent-tag-id='199' id='sdPretag1254039024sd'>ablation</sd-pretag> site (marked with yellow asterisk).</p> <p>
(I) Recoil area (% increase from pre-<sd-pretag parent-tag-id='199' id='sdPretag1270895577sd'>ablation</sd-pretag> area) for row 0, row 1, row 2 and row 3 in <sd-pretag parent-tag-id='5' id='sdPretag1732866546sd'>stretched</sd-pretag> and anchored <sd-pretag parent-tag-id='62' id='sdPretag447642075sd'>discs</sd-pretag>. Data is plotted as mean ± S.E.M, n=8- 13 <sd-pretag parent-tag-id='62' id='sdPretag1709890951sd'>discs</sd-pretag>/condition.</p> <p>Error bars indicate S.E.M.; * p<0.05 and **p<0.01 with <em>t</em>-test. Scale bars, 5 μm (A,E,G), 3 μm (C,H), 2 μm (H: inset).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=18693
|
[
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"original_type": "protein",
"role": "assayed",
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"uniprot_ids": [
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]
},
{
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"Q24298"
]
}
] |
||
10.1101/241497
|
Polarization of Myosin II refines tissue material properties to buffer mechanical stress.
|
2018
|
Figure 6
|
<sd-panel> <p><strong>Figure 6: <sd-pretag parent-tag-id='177' id='sdPretag1525016991sd'>Myosin</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1517189791sd'>polarity</sd-pretag> is <sd-pretag parent-gene-tag-id='244' parent-protein-tag-id='358' id='sdPretag1167324127sd'>Rok</sd-pretag> independent.</strong></p> <p>(A) <sd-pretag role='component' id='sdPretag707153966sme' type='geneprod' >RokK116a</sd-pretag>::<sd-pretag parent-tag-id='217' id='sdPretag1385210793sd'>Venus</sd-pretag> localization in anchored, <sd-pretag parent-tag-id='5' id='sdPretag1380240158sd'>stretched</sd-pretag>, relaxed third instar <sd-pretag parent-tag-id='219' id='sdPretag1968090875sd'>imaginal discs</sd-pretag>.
(B) <sd-pretag role='assayed' id='sdPretag795607718sme' type='geneprod' ><em>Drok2</em></sd-pretag> mitotic clones (<sd-pretag parent-tag-id='223' id='sdPretag1056404048sd'>RFP</sd-pretag>-) in <sd-pretag parent-tag-id='65' id='sdPretag1754604173sd'>Sqh</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1860725212sd'>GFP</sd-pretag> third instar <sd-pretag parent-tag-id='219' id='sdPretag1745085843sd'>imaginal disc</sd-pretag>. Inset demonstrates <sd-pretag parent-tag-id='65' id='sdPretag1597438625sd'>Sqh</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag2137862641sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1019133805sd'>polarity</sd-pretag> inside <sd-pretag role='intervention' id='sdPretag2004617883sme' type='geneprod' ><em>Drok2</em></sd-pretag> clone.</p> <p>(C) <em><sd-pretag parent-tag-id='65' id='sdPretag2107270397sd'>Sqh</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1681484982sd'>GFP</sd-pretag>, enGal4><sd-pretag parent-tag-id='233' id='sdPretag219115353sd'>UAS</sd-pretag>-<sd-pretag parent-gene-tag-id='244' parent-protein-tag-id='358' id='sdPretag2083755127sd'>rok</sd-pretag> <sd-pretag role='component' id='sdPretag526493842sme' type='cell' >RNAi</sd-pretag>, <sd-pretag parent-tag-id='233' id='sdPretag232011706sd'>UAS</sd-pretag>-<sd-pretag role='reporter' id='sdPretag263424923sme' type='geneprod' >Act5CRFP</sd-pretag></em> <sd-pretag parent-tag-id='62' id='sdPretag629583736sd'>discs</sd-pretag> subjected to <sd-pretag parent-tag-id='5' id='sdPretag737992199sd'>stretching</sd-pretag>; inset demonstrates <sd-pretag parent-tag-id='65' id='sdPretag1532265098sd'>Sqh</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag858192080sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1653918375sd'>polarity</sd-pretag> in <sd-pretag parent-gene-tag-id='244' parent-protein-tag-id='358' id='sdPretag1584532998sd'><em>rok</em></sd-pretag> <sd-pretag role='component' id='sdPretag2000521973sme' type='cell' >RNAi</sd-pretag> cells (marked by red.
(D) <sd-pretag parent-tag-id='13' id='sdPretag1469896683sd'><em>zip</em></sd-pretag> <sd-pretag role='component' id='sdPretag1361306004sme' type='cell' >RNAi</sd-pretag> clones (labeled as <sd-pretag parent-tag-id='13' id='sdPretag256814616sd'>zip</sd-pretag> iR, indicated in red) in <sd-pretag parent-tag-id='5' id='sdPretag1852974309sd'>stretched</sd-pretag> third instar <sd-pretag role='intervention' id='sdPretag1130509444sme' type='geneprod' >RokK116a</sd-pretag>::<sd-pretag parent-tag-id='217' id='sdPretag391038354sd'>Venus</sd-pretag> <sd-pretag parent-tag-id='62' id='sdPretag1449836423sd'>discs</sd-pretag>.</p> <p>Error bars indicate S.E.M.; * p<0.05 and **p<0.01 with <em>t</em>-test. Scale bars, 2 μm (D: inset), 5 μm (A,D, B: inset, C: inset), 10 μm (B,C).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=18694
|
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"uniprot_ids": [
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]
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{
"ext_dbs": "NCBI gene",
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"text": "rok",
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"X2JE40",
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},
{
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{
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{
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{
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},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q9VXE3",
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] |
||
10.1101/241497
|
Polarization of Myosin II refines tissue material properties to buffer mechanical stress.
|
2018
|
Figure 7
|
<sd-panel> <p><strong>Figure 7: <sd-pretag role='component' id='sdPretag403549348sme' type='geneprod' >MyoII</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag871385817sd'>polarity</sd-pretag> is required for <sd-pretag parent-tag-id='38' id='sdPretag1315466700sd'>wing disc</sd-pretag> shape maintenance via <sd-pretag parent-tag-id='278' id='sdPretag1214714639sd'>Dia</sd-pretag>-dependent <sd-pretag role='component' id='sdPretag1762985222sme' type='undefined' >actin</sd-pretag> polymerization.
</strong>(A) <sd-pretag parent-tag-id='62' id='sdPretag1762535164sd'>Discs</sd-pretag> expressing <sd-pretag parent-tag-id='65' id='sdPretag2043553444sd'>Sqh</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag103214611sd'>GFP</sd-pretag> and <em>enGal4><sd-pretag parent-tag-id='233' id='sdPretag1136293293sd'>UAS</sd-pretag>-<sd-pretag parent-tag-id='278' id='sdPretag901876629sd'>dia</sd-pretag> <sd-pretag role='component' id='sdPretag792399764sme' type='cell' >RNAi</sd-pretag>, <sd-pretag parent-tag-id='233' id='sdPretag293136107sd'>UAS</sd-pretag>-<sd-pretag parent-tag-id='262' id='sdPretag1784125929sd'>Act5c</sd-pretag>-<sd-pretag parent-tag-id='223' id='sdPretag26391220sd'>RFP</sd-pretag></em> that were <sd-pretag parent-tag-id='5' id='sdPretag1035472444sd'>stretched</sd-pretag>; <sd-pretag parent-tag-id='233' id='sdPretag666846415sd'>UAS</sd-pretag>-<sd-pretag parent-tag-id='262' id='sdPretag1196386796sd'>Act5C</sd-pretag>::<sd-pretag parent-tag-id='223' id='sdPretag536158316sd'>RFP</sd-pretag> marks <sd-pretag parent-tag-id='278' id='sdPretag367474921sd'><em>dia</em></sd-pretag> iR (posterior side) in red.</p> <p>(A") Insets comparing <sd-pretag parent-tag-id='65' id='sdPretag300046236sd'>Sqh</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag976730759sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag854161480sd'>polarity</sd-pretag> in <sd-pretag parent-tag-id='5' id='sdPretag840600867sd'>stretched</sd-pretag> <em>enGal4><sd-pretag parent-tag-id='278' id='sdPretag45223544sd'>dia</sd-pretag> iR, <sd-pretag parent-tag-id='233' id='sdPretag383509132sd'>UAS</sd-pretag>- <sd-pretag parent-tag-id='262' id='sdPretag1219097122sd'>Act5c</sd-pretag>-<sd-pretag parent-tag-id='223' id='sdPretag1061200481sd'>RFP</sd-pretag></em> <sd-pretag parent-tag-id='62' id='sdPretag1984523735sd'>discs</sd-pretag>; panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (<sd-pretag parent-tag-id='278' id='sdPretag867196736sd'><em>dia</em></sd-pretag> iR) side.
(B) Tissue marked with <sd-pretag parent-tag-id='63' id='sdPretag620958045sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1206337816sd'>GFP</sd-pretag> and <sd-pretag parent-tag-id='65' id='sdPretag24388850sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag459910684sd'>mCherry</sd-pretag>, expressing <em><sd-pretag parent-tag-id='278' id='sdPretag484050601sd'>dia</sd-pretag>-</em><sd-pretag role='component' id='sdPretag824935471sme' type='cell' >RNAi</sd-pretag> (<sd-pretag parent-tag-id='278' id='sdPretag1256334301sd'><em>dia</em></sd-pretag> iR) with hhGal4 driver and subjected to <sd-pretag parent-tag-id='5' id='sdPretag1419396904sd'>stretching</sd-pretag>. Dotted line indicates anterior-posterior (A-P) compartment boundary.</p> <p>(B") Inset demonstrates lack of <sd-pretag parent-tag-id='65' id='sdPretag1111500028sd'>Sqh</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag626241871sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1141676815sd'>polarity</sd-pretag> in <em><sd-pretag parent-tag-id='278' id='sdPretag1957562151sd'>dia</sd-pretag>-</em><sd-pretag role='component' id='sdPretag654212593sme' type='cell' >RNAi</sd-pretag> cells.
(C-D), Quantification of <sd-pretag parent-tag-id='63' id='sdPretag1499651012sd'>E-cad</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag875485409sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1405147311sd'>polarity</sd-pretag> (C) and <sd-pretag parent-tag-id='65' id='sdPretag1117279513sd'>Sqh</sd-pretag>::<sd-pretag parent-tag-id='66' id='sdPretag977488071sd'>mCherry</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag499314128sd'>polarity</sd-pretag> (D) in experiment described in (B); <sd-pretag parent-tag-id='183' id='sdPretag299371494sd'>polarity</sd-pretag> is measured as mean <sd-pretag parent-tag-id='302' id='sdPretag896340560sd'>fluorescent intensity</sd-pretag> on horizontal <sd-pretag role='component' id='sdPretag81656786sme' type='subcellular' >junctions</sd-pretag> relative to vertical <sd-pretag role='component' id='sdPretag412559957sme' type='subcellular' >junctions</sd-pretag> (see Experimental Procedures for details); n=4 <sd-pretag parent-tag-id='38' id='sdPretag1706911205sd'>wing discs</sd-pretag>.
(E) <sd-pretag parent-tag-id='5' id='sdPretag1660797088sd'>Stretched</sd-pretag> <sd-pretag parent-tag-id='62' id='sdPretag1606929017sd'>discs</sd-pretag> expressing <sd-pretag parent-tag-id='65' id='sdPretag931507214sd'>Sqh</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag470652348sd'>GFP</sd-pretag> and <em>enGal4><sd-pretag parent-tag-id='233' id='sdPretag1768987393sd'>UAS</sd-pretag>-<sd-pretag parent-tag-id='312' id='sdPretag1131506711sd'>cofilin</sd-pretag></em> (upper panel) <em>or <sd-pretag parent-tag-id='233' id='sdPretag546469045sd'>UAS</sd-pretag>-<sd-pretag parent-tag-id='314' id='sdPretag351900386sd'>AIP1</sd-pretag></em> (lower panel) <sd-pretag role='component' id='sdPretag817635949sme' type='cell' ><em>RNAi</em></sd-pretag> and <em><sd-pretag parent-tag-id='233' id='sdPretag440242436sd'>UAS</sd-pretag>-<sd-pretag parent-tag-id='262' id='sdPretag778185918sd'>Act5c</sd-pretag>-<sd-pretag parent-tag-id='223' id='sdPretag842807886sd'>RFP</sd-pretag></em> (marks posterior side in red).
(E"), Insets comparing <sd-pretag parent-tag-id='65' id='sdPretag161047360sd'>Sqh</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag722287215sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag915538197sd'>polarity</sd-pretag> in <sd-pretag parent-tag-id='5' id='sdPretag1187997428sd'>stretched</sd-pretag> <sd-pretag parent-tag-id='62' id='sdPretag1520034221sd'>discs</sd-pretag> described in (E); panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (<sd-pretag parent-tag-id='278' id='sdPretag630092429sd'><em>dia</em></sd-pretag> iR) side. (F) <sd-pretag parent-tag-id='65' id='sdPretag277720089sd'>Sqh</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1301421524sd'>GFP</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag1274074507sd'>polarity</sd-pretag> in enGal4> <sd-pretag parent-tag-id='278' id='sdPretag266191406sd'><em>dia</em></sd-pretag> iR, <sd-pretag parent-tag-id='233' id='sdPretag552414840sd'>UAS</sd-pretag>-<sd-pretag parent-tag-id='262' id='sdPretag382371498sd'>Act5C</sd-pretag>::<sd-pretag parent-tag-id='223' id='sdPretag2045480985sd'>RFP</sd-pretag> <sd-pretag parent-tag-id='62' id='sdPretag905833183sd'>discs</sd-pretag> labeled with <sd-pretag parent-tag-id='333' id='sdPretag817908019sd'>Phalloidin</sd-pretag>-647; Inset (Phall-647) zooms in on cell shape in anterior and posterior <sd-pretag parent-tag-id='62' id='sdPretag81147511sd'>disc</sd-pretag> compartments.
(G) <sd-pretag parent-tag-id='62' id='sdPretag939886570sd'>Discs</sd-pretag> expressing <sd-pretag role='intervention' id='sdPretag1179101322sme' type='geneprod' ><em>nub</em>Gal4</sd-pretag> and <sd-pretag parent-gene-tag-id='344' parent-protein-tag-id='345' id='sdPretag786058054sd'>Cd8</sd-pretag>::<sd-pretag parent-gene-tag-id='16' parent-protein-tag-id='64' id='sdPretag1436895191sd'>GFP</sd-pretag> (control) or <sd-pretag parent-tag-id='278' id='sdPretag775403991sd'><em>dia</em></sd-pretag> iR (KK) labeled with <sd-pretag parent-tag-id='342' id='sdPretag1190144740sd'>DAPI</sd-pretag>; ellipse is fitted into the pouch region (red dotted line) and <sd-pretag parent-tag-id='353' id='sdPretag1985425272sd'>aspect ratio</sd-pretag> determined as long ellipse axis (b) divided by short ellipse axis (a) (white lines).
(H) Box plot showing distribution of <sd-pretag parent-tag-id='38' id='sdPretag1947484151sd'>wing disc</sd-pretag> pouch <sd-pretag parent-tag-id='353' id='sdPretag2071666951sd'>aspect ratios</sd-pretag> in conditions described in (G), n=19 (ctrl) and 16 (<sd-pretag parent-tag-id='278' id='sdPretag1417087006sd'>dia</sd-pretag> iR) <sd-pretag parent-tag-id='355' id='sdPretag691264275sd'>wing pouches</sd-pretag>; horizontal line indicates median.
(I) Model of Diaphanous-<sd-pretag parent-tag-id='177' id='sdPretag1915766774sd'>Myosin</sd-pretag> force buffering pathway. Tissue (<sd-pretag parent-tag-id='63' id='sdPretag1656576148sd'>E-cad</sd-pretag> marks cell <sd-pretag role='component' id='sdPretag760639647sme' type='subcellular' >junctions</sd-pretag> in green) subjected to mechanical <sd-pretag parent-tag-id='5' id='sdPretag279195075sd'>stretching</sd-pretag> <sd-pretag parent-tag-id='183' id='sdPretag55898703sd'>polarizes</sd-pretag> <sd-pretag role='component' id='sdPretag152121062sme' type='geneprod' >MyoII</sd-pretag> and <sd-pretag role='assayed' id='sdPretag933513318sme' type='geneprod' >Actin</sd-pretag> (in red) in <sd-pretag parent-tag-id='278' id='sdPretag2074753337sd'>Dia</sd-pretag>-dependent manner. This mechanism is required to limit tension induced cell deformation and thus maintain <sd-pretag parent-tag-id='38' id='sdPretag1120533580sd'>wing disc</sd-pretag> pouch shape. Error bars indicate S.E.M.; * p<0.05 and **p<0.01 with <em>t</em>-test. Scale bars, 5 μm (A",B",E", F: inset), 10 μm (A,B,E), 30 μm (F, G).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=18695
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] |
||
10.15252/embj.2022111185
|
A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation
|
2022
|
Figure 2
|
<sd-panel><p><strong>Figure 2. ApoSOD1<sup>2SH</sup> undergoes liquid-liquid phase separation.</strong></p> <p><strong>A.</strong> SOD1<sup>2SH</sup> undergoes LLPS on the removal of metal ion cofactors.</p> <p><strong>B.</strong> DIC microscopic image of WTSOD1<sup>2SH</sup> diluted to 100 µM in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated 37 °C, 180 RPM for 30 min shows no droplet formation; scale bar: 100 µm; inset shows Alexa Fluor 488 maleimide labelled WTSOD1<sup>2SH</sup>; scale bar: 5 µm</p> <p><strong>C.</strong> Solution turbidity plot (absorbance at 600 nm) shows that WTSOD1<sup>2SH</sup> (at concentrations ranging from 25 to 200 µM does not undergo LLPS in presence or absence of heparin, LLPS inducer; data is represented as mean ± SD (from 3 biological replicates).</p> <p><strong>D.</strong> DIC microscopic image of ApoSOD1<sup>2SH</sup> under condensate inducing conditions (37 C, 180 RPM) at a concentration of 100 µM incubated 100 mM NaCl; scale bar:100 µm (inset on bottom left shows Alexa Fluor 488 maleimide labeled protein condensates; scale bar: 5 µm.</p> <p><strong>E.</strong> Fluorescent microscopic images of Alexa Fluor 488 maleimide labeled ApoSOD1<sup>2SH</sup> condensates diluted in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated (180 RPM, 37 C) in absence (left) and presence (right) of heparin at 30 min time-point; scale bar: 100 µm.</p> <p><strong>F</strong>. Solution turbidity measurements with ApoSOD1<sup>2SH</sup> (absorbance at 600 nm) shows that while in the absence of heparin the turbidity increases with time, the presence of heparin results in faster LLPS; data is represented as mean ± SD (from 3 biological replicates).</p> <p><strong>G</strong>. Comparison of ApoSOD1<sup>2SH</sup> droplet numbers per microscopic field of view (view area 0.001963 mm<sup>2</sup>) calculated from 5 different images of droplets incubated with and without heparin at 2 h timepoint. Data is represented as mean ±SD. Statistical significance was established using unpaired non-parametric t-test (****p< 0.0001).</p> <p><strong>H</strong>. Liquid nature of ApoSOD1<sup>2SH</sup> showing droplet fusion and surface wetting; scale bar: 5 µm. <strong>I</strong>. Amplitude normalized FCS curves of ApoSOD1<sup>2SH</sup> (in blue) of dilute phase (DP) and condensed phase (CP). Intense color indicates CP and light variant indicates DP. The increase in τ<sub>D</sub> (as shown by the arrow) translates to a decrease in D (please refer to the Equation 2 in Methods section)</p> <p><strong>J</strong>. Scheme depicting that presence of Zn and not Cu in pre-incubation mixture inhibits LLPS.</p> <p><strong>K.</strong> DIC microscopic images of ApoSOD1<sup>2SH</sup> in presence of Zn (top) showing no droplet formation and Cu (below) showing the presence of droplets; scale bar: 100 µm.</p> <p><strong>L</strong>. Solution turbidity plot (absorbance at 600 nm) of ApoSOD1<sup>2SH</sup> subjected to increasing concentrations of Cu and Zn; turbidity decreases in a dose dependent manner for Zn while no significant change is observed in presence of Cu; data is represented as mean ± SD (from 3 biological replicates).</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=50928
|
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||
10.15252/embj.2022111185
|
A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation
|
2022
|
Figure 3
|
<sd-panel><p><strong>Figure 3. <sd-pretag id="sdPretag378742780sm" category="disease">ALS</sd-pretag> mutants with compromised Zn binding undergo <sd-pretag id="sdPretag2052327932sm" type="small" role="intervention">LLPS</sd-pretag>.</strong></p> <p><strong>A.</strong> <sd-pretag id="sdPretag1084217561sm" category="assay">DIC microscopic</sd-pretag> images of <sd-pretag id="sdPretag854634083sm" type="cell" role="component">I113T</sd-pretag> <sd-pretag id="sdPretag600498907sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> (top) and G85R <sd-pretag id="sdPretag1678170412sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> (bottom) droplets incubated in the absence and presence of Zn and Cu respectively (scale bar: 100 µm); insets on bottom left show Alexa <sd-pretag id="sdPretag1466046777sm" category="assay">Fluor</sd-pretag> <sd-pretag id="sdPretag924645777sm" role="reporter">488</sd-pretag> <sd-pretag id="sdPretag1350141766sm" type="small" role="assayed">maleimide</sd-pretag> labeled protein condensates for I113T <sd-pretag id="sdPretag552610791sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> and G85R <sd-pretag id="sdPretag1653166638sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> respectively; scale bar: 5 µm. Droplet dissolution was observed for <sd-pretag id="sdPretag518659482sm" type="cell" role="component">I113T</sd-pretag> <sd-pretag id="sdPretag1006282531sm" type="geneprod" role="intervention">SOD1<sup>2SH</sup></sd-pretag> on addition of Zn while droplets persisted on addition of Cu. Red arrowhead indicates fusion of <sd-pretag id="sdPretag7227134sm" type="subcellular" role="component">liquid droplets</sd-pretag>.</p> <p><strong>B</strong>. Amplitude normalized <sd-pretag id="sdPretag234595380sm" category="disease">FCS</sd-pretag> curves of <sd-pretag id="sdPretag1535758800sm" type="cell" role="component">I113T</sd-pretag> <sd-pretag id="sdPretag757141289sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> (in green) and G85R <sd-pretag id="sdPretag866290539sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> (in grey) of dilute phase (DP) and condensed phase (CP) showing an increase in diffusion time from DP to CP. The intense color indicates CP and light variant indicates DP.</p> <p><strong>C</strong>. On metal <sd-pretag id="sdPretag1650966065sm" type="tissue" role="component">cofactor</sd-pretag> removal, ApoG37R <sd-pretag id="sdPretag1538477926sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> formed condensates which ceased to exist when protein was incubated with Zn, while condensates persisted on Cu addition (left to right); scale bar: 100 µm.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=50929
|
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},
{
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] |
||
10.15252/embj.2022111185
|
A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation
|
2022
|
Figure 4
|
<sd-panel><p><strong>Figure 4. Zn <sd-pretag id="sdPretag2057217960sm" category="disease">drives disorder</sd-pretag> to order transition in <sd-pretag id="sdPretag1800314500sm" type="geneprod" role="intervention">SOD1</sd-pretag>.</strong></p> <p><strong>A</strong>. Scheme shows Zn <sd-pretag id="sdPretag1924924807sm" category="disease">dependent disorder</sd-pretag> to order transition in monomeric <sd-pretag id="sdPretag198005722sm" type="geneprod" role="intervention">SOD1</sd-pretag>.</p> <p><strong>B</strong>. Plot of the log<sub>e</sub> of the hydrodynamic radius (r<sub>H</sub>) versus the log <sub>e</sub> of the number of residues in the polypeptide chain. The line fitted to these data for the native folded proteins (yellow dashed) has a slope of 0.29 ± 0.02 and a y-axis intercept of 1.56 ± 0.1, while the other fitted to the chemically denatured protein (grey dashed) data has a slope of 0.57 ± 0.02 and a y-axis intercept of 0.79 ± 0.07. Literature data have been used for folded and chemically denatured proteins while we employed <sd-pretag id="sdPretag1382073577sm" category="disease">FCS</sd-pretag> to calculate the r<sub>H</sub> of all <sd-pretag id="sdPretag1263029368sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> variants with (solid shapes) and without Zn (hollow shapes). The hollow grey circle of G85R <sd-pretag id="sdPretag1316837669sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> is directly under hollow green I113T <sd-pretag id="sdPretag389344958sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> in the log-log plot.</p> <p><strong>C</strong>. Variation of diffusion coefficients of de-metalated protein variants determined from <sd-pretag id="sdPretag463130592sm" category="assay">FCS</sd-pretag> measurement with increasing Zn concentration (inset shows a comparison of binding constants between mutants and Zn; <sd-pretag id="sdPretag14273761sm" type="geneprod" role="intervention">ApoSOD1<sup>2SH</sup></sd-pretag> and ApoG37R <sd-pretag id="sdPretag823684272sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> has a higher binding affinity to Zn than <sd-pretag id="sdPretag1424413706sm" type="geneprod" role="assayed">ApoI113T SOD1<sup>2SH</sup></sd-pretag> and ApoG85R <sd-pretag id="sdPretag1658475498sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag>). Data is represented as mean ± SD (from 3 biological replicates).</p> <p><strong>D</strong>. Disordered/ extended conformation content decreases in <sd-pretag id="sdPretag74532729sm" type="geneprod" role="assayed">ApoSOD1<sup>2SH</sup></sd-pretag> and ApoI113T <sd-pretag id="sdPretag2036479192sm" type="geneprod" role="assayed">SOD1<sup>2SH</sup></sd-pretag> with the addition of Zn calculated from FTIR spectra; data is represented as mean ± SD (from 3 biological replicates).</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=50930
|
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||
10.15252/embj.2022111185
|
A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation
|
2022
|
Figure 5
|
<sd-panel><p><strong>Figure 5. Characterization of <sd-pretag id="sdPretag2047104828sm" type="geneprod" role="intervention">SOD1<sup>2SH</sup></sd-pretag> variants and molecular interactions in the condensed phase using molecular dynamics simulations.</strong></p> <p><strong>A, B.</strong> (A) Per-residue <sd-pretag id="sdPretag1529753583sm" category="assay">RMSF</sd-pretag> profiles of <sd-pretag id="sdPretag1279756786sm" type="geneprod" role="intervention">ApoSOD1<sup>2SH</sup></sd-pretag> & its mutants computed from 5 µs simulations performed for each variant. (B) Per-residue <sd-pretag id="sdPretag1184248191sm" category="assay">RMSF</sd-pretag> profiles of <sd-pretag id="sdPretag796374782sm" type="geneprod" role="assayed">ApoSOD1<sup>2SH</sup></sd-pretag>, <sd-pretag id="sdPretag2048431571sm" type="geneprod" role="assayed">ApoSOD1<sup>S</sup></sd-pretag><sup>-S</sup>, ZnSOD1<sup>S-S</sup> and ZnSOD1<sup>2SH</sup> computed from 5 µs simulations performed for each variant. Error bars in <strong>A</strong> and <strong>B</strong> correspond to standard errors which are estimated by block averaging over 10 (500 ns each) blocks.</p> <p><strong>C</strong>. Salt-<sd-pretag id="sdPretag1443532196sm" type="tissue" role="component">bridge</sd-pretag> analysis of <sd-pretag id="sdPretag1750460591sm" type="geneprod" role="assayed">ApoG85R SOD1<sup>2SH</sup></sd-pretag> (dist. cutoff <0.5 nm = salt-bridge).</p> <p><strong>D</strong>. Snapshots from the ApoG85R SOD1<sup>2SH</sup> trajectory showing the formation of non-native salt-bridges which are likely to be detrimental for Zn-binding.</p> <p><strong>E.</strong> Density profile of <sd-pretag id="sdPretag906280624sm" type="geneprod" role="intervention">ApoSOD1<sup>2SH</sup></sd-pretag> and its loop-deletion variants computed from the CG slab simulations. The condensed phase is centered in the middle of the simulation box.</p> <p><strong>F.</strong> Slab snapshots from CG simulations of <sd-pretag id="sdPretag1354919008sm" type="geneprod" role="intervention">ApoSOD1<sup>2SH</sup></sd-pretag> and its variants at 275 K (blue beads represent the loop IV, red beads represent loop <sd-pretag id="sdPretag859954633sm" type="geneprod" role="intervention">VII</sd-pretag>, orange beads represent the other domains.)</p> <p><strong>G.</strong> Pairwise intermolecular <sd-pretag id="sdPretag1190620193sm" type="subcellular" role="component">contact</sd-pretag> formation (normalized by maximum probability) in the condensed phase as a function of residue index.</p> <p><strong>H.</strong> Saturation concentration measured in the CG phase coexistence simulations for <sd-pretag id="sdPretag729969873sm" type="geneprod" role="intervention">ApoSOD1<sup>2SH</sup></sd-pretag> and its variants including <sd-pretag id="sdPretag2068814113sm" category="disease">ALS</sd-pretag> mutants at 275 K. The error bars correspond to SD, which are estimated from block averages over 4 blocks.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=50931
|
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"text": "SOD1",
"type": "geneprod",
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]
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] |
||
10.15252/embj.2022111185
|
A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation
|
2022
|
Figure 6
|
<sd-panel><p><strong>Figure 6. Maturation of liquid droplets precedes aggregation.</strong></p> <p><strong>A</strong>. Cartoon scheme showing the maturation of protein droplet with time (region1: diffused region outside droplet, region 2: low intensity portion inside the droplet, region 3: highly intense portion inside droplet appears during maturation) at different time points.</p> <p><strong>B</strong>. Diffusion coefficient of region 1 and 2 (within droplet) was measured using point FCS as shown in the scheme.</p> <p><strong>C.</strong> Upper panel shows fluorescence images of the maturation of Alexa Fluor 488 labeled ApoSOD1<sup>2SH</sup> droplets with time, samples were incubated at 37 C up to ~25 hours. Images are taken at indicated time point: 0* hour (the time after incubation required for droplet formation), 12 hours and >24 hours; images below show magnified scale bar: 5 µm. The middle panel shows magnified view of the marked region from corresponding images on the upper panel. Red arrowhead indicates matured (solid) portion inside the droplet. Lower panel shows coexistence of liquid and solid phase inside droplet; scale bar: 10 µm. Intensity plot (bottom right) shows the distribution of high intensity region within matured droplet.</p> <p><strong>D</strong>. Diffusion coefficient obtained from point FCS measurement at different regions of the droplet during maturation course; 0* (x-axis label) indicates the time after incubation required for droplet formation. Data is shown mean ± SD (from 3 biological replicates). Statistical significance was measured using paired t-test; ns denotes non-significant; **p< 0.01; ***p< 0.001</p> <p><strong>E</strong>. ThT fluorescence assay plot showing aggregation kinetics of all SOD1<sup>2SH</sup> variants. ApoSOD1<sup>2SH</sup>, I113T SOD1<sup>2SH</sup> and G85R SOD1<sup>2SH</sup> show high ThT fluorescence.</p> <p><strong>F</strong>. AFM micrographs of ApoSOD1<sup>2SH</sup>, I113T SOD1<sup>2SH</sup> and G85R SOD1<sup>2SH</sup> show fibrillar aggregates after prolonged incubation at 37 ℃, 180 RPM in absence of Zn (left panel); AFM micrographs of ApoSOD1<sup>2SH</sup>, I113T SOD1<sup>2SH</sup> and G85R SOD1<sup>2SH</sup> incubated with Zn (at 1:5 protein: Zn molar ratio) show presence of oligomers for ApoSOD1<sup>2SH</sup>, I113T SOD1<sup>2SH</sup> and short fibrils for G85R SOD1<sup>2SH</sup> (right panel); scale bar: 200 nm.</p> <p><strong>G</strong>. Cell proliferation of SHSY5Y human neuroblastoma cells assessed by MTT assay post 12 h treatment with 5 µM ApoSOD1<sup>2SH</sup>, I113T SOD1<sup>2SH</sup> and G85R SOD1<sup>2SH</sup> condensates and aggregates (fibrils). Cell culture medium (DMEM) was used as negative control and untreated SHSY5Y cells were used as positive control. Data are shown as mean <span class="math inline">±</span> SEM (from 3 biological replicates). P values were determined by two tailed unpaired t tests; *p<0.05, **p< 0.01; ***p< 0.001, ****p< 0.0001.</p> <p><strong>H</strong>. Dot plots showing flow cytometric analysis of annexin V and propidium iodide (PI) staining of apoptotic SHSY5Y neuroblastoma cells following a 12 h treatment with 5 µM ApoSOD1<sup>2SH</sup> condensates, I113T SOD1<sup>2SH</sup> condensates, G85R SOD1<sup>2SH</sup> condensates, ApoSOD1<sup>2SH</sup> fibrils, I113T SOD1<sup>2SH</sup> fibrils and G85R SOD1<sup>2SH</sup> fibrils respectively (see Methods for details). High cytotoxicity was observed for SOD1<sup>2SH</sup> condensates.</p></sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=50932
|
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{
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{
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] |
||
27013495
|
10.15252/embr.201541392
|
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis
|
2016
|
Figure 1
|
<p><strong>Figure 1 </strong><em>The deubiquitinase Usp27x interacts with </em><em>Bim</em><em><sub>EL</sub></em></p>
<p><strong>A</strong> 293FT cells were transfected with 3xFlag-Usp27x (pFCMV7.1 vector backbone) together with a construct driving expression of untagged Bim<sub>EL</sub> or 3xHA-Bim<sub>EL</sub>(both pMIG vector backbone). Cells were lysed, and 3xHA-Bim<sub>EL</sub> was immunoprecipitated with anti-HA-antibodies. IP products were tested by Western blotting for the presence of Bim and FLAG-Usp27x probing with antibodies against Bim or against the FLAG-peptide. See also Appendix Fig. S1B. Western blots show representative of n≥3 independent experiments.</p>
<p><strong>B</strong> Usp27x binds a mutant of Bim incapable of binding to anti-apoptotic Bcl-2-proteins. 293FT cells transfected with constructs encoding 3xFLAG-Usp27x and 3xHA-tagged Bim<sub>EL</sub> (see A, 2µg each) or 3xHA-tagged Bim<sub>EL</sub>∆∆ (a mutant with two mutations in the BH3-domain, incapable of binding antiapoptotic Bcl-2 proteins [50]) were immunoprecipitated from whole cell extracts using anti-HA-resin. Bim and Usp27x were detected with anti-HA- and anti-FLAG-antibodies as indicated; antiapoptotic proteins: Mcl-1, Bcl-XL, Bcl-2. The caspase-inhibitor Q-VD-OPh (QVD) was added to the cultures described in A and B to inhibit Bim-induced apoptosis. Western blots are representative of n=3 independent experiments.</p>
<p><strong>C</strong> <em>Usp27x interacts with endogenous </em><em>Bim</em><em><sub>EL</sub></em><em> independently of its catalytic activity</em></p>
<p>293FT cells either carrying 3xFlag-Usp27x (293FT-TetR-3xFlag-usp27x) or the catalytic inactive mutant 3xFlag-Usp27xC87A under the control of the Tet-repressor were treated for 24h with dox to induce expression of Usp27x or Usp27xC87A. In all conditions PMA (to induce Bim-ubiquitination, 16.2 nM), and Q-VD-OPh (to inhibit apoptosis, 10 µM, see Fig. 3) were added at the time of Usp27x-induction. MG132 (to prevent Bim-degradation, 40 µM) was added in all conditions 4h prior to cell lysis. 3xFlag tagged Usp27x or Usp27xC87A was immunoprecipitated from whole cell lysates using anti-Flag resin. Interaction with Bim<sub>EL</sub> or β-TrCP was detected by Western blotting using anti-Bim or anti-β-TrCP antibodies. Western blots are representative of n≥3 independent experiments.</p>
<p><strong>D</strong> <em>Usp27x expression does not inhibit interaction of </em><em>Bim</em><em><sub>EL</sub></em><em> to β-</em><em>TrCP</em></p>
<p>293FT-TetR-3xFlag-Usp27x cells were transfected with pMIG-3xHA-Bim<sub>EL</sub> in the presence of both PMA and QVD. At the same time dox (to induce 3xFlag-Usp27x) was added as indicated and 20h later cells were treated with MG132 [40µM] for additional 4h.Cells were lysed and 3xHA-Bim<sub>EL</sub> was immunoprecipitated as described above. As a control HA-matrix was used without lysate to rule out any unspecific signal that might come from the immobilized anti HA-antibody (beads). Western blots show representative of n=2 independent experiments.</p>
<p><strong>E</strong><em> Binding of Usp27x to </em><em>Bim</em><em><sub>EL</sub></em><em> can be blocked by the MEK-inhibitor UO126 </em></p>
<p>293FT-TetR-3xFlag-Usp27x cells (see C) were treated for 24h with dox to induce expression of Usp27x. In all conditions PMA [16.2 nM], and Q-VD-OPh [10 µM] were added at the time of Usp27x-induction. As a control, cells were pretreated with UO126 [10µM] for 30 minutes to block the PMA stimulated ERK pathway before the addition of dox plus PMA plus QVD. 3xFlag tagged Usp27x was immuno-precipitated from whole cell lysates using anti-Flag resin. Interaction with Bim<sub>EL</sub> was detected by Western blotting using anti-Bim antibodies. As a control Flag-matrix (beads) was used alongside the IP under same conditions but without addition of protein lysates. Western blots are representative of n=4 independent experiments (see also Appendix Fig. S2B).</p>
<p><strong>F</strong> <em>Binding of Usp27x </em><em>to </em><em>Bim</em><em><sub>EL</sub> does not require </em><em>β-</em><em>TrCP</em></p>
<p>293FT-TetR-3xFlag-Usp27x cells were transfected with control siRNA (siCo3) or siRNA specific for β-TrCP. 48h later cells were stimulated with PMA plus dox plus QVD for additional 24h. After cell lysis, Flag-Usp27x was immuno-precipitated using anti-Flag-matrix, and Bim<sub>EL</sub> and β-TrCP bound to Usp27x were identified by Western blotting. Blots are representative of n=2 independent experiments.</p>
<p><strong>G</strong> <em>Usp27x binds specifically to </em><em>Bim</em><em><sub>EL</sub></em></p>
<p>293FT-TetR-3xFlag-Usp27x cells or 293FT-3xFlag-Usp27x cells with a specific deletion of the Bim<sub>EL</sub> protein were treated with dox, PMA and QVD as in C. Cells were lysed, and lysates were subjected to anti-Flag immunoprecipitation. 3xFlag-Usp27x was detected using anti-FLAG antibodies. Blots are representative of n=3 independent experiments.</p>
|
https://api.sourcedata.io/file.php?figure_id=6904
|
[
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}
] |
|
27013495
|
10.15252/embr.201541392
|
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis
|
2016
|
Figure 2
|
<p><strong>Figure </strong><strong>2</strong> <em>Expression of Usp27x stabilizes </em><em>the expression of the phosphorylated form of </em><em>Bim</em><em><sub>EL</sub> and </em><em>reduces </em><em>Bim</em><em>-ubiquitination in PMA-stimulated </em><em>293FT </em><em>cells</em></p>
<p><strong>A</strong> Usp27x inhibits the PMA-induced degradation of Bim<sub>EL</sub> in 293FT cells. 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells were treated with dox to induce 3xFlag-Usp27x or Usp27xC87A, or with PMA [16.2 nM] (to induce Bim<sub>EL</sub> degradation), or with the combination of dox, PMA and QVD [10µM] (to inhibit apoptosis) as indicated for 24 h (left) or 48 h (right). Cell lysates were analysed by Western blotting. The blots are representative of n=4 similar experiments. See also Appendix Fig. S5B. * Probably modified form of wild-type 3xFlag-Usp27x (such as dimer, ubiquitinated Usp27x or a stable complex of Usp27x with another interacting partner); this form is only seen if high expression levels of catalytically active Usp27x are reached (see Appendix Fig. S5A where Usp27x was transfected into 293FT cells) or if wt Usp27x is enriched during immunoprecipitation.** Probably degradation product of 3xFlag-Usp27x. These forms of Usp27x were not seen in every experiment.</p>
<p><strong>B</strong> 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells treated as in A were analysed using conditions of higher resolution. A band very likely corresponding to phosphorylated Bim<sub>EL</sub> is detectable. Similar results were obtained in n=2 separate experiments.</p>
<p><strong>C</strong> <em>Usp27x stabilizes expression of p-</em><em>Bim</em><em><sub>EL</sub> (Ser69) </em><em>in Bim</em><em>-degrading conditions without reducing β-</em><em>TrCP</em><em> levels</em></p>
<p>293FT-TetR-3xFlagUsp27x were treated with dox, PMA [16.2 nM], UO126 [10µM] or QVD [10µM] as indicated for 24h, and levels of phosphorylated Bim<sub>EL</sub> at serine69 were analysed by Western blotting using a phospho-Bim (Ser69) specific antibody. Scans are from the same membrane and exposure times. Similar results were obtained in n=2 separate experiments.</p>
<p><strong>D </strong><em>UO126 reduces phosphorylation-associated shift of </em><em>Bim</em><em><sub>EL</sub></em></p>
<p>293FT-TetR-3xFlagUsp27x were treated for 24h with the indicated drug combinations and Bim was detected by Western blotting. UO126 was added 30 minutes prior other stimulation. Similar results were obtained in n=3 separate experiments (see Appendix Fig. S5D).</p>
<p><strong>E</strong> <em>Usp27x-expression reduces ubiquitination of </em><em>Bim</em></p>
<p>293FT-TetR-3xFlagUsp27x cells were transfected with a vector coding for 6His-ubiquitin (left) or GFP (right). After 24h cells were treated with PMA and QVD, and 3xFlag-Usp27x was induced by dox at the same time. After additional 20h cells were treated for 4h with MG132 (40 µM) to block proteasomal degradation of ubiquitinated proteins. Cells were lysed under denaturing conditions and His-ubiquitin-labelled proteins or proteins from only GFP expressing cells were purified by Ni<sup>2+</sup>-NTA affinity chromatography. Ubiquitinated Bim (Bim-Ub<sub>n</sub>) was detected by Western blot using anti-Bim antibodies (left). Similar results were obtained in n=3 separate experiments (another experiment including a Coomassie stain is shown in Appendix Fig. S3E). *Indicates non-His-ubiquitinated Bim<sub>EL</sub> that was bound unspecifically to the Ni<sup>2+</sup>-agarose beads.</p>
|
https://api.sourcedata.io/file.php?figure_id=6905
|
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},
{
"ext_dbs": "Uniprot///Uniprot///Uniprot///Uniprot",
"ext_ids": "P62987///P62979///P0CG48///P0CG47",
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"ext_tax_names": "Homo sapiens///Homo sapiens///Homo sapiens///Homo sapiens",
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}
] |
|
27013495
|
10.15252/embr.201541392
|
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis
|
2016
|
Figure 3
|
<p><strong>Figure </strong><strong>3</strong> <em>Usp27x e</em><em>xpression sensitizes 293</em><em>F</em><em>T cells to apoptosis induction by stimulation with PMA</em></p>
<p><strong>A</strong> 293FT-TetR-3xFlag-Usp27x or 3xFlag-Usp27xC87A, a polyclonal derivative where the Bim-locus had been targeted by CRISPR/Cas9- and four single clones obtained by serial dilution from the polyclonal line were treated as indicated (PMA [16.2 nM], QVD [10 µM]). Apoptosis was measured after 24 h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=14 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xC87A, and 3xFlag-Usp27xBim2KO clone #14), n=7 (3xFlag-Usp27xBim2KO clone #11) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown.</p>
<p><strong>B </strong>The same cells as in A (except Usp27xC87A mutant) were treated as in A and stained for active Bax. Data (means/SEM) are from n=8 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xBim2KO clone #11 and #14) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown. Expression levels of Bim, β-TrCP and Flag-Usp27x of the different cell lines are shown in Appendix Fig. S5E.</p>
|
https://api.sourcedata.io/file.php?figure_id=6906
|
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] |
|
27013495
|
10.15252/embr.201541392
|
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis
|
2016
|
Figure 4
|
<p><strong>Figure </strong><strong>4</strong> <em>Usp27x </em><em>but not Usp22 </em><em>can counter </em><em>Bim</em><em> destabilization through the </em><em>Raf</em><em>-ERK pathway </em></p>
<p><strong>A</strong> Lines based on the human melanoma cell line 1205Lu (BRAF-V600E-positive) were made to carry GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 under the control of a doxycycline (dox)-inducible promoter. GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 were induced with dox for 24 or 48h. Levels of Bim were determined by Western blotting. The two left panels are from the same membrane and exposure times. Similar results were obtained in n=4 separate experiments (left) and n=3 experiments (right). See also Appendix Fig. S6A where 3xFlag-Usp22 is also shown to be unable to increase Bim levels.</p>
<p><strong>B</strong> A cell line derived from the BRAF-V600E-positive human melanoma line WM1158 was generated to carry inducible GFP-Usp27x. GFP-Usp27x was induced, and Bim was detected as in A. Similar results were obtained in n=3 separate experiments.</p>
<p><strong>C</strong> Derivatives of the NSCLC cell line HCC827 (expressing constitutively active EGFR) were generated that only express the TetR-repressor or carrying in addition either GFP-Usp27x or GFP-Usp27xC87A. Cells were treated with dox for 72h. Bim was detected by Western blotting. Similar results were obtained in n=2 separate experiments. GFP-Usp27x, GFP-Usp27xC87A, or GFP-Usp22 expression (A-C) was detected using an antibody against GFP.</p>
<p><strong>D</strong> Caco2 cells carrying a dox inducible HA-BRAF-V600E [37] (left two lanes) or the same cells stably expressing 3xFlag-Usp27x were treated with dox for 48h to induce expression of HA-BRAF-V600E. Bim was detected by Western blotting. The amount of Bim<sub>EL</sub> was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100% (normalized to the tubulin signal for each condition). Similar results were obtained in n=3 separate experiments with varying induction times. See also Appendix Fig. S6D.</p>
|
https://api.sourcedata.io/file.php?figure_id=6907
|
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|
27013495
|
10.15252/embr.201541392
|
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis
|
2016
|
Figure 5
|
<p><strong>Figure </strong><strong>5</strong> <em>Endogenous </em><em>Usp27x stabilizes </em><em>Bim</em><em> in Caco2 </em><em>and 293FT cells</em></p>
<p><strong>A </strong>Loss of endogenous Usp27x enhances destabilization of Bim<sub>EL</sub> in response to BRAF-V600E</p>
<p>Caco2 cells carrying inducible HA-BRAF-V600E were transfected with control siRNA (siCo3) or siRNA directed against Usp27x (combination of three siRNAs targeting Usp27x mRNA) for 24h prior to BRAF-V600E expression with dox for 2h (left) or 4h (right). Bim<sub>EL</sub> was detected by Western blotting. Similar results were also observed in n=2 more experiments after 3h induction (not shown, see Appendix Fig. S6E for siRNA efficacy).</p>
<p><strong>B</strong> 293FT cells deficient for Usp27x show enhanced destabilization of Bim<sub>EL</sub> in response to PMA</p>
<p>293FT wt cells or 293FT-Usp27xKO (clone 2/10, generated using CRISPR-Cas-9) were treated with PMA [16.2 nM] for 16h or 24h to induce Bim<sub>EL</sub> degradation. Western blots show protein levels of n=3 independently performed experiments (similar results were obtained in one more experiment, not shown). See Appendix Fig. S6G for verification of the Usp27xKO cell line by DNA sequence analysis.</p>
|
https://api.sourcedata.io/file.php?figure_id=6908
|
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|
27013495
|
10.15252/embr.201541392
|
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis
|
2016
|
Figure 6
|
<p><strong>Figure </strong><strong>6</strong> <em>Usp27x stabilizes </em><em>Bim</em><em> protein levels</em><em> in the ERK-degradation pathway</em><em> in </em><em>293FT cells, </em><em>1205Lu melanoma and HCC827 NSCLC </em><em>cells</em></p>
<p><strong>A</strong> 293FT-TetR-3xFlagUsp27x cells were treated with PMA [16.2 nM] plus QVD [10 µM] and 3xFlag-Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide (CHX). Cells were harvested at the indicated time points, and levels of Bim<sub>EL</sub> were determined by Western blotting. Similar results were obtained in n=3 separate experiments. Cut membranes shown were from one membrane with same exposure time. For each condition Bim<sub>EL</sub>-levels were quantified and normalized to the tubulin-signal. Percent Bim<sub>EL</sub> gives the expression relative to the starting point.</p>
<p><strong>B</strong> 1205Lu melanoma cells carrying dox-inducible GFP-Usp27x were treated with dox for 24h. Cycloheximide (CHX, 1µg/ml) was then added. Cells were harvested at the indicated time points, and levels of Bim<sub>EL</sub> were determined by Western blotting. GFP-Usp27x was detected with anti-GFP-antibodies. Similar results were obtained in n=3 separate experiments. A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox-inducible GFP-Usp27xC87A mutant. For each condition Bim<sub>EL</sub>-levels were quantified and normalized to the tubulin-signal. Percent Bim<sub>EL</sub> gives the expression relative to the starting point set to 100%. The starting point for the non-Usp27x/C87A inducing cells (-dox) is shown in lane 1 for experiment one and two (-dox, - CHX). Cut membranes shown were from one membrane with same exposure time. Same results were obtained for WM1158 melanoma cells (data not shown).</p>
<p><strong>C</strong> HCC827 cells were induced to express GFP-Usp27x with dox as indicated. 24h later cycloheximide (CHX, 1µg/ml) was added for the indicated time. Bim<sub>EL</sub> was detected after the indicated times of treatment by Western blotting. For each condition Bim<sub>EL</sub>-levels were quantified and normalized to the tubulin-signal. Percent Bim<sub>EL</sub> gives the expression relative to the starting point. Similar results were obtained in n=2 separate experiments. Cut membranes shown were from one membrane with same exposure time.</p>
|
https://api.sourcedata.io/file.php?figure_id=6909
|
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"ext_tax_names": "Homo sapiens",
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"mapping_status": "mapped",
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]
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]
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"role": "intervention",
"text": "Usp27x",
"type": "geneprod",
"uniprot_ids": [
"Q8CEG8"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "O43521",
"ext_tax_ids": "9606",
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"text": "BimEL",
"type": "geneprod",
"uniprot_ids": [
"O43521"
]
}
] |
|
27013495
|
10.15252/embr.201541392
|
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis
|
2016
|
Figure 7
|
<p><strong>Figure </strong><strong>7</strong> <em>Usp27x sensitizes 1205Lu melanoma and HCC827 NSCLC cells to apoptosis induction by inhibition of the </em><em>Raf</em><em>-ERK-pathway</em></p>
<p><strong>A</strong> 1205Lu melanoma cells carrying dox-inducible GFP, GFP-Usp27x or GFP-Usp27xC87A were treated with doxycycline (dox) as indicated. After 48 h UO126 (10µM) was added for another 48 h. Apoptosis was measured by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=3 (GFP) or from n≥6 separate experiments (GFP-Usp27x and GFP-Usp27xC87A). P-values (t-test) for statistically significant differences are shown. N.s., p>0.05. The addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). A stain for active Bax is shown in Appendix Fig. S7B.</p>
<p><strong>B</strong> HCC827 NSCLC cells carrying dox-inducible GFP-Usp27xC87A, GFP-Usp27x, or two separate lines established from these GFP-Usp27x-cells where the Bim-locus had been targeted for deletion using CRISPR/Cas9 (Bim-KO), were treated with combinations of dox and the EGFR-inhibitor gefitinib as indicated. Apoptosis was measured after 72h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=≥15 (maternal GFP-Usp27x line) or from n≥5 (for Bim1KO) or from n≥6 (for Bim2KO), or n=4 (for GFP-Usp27xC87A) separate experiments. P-values (t-test) for statistically significant differences are shown. Again, the addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). The inset shows Bim-knock-out-efficiency (n=2).</p>
|
https://api.sourcedata.io/file.php?figure_id=6910
|
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"mapping_status": "mapped",
"ncbi_gene_id": "54651",
"original_type": "gene",
"role": "intervention",
"text": "Usp27x",
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"P42574"
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"mapping_status": "mapped",
"ncbi_gene_id": "10018",
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"text": "Bim",
"type": "geneprod",
"uniprot_ids": [
"O43521",
"A0A0C4DH20",
"E9PAM9",
"H7BZE5"
]
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"O43521",
"A0A0C4DH20",
"E9PAM9",
"H7BZE5"
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"uniprot_ids": [
"Q8CEG8"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "54651",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "54651",
"original_type": "gene",
"role": "intervention",
"text": "Usp27x",
"type": "geneprod",
"uniprot_ids": [
"Q8CEG8"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "54651",
"ext_tax_ids": "10090",
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"uniprot_ids": [
"Q8CEG8"
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},
{
"ext_dbs": "Uniprot",
"ext_ids": "O43521",
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"mapping_status": "mapped",
"ncbi_gene_id": null,
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]
},
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"ext_dbs": "Uniprot",
"ext_ids": "P42574",
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"mapping_status": "mapped",
"ncbi_gene_id": null,
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"role": "assayed",
"text": "caspase-3",
"type": "geneprod",
"uniprot_ids": [
"P42574"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P42574",
"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": "Caspase-3",
"type": "geneprod",
"uniprot_ids": [
"P42574"
]
}
] |
|
27013495
|
10.15252/embr.201541392
|
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis
|
2016
|
Figure 8
|
<p><strong>Figure </strong><strong>8 </strong><em>Loss of Usp27x </em><em>reduces gefitinib-induced apoptosis in </em><em>HCC827 NSCLC</em></p>
<p>HCC827 NSCLC cells and two separate polyclonal lines established from these cells where the Usp27x-locus had been targeted for deletion using CRISPR/Cas9 with two different guide RNA against Usp27x (Usp27xKO-1 and Usp27xKO-4), were treated with gefitinib [10µM] for 48h. Apoptosis was measured by staining for active caspase-3 (A) or by staining for activated Bax (antibody: 6A7 clone) followed by flow cytometric analysis. Data shown in A, B (means/SEM) are from n=4 separate experiments. P-values (t-test) for statistically significant differences are shown.</p>
<p> </p>
|
https://api.sourcedata.io/file.php?figure_id=6911
|
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"mapping_status": "mapped",
"ncbi_gene_id": "389856",
"original_type": "gene",
"role": "intervention",
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"A6NNY8"
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{
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"original_type": "gene",
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"text": "Usp27x",
"type": "geneprod",
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"A6NNY8"
]
},
{
"ext_dbs": "NCBI gene",
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"mapping_source": "ncbi_gene",
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"ncbi_gene_id": "389856",
"original_type": "gene",
"role": "intervention",
"text": "Usp27x",
"type": "geneprod",
"uniprot_ids": [
"A6NNY8"
]
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{
"ext_dbs": "Uniprot",
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"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Bax",
"type": "geneprod",
"uniprot_ids": [
"Q07812"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P42574",
"ext_tax_ids": "9606",
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"ncbi_gene_id": null,
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"text": "caspase-3",
"type": "geneprod",
"uniprot_ids": [
"P42574"
]
}
] |
|
27485122
|
10.15252/emmm.201606520
|
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain
|
2016
|
Figure 1
|
<p><strong>Figure 1.</strong> <strong>Efficiency an</strong><strong>d</strong><strong> specificity of</strong><strong><em> in vivo</em></strong><strong> down</strong><strong>-</strong><strong> and upregulation of miR-132. </strong></p>
<p>A. Experimental scheme of antagomiR-132 and miR-132 mimic injections into the lateral ventricle of 2-month old APPPS1 mice. B. Semi-quantitative PCR of miR-132 and negative control miRNAs in the hippocampus of antagomiR-132-injected mice (ant-132) in comparison to control-injected animals (aCSF and scramble) at six months of age. Sample size, n=9 per group. C. FISH of miR-132 and negative control miR-124 in the hippocampus of ant-132- and scramble-injected mice. Scramble probes were used as FISH negative controls. Scale bar, 500μm. D. Semi-quantitative PCR of miR-132 and miR-212 in the hippocampus of 3-month old miR-132 mimic-injected mice (miR-132) compared to animals injected with a negative control oligonucleotide (Ctr). Sample size, n=6 per group. Values were normalized to scramble- (B) or control-injected group (D) and presented as mean ± SEM. In B, one-way ANOVA was used, while in D, Student’s t-test was applied.</p>
|
https://api.sourcedata.io/file.php?figure_id=9398
|
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},
{
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"text": "miR-132",
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"mapping_source": "unmapped",
"mapping_status": "unmapped",
"ncbi_gene_id": "387150",
"original_type": "gene",
"role": "intervention",
"text": "miR-132",
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},
{
"ext_dbs": "NCBI gene",
"ext_ids": "387208",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "unmapped",
"mapping_status": "unmapped",
"ncbi_gene_id": "387208",
"original_type": "gene",
"role": "assayed",
"text": "miR-212",
"type": "geneprod",
"uniprot_ids": []
}
] |
|
27485122
|
10.15252/emmm.201606520
|
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain
|
2016
|
Figure 2
|
<p><strong>Figure 2. Effect of miR-132 on Aβ accumulation. </strong></p>
<p>A. Amyloid staining (6E10) combined with miR-132 FISH in ant-132- and scramble-injected mice at six months of age. Scale bar, 500 μm. B. Quantification of amyloid burden in hippocampus and cortex of ant-132-injected and control animals (see Materials and Methods). Sample size, n=4 per group. C, D. ELISA of soluble (TBS) and insoluble (Formic acid) Aβ<sub>40</sub> and Aβ<sub>42 </sub>levels in the hippocampus of ant-132- (C) and miR-132-injected (D) animals at six and three months of age, respectively. Sample size, n=6 per group. In B, C and D, values were normalized to scramble- (B, C) or control-injected group (D) and presented as mean ± SEM. Student’s t-test was used.</p>
|
https://api.sourcedata.io/file.php?figure_id=9399
|
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}
] |
|
27485122
|
10.15252/emmm.201606520
|
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain
|
2016
|
Figure 3
|
<p><strong>Figure 3. Regulatory effect of miR-132 on </strong><strong>TAU</strong><strong> expression and phosphorylation. </strong></p>
<p>Western blot analysis of total TAU and pTAU (AT8, AT270) levels upon miR-132 knockdown (A) or overexpression (B) at six and three months of age, respectively. Sample size, n=9 per group (A) and n=6 per group (B). Values were normalized to the respective control groups and presented as mean ± SEM. Student’s t-test was used.</p>
|
https://api.sourcedata.io/file.php?figure_id=9400
|
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] |
|
27485122
|
10.15252/emmm.201606520
|
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain
|
2016
|
Figure 5
|
<p><strong>Figure 5. miR-132 regulation over ITPKB. </strong></p>
<p>A. Luciferase reporter assay of wild-type (wt) and mutant (mut) ITPKB 3’UTR in HEK293 cells co-transfected with a synthetic miR-132 (miR-132) or a negative control (Neg Ctr) oligonucleotide. B. ELISA of Aβ<sub>40</sub> and Aβ<sub>42 </sub>levels in HEK293-APP<sup>swe</sup> cells transfected with a miR-132 antisense oligonucleotide (miR-132 inh), an siRNA against ITPKB (ITPKB siRNA) or both. The assays in A and B were performed in three independent experiments, each in triplicates. C. Western blot analysis of ITPKB knock down in APPPS1 hippocampus using an siRNA oligonucleotide against ITPKB. Sample size, n=6 per group. Values were normalized to control-injected group (Ctr) and presented as mean ± SEM. D. ELISA of insoluble (Formic acid-soluble) Aβ<sub>40</sub> and Aβ<sub>42 </sub>levels in the hippocampus of ITPKB siRNA- and control-injected animals at three months of age. Sample size, n=6 per group. Values were normalized to control group and presented as mean ± SEM. E, F. Western blot analysis of ITPKB levels upon miR-132 down- (E) or up-regulation (F) at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to control groups and presented as mean ± SEM. In A, C-F Student’s t-test was used, while in B one-way ANOVA was employed.</p>
|
https://api.sourcedata.io/file.php?figure_id=9402
|
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] |
|
27485122
|
10.15252/emmm.201606520
|
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain
|
2016
|
Figure 6
|
<p><strong>Figure 6</strong><strong>. Effect of miR-132 regulation on BACE1 and ERK1/2 activity. </strong></p>
<p>Western blot analysis of CTFβ (A), sAPPβ-swe (B) and phosphorylated ERK1/2 (pERK1/2) (C) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1 hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student’s t-test was used.</p>
|
https://api.sourcedata.io/file.php?figure_id=9403
|
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"text": "APPβ",
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] |
|
27485122
|
10.15252/emmm.201606520
|
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain
|
2016
|
Figure 7
|
<p><strong>Figure </strong><strong>7</strong><strong>. miR-132/ITPKB expression profile in human AD prefrontal cortex. </strong></p>
<p>A. Double immunofluorescence staining of amyloid plaques (6E10) and ITPKB in AD prefrontal cortex. Scale bar, 50 μm. Arrowheads indicate ITPKB immunopositivity. B. miR-132 FISH coupled with double immunofluorescence against hyperphosphorylated TAU (AT8)-containing neurofibrillary tangles (NFTs) and ITPKB in AD prefrontal cortex. miR-124 was used as a control. Scale bar, 50 μm. C. Quantification of miR-132, ITPKB and hyperphosphorylated TAU (pTAU) signal in single neurons. Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed (see Materials and Methods). Sample size (AD patients), n=3. Values are presented as mean ± SEM. Two-way Anova was used. Quantifications are summarized in the table provided. Arrow directions refer to the comparison of each normalized signal to the same signal in the other group (in the “low miR-132” group comparisons are made to the “high miR-132” group and <em>vice versa</em>).</p>
|
https://api.sourcedata.io/file.php?figure_id=9404
|
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"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "TAU",
"type": "geneprod",
"uniprot_ids": [
"P10636"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P10636",
"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": "TAU",
"type": "geneprod",
"uniprot_ids": [
"P10636"
]
}
] |
|
27485122
|
10.15252/emmm.201606520
|
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain
|
2016
|
Figure 8
|
<p><strong>Figure 8. m</strong><strong>iR-132/ITP</strong><strong>KB pathway assessment in human AD hippocampus. </strong></p>
<p>Semi-quantitative PCR of miR-132 and western blot analysis of ITPKB, phosphorylated (pERK1/2) and total ERK1/2, phosphorylated (AT8 , AT270) and total TAU, phosphorylated sphingosine kinase 1 (pSphK1), BACE1, full length APP (flAPP), APP CTFs and sAPPβ in human AD hippocampi (AD) compared to non demented control samples (ND). Sample size, n=39 for AD and n=15 for ND. Values were normalized to ND and presented as mean ± SEM. The Student’s t-test was used.</p>
|
https://api.sourcedata.io/file.php?figure_id=9405
|
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"mapping_source": "unmapped",
"mapping_status": "unmapped",
"ncbi_gene_id": "406921",
"original_type": "gene",
"role": "assayed",
"text": "miR-132",
"type": "geneprod",
"uniprot_ids": []
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P05067",
"ext_tax_ids": "9606",
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"role": "assayed",
"text": "APP",
"type": "geneprod",
"uniprot_ids": [
"P05067"
]
},
{
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"ext_ids": "P05067",
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"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "APPβ",
"type": "geneprod",
"uniprot_ids": [
"P05067"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P56817",
"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": "BACE1",
"type": "geneprod",
"uniprot_ids": [
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]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P27987",
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"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "ITPKB",
"type": "geneprod",
"uniprot_ids": [
"P27987"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P27361",
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"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "ERK1",
"type": "geneprod",
"uniprot_ids": [
"P27361"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P10636",
"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": "TAU",
"type": "geneprod",
"uniprot_ids": [
"P10636"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q9NYA1",
"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": "SphK1",
"type": "geneprod",
"uniprot_ids": [
"Q9NYA1"
]
}
] |
|
10.15252/embj.2021107776
|
Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis
|
2021
|
Figure 2
|
<p><strong>Figure 2. Validation of selected host-virus protein-protein interactions.</strong></p><p>A Pulldown and Western blot analysis validated the interaction between the viral protein ORF3a and its interactors, VPS11 and CLCC1, and the binding of S protein to SPCS2. HEK293T cells with SFB-tagged bait expression were collected and lysed. Cell lysates were subjected to pulldown assay using S-protein beads. Western blot analysis was conducted with the indicated antibodies. Cells transfected with vector or construct encoding control genes were included as controls in these experiments.</p><p>B Immunostaining analysis of protein localization. U2OS cells were transfected with construct encoding ORF3a. Cells were fixed and stained with the indicated antibodies. The green signal is CLCC1 or lysosome marker LAMP1, the red signal is flag (for SFB-ORF3a), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.</p><p>C Overlap of HCIPs identified from SFB-tandem affinity purification and second-generation biotin ligase (BioID2) labeling experiments.</p><p>D Overlap of HCIPs reported previously and those identified in the current study.</p>
|
https://api.sourcedata.io/file.php?figure_id=45042
|
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"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "ORF3a",
"type": "geneprod",
"uniprot_ids": [
"P0DTC3"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q96S66",
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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": "CLCC1",
"type": "geneprod",
"uniprot_ids": [
"Q96S66"
]
},
{
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"ext_ids": "P0DTC2",
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"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "S protein",
"type": "geneprod",
"uniprot_ids": [
"P0DTC2"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P0DTC2",
"ext_tax_ids": "2697049",
"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "S-protein",
"type": "geneprod",
"uniprot_ids": [
"P0DTC2"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q15005",
"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": "SPCS2",
"type": "geneprod",
"uniprot_ids": [
"Q15005"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q9H270",
"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": "VPS11",
"type": "geneprod",
"uniprot_ids": [
"Q9H270"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "43740569",
"ext_tax_ids": "2697049",
"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "43740569",
"original_type": "gene",
"role": "intervention",
"text": "ORF3a",
"type": "geneprod",
"uniprot_ids": [
"A0A679G5L0",
"A0A6V7ALA6",
"A0A6V7ALH5"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P0DTC3",
"ext_tax_ids": "2697049",
"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "ORF3a",
"type": "geneprod",
"uniprot_ids": [
"P0DTC3"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q96S66",
"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": "CLCC1",
"type": "geneprod",
"uniprot_ids": [
"Q96S66"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P11279",
"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": "LAMP1",
"type": "geneprod",
"uniprot_ids": [
"P11279"
]
}
] |
||
10.15252/embj.2021107776
|
Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis
|
2021
|
Figure 5
|
<p><strong>Figure 5. Comparison of interactomes of seven NSP1 proteins from seven human coronaviruses.</strong></p><p>A Comparisons of NSP1 proteins from seven human coronaviruses.</p><p>B Principal Component Analysis of the seven HCIP lists with bait NSP1 from different human coronaviruses.</p><p>C Heat map of the NSP1 HCIPs obtained using SFB- TAP. NSP1 HCIPs were compared among seven human coronaviruses using the preys' spectral counts. Six areas of the heat map are manually selected, enlarged and labeled as (1), (2), (3), (4), (5), and (6). These groups of proteins are enriched by NSP1 protein specific from one or limited human coronaviruses. Functional characterization of each protein is shown with the red circles below the enlarged images.</p><p>D Pulldown and Western blot validation of the interaction between human coronavirus NSP1 proteins and the human proteins PYCR1/PYCR2. Cells transfected with vector or construct encoding GFP were included as controls in these experiments.</p>
|
https://api.sourcedata.io/file.php?figure_id=45048
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P32322",
"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": "PYCR1",
"type": "geneprod",
"uniprot_ids": [
"P32322"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q96C36",
"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": "PYCR2",
"type": "geneprod",
"uniprot_ids": [
"Q96C36"
]
}
] |
||
10.15252/embj.2021107776
|
Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis
|
2021
|
Figure 6
|
<p><strong>Figure 6. Comparison of interactomes of N proteins from different human coronaviruses.</strong></p><p>A Comparison of N proteins from seven human coronaviruses.</p><p>B Principal Component analysis of the seven high-confidence interacting protein (HCIP) lists with bait N protein from different human coronaviruses.</p><p>C Heat map of the N protein HCIPs identified by SFB-tandem affinity purification. N protein HCIPs were compared among seven human coronaviruses using the preys' spectral counts. Three areas of the heat map are manually selected, enlarged and labeled as (1), (2), and (3), which are enriched by all N proteins of the seven human coronaviruses. Functional characterization for each protein is shown with the red circles below the enlarged images.</p><p>D The human proteins which binding to N proteins of human coronaviruses were integrated and analyzed by STRING to illustrate the protein-protein interaction among the HCIPs. The minimum required interaction score setting in STRING is 0.9. Different function group or complex were colored differently. The filled circles are proteins, and the lines are the interactions.</p>
|
https://api.sourcedata.io/file.php?figure_id=45050
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P0DTC9",
"ext_tax_ids": "2697049",
"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "N protein",
"type": "geneprod",
"uniprot_ids": [
"P0DTC9"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P15130",
"ext_tax_ids": "11137",
"ext_tax_names": "Human coronavirus 229E",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "N protein",
"type": "geneprod",
"uniprot_ids": [
"P15130"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6Q1R8",
"ext_tax_ids": "277944",
"ext_tax_names": "Human coronavirus NL63",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "N proteins",
"type": "geneprod",
"uniprot_ids": [
"Q6Q1R8"
]
}
] |
||
10.15252/embj.2021107776
|
Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis
|
2021
|
Figure 7
|
<p><strong>Figure 7. Analysis of HCIPs showing specific interaction with one or more N proteins of human coronaviruses.</strong></p><p>A Preys identified showing significant enrichment by N proteins specifically from one or more human coronaviruses. PSM, peptide-spectrum match.</p><p>B Pulldown and Western blot validation of the interaction between N protein and G3BP1/G3BP2. Cells transfected with vector or construct encoding GFP or N protein from different human coronaviruses were compared in these experiments.</p><p>C,D Effect of SARS-CoV-2 N protein on stress granule formation. A549 (C) and MCF10A (D) cells were transfected with pinducer20-N protein of SARS-CoV-2. All the cells are treated with sodium arsenite but with or without Dox to induce N protein expression. Cells were fixed and stained with the indicated antibodies. The green signal is G3BP1, the red signal is HA (for N protein), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.</p><p>E Quantification of the stress granule formation in A549 and MCF10A cells. Total of 30 cells were counted and a student's t test was used for the statistical analysis (***, p<0.001). Box limits represent 25<sup>th</sup> percentile and 75<sup>th</sup> percentile; horizontal line represents median. Whiskers display min. to max. values.</p><p><strong>Expanded View Figure Legends</strong></p>
|
https://api.sourcedata.io/file.php?figure_id=45051
|
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"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
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"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "N proteins",
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]
},
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]
},
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},
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"ext_ids": "Q9UN86",
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"text": "G3BP2",
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},
{
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"ext_ids": "P0DTC9",
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"role": "assayed",
"text": "N protein",
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"uniprot_ids": [
"P0DTC9"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "43740575",
"ext_tax_ids": "2697049",
"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
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"role": "intervention",
"text": "N protein",
"type": "geneprod",
"uniprot_ids": [
"P0DTC9",
"A0A6B9VLF5",
"A0A6C0T6Z7",
"A0A6V7AMQ7",
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]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "43740575",
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"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "43740575",
"original_type": "gene",
"role": "intervention",
"text": "N protein",
"type": "geneprod",
"uniprot_ids": [
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"A0A6B9VLF5",
"A0A6C0T6Z7",
"A0A6V7AMQ7",
"A0A6V7AN48"
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},
{
"ext_dbs": "NCBI gene",
"ext_ids": "43740575",
"ext_tax_ids": "2697049",
"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "43740575",
"original_type": "gene",
"role": "intervention",
"text": "N protein",
"type": "geneprod",
"uniprot_ids": [
"P0DTC9",
"A0A6B9VLF5",
"A0A6C0T6Z7",
"A0A6V7AMQ7",
"A0A6V7AN48"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q13283",
"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": "G3BP1",
"type": "geneprod",
"uniprot_ids": [
"Q13283"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P0DTC9",
"ext_tax_ids": "2697049",
"ext_tax_names": "Severe acute respiratory syndrome coronavirus 2",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "N protein",
"type": "geneprod",
"uniprot_ids": [
"P0DTC9"
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}
] |
||
10.15252/embr.202050922
|
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness
|
2021
|
Figure 1
|
<sd-panel> <p><strong>Figure 1. LSD1 is secreted from gastric cancer cells through small extracellular vesicles (sEVs).</strong></p> <p>(A) Sphere formation in MGC-803 cells incubated with fresh medium or conditioned medium from MGC-803 cells for 7 days. The number of spheres was quantified and indicated on the right. Scale bar = 100 µm (n = 3 biological replicates; mean ± standard error of mean (SEM); *<em>P</em> = 0.0146; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(B) Sphere formation in MGC-803 cells with indicated treatment. The number of spheres was quantified and indicated on the right. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; no significant differences; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(C) Sphere formation in MGC-803 cells incubated with an equal volume of phosphate-buffered saline, supernatant after differential centrifugation, or sEVs. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; ns = no significant difference; *<em>P</em> = 0.0390; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(D) Sphere formation in MGC-803 cells treated with sEVs (20 μg/mL) from five gastric cancer cell lines as indicated (n = 3 biological replicates; mean ± SEM; ***<em>P</em> = 0.0007 <em>(MGC-803)</em>, **<em>P</em> = 0.0061 <em>(HGC-27)</em>, and **<em>P</em> = 0.0018 <em>(BGC-823)</em>; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(E) Expression levels of LSD1 in MGC-803, MKN-45, HGC-27, BGC-823, and NCI-N87 cell lines and their corresponding sEVs. The samples with equal amounts of proteins were loaded (n = 3 biological replicates; mean ± SEM; compared with the NCI-N87; *<em>P</em> = 0.0432 <em>(cell/MGC-803)</em>, *<em>P</em> = 0.0300 <em>(cell/HGC-27)</em>, **<em>P</em> = 0.0306 <em>(cell/BGC-823)</em>, **<em>P</em> = 0.0018 <em>(sEVs/MGC-803)</em>, **<em>P</em> = 0.0029 <em>(sEVs/HGC-27)</em>, and ***<em>P</em> = 0.0003 <em>(sEVs/BGC-823)</em>; two-tailed unpaired Student's <em>t</em>-test; GAPDH was used as a loading control for cell lysis; CD9 was used as a loading control for sEV lysis).</p> <p>(F) Establishment of <em>LSD1</em> knockout (KO) MGC-803 cell line. Con indicates MGC-803 cells, while KO indicates <em>LSD1</em> KO MGC-803 cells.</p> <p>(G and H) Transmission electron microscopy images (G) and the size distribution (H) of sEVs from MGC-803 and <em>LSD1</em> KO MGC-803 cells. Scale bar = 100 nm.</p> <p>(I) Expression levels of LSD1, CD63, CD9, TSG101, and calnexin in sEVs from MGC-803 and <em>LSD1</em> KO MGC-803 cells. sEVs were extracted using two different extraction methods (ultracentrifugation (UC) and commercial kit). Calnexin is an sEV negative marker. UC, sEVs isolated using the ultracentrifugation method; Kit, sEVs isolated using the commercial kit.</p> <p>(J) Expression levels of LSD1, CD63, and CD9 in sEVs with indicated treatment.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41286
|
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"ext_tax_names": "Homo sapiens",
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"mapping_status": "mapped",
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"original_type": "protein",
"role": "assayed",
"text": "LSD1",
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"O60341"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P08962",
"ext_tax_ids": "9606",
"ext_tax_names": "Homo sapiens",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
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"text": "CD63",
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"P08962"
]
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{
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"ext_tax_names": "Homo sapiens",
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"O60341"
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] |
||
10.15252/embr.202050922
|
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness
|
2021
|
Figure 2
|
<sd-panel> <p><strong>Figure 2. LSD1-containing small extracellular vesicles (sEVs) fuse to the recipient cell and deliver LSD1.</strong></p> <p>(A) Confocal microscopy image analysis of sEV fusion to MGC-803 cells. The MGC-803 (left side) and MKN-45 (right side) cells were treated with sEVs derived from MGC-803 cells (Con sEVs) or <em>LSD1</em> knockout (KO) MGC-803 cells (KO sEVs) and stained with PKH26 for 12 h. Additionally, the cell membrane was stained with Dio, while the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 100 µm.</p> <p>(B) Immunofluorescence confocal microscopy analysis of LSD1 (green) in <em>LSD1</em> KO MGC-803 cells (left panel) and <em>LSD1</em> KO MKN-45 cells (right panel) incubated with 20 μg/mL Con sEVs and KO sEVs for 12 h. The cell membrane was stained with PKH26, while the nuclei were stained with DAPI. Scale bar = 100 µm.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41289
|
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"role": "intervention",
"text": "LSD1",
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"O60341",
"A0A8I5KSH0",
"A0A8I5KXU4",
"R4GMQ1"
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"text": "LSD1",
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"O60341",
"A0A8I5KSH0",
"A0A8I5KXU4",
"R4GMQ1"
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{
"ext_dbs": "NCBI gene",
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"role": "intervention",
"text": "LSD1",
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"O60341",
"A0A8I5KSH0",
"A0A8I5KXU4",
"R4GMQ1"
]
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{
"ext_dbs": "Uniprot",
"ext_ids": "O60341",
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"ext_tax_names": "Homo sapiens",
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}
] |
||
10.15252/embr.202050922
|
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness
|
2021
|
Figure 3
|
<sd-panel> <p><strong>Figure 3. LSD1 facilitates stemness and promotes the accumulation of SOX2.</strong></p> <p>(A) Expression level of LSD1 (KDM1A) in gastric cancer and non-cancerous tissues from UALCAN datasets (STAD, stomach adenocarcinoma; central band, boxes, and whiskers of the boxplot represent the median, first quartile, third quartile, minimum, and maximum values, respectively).</p> <p>(B) Expression level of LSD1 (KDM1A) in gastric cancer or non-cancerous tissues from Gene Expression Profiling Interactive Analysis (GEPIA) datasets (central band, boxes, and whiskers of the boxplot represent the median, first quartile, third quartile, minimum, and maximum values, respectively).</p> <p>(C) Overall survival analysis using GEPIA datasets (log-rank test; the solid line represents the survival curve, while the dashed line represents the 95% confidence interval).</p> <p>(D) <em>In vitro</em> limiting dilution assay with MGC-803 and <em>LSD1</em> knockout (KO) MGC-803 cells (the solid line represents the sphere formation ability curve, while the dashed line represents the 95% confidence interval).</p> <p>(E) Sphere formation assay results of MGC-803 and <em>LSD1</em> KO MGC-803 cells. Scale bar = 100 µm (n = 3 biological replicates; mean ± standard error mean (SEM); **<em>P</em> = 0.0058; two-tailed unpaired Student's <em>t-</em>test).</p> <p>(F) Expression levels of LSD1, Nanog, OCT4, SOX2, and CD44 in <em>LSD1</em> KO MGC-803 cells.</p> <p>(G) Correlation between <em>LSD1</em> and <em>SOX2</em> mRNA levels analyzed using the GEPIA dataset (Pearson's test).</p> <p>(H) Correlation between LSD1 and SOX2 in 172 gastric cancer tissues (Pearson's test).</p> <p>(I) The mRNA levels of <em>SOX2</em> in different cells were detected using quantitative real-time polymerase chain reaction (n = 3 biological replicates; mean ± SEM).</p> <p>(J) Stability of SOX2 in MGC-803 and <em>LSD1</em> KO MGC-803 cells treated with cycloheximide (20 μM) at the indicated times.</p> <p>(K) Relative intensity of SOX2 in (J) (n = 3 biological replicates, mean ± SEM).</p> <p>(L) Immunoprecipitation of Kme1/2 and ubiquitin (Ub) with SOX2 in the presence or absence of LSD1. The cells were treated with MG132 (10 μM) for 8 h before analysis.</p> <p>(M) Reverse immunoprecipitation of Kme1/2 on SOX2.</p> <p>(N) Immunoprecipitation of Kme1/2 on SOX2 (WT indicates HEK293T cells co-transfected with LSD1-WT and SOX2-WT; Mut indicates HEK293T cells co-transfected with LSD1-WT and SOX2-Mut; WT, wild type; Mut, mutant).</p> <p>(O) Stability of SOX2 in HEK293T cells co-transfected with different plasmids (WT, wild type; SOX2-Mut, K42R and K117R mutations; LSD1-Mut: K661A mutation).</p> <p>(P) Relative intensity of SOX2 in (O) (n = 3 biological replicates; mean ± SEM; **<em>P</em> = 0.0055; two-tailed unpaired Student's <em>t-</em>test).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41290
|
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{
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"text": "LSD1",
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]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q9H9S0",
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"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Nanog",
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},
{
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]
},
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},
{
"ext_dbs": "NCBI gene",
"ext_ids": "6657",
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"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "6657",
"original_type": "gene",
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"A0A0U3FYV6"
]
},
{
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"ncbi_gene_id": "6657",
"original_type": "gene",
"role": "intervention",
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},
{
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] |
||
10.15252/embr.202050922
|
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness
|
2021
|
Figure 4
|
<sd-panel> <p><strong>Figure 4. LSD1-containing small extracellular vesicles (sEVs) promote cancer cell stemness <em>in vitro.</em></strong></p> <p>(A) Sphere formation assay of MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or <em>LSD1</em> knockout (KO) MGC-803 cells for 7 days (n = 3 biological replicates; mean ± standard error of mean (SEM); **<em>P</em> = 0.0090 (MGC-803) and **<em>P</em> = 0.0012 (MKN-45); two-tailed unpaired Student's <em>t-</em>test; scale bar = 100 µm).</p> <p>(B) <em>In vitro</em> limiting dilution assays performed using MGC-803 (upper panel) and MKN-45 (bottom panel) cells incubated with 20 μg/mL sEVs from MGC-803 or <em>LSD1</em> KO MGC-803 cells for 14 days (the solid line represents the sphere formation ability curve, while the dashed line represents the 95% confidence interval. The circles and triangles represent data from different groups).</p> <p>(C) Expression levels of LSD1, OCT4, and SOX2 in MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or <em>LSD1</em> KO MGC-803 cells for 48 h.</p> <p>(D) Quantification of the results of (C) (n = 3 biological replicates; mean ± SEM; *<em>P</em> = 0.0385 (LSD1), *<em>P</em> = 0.0160 (OCT4), and ***<em>P</em> = 0.0006 (SOX2) for MGC-803; *<em>P</em> = 0.0299 (LSD1), *<em>P</em> = 0.0147 (OCT4), and *<em>P</em> = 0.0258 (SOX2) for MKN-45; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(E) Expression level of CD44 in MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or <em>LSD1</em> KO MGC-803 cells for 48 h.</p> <p>(F) Sphere formation assay results of MGC-803 (upper panel) and MKN-45 (bottom panel) cells incubated with 20 μg/mL sEVs from HEK293T cells (left panel), WT-LSD1 sEVs (middle panel), and LSD1 K661A sEVs (right panel) for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **<em>P</em> = 0.0021 (MGC-803) and **<em>P</em> = 0.0020 (MKN-45); two-tailed unpaired Student's <em>t</em>-test).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41291
|
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"ncbi_gene_id": "23028",
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"A0A8I5KXU4",
"R4GMQ1"
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{
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{
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{
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]
},
{
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"mapping_status": "mapped",
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"original_type": "protein",
"role": "assayed",
"text": "OCT4",
"type": "geneprod",
"uniprot_ids": [
"Q01860"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q01860",
"ext_tax_ids": "9606",
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"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
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"text": "OCT4",
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]
},
{
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{
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},
{
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] |
||
10.15252/embr.202050922
|
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness
|
2021
|
Figure 5
|
<sd-panel> <p><strong>Figure 5. LSD1-containing small extracellular vesicles (sEVs) promote gastric cancer cell stemness <em>in vivo</em> and <em>clinic</em>al samples.</strong></p> <p>(A and B) <em>In vivo</em> limiting dilution assay results of MGC-803 cells treated with phosphate-buffered saline, BBI608, sEVs from control cells (Con sEVs), and sEVs from <em>LSD1</em> knockout (KO) cells (KO sEVs) as indicated for 28 days in BALB/c-nu mice. Representative images of tumors excised from the mice (A) and the frequency of tumor formation (B) are shown (the solid line represents the sphere formation ability curve, while the dashed line represents the 95% confidence interval. The circles and triangles represent data from different groups).</p> <p>(C) Tumor volume of each group subjected to <em>in vivo</em> limiting dilution assay with 5×10<sup>6</sup> cells with indicated treatment (n = 6 biological replicates; mean ± standard error of mean(SEM), *<em>P</em> = 0.0441 and ***<em>P</em> = 0.0002; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(D) Tumor weight of each group subjected to <em>in vivo</em> limiting dilution assay (n = 6 biological replicates; mean ± SEM; ***<em>P</em> < 0.0001, *<em>P</em> = 0.0285, *<em>P</em> = 0.0162, *<em>P</em> = 0.0498, and *<em>P</em> = 0.0049; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(E) Expression levels of LSD1, CD44, SOX2, and OCT4 in tumor tissues subjected to <em>in vivo</em> limiting dilution assay performed with 5×10<sup>6</sup> cells.</p> <p>(F) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to <em>in vivo</em> limiting dilution assay. Scale bar = 50 µm (n = 3 biological replicates; mean ± SEM; **<em>P</em> = 0.0024, **<em>P</em> = 0.0088, and *<em>P</em> = 0.0342; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(G) Representative images of second-generation tumors (left) and the tumor formation rate (right) in each group.</p> <p>(H) Second-generation tumor weight in each group (n = 6 biological replicates; mean ± SEM; *<em>P</em> = 0.0091; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(I and J) Expression levels of LSD1 in sEVs isolated from the plasma. CD9 and CD63 were used as markers of sEVs. CD9 was used as a loading control for sEV lysis (n = 3 biological replicates; mean ± SEM; *<em>P</em> = 0.0256 <em>(patient 1)</em>, **<em>P</em> = 0.0068 <em>(patient 2)</em>, **<em>P</em> = 0.0013 <em>(patient 3)</em>, **<em>P</em> = 0.0031 <em>(patient 4)</em>, ***<em>P</em> = 0.0009 <em>(patient 5)</em>, **<em>P</em> = 0.0012 <em>(patient 6)</em>, ***<em>P</em> = 0.0007 <em>(patient 7)</em>, ***<em>P</em> = 0.0010 <em>(patient 8)</em>, ***<em>P</em> = 0.0001 <em>(patient 9)</em>, and *<em>P</em> = 0.0461 <em>(patient 10)</em>; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(K) Sphere formation assay results of MGC-803 cells treated with sEVs as indicated for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **<em>P</em> = 0.0019 and **<em>P</em> = 0.0050; two-tailed unpaired Student's <em>t</em>-test).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41292
|
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]
},
{
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"text": "CD63",
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"text": "LSD1",
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] |
||
10.15252/embr.202050922
|
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness
|
2021
|
Figure 6
|
<sd-panel> <p><strong>Figure 6. LSD1-containing small extracellular vesicles (sEVs) induce oxaliplatin resistance in gastric cancer cells.</strong></p> <p>(A-D) Proliferation assay results of MGC-803 (A), MKN-45 (B), NCI-N87 (C), and <em>LSD1</em> knockout (KO) MGC-803 (D) cells treated with oxaliplatin along with indicated treatments (n = 3 biological replicates, mean ± standard error of mean (SEM); *<em>P</em> = 0.0204 (MGC-803), *<em>P</em> = 0.0498 (MKN-45), *<em>P</em> = 0.0473 (NCI-N87), and *<em>P</em> = 0.0019 (<em>LSD1</em> KO MGC-803); two-tailed unpaired Student's <em>t</em>-test).</p> <p>Representative images of tumors (E) and the tumor weight (F) of mice treated with oxaliplatin in the presence or absence of sEVs (n = 3 biological replicates; mean ± SEM; *<em>P</em> = 0.0488; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(G) Tumor growth rate in mice treated with oxaliplatin in the presence or absence of sEVs. Tumor growth rate was measured according to tumor volume (n = 3 biological replicates; mean ± SEM; *<em>P</em> = 0.0439; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(H) Proliferation assay results of MGC-803 cells treated with oxaliplatin in the presence or absence of sEVs from the plasma of patients with gastric cancer or healthy individuals (n = 3 biological replicates; mean ± SEM; *<em>P</em> = 0.0487; two-tailed unpaired Student's <em>t</em>-test).</p> <p>(I) Schematic model for the shuttling of LSD1 from parent cells to recipient cells using sEVs as vehicles. The sEV-delivered LSD1 promotes recipient gastric cancer stemness and chemoresistance.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=41293
|
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"text": "LSD1",
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] |
||
10.15252/embr.202051723
|
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth
|
2021
|
Figure 2
|
<p><strong>Figure 2. DVL3 genomic binding in MDA-MB-468 and MCF7 cells</strong></p><p><strong>(A) An assembly of IgG (first row) and DVL3 (second row) ChIP-Seq data in MDA-MB-468 for the <em>ARAP1, RFX5-GBAT2, WDR74, POU2F3, SNORD3D</em> and <em>HLA-A</em> genes, visualized by IGV.</strong></p><p><strong>(B) An assembly of IgG (first row) and DVL3 (second row) ChIP-Seq data in MCF7 for the <em>ARAP1, RFX5-GBAT2, ZNF672, SSC5D, HLA-A</em> and <em>HLA-E</em> genes, visualized by IGV.</strong></p><p><strong>(C) ChIP-qPCRs at <em>ARAP1, RFX5-GBAT2, WDR74</em> and <em>HAUS5</em> promoters for IgG and DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).</strong></p><p><strong>(D) ChIP-qPCRs at <em>RFX5-GBAT2, COX14, HLA-E</em> and <em>PSMB8</em> promoters for IgG and DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3).</strong></p><p><strong>(E) RT-qPCR-based analysis of expression changes of <em>RFX5, GBAT2, WDR74, HCG15, POU2F3, SNORD3B, APOC1P1, HLA-A, TAP1, PSMD8, PDG1</em> and <em>JUNB</em> genes in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells.</strong></p><p><strong>(F) RT-qPCR-based analysis of expression changes of <em>RFX5, GBAT2, PCAT6, ZNF672, SSC5D, APOC1P1, HLA-A, HLA-F</em> and <em>TAP1</em> genes in MCF7 (NTC, shA DVL3, and shB DVL3) cells.</strong></p><p><strong>Data information: For (A) and (B) each column is 4000 bp wide. The third rows show the gene nearest to the ChIP-Seq alignment including its location, and orientation. The medium thick dark lines are the UTRs of the gene and the thicker dark regions are exons followed by thin lines with arrows which are the introns. C and D panels show a representative ChIP-qPCR of three independent ChIP-qPCR experiments. For C and D panels an independent t test was used for statistical comparison. * p < 0.05; n=3, technical replicates;</strong> data show means ± SD<strong>. For E and F panels transcript levels were normalized to actin transcript levels, an independent t test was used for statistical comparison. * p < 0.05;</strong> data show means ± SD, <strong>n=3, biological replicates.</strong></p>
|
https://api.sourcedata.io/file.php?figure_id=39842
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10.15252/embr.202051723
|
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth
|
2021
|
Figure 4
|
<p><strong>Figure 4. Localization of H3K4me3 mark respect to DVL3 genomic binding in MDA-MB-468 and MCF7 cells</strong></p><p><strong>(A) An assembly of IgG (first row), DVL3 (second row) and H3K4me3 peak (third row) ChIP-Seq data in MDA-MB-468 for the</strong> <em><strong>ARAP1,</strong></em> <strong><em>RFX5-GBAT2, APOC1P1, PSMB8, TAP1</em> and <em>HLA-A</em> genes, visualized by IGV. The green rectangular block is the location of the statistically-qualified peak of H3K4me3 in MDA-MB-468.</strong></p><p><strong>(B) An assembly of IgG (first row), DVL3 (second row) and H3K4me3 (third row) ChIP-Seq data in MCF7 for the <em>HAUS5, APOC1P1, ZNF672, HLA-A</em> and <em>HLA-E</em> genes, visualized by IGV.</strong></p><p><strong>(C) ChIP-qPCRs at <em>ARAP1, RFX5-GBAT2, KDM5B, APOC1P1</em>, and <em>PSMB8</em> promoters for IgG and H3K4me3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).</strong></p><p><strong>(D) ChIP-qPCRs at <em>RFX5-GBAT2, PCAT6, APOC1P1</em> and <em>HAUS5</em> promoters for IgG and H3K4me3 in MCF7 (NTC, shA DVL3, and shB DVL3).</strong></p><p><strong>(E) Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and H3K4me3 ChIP-Seq</strong></p><p><strong>(F) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and H3K4me3 ChIP-Seq</strong></p><p><strong>(G) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and FAIRE Seq.</strong></p><p><strong>Data information: For (A) and (B) each column is 4000 bp wide. The fourth rows show the gene nearest to the ChIP-Seq alignment, including its location and orientation. C and D panels show a representative ChIP-qPCR of three independent ChIP-qPCR experiments. For C and D panels, an independent t-test was used for statistical comparison. * p < 0.05;</strong> data show means ± SD, <strong>n=3, technical replicate.</strong></p>
|
https://api.sourcedata.io/file.php?figure_id=39846
|
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||
10.15252/embr.202051723
|
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth
|
2021
|
Figure 5
|
<p><strong>Figure 5. Localization of KMT2D and epigenetic enhancer marks with respect to DVL3 genomic binding in MDA-MB-468 and MCF7 cells</strong></p><p><strong>(A) An assembly of IgG (first row), DVL3 (second row) and KMT2D (third row) ChIP-Seq data in MCF7 for the <em>HAUS5, WDR74, YWHAQ, CDNF/HSPA14</em> and <em>PRDM10</em> genes, visualized by IGV. The bar below the DVL3 peak is the location of the statistically-qualified peak of KMT2D in MCF7.</strong></p><p><strong>(B) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and KMT2D ChIP-Seq.</strong></p><p><strong>(C) ChIP-qPCRs at <em>ARAP1, RFX5-GBAT2</em>, and <em>PSMB8</em> promoters for IgG and KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).</strong></p><p><strong>(D) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MDA-MB-468 for the <em>PRDM10, TRIM26, BTN2A1, PNRC2</em>, and <em>MR1</em> genes, visualized by IGV. The green rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MDA-MB-468.</strong></p><p><strong>(E) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MCF7 for the <em>WDR74, YWHAQ, CDNF/HSPA14, BTN2A1</em>, and <em>MR1</em> genes, visualized by IGV. The turquoise and purple rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MCF7, respectively.</strong></p><p><strong>(F) Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and H3K27ac ChIP-Seq.</strong></p><p><strong>(G) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and H3K27ac ChIP-Seq.</strong></p><p><strong>(H)Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and H4K8ac ChIP-Seq</strong></p><p><strong>(I) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and H4K8ac ChIP-Seq.</strong></p><p><strong>(J) Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and super-enhancer targets in basal breast carcinoma (BRCA)</strong></p><p><strong>(K) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and super-enhancer targets in luminal breast carcinoma.</strong></p><p><strong>Data information: Panel C shows a representative ChIP-qPCR of three independent ChIP-qPCR experiments. For panel C, an independent t-test was used for statistical comparison. * p < 0.05;</strong> data show means ± SD, <strong>n=3, technical replicate. For (A), (D) and (E) each column is 4000 bp wide. The fourth rows (panel A) and the fifth rows (panel D and E) show the gene nearest to the ChIP-Seq alignment, including its location and orientation.</strong></p>
|
https://api.sourcedata.io/file.php?figure_id=39848
|
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||
10.15252/embr.202051723
|
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth
|
2021
|
Figure 6
|
<p><strong>Figure 6. The effects of silencing DVL3 on KMT2D expression and subcellular localization</strong></p><p><strong>(A) DVL3 co-immunoprecipitates KMT2D in MDA-MB-468 cells (upper panel) and MCF7 cells (lower panel). IgG heavy chain (Hc) was blotted for as a control for equal antibody capture for immunoprecipitation and whole cell extracts (WCE) as a positive control.</strong></p><p><strong>(B) MDA-MB-468 histone extracts from NTC, shA DVL3 and shB DVL3 subjected to immunoblot analysis for H3K4me3 and H3.</strong></p><p><strong>(C) An assembly of IgG (first row), DVL3 (second row) ChIP-Seq in MDA-MB-468 cells, IgG (third row), and, DVL3 (fourth row) ChIP-Seq data in MCF7 for the KMT2D gene, visualized by IGV. Each column is 20000 bp wide. The fifth row show the location and orientation of the KMT2D gene.</strong></p><p><strong>(D) RT-qPCR-based analysis of expression changes of DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells.</strong></p><p><strong>(E) RT-qPCR-based analysis of expression changes of KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells.</strong></p><p><strong>(F) RT-qPCR-based analysis of expression changes of DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3) cells.</strong></p><p><strong>(G) RT-qPCR-based analysis of expression changes of KMT2D in MCF7 (NTC, shA DVL3, and shB DVL3) cells.</strong></p><p><strong>(H) Immunofluorescence of KMT2D localization in MDA-MB-468 cells. The cells were probed with KMT2D antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of KMT2D (red) and nuclear staining (blue) is shown as KMT2D/DAPI, merge of KMT2D (red) and actin (green) is shown as KMT2D/ACTIN, and, merge of KMT2D (red), nuclear staining (blue) and actin (green) is shown as MERGE for NTC, shA DVL3 and shB DVL3 cells. Scale bar: 10µm</strong></p><p><strong>(I) Sequential ChIP (ChIP-reChIP) to examine occupancy of DVL3 and KMT2D at RFX5, HAUS5, COX14, APOC1P1, WDR74, JUNB, and, HLA-A promoters analyzed by end-point PCR.</strong></p><p><strong>(J) Sequential ChIP (ChIP-reChIP) to examine occupancy of DVL3 and H3K4me3 at RFX5, APOC1P1, JUNB, and, HLA-A promoters analyzed by end-point PCR.</strong></p><p><strong>Data information: For D-G panels transcript levels were normalized to actin transcript levels and an independent t test was used for statistical comparison. * p < 0.05; data show means ± SD, n=3 biological replicates.</strong></p>
|
https://api.sourcedata.io/file.php?figure_id=39850
|
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||
10.15252/embr.202051723
|
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth
|
2021
|
Figure 7
|
<p><strong>Figure 7. The effects of silencing DVL3 on the formation and progression of spheroid and orthotopic xenograft tumor models.</strong></p><p><strong>(A) Quantification of spheroids' area in MDA-MB-468 cell line (NTC vs. shDVL3). The data are presented as the mean ± SD, n = 3 showing statistical difference after 32h *p < 0.05.</strong></p><p><strong>(B) Quantification of the spheroids' area in the MCF7 cell line (NTC, shA DVL3 and shB DVL3). The data are presented as the mean ± SD, n = 3 showing statistical difference after 100h *p < 0.05.</strong></p><p><strong>(C) IVIS images showing representative tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal.</strong></p><p><strong>(D) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, Average luminescence was used to measure volume of tumor; n ≥ 9.</strong></p><p><strong>(E) Tumor pictures of tumors resulting from the injections of MDA-MB-468 (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Scale shown in cm.</strong></p><p><strong>(F) Final weight of tumors resulting from the injections of MDA-MB-468 (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, One-way ANOVA was used for statistical comparison, n ≥ 9.</strong></p><p><strong>(G) IVIS images showing representative tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal.</strong></p><p><strong>(H) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Average luminescence was used to measure volume of tumor. n=7.</strong></p><p><strong>Data information: For all panels: T-Test was used for statistical comparison, unless is specificated, data show means ± SD, * indicates significant difference from control p˂0.05.</strong></p>
|
https://api.sourcedata.io/file.php?figure_id=39851
|
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||
10.15252/embr.202051723
|
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth
|
2021
|
Figure 8
|
<p><strong>Figure 8. Nuclear DVL3 influences gene expression and spheroid formation</strong></p><p><strong>(A) Immunofluorescence of HA-tagged DVL3 localization in MDA-MB-468 cells. The cells were probed with an HA antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of actin (green) and nuclear staining (blue) is shown as ACTIN/DAPI, merge of DVL3 (red) and nuclear staining (blue) is shown as HA/DAPI, merge of DVL3 (red) and actin (green) is shown as HA/ACTIN, and, merge of DVL3 (red), nuclear staining (blue) and actin (green) is shown as MERGE for empty vector (EV), HA-DVL3-WT and HA-DVL3-NESm transient transfected cells. Scale bar: 10µm.</strong></p><p><strong>(B) RT-qPCR-based analysis of expression changes of DVL3 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells.</strong></p><p><strong>(C) RT-qPCR-based analysis of expression changes of KMT2D in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells.</strong></p><p><strong>(D) RT-qPCR-based analysis of expression changes of <em>ARAP1, RFX5, GBAT2, WDR74, SNORD3D, HAUS5, TAP1, HLA-A, PDG1</em>, and, <em>MR1</em> in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells.</strong></p><p><strong>(E) Images and quantification of spheroid areas in stable MDA-MB-468 cell line (EV, HA-DVL3-WT and HA-DVL3-NESm). The data are presented as the mean ± SD, n = 8 showing statistical difference after 32h for HA-DVL3-WT respect to EV and48h for HA-DVL3-NESm respect to EV *p < 0.05. Independent t tests were performed per time point for statistical comparison. Scale bar: 300µm.</strong></p><p><strong>(F) Proposed model of the role of DVL3, colocalizing with transcription factors such as ZBTB7B and chromatin modifying enzymes as KMT2D in transcriptional regulatory region and regulate transcription.</strong></p><p><strong>Data information: For B-D panels transcript levels were normalized to actin transcript levels and an independent t test was used for statistical comparison. * p < 0.05; data show means ± SD, n=3.</strong></p><p><strong><br></strong></p><p><strong>Expanded View Figure legends</strong></p>
|
https://api.sourcedata.io/file.php?figure_id=39852
|
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||
27220849
|
10.15252/embj.201593169
|
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles
|
2016
|
Figure 2
|
<p><strong>Fig</strong><strong>ure</strong><strong> 2.</strong><strong> SPOP localizes to nuclear speckles or other nuclear bodies</strong><strong>.</strong> Confocal microscopy images of fixed NIH 3T3 cells transiently expressing <strong>(A) </strong>full length HA-SPOP<strong>, (B) </strong>GFP-Gli3<sup>1</sup><sup>455</sup>, and <strong>(C) </strong>HA-SPOP+GFP-Gli3<sup>1-455</sup> are shown. DAPI was used to stain the nucleus, SPOP localization was identified using a anti-HA antibody, nuclear speckle localization was identified using a anti-SC-35 antibody, and Gli3<sup>1-455</sup> localization was identified via GFP fluorescence. SPOP co-localizes with a marker for nuclear speckles or with the substrate Gli3<sup>1</sup><sup>455</sup>. The areas with overlapping HA- and GFP signal contain 75% of the punctate HA signal, and 100% of the punctate GFP signal.</p>
|
https://api.sourcedata.io/file.php?figure_id=8197
|
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},
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] |
|
27220849
|
10.15252/embj.201593169
|
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles
|
2016
|
Figure 3
|
<p><strong>Figure 3. </strong><strong>SPOP nuclear bodies have liquid-like character. </strong><strong>(</strong><strong>A</strong><strong>-</strong><strong>B</strong><strong>)</strong> NIH 3T3 cells were <strong>(A)</strong> co-transfected with constructs expressing full length SPOP and GFP-Gli3<sup>1</sup><sup>455</sup> or <strong>(B)</strong> transfected with a construct expressing SC-35-GFP, and GFP fluorescence was monitored in live cells. <strong>(A)</strong> Snapshots were taken from individual timepoints as noted in the Figure to show photobleaching (at 0 s), recovery, and nuclear body fusion events. The arrow in each panel points to the body that was photobleached and subsequently fuses with another body. These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery. <strong>(B)</strong> Snapshots at indicated timepoints show a nuclear speckle fusion event. Additional images are shown in Fig EV1. <strong>(</strong><strong>C</strong><strong>)</strong> Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec. Data from 45 individual cells and FRAP events were corrected for background, normalized to the minimum and maximum intensity, and the mean is shown with error bars representing the standard error of the mean. The mean characteristic recovery time is indicated ± SEM. <strong>(</strong><strong>D-</strong><strong>E)</strong> Nuclear bodies are close to spherical. <strong>(</strong><strong>D</strong><strong>)</strong> Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA-SPOP (151 cells) <strong>(</strong><strong>D</strong><strong>)</strong>, or GFP-Gli3<sup>1-455</sup> and HA-SPOP (155 cells) <strong>(</strong><strong>E</strong><strong>) </strong>were calculated as described previously (Brangwynne <em>et al</em>, 2011). Representative individual images of cells are shown as insets in each panel.</p>
|
https://api.sourcedata.io/file.php?figure_id=8198
|
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"uniprot_ids": [
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{
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"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
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"mapping_status": "mapped",
"ncbi_gene_id": "20747",
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"text": "SPOP",
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"uniprot_ids": [
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] |
|
27220849
|
10.15252/embj.201593169
|
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles
|
2016
|
Figure 4
|
<p><strong>Fig</strong><strong>ure</strong> <strong>4</strong><strong>. S</strong><strong>elf-association defective S</strong><strong>POP </strong><strong>mutants do not</strong> <strong>localize</strong><strong> to nuclear speckles. (A) </strong>SEC chromatograms of given loading concentrations of SPOP<sup>28-359</sup> are shown. The concentrations were normalized to the monomer molecular weight, i.e., identical concentrations of dimeric SPOP ΔBACK (see Fig EV2) and oligomeric SPOP<sup>28-359</sup> contain identical numbers of protomers. <strong>(B)</strong> SEC chromatograms for SPOP constructs defective in self-association in one or both oligomerization domains are shown. Proteins were injected at the same loading concentration (533 µM) and the elution volume of globular molecular weight standards is noted above the graph. <strong>(C)</strong> SEC chromatograms of SPOP<sup>28-359</sup> (200 µM), SPOP mutBACK (718 µM), and mixtures of the two (ratios given in the Figure, SPOP at 200 µM) are shown. The elution volume of globular molecular weight standards is noted above the graph. <strong>(</strong><strong>D</strong><strong>) </strong>Constructs for expressing full-length HA-SPOP or HA-SPOP mutants capable of oligomerization through only one (or neither) of the self-association domains were transfected into NIH 3T3 cells. DAPI was used to stain the nucleus and SPOP localization was identified using an anti-HA antibody. Experiments were performed at least twice on four biological samples. Multiple cells were examined and representative cells are shown. For additional images see Appendix Fig S3. Scale bar represents 5 µm.</p>
|
https://api.sourcedata.io/file.php?figure_id=8199
|
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{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
}
] |
|
27220849
|
10.15252/embj.201593169
|
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles
|
2016
|
Figure 5
|
<p><strong>Fig</strong><strong>ure</strong> <strong>5</strong><strong>. SPOP forms higher-order oligomeric species in cells. (A)</strong> Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide. All SPOP proteins bound substrate with an affinity similar to that of SPOP<sup>28-359</sup> (4-8 µM, see Table 2). Experimental data are shown as circles; the solid lines are fits to eq. 2 (Roehrl <em>et al</em>, 2004).<strong> (B) </strong><em>In vitro</em> crosslinking assays were performed for SPOP<sup>28-359</sup> and each mutant at 30 μM protein with the amide-specific BS3 crosslinker. Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non-specific crosslinking of protein species. <strong>(C)</strong> Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP, SPOP mutBACK, SPOP mutBTB, or SPOP mutBTB-BACK. SPOP<sub>1</sub>, SPOP<sub>2</sub> and SPOP<sub>n</sub> identify SPOP monomers, dimers and larger species, respectively. For loading levels see Appendix Fig S4.</p>
|
https://api.sourcedata.io/file.php?figure_id=8200
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
}
] |
|
27220849
|
10.15252/embj.201593169
|
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles
|
2016
|
Figure 6
|
<p><strong>Fig</strong><strong>ure</strong> <strong>6</strong><strong>.</strong> <strong>The </strong><strong>SPOP oligomerization domains </strong><strong>dimerize </strong><strong>with different affinities</strong><strong>. </strong>(<strong>A-C</strong>) The BTB domain dimerizes with nanomolar affinity. (<strong>A</strong>) SV-AUC data for 0.2 nM AF488-SPOP ΔBACK. Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation (circles). The data were subjected to standard <em>c(s)</em> analysis in SEDFIT (Schuck, 2000) and the results of this fit are shown (solid lines, rainbow color scheme). <strong>(</strong><strong>B</strong><strong>) </strong>Sedimentation coefficient distributions for a selection of values from the dilution series are shown. <strong>(</strong><strong>C</strong><strong>) </strong>The isotherm of weighted-average s-values vs. concentration (black circles), was fit to a dimer self-association model and reveals a <em>K</em><em><sub>D</sub></em> of 1.11 nM, [95% confidence interval 0.88, 1.40 nM] (solid line). (<strong>D</strong>) The BACK domain dimerizes with micromolar affinity. Experimental weight-average molar mass (M<sub>w</sub>) from CG-MALS for SPOP mutBTB (black circles) was fitted to a self-association model (see text for details) for BACK-BACK dimerization (black line) yielding a <em>K</em><em><sub>D</sub></em> of 36 ± 3 μM (average and standard deviation from 3 independent experiments). Fits to the monomer/dimer equilibrium model and to alternative monomer/trimer, tetramer and pentamer equilibrium models are depicted as black solid line and as dashed and grey lines, respectively. Residuals for all models are depicted in the lower panel.</p>
|
https://api.sourcedata.io/file.php?figure_id=8201
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
}
] |
|
27220849
|
10.15252/embj.201593169
|
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles
|
2016
|
Figure 7
|
<p><strong>Fig</strong><strong>ure</strong> <strong>7</strong><strong>. Indefinite SPOP self-association fits to</strong><strong> an isodesm</strong><strong>ic model. (</strong><strong>A</strong><strong>) </strong>Cartoon schematic depicting the proposed isodesmic self-association model. <em>K</em><em><sub>D</sub></em><em><sub>1</sub></em> and <em>K</em><em><sub>D</sub></em><em><sub>2</sub></em> represent the domain-mediated dimerization affinities for the BTB domain and BACK domains, respectively. <em>K</em><em><sub>D</sub></em><em><sub>2</sub></em> is identical for all association steps independent of oligomer size. At large oligomer sizes, <em>K</em><em><sub>D</sub></em><em><sub>2</sub></em> may increase due to entropic penalties, resulting in <em>K</em><em><sub>D</sub></em><em><sub>2</sub></em>*. The N- and C-termini contain neither defined domains nor low-complexity sequences but may add additional self-association behavior as evidenced by aggregation; these interactions were not dissected due to the poor reversibility of aggregation (Fig EV2B). <strong>(</strong><strong>B</strong><strong>) </strong>Experimental weight-average molar mass (M<sub>w</sub>) from CG-MALS for SPOP<sup>28-359</sup> (orange circles) was fitted to an isodesmic self-association model in which SPOP dimers are the self-associating unit (orange line). The largest SPOP oligomer taken into account was an undecamer of SPOP dimers ((SPOP<sub>2</sub>)<sub>11</sub>). The fits from three independent experiments yielded a <em>K</em><em><sub>D</sub></em><em><sub>2</sub></em> of 2.4 ± 0.4 µM. Lines for fits of the data to self-association models that assume formation of individual oligomeric species instead of isodesmic self-association are shown for reference (gray lines). <strong>(C)</strong> Experimental weight-average molar mass (M<sub>w</sub>) from CG-MALS data (circles) and fits (solid lines) are depicted for each SPOP construct assayed, showing that a progressive increase in SPOP oligomer size is observed only when both self-association domains are interaction-competent. This Figure is comprised of data shown in Figures 6D, 7B, and Appendix Fig S5A for direct comparison. <strong>(D) </strong>Ribbon diagram illustrating structural model of an octamer SPOP assembly. In a 27 µM SPOP solution three percent of the oligomeric assemblies would be equal in size or larger. The domains are colored as in Figures 1 and 7A (MATH, green; BTB, red; BACK, blue), with alternating monomers shown in gray for clarity. The model was built using two available crystal structures (PDB ID 3HQI (Zhuang <em>et al</em>, 2009) and 4HS2 (van Geersdaele <em>et al</em>, 2013)), and no further assumptions.</p>
|
https://api.sourcedata.io/file.php?figure_id=8202
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "20747",
"ext_tax_ids": "10090",
"ext_tax_names": "Mus musculus",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "20747",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8",
"Q9DBZ2"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
}
] |
|
27220849
|
10.15252/embj.201593169
|
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles
|
2016
|
Figure 8
|
<p><strong>Fig</strong><strong>ure</strong> <strong>8</strong><strong>. </strong><strong>SPOP forms dynamic higher-order oligomers t</strong><strong>hat can be analyzed quantitatively</strong><strong>. </strong><strong>(A)</strong> Native MS spectrum of 30 µM SPOP<sup>28-359</sup> confirms that SPOP self-associates through addition of dimers. The increase in mass indicates that the dimer is the stable building block within the assemblies. <strong>(</strong><strong>B</strong><strong>)</strong> Graphical representation of the SPOP concentration of each oligomeric species within a 27 µM SPOP<sup>28-359</sup> sample. The cartoons’ sizes are scaled based upon the fraction present within the total sample. <strong>(</strong><strong>C</strong><strong>)</strong> Concentration-dependence of the SPOP oligomer size distribution (mole fraction) from isodesmic modeling of CG-MALS data.</p>
|
https://api.sourcedata.io/file.php?figure_id=8203
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "Q6ZWS8",
"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": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q6ZWS8"
]
}
] |
|
27220849
|
10.15252/embj.201593169
|
Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles
|
2016
|
Figure 9
|
<p><strong>Fig</strong><strong>ure</strong> <strong>9</strong><strong>. </strong><strong>The s</strong><strong>elf-association deficient </strong><strong>SPOP</strong><strong> mutant</strong><strong> mutBTB</strong><strong> ha</strong><strong>s a</strong> <strong>substrate degradation</strong><strong> defect</strong> <strong><em>in vivo</em></strong><strong>. </strong>UAS transgenes were expressed under the control of an epithelial driver <em>C765-Gal4</em> in Drosophila melanogaster. <strong>(A)</strong> UAS vector served as a control and yielded a normal wing. Longitudinal veins are denoted by numbers 1-5. Expression of <strong>(B)</strong> SPOP WT resulted in a modest Hh loss-of function phenotype with fusion of LV3 and LV4 (arrow). <strong>(C)</strong> mutBTB acts in a dominant-negative manner, as evidenced by a modest Hh gain-of function phenotype with expansion of the LV3-LV4 intervein space. Expression of <strong>(D)</strong> mutBACK and <strong>(E)</strong> mutBTB-BACK does not induce a wing patterning defect. ~50 animals each were analyzed from two independent crosses.</p>
|
https://api.sourcedata.io/file.php?figure_id=8204
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "41704",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "41704",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q9VFP2"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "41704",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "41704",
"original_type": "gene",
"role": "intervention",
"text": "SPOP",
"type": "geneprod",
"uniprot_ids": [
"Q9VFP2"
]
}
] |
|
30814124
|
10.15252/embj.201899122
|
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1
|
2019
|
Figure 1
|
<sd-panel> <p><strong>Figure 1. Effect of <em>Nanog</em> on erythropoietic <sd-pretag id="sdPretag2084827341sm" type="tissue" role="component">development</sd-pretag>.</strong></p> <p><strong>A.</strong> Dox-induced prolongation of <sd-pretag id="sdPretag1507131232sm" type="geneprod" role="assayed"><em>Nanog</em></sd-pretag> expression in <em>Nanog<sup>tg</sup></em> <sd-pretag id="sdPretag1820753938sm" type="organism" role="component">embryos</sd-pretag> up to E9.5 results in lack of <sd-pretag id="sdPretag5916838sm" type="tissue" role="component">blood</sd-pretag> (left) and downregulation of erythropoietic gene expression. The center and right panels show whole-<sd-pretag id="sdPretag1060806011sm" category="assay">mount in situ hybridization</sd-pretag> for <em><sd-pretag id="sdPretag1918800379sm" type="geneprod" role="component">Hbb</sd-pretag>-<sd-pretag id="sdPretag894845629sm" type="geneprod" role="component">bh1</sd-pretag></em> (in <sd-pretag id="sdPretag323880336sm" type="organism" role="component">embryos</sd-pretag> with intact yolk sacs) and for the long non-coding RNA <em>Redrum</em>. Asterisks mark the <sd-pretag id="sdPretag1828152750sm" type="tissue" role="component">aorta</sd-pretag>-gonad-mesonephros region (AGM) and arrows the tailbud. <sd-pretag id="sdPretag1691482308sm" type="organism" role="component">Embryos</sd-pretag> of the same genotype but not treated with dox were used as controls (-dox). Scale bars, 500 µm.</p> <p><strong>B.</strong> <sd-pretag id="sdPretag567352197sm" category="assay">Endomucin staining</sd-pretag> of <sd-pretag id="sdPretag1471376722sm" type="tissue" role="component">vessels</sd-pretag> in control (-dox) or treated (+dox) E9.5 <em>Nanog<sup>tg</sup></em> <sd-pretag id="sdPretag1231158729sm" type="organism" role="component">embryos</sd-pretag>. On the right, higher magnifications of the boxed areas. Scale bar, 500 µm.</p> <p><strong>C.</strong> Representative <sd-pretag id="sdPretag1947123662sm" category="assay">FACS</sd-pretag> plot of the distribution of the <sd-pretag id="sdPretag1605200376sm" type="geneprod" role="assayed">CD71</sd-pretag> and <sd-pretag id="sdPretag619365344sm" type="geneprod" role="assayed">Ter119</sd-pretag> populations in dissected yolk sacs from untreated and dox-treated E9.5 <sd-pretag id="sdPretag329129701sm" type="organism" role="component"><em>Nanog<sup>tg</sup></em> embryos</sd-pretag>.</p> <p><strong>D.</strong> Quantification of the <sd-pretag id="sdPretag586325561sm" type="geneprod" role="assayed">CD71</sd-pretag>+ <sd-pretag id="sdPretag2131727360sm" type="geneprod" role="assayed">Ter119</sd-pretag>+ population in controls (-dox, black dots; n=8) and <em>Nanog</em> expressing (+dox, red dots; n=7) E9.5 yolk sacs. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 <em>Nanog<sup>tg</sup></em> <sd-pretag id="sdPretag561714682sm" type="organism" role="component">embryos</sd-pretag>. ***<em>P.</em> < 0.0005; Student's t-test. Horizontal line represents mean values and error bars standard deviation (SD).</p> <p><strong>E.</strong> Representative <sd-pretag id="sdPretag508182635sm" category="assay">FACS</sd-pretag> plots showing the distribution of <sd-pretag id="sdPretag583483590sm" type="geneprod" role="assayed">cKit</sd-pretag> and <sd-pretag id="sdPretag519749768sm" type="geneprod" role="assayed">CD41</sd-pretag> populations in yolk sacs from untreated controls (-dox) and <em>Nanog</em> expressing (+dox) E9.5 <em>Nanog<sup>tg</sup></em> <sd-pretag id="sdPretag1148027166sm" type="organism" role="component">embryos</sd-pretag>.</p> <p><strong>F.</strong> Quantification of different progenitor populations in yolk sacs from control (-dox, black dots; n=8) and <em>Nanog</em> expressing (+dox, red dots; n=7) E9.5 <sd-pretag id="sdPretag482923876sm" type="organism" role="component">embryos</sd-pretag>. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 <em>Nanog<sup>tg</sup></em> <sd-pretag id="sdPretag197068185sm" type="organism" role="component">embryos</sd-pretag>. Horizontal line represents mean values and error bars SD.</p> <p><strong>G.</strong> Differences in the expression levels of <em>Nanog</em> and selected <sd-pretag id="sdPretag1256230305sm" type="subcellular" role="component">hematopoietic</sd-pretag> genes in the <sd-pretag id="sdPretag1652173870sm" type="geneprod" role="assayed">CD71</sd-pretag>+ <sd-pretag id="sdPretag109339859sm" type="geneprod" role="assayed">Ter119</sd-pretag>+ population of control (-dox; n=7) and <em>Nanog</em>-expressing (+dox; n=4) E9.5 <sd-pretag id="sdPretag1108036012sm" type="organism" role="component">embryos</sd-pretag>. **<em>P</em> < 0.005, ***<em>P</em> < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=24811
|
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] |
|
30814124
|
10.15252/embj.201899122
|
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1
|
2019
|
Figure 2
|
<sd-panel> <p><strong>Figure 2. Wild type <sd-pretag id="sdPretag657358521sm" type="cell" role="component">ES</sd-pretag> cells rescue erythroid maturation in chimeric <sd-pretag id="sdPretag1657512790sm" type="organism" role="component">embryos</sd-pretag>.</strong></p> <p><strong>A.</strong> Experimental design for chimera generation, and contribution of GFP <sd-pretag id="sdPretag1843076643sm" type="cell" role="component">cells</sd-pretag> to chimeric <sd-pretag id="sdPretag844360399sm" type="organism" role="component">embryo</sd-pretag> (right hand side panels).</p> <p><strong>B.</strong> Freshly dissected dox-treated <em><sd-pretag id="sdPretag646376358sm" type="organism" role="component">Nanog<sup>tg</sup></sd-pretag></em> E10.5 <sd-pretag id="sdPretag417300322sm" type="organism" role="component">embryos</sd-pretag> without (control) and with (chimera) contribution of <sd-pretag id="sdPretag802510147sm" type="cell" role="component">wt</sd-pretag>-<sd-pretag id="sdPretag1841602503sm" type="cell" role="component">ES<sup>GFP</sup></sd-pretag> cells (left, brightfield; right, <sd-pretag id="sdPretag1665030817sm" type="geneprod" role="reporter">GFP</sd-pretag>). Arrows mark the presence of <sd-pretag id="sdPretag1164082919sm" type="tissue" role="component">blood</sd-pretag> in chimeric <sd-pretag id="sdPretag2048717526sm" type="organism" role="component">embryos</sd-pretag> that is absent from controls. Scale bar, 500 µm.</p> <p><strong>C.</strong> Representative <sd-pretag id="sdPretag349372157sm" category="assay">FACS</sd-pretag> plots showing of red <sd-pretag id="sdPretag416596690sm" type="cell" role="component">blood</sd-pretag><sd-pretag id="sdPretag1350746686sm" role="component"> cell</sd-pretag> maturation as determined by <sd-pretag id="sdPretag989877582sm" type="geneprod" role="assayed">CD71</sd-pretag>/<sd-pretag id="sdPretag604140887sm" type="geneprod" role="assayed">Ter119</sd-pretag> staining in single dox-treated E10.5 control (left) and chimeric (right) <sd-pretag id="sdPretag402968295sm" type="organism" role="component">embryos</sd-pretag>.</p> <p><strong>D-F.</strong> Quantification of the <sd-pretag id="sdPretag25136696sm" type="geneprod" role="assayed">CD71</sd-pretag>+ <sd-pretag id="sdPretag927075079sm" type="geneprod" role="assayed">Ter119</sd-pretag>+ population in single dox-treated E10.5 <sd-pretag id="sdPretag387530060sm" type="organism" role="component">embryos</sd-pretag> (<strong>D</strong>; control, n=18; chimera, n=10), untreated <sd-pretag id="sdPretag571501880sm" type="organism" role="component">embryos</sd-pretag> (<strong>E</strong>; control, n=7; chimera, n=12), and in <sd-pretag id="sdPretag1469487533sm" type="geneprod" role="reporter">GFP</sd-pretag>- cells (not derived from wild type <sd-pretag id="sdPretag2051051136sm" type="cell" role="component">ES cells</sd-pretag>) from dox-treated <sd-pretag id="sdPretag1188197145sm" type="organism" role="component">embryos</sd-pretag> (<strong>F</strong>; control, n=18; chimera, n=10). ***<em>P</em> < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=24813
|
[
{
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"ext_tax_names": "Mus musculus",
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"mapping_status": "mapped",
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"A2RS90",
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{
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{
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}
] |
|
30814124
|
10.15252/embj.201899122
|
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1
|
2019
|
Figure 3
|
<sd-panel> <p><strong>Figure 3. <em>Nanog</em>-knockout <sd-pretag id="sdPretag2081539371sm" type="cell" role="component">ES cells</sd-pretag> show increased potential to generate erythroid precursors.</strong></p> <p><strong>A.</strong> Quantification of colony-forming units generated by wild type (wt) and knockout (<em>Nanog<sup>-/-</sup></em>) <sd-pretag id="sdPretag1735020546sm" type="cell" role="component">ES cells</sd-pretag> after culture of EBs for 5 (D5), 6 (D6), or 7 (D7) days and plating disaggregated cells in different hemogenic-promoting conditions). Panels on the left show representative images of <sd-pretag id="sdPretag1499195442sm" type="organism" role="component">mouse</sd-pretag> hematopoietic colonies obtained after 12 days of culture in specific media. <sd-pretag id="sdPretag1262206135sm" type="geneprod" role="reporter">CFU</sd-pretag>-<sd-pretag id="sdPretag277418897sm" type="geneprod" role="assayed">GEMM</sd-pretag>, progenitors giving rise to <sd-pretag id="sdPretag374727132sm" type="cell" role="component">granulocytes</sd-pretag>, <sd-pretag id="sdPretag122866724sm" type="cell" role="component">erythrocytes</sd-pretag>, <sd-pretag id="sdPretag954704332sm" type="cell" role="component">monocytes</sd-pretag>, and <sd-pretag id="sdPretag682410745sm" type="cell" role="component">megakaryocytes</sd-pretag>; BFU-E, burst forming units-erythroid; Ery-P, colony forming primitive erythroid; <sd-pretag id="sdPretag692514735sm" type="geneprod" role="reporter">CFU</sd-pretag>-GM, <sd-pretag id="sdPretag1052226290sm" type="cell" role="component">granulocyte</sd-pretag>-<sd-pretag id="sdPretag2051476650sm" type="cell" role="component">monocyte</sd-pretag> precursors; CFU-M, <sd-pretag id="sdPretag503470675sm" type="cell" role="component">monocyte</sd-pretag> precursors; CFU-G, <sd-pretag id="sdPretag1613973495sm" type="cell" role="component">granulocyte</sd-pretag> precursors. No <sd-pretag id="sdPretag1218484498sm" type="geneprod" role="reporter">CFU</sd-pretag>-<sd-pretag id="sdPretag156494414sm" type="geneprod" role="assayed">GEMM</sd-pretag> are detected at D5 and no BFU-E at D7. For both wt and knockout cells, n=3 each with 3 technical replicates. *<em>P</em> < 0.05, **<em>P</em> < 0.005, ***<em>P</em> < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.</p> <p><strong>B.</strong> <sd-pretag id="sdPretag2130711992sm" category="assay">RT</sd-pretag>-<sd-pretag id="sdPretag1085317516sm" category="assay">qPCR</sd-pretag> determination of the relative expression of <em>Brachyury</em> and selected hematopoietic genes in control (wt, right) and knockout (<em>Nanog<sup>-/-</sup></em>, left) <sd-pretag id="sdPretag703156415sm" type="cell" role="component">ES cells</sd-pretag> (n=3) during 10 days of EB differentiation in hematopoietic-cytokine-enriched medium. Black arrowheads indicate the peak of <em>Brachyury</em> expression and white arrowheads the time of maximum hematopoietic-gene expression.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=24815
|
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"role": "assayed",
"text": "Brachyury",
"type": "geneprod",
"uniprot_ids": [
"P20293",
"Q78ZW9"
]
}
] |
|
30814124
|
10.15252/embj.201899122
|
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1
|
2019
|
Figure 4
|
<sd-panel> <p><strong>Figure 4. Induced <sd-pretag id="sdPretag1232777088sm" type="geneprod" role="component"><em>Nanog</em></sd-pretag> expression blocks erythroid maturation in adult <sd-pretag id="sdPretag1423279583sm" type="organism" role="component">mice</sd-pretag>.</strong></p> <p><strong>A.</strong> Experimental design for the treatment of adult <sd-pretag id="sdPretag802280249sm" type="organism" role="component"><em>Nanog<sup>tg</sup></em> mice</sd-pretag>.</p> <p><strong>B.</strong> Representative <sd-pretag id="sdPretag507414527sm" category="assay">FACS</sd-pretag> plots showing the distribution of different populations distinguished by <sd-pretag id="sdPretag805677553sm" type="geneprod" role="assayed">CD71</sd-pretag>/<sd-pretag id="sdPretag1401212265sm" type="geneprod" role="assayed">Ter119</sd-pretag> staining in whole <sd-pretag id="sdPretag92308811sm" type="cell" role="component">bone marrow</sd-pretag><sd-pretag id="sdPretag364339155sm" type="cell" role="component"> from</sd-pretag> untreated (-dox) or treated (+dox) adult <sd-pretag id="sdPretag1785729446sm" type="organism" role="component">mice</sd-pretag>. S0 (double negative cell), S1 (<sd-pretag id="sdPretag440225755sm" type="cell" role="component">proerythroblast</sd-pretag>), S2 (basophylic <sd-pretag id="sdPretag1299162844sm" type="cell" role="component">erythroblast</sd-pretag>), S3 (<sd-pretag id="sdPretag1599926112sm" type="subcellular" role="component">polychromatic</sd-pretag><sd-pretag id="sdPretag818300963sm" role="component"> erythroblast</sd-pretag>), and S4 (<sd-pretag id="sdPretag1664583949sm" type="subcellular" role="component">orthochromatic</sd-pretag> <sd-pretag id="sdPretag1732204299sm" type="cell" role="component">erythroblast</sd-pretag>) are different stages of <sd-pretag id="sdPretag911009302sm" type="tissue" role="component">blood</sd-pretag> maturation.</p> <p><strong>C.</strong> Quantification of the S1-S4 erythroid populations (-dox, n=4; +dox, n=5). *<em>P</em> < 0.05, **<em>P</em> < 0.005, ***<em>P</em> < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.</p> <p><strong>D.</strong> Representative <sd-pretag id="sdPretag730272768sm" category="assay">FACS</sd-pretag> plots showing the distribution of <sd-pretag id="sdPretag472201152sm" type="geneprod" role="assayed">CD16</sd-pretag>/32 and <sd-pretag id="sdPretag2070416205sm" type="geneprod" role="assayed">CD34</sd-pretag> <sd-pretag id="sdPretag1934881672sm" type="cell" role="component">hematopoietic</sd-pretag> precursors sorted from the <sd-pretag id="sdPretag1748875495sm" type="geneprod" role="component">cKit</sd-pretag>+<sd-pretag id="sdPretag786868369sm" type="geneprod" role="component">Sca1</sd-pretag>-LIN- <sd-pretag id="sdPretag1508394217sm" type="cell" role="component">bone marrow</sd-pretag> of untreated (-dox) or treated (+dox) adult <sd-pretag id="sdPretag1844174607sm" type="organism" role="component"><em>Nanog<sup>tg</sup></em> mice</sd-pretag>.</p> <p><strong>E.</strong> Quantification of precursor populations based on <sd-pretag id="sdPretag461959726sm" type="geneprod" role="intervention">CD16</sd-pretag>/32 and <sd-pretag id="sdPretag1017023869sm" type="geneprod" role="intervention">CD34</sd-pretag> sorting, as total number of cells per individual femur (-dox, n=5; +dox, n=6). *<em>P</em> < 0.05, **<em>P</em> < 0.005; Student's t-test. Horizontal line represents mean values and error bars SD.</p> <p><strong>F.</strong> <sd-pretag id="sdPretag142028177sm" category="assay">RT</sd-pretag>-<sd-pretag id="sdPretag858043317sm" category="assay">qPCR</sd-pretag> quantification of the relative expression of <sd-pretag id="sdPretag162986534sm" type="subcellular" role="component">hematopoietic</sd-pretag> genes in <sd-pretag id="sdPretag163431197sm" type="cell" role="component">megakaryocyte</sd-pretag>-erythroid progenitors (MEP) (-dox, n=8; +dox, n=5). *<em>P</em> < 0.05, ****<em>P</em> < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.</p> <p><strong>G.</strong> Experimental design for the transplant of <em>Nanog<sup>tg</sup></em> <sd-pretag id="sdPretag1743508719sm" type="cell" role="component">bone marrow</sd-pretag> to wild type recipients and treatment of chimeric <sd-pretag id="sdPretag725362561sm" type="organism" role="component">mice</sd-pretag>.</p> <p><strong>H.</strong> Contribution of <em>Nanog<sup>tg</sup></em> transplanted <sd-pretag id="sdPretag617602030sm" type="cell" role="component">bone marrow cells</sd-pretag> to peripheral <sd-pretag id="sdPretag1548551632sm" type="tissue" role="component">blood</sd-pretag> before (left) and after (right) dox treatment. Percentage of host derived <sd-pretag id="sdPretag415105716sm" type="cell" role="component">cells</sd-pretag> (CD45.1+) are shown in black, and of donor <sd-pretag id="sdPretag441338398sm" type="cell" role="component">derived cells</sd-pretag> (CD45.1/<sd-pretag id="sdPretag1762966466sm" type="geneprod" role="intervention">CD45</sd-pretag>.2 double +) in red. Individual <sd-pretag id="sdPretag1799566704sm" type="organism" role="component">mice</sd-pretag> are indicated on the x-axis (n=7).</p> <p><strong>I.</strong> Contribution of <em>Nanog<sup>tg</sup></em> transplanted cells to LSK, CMP, <sd-pretag id="sdPretag1212659958sm" type="molecule" role="reporter">GMP</sd-pretag> and <sd-pretag id="sdPretag185803334sm" type="geneprod" role="assayed">MEP</sd-pretag> populations purified from <sd-pretag id="sdPretag1769408440sm" type="cell" role="component">bone marrow</sd-pretag>. Percentage of host derived cells (<sd-pretag id="sdPretag1782062543sm" type="geneprod" role="assayed">CD45</sd-pretag>.1+) are show in black, and of donor derived cells (CD45.1/<sd-pretag id="sdPretag2015205461sm" type="geneprod" role="component">CD45</sd-pretag>.2 double +) in blue. Individual <sd-pretag id="sdPretag2054775308sm" type="organism" role="component">mice</sd-pretag> are indicated on the x-axis (n=7).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=24817
|
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] |
|
30814124
|
10.15252/embj.201899122
|
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1
|
2019
|
Figure 5
|
<p><strong>Figure 5. Direct transcriptional regulation of <em>Tal1</em> expression by <em>Nanog</em></strong></p><p><strong>A.</strong> Expected and observed number of mesodermal (Flk1+) cells of the E7.0 mouse embryo expressing <em>Nanog</em> and selected mesodermal or hematopoietic gene expression, based on single cell RNA-seq data (Scialdone et al., 2016). Statistical significance was calculated with a hypergeometric test.</p><p><strong>B.</strong> PCA showing the distribution of Flk1+ E7.0 mesoderm cells expressing <em>Nanog</em> (green) or <em>Tal1</em> (red). The single cell expressing both genes is shown in yellow and indicated by an arrow.</p><p><strong>C.</strong> E6.5 <em>Nanog<sup>tg</sup></em> embryos after 8 hours ex-utero culture in the presence (+dox) or absence (-dox) of doxycycline. Scale bar, 100 µm.</p><p><strong>D.</strong> RT-qPCR quantification of the relative expression of <em>Nanog</em>, <em>Tal1</em>, and <em>Klf1</em> in individual untreated embryos (-dox) or treated embryos (+dox) (n=5). **<em>P</em> < 0.005, ***<em>P</em> < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.</p><p><strong>E.</strong> Whole mount in situ hybridization of <em>Tal1</em> in E7.5 untreated (-dox) or in utero treated (+dox) <em>Nanog<sup>tg</sup></em> embryos. Scale bar, 100 µm.</p><p><strong>F.</strong> UCSC browser view of the <em>Tal1/Stil1</em> region (mm9; chr4:114,705,753-114,756,741), indicating the presence of the NANOG binding peak, determined by ChIP-seq, in EpiLCs (2 replicates are shown) but not in ES cells (Murakami et al., 2016); the binding peak was deleted by CRISPR/Cas9 genome editing (scissors).</p><p><strong>G.</strong> RT-qPCR determination of relative expression in wild type and CRISPR-deleted embryos (n=5) of <em>Tal1</em> (wt, n=19; deleted, n=13), <em>Klf1</em> (wt, n=3; deleted, n=6), <em>Gfi1b</em> (wt, n=10; deleted, n=8), <em>Runx1</em> (wt, n=13; deleted, n=5) and <em>Stil</em> (wt, n=19; deleted, n=13). **<em>P</em> < 0.005, Student's t-test. Horizontal line represents mean values and error bars SD.</p><p><strong>H</strong>. Experimental design for ES to EpiL cell differentiation of <em>Nanog<sup>tg</sup></em> cells and two independent clones (<em>Nanog<sup>tg</sup>;dTal1<sup>del#1</sup></em> and <em>Nanog<sup>tg</sup>;dTal1<sup>del#2</sup></em>) where the binding site for NANOG distal to <em>Tal1</em> has been deleted (left). On the right, relative expression of <em>Tal1</em> determined by RT-qPCR for each ES cell line (ESC; n=9 for all three lines) and EpiL cells without (EpiLC; <em>Nanog<sup>tg</sup></em> and <em>Nanog<sup>tg</sup>;dTal1<sup>del#1</sup>,</em> n=8; <em>Nanog<sup>tg</sup>;dTal1<sup>del#2</sup>,</em> n=6) or with dox treatment (EpiLC +dox; n=9 for all three lines). The genotype of the cell lines is indicated below. Values were normalized to <em>Nanog<sup>tg</sup></em> ESC. *<em>P</em> < 0.05, **<em>P</em> < 0.01, ns = not significant; ANOVA with Fisher post-test. Horizontal line represents mean values and error bars standard error of the mean (SEM).</p><p><strong><br></strong></p><p><strong>EXPANDED VIEW FIGURE LEGENDS</strong></p>
|
https://api.sourcedata.io/file.php?figure_id=24819
|
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] |
|
27113760
|
10.15252/embr.201541569
|
Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy
|
2016
|
Figure 1
|
<p>Figure 1. Shh release and its pathway activation in response to epileptic stimulations.</p>
<p>Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in pilocarpine (A,B) or kindling (D,E) models. For D and E, samples obtained from mice evoked with a single kindling stimulation to induce seizure activity as evidenced in EEG. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F. (G) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus (SE) induced by pilocarpine (n=7-10). (H,I) Shh levels assayed by ELISA in the medium of slices (H, n=9) or hippocampal neurons (I, n=6) incubated with picrotoxin (Pic) or Mg2+-free (0Mg) for the indicated times. (J) Representative western-blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times (n=13-19). α-tubulin (α-Tub), a loading control. Data were mean + SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Control (Ctrl) with student’s t test.</p>
|
https://api.sourcedata.io/file.php?figure_id=7162
|
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|
27113760
|
10.15252/embr.201541569
|
Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy
|
2016
|
Figure 3
|
<p>Figure 3. Shh inhibits glutamate transporter activities and increases the extracellular glutamate levels.</p><p>(A) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents. n=4-6. (B) The paired-pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle (Ctrl) or Shh (13-14 slices, 6 rats). Insets: representative traces recorded in response to paired pulse stimuli with different intervals. Statistics of 3H-glutamate uptake by neurons treated with indicated agents (C, n=10) or transfected with two lentivirus-based RNAi against EAAC1 (R2 and R3) or nonsense RNAi (Non) in response to vehicle (Ctrl) or Shh (D, n=3-6). No difference between R2 or R3 in Ctrl and those in Shh. Inset of (D): representative western-blots for EAAC1. Aspartate (Asp)-evoked currents at -70 mV from hippocampal neurons transfected with RNAi or Non (E, n=17-23) or treated with indicated agents (F, n=16-18). (G) Upper, representative western-blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower, 3H-glutamate uptake by cells transfected with the indicated vectors. n=9. (H,I) Shh effects on 3H-glutamate uptake (H, n=6) or Asp-evoked currents (I, n=17-23) of neurons with or without pertussis toxin (PTX) pretreatment. For E,F,I, the uppers: representative traces. Shh: 500 ng/mL. Data were mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl or Non (Ctrl) with student’s t test.</p>
|
https://api.sourcedata.io/file.php?figure_id=7164
|
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] |
|
27113760
|
10.15252/embr.201541569
|
Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy
|
2016
|
Figure 4
|
<p>Figure 4. Inhibiting Shh pathway suppresses epileptogenesis in mouse epilepsy models.</p>
<p>Effects of Cyclo (A-C, n=14), 5E1 (D-F, n=16-20), or ablation of Smo in CaMKIIα-positive neurons (G-I), or ablation of Smo in Aldh1l1-positive astrocytes (J-L) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice. In G-I, Ctrl: Smofl/fl induced by tamoxifen; CaMK: Smofl/flCaMKIIα-CreERT2 induced by tamoxifen, n=14-20. In J-L, Ctrl: Smo+/fl; Aldh1l1: Smo+/flAldh1l1-Cre, n=19-20. (M) Frequency of spontaneous seizures (class 4-5) in mice administrated with Cyclo or vehicle (Ctrl) at 4th, 6th and 8th week after pilocarpine SE induction. n=22-23. (N) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction. n=22-23. Cyclo: 10 mg/kg. 5E1: 900 ng/mouse. Data were mean ± SEM from at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl with student’s t test. (O) Schematic diagram depicting a working model for Shh-regulation of epileptogenesis. Epileptic activity triggers sequential responses, including Shh release, inhibition of glutamate transporter activity, and increase in extracellular glutamate, leading to epilepsy development.</p>
|
https://api.sourcedata.io/file.php?figure_id=7165
|
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] |
|
10.15252/emmm.202013466
|
CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance
|
2021
|
Figure 2
|
<p><strong>Figure 2. Genome-wide CRISPR Activation Screen Identifies BRAFi-Resistance Genes in Cutaneous Melanoma</strong></p><p>(A) CRISPR-SAM workflow. Cell library was exposed to DMSO (solvent of BRAFi) or BRAFi 2 µM (vemurafenib (Vem) or PLX8394 (PB, Paradox Breaker)) during 14 days. 40x10<sup>6</sup> cells per arm. Experiments were done in duplicate.</p><p>(B) Plot showing sgRNAs detected in BRAFi-resistant cells <em>versus</em> in control cells (cell library exposed to DMSO) with a fold change >1.5. Raw data are available in Table EV5. (BRAFi for PBV). Appendix Figure S1 depicted the comparison Vem <em>vs</em> PB.</p><p>(C) sgRNAs counts in BRAFi-resistant cells <em>versus</em> in DMSO-exposed cells for <em>EGFR</em> and <em>BIRC3</em>. (BRAFi for PBV, Appendix Figure S1)</p><p>(D) Determination of differentiation states of 12 human BRAF(V600) melanoma cell lines from CCLE according to (Tsoi <em>et al</em>, 2018).</p><p>(E) BRAF(V600) melanoma cell lines from CCLE were distributed in two groups (Sensitive and "intrinsically" Resistant, n=6 cell lines per group) according to their BRAFi IC<sub>50</sub> (µM, half maximal inhibitory concentrations) (Barretina <em>et al</em>, 2012). Error bars reflect mean <span class="underline">+</span> s.d..</p><p>(F) Volcano plot showing the expression levels of BRAFi-resistant genes (identified in 501Mel by CRISPR-SAM screen) in 12 human melanoma cell lines. The fold change corresponds to the ratio of expression levels found in resistant and sensitive cell lines. In red: selected genes. <em>EGFR</em> is considered as positive control and <em>SMAD3</em>, <em>BIRC3</em> and <em>SLC9A5</em>; selected as favorite genes for the next steps. <em>SMAD3</em>, <em>BIRC3</em>, <em>SLC9A5</em> and <em>AFAP1</em> are BRAFi-resistance genes and potent tumor-promoting genes (Fig. 1). Raw data are available in Table EV5. (BRAFi for PBV, Appendix Figure S1)</p><p>(G) Heatmap recapitulating the expression levels of the selected hits (red dots in Fig. 2F) in BRAFi-resistant and -sensitive cell lines. Markers of differentiation (<em>MITF</em>, <em>MLANA</em>, <em>OCA2</em>, <em>TYRP1</em>, <em>TYR</em>). In red: resistance genes already published (<em>NRP1</em>, <em>AXL</em>, <em>ZEB1</em>, <em>LPAR1</em>). Scale corresponds to Z scores.</p>
|
https://api.sourcedata.io/file.php?figure_id=39258
|
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||
10.15252/emmm.202013466
|
CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance
|
2021
|
Figure 4
|
<p><strong>Figure 4. BRAFi-Resistance Genes Promote Tumor Growth</strong></p><p>(A) Workflow to identify genes involved in tumor growth from BRAFi-persister cells. Cell populations were subcutaneously grafted on nude mice flanks (3x10<sup>6</sup> cells per mouse); Vem-persister cells (n=11 mice) and PLX8394 (PB)-persister cells (n=12 mice) (Tables EV2). (BRAFi for PBV, Appendix Figure S1)</p><p>(B) Tumor growth curves from BRAFi-persister cells (monitored during 5 months) (Table EV2). V for Vem-resistant cells and PB for PLX8394-resistant cells.</p><p>(C) Distribution of sgRNAs in BRAFi-resistant cells (before xenograft) and in the 7 tumors emerging from the BRAFi-resistant cells (log<sub>10</sub>(sgRNAs counts)). Blue and red lines indicated the enrichment ≥10 fold or ≥100 fold (tumors <em>versus</em> BRAFi-resistant cells (<em>in vitro</em>)). Raw data are available in Table EV7.</p><p>(D) From the seven tumors arising from BRAFi-resistant cells, the common genes (enriched) have been extracted. Ninety-seven genes including <em>SMAD3</em>, <em>BIRC3</em> and <em>SLC9A5</em> are detected in all these 7 tumors. Raw data are available in Table EV7.</p><p>(E) sgRNAs counts in tumors <em>versus</em> sgRNA detected in BRAFi-resistant cell library (<em>in vitro</em>) (respectively, black and orange points) for selected candidates. Each black point corresponds to one tumor. <em>EGFR</em> was the most potent BRAFi-resistant gene (Fig.2). Two replicates have been shown for CRISPR-SAM cell library (orange points). <em>SMAD3</em>, <em>BIRC3</em> and <em>SLC9A5</em> were identified as hits in the three screens (Fig.1, 2 and 4).</p>
|
https://api.sourcedata.io/file.php?figure_id=39262
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||
10.15252/emmm.202013466
|
CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance
|
2021
|
Figure 5
|
<p><strong>Figure 5. Functional Validation of Genes Involved in BRAFi-Resistance and Relapse</strong></p><p>(A) PCA analysis of melanoma cell lines in function of their dedifferentiation states (generated by the webtool <a href="http://systems.crump.ucla.edu/dediff/index.php">http://systems.crump.ucla.edu/dediff/index.php</a>).</p><p>(B) SMAD3 expression increases with melanoma cell dedifferentiation. PCA analysis of <em>SMAD3</em> expression in melanoma cell lines in function of their dedifferentiation states. Scale: red color corresponds to a high <em>SMAD3</em> expression level (unit of the scale : Log2 FPKM).</p><p>(C) <em>SMAD3</em> expression in these four subtypes of melanoma cells (U, undifferentiated; NC, neural crest-like; T, transitory; M, melanocytic). Number in each group: U = 10, NC = 14, T = 12, M = 17. Error bars reflect mean <span class="underline">+</span> s.d.. Multiple comparisons have been done using ordinary one-way ANOVA **: p<0.01, ***p: <0.001.</p><p>(D) SMAD3 gain-of-function increases BRAFi-resistance. Determination of BRAFi half maximal inhibitory concentrations (IC<sub>50</sub> values) for 501Mel cell lines (Log[Vem] (nM)). Parental 501Mel cells (n=5), 501Mel cells expressing dCas9 and HSF1-p65-MS2 (named here 501Mel 2+, n=5), the 501Mel 2+ cells expressing a control guide (backbone, n=5) and the 501Mel 2+cells overexpressing <em>SMAD3</em> (n=3 biologically independent experiments). A representative experiment has been chosen. Dotted line is used to determine the IC<sub>50</sub> values.</p><p>(E) <em>SMAD3</em> and <em>SLC9A5</em> gain-of-function increases invasion capability of melanoma cells. Invasion assays for engineered cells lines (melanoma spheroids): CTR; 501Mel 2+, SMAD3; 501Mel 2+cells overexpressing <em>SMAD3</em>. Two other cell lines overexpressing <em>SLC9A5</em> or <em>BIRC3</em> have been tested. Explanation for ratio calculation is detailed in Fig. EV3A. Results obtained from two biologically independent experiments. (n=5, 4, 6 and 3 spheroids, respectively). Error bars reflect mean <span class="underline">+</span> s.d.. Multiple comparisons have been done using ordinary one-way ANOVA, *: <strong>p<</strong>0.05, ***p: <0.001.</p><p>(F) Characterization of SKMel28 sensitive- and resistant- cell lines (SKMel28S and SKMel28R). Determination of BRAFi half maximal inhibitory concentrations (IC<sub>50</sub> values) (Log[Vem] (nM)). A representative experiment has been chosen among two experiments. Dotted line is used to determine the IC<sub>50</sub> values.</p><p>(G) <em>SMAD3</em> depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib) on BRAFi-resistant cells (SKMel28R). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib; BRAFi). <strong>n=</strong>3 biologically independent experiments. Each histogram represents the mean <strong><span class="underline">+</span></strong> s.d. ; Multiple comparisons have been done using ordinary one-way ANOVA, **: <strong>p<</strong>0.01.</p><p>(H) <em>SMAD3</em> depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib, BRAFi) on BRAFi-resistant cells (Me1402). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib). <strong>n=</strong>3 biologically independent experiments. Each histogram represents the mean <strong><span class="underline">+</span></strong> s.d. ; Multiple comparisons have been done using ordinary one-way ANOVA, *: p<0.05, **: <strong>p<</strong>0.01.</p><p>(I) Validation of SMAD3 knock-down by western-blot experiments in Me1402 cells exposed or not to vemurafenib (BRAFi (Vem) 5µM, 2 days). Cells were exposed to BRAFi 24hr after siRNA transfection. Vemurafenib inhibitory effect on mutated BRAF was evaluated by analysing the phospho-ERK1/2 levels. ERK and HSC70 serves as loading control.</p><p>(J) Validation of SMAD3 inhibitor (SIS3, SMAD3i). Effect of SMAD3i (10µM) on the level of phospho-SMAD3 Ser423/425 in response to TGFβ (2ng/mL, 1hr) (or solvent: HCl 4mM + Bovine serum albumin 1mg/mL) in SKMel28R cells (expressing a high endogenous level of <em>SMAD3</em> mRNA). Serum starved cells (500,000 per well) were pretreated with SMAD3i 10µM (or control solvent) during 2hr before TGFβ addition. SMAD3 and HSC70 serve as loading control for western-blot experiments.</p><p>(K) Inhibitory effect of SMAD3i (SIS3) on the SMAD-responsive luciferase activity. Vector encodes the Firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the SMAD Binding Element (SBE). Cells (10,000 per well) were pretreated with SMAD3i 10µM or control solvent during 1.5hr, and next cells were exposed to TGFβ 10ng/mL (or solvent: HCl 4mM + Bovine serum albumin 1mg/mL) for 6hr. <strong>n=</strong>3 biologically independent experiments. Each histogram represents the mean <strong><span class="underline">+</span></strong> s.d. ; Bilateral Student test (with non-equivalent variances); TGFβ <em>vs</em> TGFβ+SMAD3i *: p<0.05, **: p<0.01</p><p>(L) SMAD3, Phospho-SMAD3 Ser423/425 and BIRC3 expression levels in melanoma cell lines. Four subtypes of melanoma cells (U, undifferentiated; NC, neural crest-like; T, transitory; M, melanocytic) have been compared. 501Mel and M249 cells are melanocytic cells in contrast to dedifferentiated BRAFi-resistant cells (R). Three couples of melanoma cell lines (Sensitive (S) and (R)) have been used to illustrate the SMAD3 and BIRC3 increases in BRAFi-resistant cell lines. HEK293 cells are used as control (kidney). HSC70 serves as loading control for western-blot experiments. Each antibody has been evaluated on individual membrane (explaining the three HSC70, loading controls).</p><p>(M) The vemurafenib decreased melanoma cell density. Cell lines have been exposed to Vem (1 or 5 µM, 84h) to define two groups of cell lines (sensitive (blue) and "resistant" (red) cell lines). Normal human melanocytes (NEHM) have been used to evaluate the effect of Vem on normal cells (mean of 3 independent donors). Representative values (mean of biological triplicates) of <strong>n=</strong>3 biologically independent experiments.</p><p>(N) The SMAD3 inhibitor decreased melanoma cell density. Cell lines have been exposed to SMAD3i (0, 3, 10, 15 or 20 µM, 84h). The cell lines defined as S and R to BRAFi in the item 5M are indicated in blue and red. Normal human melanocytes (NEHM) have been used to evaluate the effect of SMAD3i on normal cells (n=3 independent donors). The SMAD3i effect on NHEMs is weak and manageable for these normal cells (NHEM). Representative values (mean of biological triplicates) of <strong>n=</strong>2 biologically independent experiments.</p><p>(O) The chemical inhibition of SMAD3 by SIS3 (SMAD3i) improved current therapy effect (BRAFi+MEKi; Vem 5µM and Cobi 1µM) on BRAFi-resistant cells (SKMel28R, M229R & M238R). Cells have been treated as detailed for Fig. 5M. Representative values (mean of biological triplicates) of <strong>n=</strong>2 biologically independent experiments.</p><p>Data information: Western-blot results are representative of at least two independent experiments. Source data and unprocessed original blots are available in Figure appendix S2 source data.</p><p>See also Fig. EV3.</p>
|
https://api.sourcedata.io/file.php?figure_id=39264
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10.15252/emmm.202013466
|
CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance
|
2021
|
Figure 6
|
<p><strong>Figure 6. The Transcription Factor AhR Drives <em>SMAD3</em> Expression</strong></p><p>(A) AhR binding sites (xenobiotic responsive element (XRE); GCGTG) in human <em>SMAD3</em> proximal promoter (-500_+1 bp).</p><p>(B) AhR activation by exogenous and endogenous ligands promotes <em>SMAD3</em> induction. 501Mel cells AhR wild-type or knock-out have been exposed to exogenous and endogenous AhR ligands; TCDD (5nM) or ITE (10µM) or the solvent (DMSO) during 10 days. <strong>n=</strong>5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean <strong><span class="underline">+</span></strong> s.d.; Multiple comparisons have been done using ordinary one-way ANOVA, <strong>***: p<</strong>0.001.</p><p>(C) AhR activation by exogenous and endogenous AhR ligands promotes <em>TIPARP</em> induction.</p><p><strong>501Mel cells have been treated as described in B. n=</strong>5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean <strong><span class="underline">+</span></strong> s.d.; Multiple comparisons have been done using ordinary one-way ANOVA, <strong>**: p<</strong>0.01, <strong>***: p<</strong>0.001.</p><p>(D) AhR antagonist (CH-223191) reduces <em>SMAD3</em> and <em>TIPARP</em> expression levels. SKMel28 cells (AhR wild-type) have been exposed to CH-223191 (5µM) or the solvent (DMSO) during 7 days. <strong>n=</strong>6 biologically independent experiments. Each histogram represents the mean <strong><span class="underline">+</span></strong> s.d.; Bilateral Student test (with non-equivalent variances): <strong>***: p<</strong>0.001.</p><p>(E) Loss of AhR reduces <em>SMAD3</em> expression levels. SMAD3 expression has been investigated in SKMel28 cells AhR wild-type (WT) or knock-out (KO). SKMel28R, have been obtained from SKMel28S by chronic exposure to non-lethal doses of BRAFi (Hugo <em>et al</em>, 2015b). R for BRAFi-resistant SKMel28 cells and S for sensitive. <strong>n=</strong>2 biologically independent experiments. Each histogram represents the mean <strong><span class="underline">+</span></strong> s.d..</p>
|
https://api.sourcedata.io/file.php?figure_id=39266
|
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] |
||
10.15252/emmm.202013466
|
CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance
|
2021
|
Figure 7
|
<p><strong>Figure 7. SMAD3 Drives Phenotype Switching and Resistance to Melanoma Therapies</strong></p><p>(A) SMAD3 signature has been established by comparing BRAFi-resistance genes and SMAD3-regulated genes identified by chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) (Ramachandran <em>et al</em>, 2018).</p><p>(B) Basal SMAD3-signature in six melanoma cell lines (S for sensitive to BRAFi and R for Resistant). <strong>n=</strong>2 biologically independent experiments. Each histogram represents the median <strong>with interquartile range.</strong></p><p>(C) Inducibility of SMAD3-signature in six melanoma cell lines exposed to TGF-β (10 ng/mL, 48h). Data were normalized to cell lines exposed to solvent (4mM HCl + 1mg/mL human BSA). Values obtained with the TGF-β stimulated M238R cell line have been set to 1. <strong>n=</strong>2 biologically independent experiments. Each histogram represents the median <strong>with interquartile range.</strong></p><p>(D) SMAD3-signature discriminates differentiation states of 53 melanoma cell lines (Tsoi <em>et al</em>, 2018). The SMAD3-signature in four subgroups of melanoma cell lines. Each point represents a cell line (n= 17, 12, 14 and 10 respectively for melanocytic, transitory, neural crest-like and undifferentiated cell lines); Each histogram represents the median with interquartile range; Multiple comparisons have been done using ordinary one-way ANOVA; <strong>**: p<</strong>0.01, <strong>***: p<</strong>0.001.</p><p>(E) Heatmap depicting mRNA levels of SMAD3-signature in BRAF(V600E) non-treated melanoma patients (dataset from TCGA; SKCM, BRAF(V600E) mutated: n=118). Three pigmentation genes (<em>MITF</em>, <em>MLANA</em> and <em>TYR</em>) have been added to highlight the differentiation states of tumors. ~20% of tumors are considered as dedifferentiated tumors (red box) with a high SMAD3-signature. Scale corresponds to Z scores.</p><p>(F) SMAD3-signature in pre-treatment biopsies from responders and non-responders to anti-PD-1 treatment (from the Hugo's cutaneous melanoma cohort, n=15 and 13 respectively) (Hugo <em>et al</em>, 2016). Each histogram represents the median with interquartile range, one tailed Mann-Whitney test; *: <strong>p=</strong>0.0324.</p><p>(G) SMAD3-signature in two groups (before BRAFi treatment or during relapse) of V600E patients from the Rizos's cohort (Rizos <em>et al</em>, 2014). (14 on 16 patients displayed an upregulation of SMAD3- signature during relapse).</p><p>(H) SMAD3 depletion decreases expression of SMAD3-regulated genes. <em>SMAD3</em> knock-down by siRNA decreased mRNA expression of BRAFi-resistance genes in SKMel28R and Me1402. <em>NRP1</em> mRNA was not detected in our experimental conditions. <strong>n=</strong>3 biologically independent experiments. Each histogram represents the mean <strong><span class="underline">+</span></strong> s.d. ; Bilateral Student test (with non-equivalent variances) *: <strong>p<</strong>0.05, **: <strong>p<</strong>0.01, ***: <strong>p<</strong>0.001. Dotted line highlights the value of 1.</p><p>(I) SMAD3 signature overlapped with mesenchymal signature in melanoma tumors (TCGA, n=459) (Akbani <em>et al</em>, 2015). Melanocytic signature highlights the differentiation states of tumors (Corre <em>et al</em>, 2018). SMAD3 signature and mesenchymal signature correlate in melanoma tumors (TCGA, n=459) (Mak <em>et al</em>, 2016). Scale corresponds to Z scores.</p><p>(J) The SMAD3 signature overlaps with the glioblastoma mesenchymal subtype (Jin <em>et al</em>, 2017). Scale corresponds to Z scores.</p><p><strong>EXPENDED VIEW FIGURE LEGENDS</strong></p>
|
https://api.sourcedata.io/file.php?figure_id=39267
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||
27585866
|
10.15252/embj.201694550
|
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment
|
2016
|
Figure 1
|
<p><strong>Figure 1. Loss of </strong><strong><em>Eed</em></strong><strong> affects H3K27 methylation and reduces proliferating cells in the intestinal epithelium. </strong></p>
<p>All analyses were performed using <em>AhCre Eed</em><em><sup>+/+</sup></em> and <em>AhCre Eed</em><em><sup>fl</sup></em><em><sup>/</sup></em><em><sup>fl</sup></em> mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.</p>
<p>A) Immunohistochemistry analysis of small intestinal sections, using the indicated antibodies.</p>
<p>B) Western blot analysis with protein extracts obtained from <em>ex vivo</em>–purified intestinal crypts and probed with the indicated antibodies.</p>
<p>C) Haematoxylin and eosin staining of sections prepared from different intestinal tracts.</p>
<p>D) Immunohistochemistry analysis of small intestinal sections, using specific antibodies for the proliferation marker KI67.</p>
<p>E) Expression analysis by qRT-PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts. Graphs show mean ± SD for three replicates.</p>
<p>All scale bars represent 100μm.</p>
<p> </p>
|
https://api.sourcedata.io/file.php?figure_id=9811
|
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|
27585866
|
10.15252/embj.201694550
|
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment
|
2016
|
Figure 2
|
<p><strong>Fig</strong><strong>ure </strong><strong>2. Loss of </strong><strong><em>Eed</em></strong><strong> perturbs cell differentiation </strong><strong>in the intestinal epithelium</strong><strong>.</strong></p>
<p>A) PAS and (B) Alcian blue staining of sections prepared from the different intestinal tracts isolated from <em>AhCre Eed</em><em><sup>+/+</sup></em> and <em>AhCre Eed</em><em><sup>fl</sup></em><em><sup>/</sup></em><em><sup>fl</sup></em> mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.</p>
<p>C) Quantification (mean ± SD) of Goblet cells in crypts and villi from the same mice sown in A and B. More than 100 crypts and villi were scored for each condition after 15 days from the first β-naphthoflavone administration.</p>
<p>D) Immunohistochemistry analyses in <em>AhCre Eed</em><em><sup>+/+</sup></em> and <em>AhCre Eed</em><em><sup>fl</sup></em><em><sup>/</sup></em><em><sup>fl</sup></em> mice injected with β-naphthoflavone and sacrificed after 15 days using a lysozyme-specific antibody (upper panels: LYZ, a Paneth cell–specific marker). Histochemical assay probing for alkaline phosphatase activity is presented in the middle panels (ALPI, an enterocyte-specific marker). Combined Ki67 immunohistochemistry and histochemical Alcian blue staining is presented in the lower panels (Alcian/KI67).</p>
<p>E) GFP expression from an <em>Lgr5-eGFP </em>transgene identifying LGR5+ ISCs in near-native agarose-embedded sections from <em>Eed</em><em>+/+</em> and <em>Eed</em><em>-/- </em>mice at 30 days from the first β-naphthoflavone administration. Cell nuclei were counterstained with DAPI.</p>
<p>All scale bars represent 100μm.</p>
|
https://api.sourcedata.io/file.php?figure_id=9812
|
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|
27585866
|
10.15252/embj.201694550
|
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment
|
2016
|
Figure 3
|
<p><strong>Figure 3. Loss of </strong><strong>Eed</strong><strong> affects cell plasticity at the bottom of the crypt.</strong></p>
<p>A) Radiation-induced intestinal regeneration: <em>AhCre Eed</em><em><sup>+/+</sup></em> and <em>AhCre Eed</em><em><sup>fl</sup></em><em><sup>/</sup></em><em><sup>fl</sup></em> mice were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 4 and 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (n=6).</p>
<p>B) Immunohistochemistry analyses in <em>AhCre Eed</em><em><sup>+/+</sup></em> and <em>AhCre Eed</em><em><sup>fl</sup></em><em><sup>/</sup></em><em><sup>fl</sup></em> mice treated as in A using H3K27me3 and KI67 specific antibodies at 8 days post-irradiation.</p>
<p>C) Quantification (mean ± SD) of KI67 positive cells per crypt (B). More than 100 crypts were scored for each condition.</p>
<p>D) <em>In vitro</em> organoids formation using crypts isolated from <em>AhCre Eed</em><em><sup>+/+</sup></em> and <em>AhCre Eed</em><em><sup>-/-</sup></em> mice 15 days after the first β-naphthoflavone injection. Organoids pictures were taken 5 days later at low and higher magnification (insets).</p>
<p>All scale bars represent 100μm.</p>
|
https://api.sourcedata.io/file.php?figure_id=9813
|
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] |
|
27585866
|
10.15252/embj.201694550
|
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment
|
2016
|
Figure 4
|
<p><strong>Figure 4. Loss of </strong><strong><em>Eed</em></strong><strong> induces transcriptional activation of genes involved in cell cycle arrest and secretory lineage specification. </strong></p>
<p>A) Volcano plot showing the expression changes with a minimal fold change of 3 (FC=3) at genome-wide levels in <em>AhCre Eed</em><em><sup>-/- </sup></em>crypts relative to <em>AhCre Eed</em><em><sup>+/+</sup></em> crypts 15 days after β-naphthoflavone administration.</p>
<p>B) Bar plot showing the percentage of H3K27me3-positive promoters among the differentially regulated genes in <em>Eed</em><em><sup>-/-</sup></em> crypts. All gene promoters and 5 independent random sampling of 167 gene promoters extracted the mouse genome are presented as specificity controls.</p>
<p>C) Intensity profiles for H3K27me3 and SUZ12 occupancy at the promoters of up-regulated genes with respect to down-regulated genes in <em>Eed</em><em><sup>-/-</sup></em> crypts.</p>
<p>D) Genomic snapshots of the <em>Cdkn2a</em> locus showing SUZ12 occupancy and H3K27me3 deposition at the promoter regions of both the p16 and the ARF isoforms.</p>
<p>E) Genomic snapshots of the RNAseq profile of the<em> Cdkn2a</em> locus in <em>AhCre Eed</em><em><sup>+/+ </sup></em>and <em>AhCre Eed</em><em><sup>-/-</sup></em> crypts 15 days after β-naphthoflavone administration.</p>
<p>F) Expression analysis by qRT-PCR (mean ± SD) of both <em>Cdkn2a</em> isoforms (<em>p16Ink4A</em> and <em>p19Arf</em>) in <em>AhCre Eed</em><em><sup>+/+ </sup></em>and <em>AhCre Eed</em><em><sup>-/-</sup></em> crypts 15 days after β-naphthoflavone administration.</p>
<p>G) Gene set enrichment analysis (GSEA) using specific secretory and enterocytic transcriptional signatures with respect to the list of up-regulated genes in <em>Eed</em><em><sup>-/-</sup></em> crypts.</p>
|
https://api.sourcedata.io/file.php?figure_id=9814
|
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] |
|
27585866
|
10.15252/embj.201694550
|
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment
|
2016
|
Figure 5
|
<p><strong>Figure 5. Loss of </strong><strong><em>Cdkn2a</em></strong><strong> restores cell proliferation in </strong><strong><em>Eed</em></strong><strong><em><sup>-/-</sup></em></strong><strong> intestinal crypts. </strong></p>
<p>A) Haematoxylin and eosin (top panels) and PAS staining (bottom panels) of sections prepared from different intestinal tracts in <em>AhCre</em><em> Cdkn2a</em><em><sup>-/-</sup> Eed</em><em><sup>+/+</sup></em> and <em>AhCre Cdkn2a</em><em><sup>-/-</sup> Eed</em><em><sup>fl</sup></em><em><sup>/</sup></em><em><sup>fl</sup></em> mice 15 days after the first β-naphthoflavone administration (n=5).</p>
<p>B) Quantification (mean ± SD) of Goblet cells along the crypt-to-villus axis from the staining shown in A. More than 100 crypts and villi were scored for each condition.</p>
<p>C) Immunhistochemistry analysis using anti-KI67 antibody of intestinal sections prepared from <em>AhCre</em><em> Cdkn2a</em><em><sup>-/-</sup> Eed</em><em><sup>+/+</sup></em> and <em>AhCre Cdkn2a</em><em><sup>-/-</sup> Eed</em><em><sup>fl</sup></em><em><sup>/</sup></em><em><sup>fl</sup></em> mice 15 days after the first β-naphthoflavone administration.</p>
<p>D) Radiation-induced intestinal regeneration: <em>AhCre</em><em> Cdkn2a</em><em><sup>-/-</sup> Eed</em><em><sup>+/+</sup></em> and <em>AhCre Cdkn2a</em><em><sup>-/-</sup> Eed</em><em><sup>fl</sup></em><em><sup>/</sup></em><em><sup>fl</sup></em> mice (n=6) were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (first panels) and Alcian Blue (last panels). Sections were also stained for immunohistochemical analysis (second and third panels) using H3K27me3 and Ki67-specific antibodies.</p>
<p>All scale bars represent 100μm.</p>
|
https://api.sourcedata.io/file.php?figure_id=9815
|
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] |
|
27585866
|
10.15252/embj.201694550
|
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment
|
2016
|
Figure 6
|
<p><strong>Figure 6. PRC2 directly maintains </strong><strong><em>Atoh1</em></strong><strong> and </strong><strong><em>Gfi1</em></strong><strong> repression</strong></p>
<p>A) Expression analysis by qRT-PCR (mean ± SD) of the indicated intestinal epithelial cell markers regulated by the Wnt and Notch signaling pathways that are involved in stem cell homeostasis, regionalization, and Goblet cell differentiation (n=3).</p>
<p>B) Genomic snapshots of the indicated genomic loci for SUZ12 occupancy and H3K27me3 deposition in intestinal crypts of wild type mice (n=3).</p>
<p>C) Expression analysis by qRT-PCR (mean ± SD) of the Goblet cell markers (<em>Atoh1</em>, <em>Gfi1</em>, <em>Spdef</em>), early secretory precursor cells (<em>Dll1</em>), NOTCH targets (<em>Hes1</em>, <em>Olfm4</em>) and intestinal stem cells (<em>Lgr5</em>, <em>Rnf43</em>) in wild type intestinal crypts purified 38hrs after treatment with the NOTCH inhibitor DBZ. DMSO served as vehicle negative control (n=4).</p>
<p>D) ChIP analysis of <em>Atoh1</em> and <em>Gfi1</em> loci using H3K27me3 specific antibody. The same crypts described in C were used. H3K27me3 levels were normalized on Histone H3 density (percentage of H3 signal). Graphs show mean ± SD.</p>
<p>E) Graphic model highlighting the role of PRC2 in restricting secretory lineage differentiation and cell cycle checkpoint activation in TA cells.</p>
|
https://api.sourcedata.io/file.php?figure_id=9816
|
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"Q3UZZ2"
]
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] |
|
10.15252/embr.201845946
|
Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA
|
2018
|
Figure 1
|
<sd-panel> <p><strong>Figure 1. Formation of condensates of FtsZ·SlmA·SBS in crowding conditions</strong>.</p> <ol type="A"> <li> <p>Scheme of the <em>E. coli</em> division elements involved in the formation of condensates. The concentrations of FtsZ, SlmA and SBS used in this study were 12, 5 and 1 μM, unless indicated otherwise.</p> </li> <li> <p>Representative confocal images of the FtsZ·SlmA·SBS condensates formed in 150 g/L dextran 500 or Ficoll 70, and absence of condensates in dilute solution. Scale bars: 20 μm, except for images at higher magnification in dextran (5 μm, second row).</p> </li> <li> <p>Turbidity of FtsZ·SlmA·SBS in buffer (n = 3), in 150 g/L dextran 500 (n = 5) or Ficoll 70 (n = 3) and in 50 g/L PEG 8 (n = 3). Data correspond to the average ± S.D.</p> </li> </ol> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=26180
|
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"P0A9A6"
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{
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{
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] |
||
10.15252/embr.201845946
|
Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA
|
2018
|
Figure 2
|
<sd-panel> <p><strong>Figure 2. Dependence of the formation of FtsZ·SlmA·SBS condensates on protein and salt concentrations.</strong></p> <ol type="A"> <li> <p>Formation of condensates as a function of FtsZ and SlmA·SBS concentration, as measured by turbidity, in working buffer (300 mM KCl). SlmA concentration was fivefold that of SBS (except at 0.5 μM SBS, where SlmA concentration was 3 μM). Data are the average of 2 independent measurements. Errors (S.D.), symmetrical, are depicted as white discs.</p> </li> <li> <p>Representative confocal images of the FtsZ·SlmA·SBS condensates at the specified salt concentrations. The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 µM, respectively. Scale bars: 5 μm.</p> </li> </ol> <p>Data information: All measurements in 150 g/L dextran 500.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=26181
|
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]
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{
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"ext_ids": "P0C093",
"ext_tax_ids": "83333",
"ext_tax_names": "Escherichia coli K-12",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
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"role": "assayed",
"text": "SlmA",
"type": "geneprod",
"uniprot_ids": [
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]
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] |
||
10.15252/embr.201845946
|
Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA
|
2018
|
Figure 3
|
<sd-panel> <p><strong>Figure 3. Dynamism of FtsZ·SlmA·SBS condensates.</strong></p> <ol type="A"> <li> <p>Representative confocal images showing final state after addition of FtsZ-Alexa 488 to FtsZ·SlmA·SBS complexes (FtsZ labeled with Alexa 647) in 150 g/L dextran. Below, images showing the stepwise diffusion of FtsZ-Alexa 488 into the condensates containing FtsZ-Alexa 647 at the indicated times in seconds (time zero, beginning of visualization for that particular condensate) and corresponding intensity profiles at selected times in the green channel. The profile in the red channel is shown as a reference and varies slightly among images. Scale bars: 5 μm (top row) and 4 μm (bottom row).</p> </li> <li> <p>Stepwise diffusion of FtsZ-Alexa 488 into FtsZ·SlmA·SBS condensates (FtsZ labeled with Alexa 647) at the indicated times in seconds (time zero, beginning of visualization for those particular condensates) in 100 g/L PEG. Scale bars: 4 μm.</p> </li> <li> <p>Assembly of FtsZ fibers upon GTP addition (0.5 mM) to FtsZ·SlmA·SBS condensates and condensates formed after FtsZ fibers disassembly at the indicated times in minutes (time zero, GTP addition) in 150 g/L dextran. Scale bars: 5 μm. Scheme of the dynamic process on the right. The number of condensates decreases upon fibers formation, and they rearrange upon GTP depletion and fibers disassembly.</p> </li> </ol> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=26183
|
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"mapping_status": "mapped",
"ncbi_gene_id": null,
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] |
||
10.15252/embr.201845946
|
Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA
|
2018
|
Figure 4
|
<sd-panel> <p><strong>Figure 4. Microfluidic encapsulation of FtsZ·SlmA·SBS in the PEG/dextran LLPS system inside microdroplets stabilized by the <em>E. coli</em> lipid mixture.</strong></p> <ol type="A"> <li> <p>Scheme of the encapsulation setup and illustration, on the right, of the distribution of species within the encapsulated LLPS system.</p> </li> </ol> <p>B-D. Representative confocal images of the microdroplets without (B) and with GTP (C and D). The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 μM, respectively (B and C) or 6, 3 and 0.5 μM respectively (D). 1 mM (C) or 2 mM GTP (D). Scale bars: 40 μm except in images on the far right (20 μm), which are either a magnification of the indicated region in the merged image (B) or independent images at higher magnification (C, D).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=26185
|
[
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"ncbi_gene_id": null,
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"text": "FtsZ",
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]
},
{
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}
] |
||
10.15252/embr.201845946
|
Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA
|
2018
|
Figure 5
|
<sd-panel> <p><strong>Figure 5. Microfluidic encapsulation of FtsZ·SlmA·SBS in the PEG/DNA LLPS system inside microdroplets and GTP induced FtsZ fiber formation.</strong></p> <ol type="A"> <li> <p>Scheme of the encapsulation procedure followed for the PEG/DNA LLPS system and illustration, on the right, of the distribution of species within the encapsulated LLPS system.</p> </li> </ol> <p>B,C. Representative confocal images of the microdroplets stabilized by the <em>E. coli</em> lipid mixture containing the biphasic PEG/DNA mixture and the FtsZ·SlmA·SBS complex without and with 2 mM GTP, respectively. Last image in (B) focuses in the lipid interface to show the high density of condensates. Scale bars: 20 μm except in images on the far right (10 μm), which are an independent image at higher magnification (B) or a magnification of the indicated region in the merged image (C).</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=26186
|
[
{
"ext_dbs": "Uniprot",
"ext_ids": "P0A9A6",
"ext_tax_ids": "83333",
"ext_tax_names": "Escherichia coli K-12",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "FtsZ",
"type": "geneprod",
"uniprot_ids": [
"P0A9A6"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P0C093",
"ext_tax_ids": "83333",
"ext_tax_names": "Escherichia coli K-12",
"mapping_source": "direct",
"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "SlmA",
"type": "geneprod",
"uniprot_ids": [
"P0C093"
]
}
] |
||
10.15252/embr.202050264
|
Extended synaptotagmin regulates contact site structure and lipid transfer function in vivo
|
2020
|
Figure 1
|
<sd-panel> <p><strong>Figure 1: Single gene encoding Esyt expressed in <em>Drosophila melanogaster.</em></strong></p> <p><strong>(A)</strong> Schematic showing the phylogeny of extended synaptotagmin in different organisms: <em>Drosophila melanogaster</em> (Dm), <em>Saccharomyces cerevesiae</em> (Tricalbin), <em>Homo sapiens</em> (Hs), <em>Mus Musculus</em> (Mm), <em>Rattus norvegicus</em> (Rn), <em>Caenorhabditis elegans</em> (Ce), <em>Danio rerio</em> (Dr) and <em>Xenopus tropicalis</em> (Xt). Bold lines - Sub nodes. Numbers on the nodes - Bootstrap value. Colours - Esyt1, Esyt2 and Esyt3 clusters.</p> <p><strong>(B)</strong> Schematic of dEsyt-PA protein domain structure aligned with mammalian Esyts and yeast tricalbins (Image generated using IBS, Illustrator for Biological sciences Version 1.0 software <a href="http://ibs.biocuckoo.org/">http://ibs.biocuckoo.org/</a>)(Liu et al., 2015). Amino acid numbers in each protein are indicated. SMP-SMP domain. C2A, B, C, D, E are the various C2 domains.</p> <p><strong>(C)</strong> Quantitative real time PCR analysis showing head enrichment of <em>dEsyt</em>; the X-axis indicates the wild type tissues from which RNA was isolated and Y-axis indicates the <em>dEsyt</em> transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).</p> <p><strong>(D)</strong> Quantitative real time PCR analysis showing <em>dEsy</em>t transcript level expression in head RNA samples from wild type and <em>s<sup>OD</sup></em>. Y-axis indicates the <em>dEsyt</em> transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).</p> <p>Data information: Bar graphs with mean ± SD are shown. Statistical tests: (C and D) Students unpaired t test (two tailed). ns - Not significant. ****p value <0.0001.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=34147
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "assayed",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
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"ext_tax_names": "Drosophila melanogaster",
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"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
},
{
"ext_dbs": "NCBI gene",
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"ext_tax_names": "Drosophila melanogaster",
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"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "assayed",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "assayed",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "35662",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "35662",
"original_type": "gene",
"role": "intervention",
"text": "sO",
"type": "geneprod",
"uniprot_ids": [
"Q27350"
]
}
] |
||
10.15252/embr.202050264
|
Extended synaptotagmin regulates contact site structure and lipid transfer function in vivo
|
2020
|
Figure 2
|
<sd-panel> <p><strong>Figure 2: <em>dEsyt</em> is dispensable for phototransduction.</strong></p> <p><strong>(A)</strong> Representative ERG trace from dark reared 0-1 day old flies of the indicated genotype stimulated by 2s flash of green light. The duration of the stimulating light is shown. Y-axis shows the amplitude of the ERG response. X-axis shows the duration of the recording.</p> <p><strong>(B)</strong> Graph showing ERG amplitude normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m.</p> <p><strong>(C)</strong> Quantification of the mean fluorescence intensity of the deep pseudopupil formed by the PIP<sub>2</sub> probe PH-GFP in the one day old flies of the indicated genotypes (n=10).</p> <p><strong>(D)</strong> Deep pseudopupil imaging of PIP<sub>2</sub> levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.</p> <p><strong>(E)</strong> Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalised to the intensity of the first image. (n=10 biological replicates). Error bars represent s.e.m.</p> <p><strong>(F)</strong> Quantification of the time course of retinal degeneration. 10 ommatidia from 5 separate flies of each genotype were scored and plotted. Y-axis is the number of intact rhabdomeres/ommatidium. The maximum value possible is 7. X-axis is the age of the flies post eclosion.</p> <p><strong>(G)</strong> Representative optical neutralization images showing rhabdomere integrity of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CL- Constant Light)</p> <p><strong>(H)</strong> Representative ON images showing the rescue of rhabdomere structural integrity in <em>dEsyt<sup>KO</sup></em> mutants on expressing the dEsyt::mCherry transgene. Genotypes of the flies are indicated on the left. Rearing conditions and the age of the flies are indicated on top.</p> <p>Data information: Scatter plots and XY plots with mean ± SD are shown. Statistical tests: (B and C) Student's unpaired t test. (E and F) Two-Way ANOVA Grouped analysis with Bonferroni post-tests to compare replicate means. ns - Not significant; **p value < 0.01; ****p value<0.0001.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=34148
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "intervention",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "intervention",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
}
] |
||
10.15252/embr.202050264
|
Extended synaptotagmin regulates contact site structure and lipid transfer function in vivo
|
2020
|
Figure 3
|
<sd-panel> <p><strong>Figure 3:Loss of <em>dEsyt</em> enhances <em>rdgB</em> mutant phenotypes.</strong></p> <p><strong>(A) (i)</strong> Representative optical neutralization (ON) images showing rhabdomere structure of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CD- Constant Dark). (ii) <strong>TEM images showing a single ommatidium from the photoreceptors of <em>rdgB<sup>9</sup></em> and <em>rdgB<sup>9</sup>;dEsyt<sup>KO</sup></em> mutants reared in 12 hr L/D cycle ( / ) for 2 days. Scale bar: 1µm (asterisk indicates degenerated rhabdomere).</strong></p> <p><strong>(B)</strong> Quantification of the time course taken for retinal degeneration. 10 ommatidia were scored from 5 flies of each genotype and plotted.</p> <p><strong>(C)</strong> Representative ERG traces showing the duration of light pulse, X-axis indicates time in msec and Y-axis indicates the average ERG amplitude in mV.</p> <p><strong>(D)</strong> Graph showing ERG amplitude of <em>rdgB<sup>9</sup></em> and <em>rdgB<sup>9</sup>;dEsyt<sup>KO</sup></em> normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m.</p> <p><strong>(E)</strong> Deep pseudopupil imaging of PIP<sub>2</sub> levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.</p> <p><strong>(F)</strong> Quantification of the mean fluorescence intensity of the PIP<sub>2</sub> probe PH-GFP from the deep pseudopupil formed by one day old flies of the indicated genotypes (n=10 biological replicates).</p> <p><strong>(G)</strong> Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalized to the intensity of the first image. (n=10 biological replicates).</p> <p>Data information: Scatter plots and XY plots with mean ± SD are shown. Statistical tests: (D and F) Student's unpaired t test. (B and G) Two-Way ANOVA Grouped analysis with Bonferroni post-tests to compare replicate means. ns - Not significant; **p value < 0.01; ***p value < 0.001; ****p value<0.0001.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=34149
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "intervention",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "intervention",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
}
] |
||
10.15252/embr.202050264
|
Extended synaptotagmin regulates contact site structure and lipid transfer function in vivo
|
2020
|
Figure 4
|
<sd-panel> <p><strong>Figure 4: dEsyt localizes to MCS and determines RDGB localization.</strong></p> <p><strong>(A)</strong> Confocal images showing the localization of endogenous dEsyt protein in photoreceptors of one day old dark reared flies probed with antibody against dEsyt. Scale bar: 5µm. <em>dEsyt<sup>KO</sup></em> shows no staining when probed with dEsyt antibody.</p> <p><strong>(B)</strong> Confocal images showing the localization of exogenously expressed dEsyt::mCherry protein expressed using the eye specific Rh1-Gal4 in one day old dark reared flies. Rh1-Gal4 is shown as a control. A single ommatidium is shown. Scale bar: 5 µm. Phalloidin marks F-actin staining and highlights rhabdomeres R1-R7.</p> <p><strong>(C)</strong> Western blot from the head extracts of wild type and <em>dEsyt<sup>KO</sup></em> probed with the antibody against RDGB. Rearing conditions, age of the flies and genotype is indicated on top of the blot. Tubulin was used as the loading control.</p> <p>(<strong>D, E</strong>) Confocal images showing the localization of RDGB in wild type and <em>dEsyt<sup>KO</sup></em> photoreceptors of flies which are (D) 1 day old-dark reared and (E) 6 days old- exposed to constant illumination. For (D) and (E) RDGB visualized using an antibody against the endogenous protein. Rhabdomeres are outlined using phalloidin which marks F-actin. Scale bar: 5 µm.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=34150
|
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{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "intervention",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
},
{
"ext_dbs": "Uniprot///Uniprot///Uniprot///Uniprot",
"ext_ids": "A0A0B4K6F9///Q7KS16///A0A0B4KGU9///Q9VC62",
"ext_tax_ids": "7227///7227///7227///7227",
"ext_tax_names": "Drosophila melanogaster///Drosophila melanogaster///Drosophila melanogaster///Drosophila melanogaster",
"mapping_source": "unknown",
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},
{
"ext_dbs": "Uniprot///Uniprot///Uniprot///Uniprot",
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{
"ext_dbs": "Uniprot///Uniprot///Uniprot///Uniprot",
"ext_ids": "A0A0B4K6F9///Q7KS16///A0A0B4KGU9///Q9VC62",
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"ext_tax_names": "Drosophila melanogaster///Drosophila melanogaster///Drosophila melanogaster///Drosophila melanogaster",
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},
{
"ext_dbs": "Uniprot///Uniprot///Uniprot///Uniprot",
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"ext_tax_ids": "7227///7227///7227///7227",
"ext_tax_names": "Drosophila melanogaster///Drosophila melanogaster///Drosophila melanogaster///Drosophila melanogaster",
"mapping_source": "unknown",
"mapping_status": "unmapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": []
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "intervention",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P43125",
"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": "RDGB",
"type": "geneprod",
"uniprot_ids": [
"P43125"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "intervention",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P43125",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
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"mapping_status": "mapped",
"ncbi_gene_id": null,
"original_type": "protein",
"role": "assayed",
"text": "RDGB",
"type": "geneprod",
"uniprot_ids": [
"P43125"
]
},
{
"ext_dbs": "Uniprot",
"ext_ids": "P43125",
"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": "RDGB",
"type": "geneprod",
"uniprot_ids": [
"P43125"
]
}
] |
||
10.15252/embr.202050264
|
Extended synaptotagmin regulates contact site structure and lipid transfer function in vivo
|
2020
|
Figure 5
|
<sd-panel> <p><strong>Figure 5:dEsyt stabilizes MCS in photoreceptors.</strong></p> <p><strong>(A) TEM images of a single ommatidium from wild type photoreceptors of flies reared in dark (i) day 1 (D1) and (iii) day 14 (D14), scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day 1 (iv) day 14. Scale bar: 200 nm. Origin boxes for magnification shown. Arrow in (Aiv) indicates the SMC forming an MCS with the microvillar PM. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules.</strong></p> <p><strong>(B) Quantification of the number of MCS per wild type photoreceptor of day 1 and day 14 old flies reared in dark (from figure 6A), Y-axis indicates the number of MCS/photoreceptor. n=30 photoreceptors from 3 separate flies (R1-R6) for (i). n=9 photoreceptors from 3 separate flies (R7) in (ii).</strong></p> <p><strong>(C) TEM images of a single ommatidium from <em>norpA<sup>P24</sup></em> photoreceptors of flies reared in dark (i) day 1 and (iii) day 14, scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day1 (iv) day 14. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM.</strong></p> <p><strong>(D) Quantification of the number of MCSs per photoreceptor (from figure 6C), X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7)</strong></p> <p><strong>(E) TEM images of a single ommatidium from (i) wild type and (iii) <em>dEsyt<sup>KO</sup></em> photoreceptors of 0-1 day old flies reared in dark, scale bar: 1µm (red asterisk indicates R7 photoreceptor) (ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM.</strong></p> <p><strong>(F) Quantification of the number of MCS per photoreceptor of wild type day 1 v/s <em>dEsyt<sup>KO</sup></em> day 1 old flies reared in dark, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7).</strong></p> <p>Data information: Scatter plots with mean ± SD are shown. Statistical tests: (B, D and F) Student's unpaired t test. ns - Not significant; ***p value <0.001.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=34151
|
[
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "intervention",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
},
{
"ext_dbs": "NCBI gene",
"ext_ids": "42929",
"ext_tax_ids": "7227",
"ext_tax_names": "Drosophila melanogaster",
"mapping_source": "ncbi_gene",
"mapping_status": "mapped",
"ncbi_gene_id": "42929",
"original_type": "gene",
"role": "intervention",
"text": "Esyt",
"type": "geneprod",
"uniprot_ids": [
"A0A0B4K6F9",
"A0A0B4KGU9",
"Q7KS16",
"Q9VC62"
]
}
] |
||
10.15252/embr.202050264
|
Extended synaptotagmin regulates contact site structure and lipid transfer function in vivo
|
2020
|
Figure 6
|
<sd-panel> <p><strong>Figure 6: dEsyt is necessary for PLCβ-dependent modulation of ER-PM MCS.</strong></p> <p><strong>(A) TEM images of a single ommatidium from photoreceptors of (i) <em>dEsyt<sup>KO</sup></em>- day 1 (iii) <em>dEsyt<sup>KO</sup></em>- day 14 flies reared in dark, scale bar: 1 µm except for (Ai): 2 µm (red asterisk indicates R7 photoreceptor). (A-ii, iv)</strong> Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. <strong>Boxes represent the origin boxes for magnification. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules.</strong></p> <p><strong>(B) Quantification of the number of MCS per photoreceptor, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor. n=9 photoreceptors from 3 separate flies for R7.</strong></p> <p><strong>(C)</strong> TEM images of a single ommatidium from photoreceptors of <strong>(i)</strong> <em>norpA<sup>P24</sup>;dEsyt<sup>KO</sup></em>- day 1 <strong>(iii)</strong> <em>norpA<sup>P24</sup>;dEsyt<sup>KO</sup></em>- day 14 flies reared in dark, scale bar: 1 µm (red asterisk indicates R7 photoreceptor). <strong>(C-ii, iv)</strong> Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. <strong>Arrows indicate the SMC forming an MCS with the microvillar PM.</strong></p> <p><strong>(D) Quantification of the number of MCS per photoreceptor, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor. (i) n=30 photoreceptors from 3 separate flies for R1-R6. (ii) n=9 photoreceptors from 3 separate flies for R7.</strong></p> <p><strong>(E)</strong> TEM images of a single ommatidium from photoreceptors of (i) <em>rdgB<sup>9</sup></em>-day1 and (iii) <em>rdgB<sup>9</sup>;dEsyt<sup>KO</sup></em> - day 1 flies reared in dark, scale bar: 1 µm (red asterisk indicates R7 photoreceptor). (E-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm.</p> <p><strong>(F) Quantification of the number of MCS per photoreceptor for R1-R6 in 1 day old dark reared wild type (from Figure 5B i), <em>rdgB<sup>9</sup></em> and <em>rdgB<sup>9</sup>;dEsyt<sup>KO</sup></em> flies. n=30 photoreceptors from 3 separate flies.</strong></p> <p>Data information: Scatter plots with mean ± SD are shown. Statistical tests: (B and D) Student's unpaired t test. (F) one-way ANOVA with post hoc Tukey's multiple pairwise comparison. Ns -not significant, **p value <0.01; ***p value <0.001.</p> </sd-panel>
|
https://api.sourcedata.io/file.php?figure_id=34152
|
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] |
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