qPCR analysis of VPS4B mRNA abundance in normal colon , adenoma and CRC samples . Adenocarcinoma ( n = 26 ) ; adenoma ( n = 42 ) ; normal colon ( n = 24 ) . Green horizontal bars indicate means and red whiskers indicate SD .

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qPCR analysis of VPS4B mRNA abundance in normal colon, adenoma and CRC samples. Adenocarcinoma (n=26); adenoma (n=42); normal colon (n=24). Green horizontal bars indicate means and red whiskers indicate SD.


Examples of immunohistochemical staining of VPS4B in normal colon and matched CRC samples as an illustration of the scoring system used for the evaluation presented in ( F ) . 3 + - very intensive staining , 2 + - medium - intensive staining , 1 + - weak staining , 0 - no staining . Scale bar , 50 µm .

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Examples of immunohistochemical staining of VPS4B in normal colon and matched CRC samples as an illustration of the scoring system used for the evaluation presented in (F). 3+ - very intensive staining, 2+ - medium-intensive staining, 1+ - weak staining, 0 - no staining. Scale bar, 50 µm.


Analysis of viability of HCT116 cells assessed 96 h after transfection with independent non - targeting siRNA ( two different duplexes used , siCTRL # 1 or # 2 ) or targeting VPS4A ( duplexes # 1 or # 2 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 3 independent experiments ± SEM . All values were normalized , averaged ( avrg ) viability of siCTRL # 1 and # 2 - transfected cells was set as 100 % , the Kruskal - Wallis test followed by Dunn ' s multiple comparison post hoc test ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 .

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Analysis of viability of HCT116 cells assessed 96 h after transfection with independent non-targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #1 or #2), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, averaged (avrg) viability of siCTRL#1 and #2-transfected cells was set as 100%, the Kruskal-Wallis test followed by Dunn's multiple comparison post hoc test; ns - non-significant (p≥0.05), **p<0.01.


Left panel , analysis of clonogenic growth of HCT116 cells assessed 15 days after transfection with non - targeting ( siCTRL # 1 ) , VPS4A - or / and VPS4B - targeting siRNAs used in various duplex combinations as indicated . Right panel , images of HCT116 clones taken at the day of clonogenic growth assessment . Data are means of 3 independent experiments ± SEM . NT - non - transfected . All values were normalized , clonogenic growth of siCTRL # 1 - transfected cells was set as 1 . One - sample t - test ; ns - non - significant ( p ≥ 0 . 05 ) , * * * * p < 0 . 0001 .

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Left panel, analysis of clonogenic growth of HCT116 cells assessed 15 days after transfection with non-targeting (siCTRL#1), VPS4A- or/and VPS4B-targeting siRNAs used in various duplex combinations as indicated. Right panel, images of HCT116 clones taken at the day of clonogenic growth assessment. Data are means of 3 independent experiments ±SEM. NT - non-transfected. All values were normalized, clonogenic growth of siCTRL#1-transfected cells was set as 1. One-sample t-test; ns - non-significant (p≥0.05), ****p<0.0001.


Viability of RKO and SW480 cells assessed as in ( A ) , growth of DLD - 1 cells assessed in BrdU incorporation assay 96 h after siRNA transfection . Various independent non - targeting ( siCTRL # 1 or # 2 ) and VPS4A - or B - targeting siRNA duplexes were used ( # 1 , # 2 , # 5 or # 1 , # 2 , respectively ) . All values were normalized , averaged ( avrg ) viability of siCTRL # 1 and # 2 - transfected cells was set as 100 % . Data are means of 3 independent experiments ± SEM . The Kruskal - Wallis test followed by Dunn ' s multiple comparison post hoc ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 , * * * p < 0 . 001 .

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Viability of RKO and SW480 cells assessed as in (A), growth of DLD-1 cells assessed in BrdU incorporation assay 96 h after siRNA transfection. Various independent non-targeting (siCTRL#1 or #2) and VPS4A- or B-targeting siRNA duplexes were used (#1, #2, #5 or #1, #2, respectively). All values were normalized, averaged (avrg) viability of siCTRL#1 and #2-transfected cells was set as 100%. Data are means of 3 independent experiments ±SEM. The Kruskal-Wallis test followed by Dunn's multiple comparison post hoc; ns - non-significant (p≥0.05), **p<0.01, ***p<0.001.


VPS4B copy number and dependency scores of selected cancer cell lines obtained in CRISPR / Cas9 and RNAi screens deposited in the DepMap portal ( https : / / depmap . org / portal / ) .

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VPS4B copy number and dependency scores of selected cancer cell lines obtained in CRISPR/Cas9 and RNAi screens deposited in the DepMap portal (https://depmap.org/portal/).


VPS4B copy number status estimated across different cell lines using TaqMan assay . The whiskers represent the minimal and maximal copy number in a given triplicate readout . The BCL2 gene localized on 18q in the close vicinity to VPS4B was analyzed as a control .

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VPS4B copy number status estimated across different cell lines using TaqMan assay. The whiskers represent the minimal and maximal copy number in a given triplicate readout. The BCL2 gene localized on 18q in the close vicinity to VPS4B was analyzed as a control.


VPS4B and VPS4A protein abundance in selected normal and cancer cell lines analyzed by immunoblotting . Vinculin and GAPDH were used as loading controls .

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VPS4B and VPS4A protein abundance in selected normal and cancer cell lines analyzed by immunoblotting. Vinculin and GAPDH were used as loading controls.


Analysis of viability of HOP62 lung cancer cells assessed 144 h after transfection with independent non - targeting siRNA ( two different duplexes used , siCTRL # 1 or # 2 ) or targeting VPS4A ( duplexes # 2 or # 5 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 4 independent experiments ± SEM . All values were normalized , cell viability of averaged ( avrg ) siCTRL # 1 - and # 2 - transfected cells was set as 100 % . Two - tailed unpaired t - test ; * * * * p < 0 . 0001 .

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Analysis of viability of HOP62 lung cancer cells assessed 144 h after transfection with independent non-targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #2 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 4 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1- and #2-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001.


Top panel , analysis of clonogenic growth of HOP62 cells assessed 14 days after transfection with various siRNA duplexes as indicated . Bottom panel , images of HOP62 clones taken at the day of clonogenic growth assessment . Data are means of 4 independent experiments ± SEM . All values were normalized , colony area of averaged ( avrg ) siCTRL # 1 and # 2 - transfected cells was set as 1 . The Mann - Whitney U test ; * * p < 0 . 01 .

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Top panel, analysis of clonogenic growth of HOP62 cells assessed 14 days after transfection with various siRNA duplexes as indicated. Bottom panel, images of HOP62 clones taken at the day of clonogenic growth assessment. Data are means of 4 independent experiments ±SEM. All values were normalized, colony area of averaged (avrg) siCTRL#1 and #2-transfected cells was set as 1. The Mann-Whitney U test; **p<0.01.


Analysis of viability of SNU410 pancreatic cancer cells assessed 168 h after transfection with independent non - targeting siRNA ( three different duplexes used , siCTRL # 1 , # 2 or # 3 ) or targeting VPS4A ( duplexes # 2 , # 4 or # 5 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 3 independent experiments ± SEM . All values were normalized , cell viability of averaged ( avrg ) siCTRL # 1 - , # 2 - and # 3 - transfected cells was set as 100 % . Two - tailed unpaired t - test ; * * * * p < 0 . 0001 .

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Analysis of viability of SNU410 pancreatic cancer cells assessed 168 h after transfection with independent non-targeting siRNA (three different duplexes used, siCTRL#1, #2 or #3) or targeting VPS4A (duplexes #2, #4 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1-, #2- and #3-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001.


Left panel , growth of HCT116 VPS4B - / - shVPS4A cells as xenografts in mice in the presence or absence of doxycycline . Day 1 indicates the first day of doxycycline administration . n = 9 for each group , each mouse bearing one tumor , ± SEM . Two - tailed unpaired t - test ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 . Right panel , scatter plot representing end - point volumes of single xenografts . Bars represent means ± SEM .

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Left panel, growth of HCT116 VPS4B-/- shVPS4A cells as xenografts in mice in the presence or absence of doxycycline. Day 1 indicates the first day of doxycycline administration. n=9 for each group, each mouse bearing one tumor, ±SEM. Two-tailed unpaired t-test; ns - non-significant (p≥0.05), **p<0.01. Right panel, scatter plot representing end-point volumes of single xenografts. Bars represent means ±SEM.


Immunoblotting analysis of VPS4A abundance in xenograft samples from untreated and doxycycline - treated mice ( 6 separate xenograft samples for each group ) . Lysates of HCT116 VPS4B - / - cells transfected with control or VPS4A - targeting siRNA marked VPS4A protein detection . GAPDH served as a loading control .

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Immunoblotting analysis of VPS4A abundance in xenograft samples from untreated and doxycycline-treated mice (6 separate xenograft samples for each group). Lysates of HCT116 VPS4B-/- cells transfected with control or VPS4A-targeting siRNA marked VPS4A protein detection. GAPDH served as a loading control.


Growth of HCT116 VPS4B - / - shCTRL # 1 cells as xenografts in mice in the presence or absence of doxycycline . The arrow indicates the first day of doxycycline ( Dox ) administration . n = 2 mice for each group , each mouse bearing two tumors , ± SEM .

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Growth of HCT116 VPS4B-/- shCTRL#1 cells as xenografts in mice in the presence or absence of doxycycline. The arrow indicates the first day of doxycycline (Dox) administration. n=2 mice for each group, each mouse bearing two tumors, ±SEM.


Heatmap visualizing expression of genes related to inflammatory response ( left panel ) and positive regulation of cell death ( right panel ) generated from the GO analysis of biological processes across different transfection conditions with at least 3 biological replicates .

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Heatmap visualizing expression of genes related to inflammatory response (left panel) and positive regulation of cell death (right panel) generated from the GO analysis of biological processes across different transfection conditions with at least 3 biological replicates.


Immunoblotting analysis of the canonical and noncanonical branches of the NF - κB pathway and mediators of caspase - dependent cell death . Lysates of HCT116 VPS4B - / - cells were collected 66 h after transfection with siRNA ( siCTRL # 1 or different siVPS4A duplexes : # 2 , # 4 or # 5 ) . Lysates of HCT116 VPS4B + / + and non - transfected HCT116 VPS4B - / - cells were used to monitor the basal pathway activity . Representative blot from 10 experiments is shown . NT - non - transfected ; p - RelA - phospho - RelA ; p - IκBα - phospho - IκBα ; cl - cleaved caspases or PARP - 1 . GAPDH or vinculin served as loading controls .

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Immunoblotting analysis of the canonical and noncanonical branches of the NF-κB pathway and mediators of caspase-dependent cell death. Lysates of HCT116 VPS4B-/- cells were collected 66 h after transfection with siRNA (siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Lysates of HCT116 VPS4B+/+ and non-transfected HCT116 VPS4B-/- cells were used to monitor the basal pathway activity. Representative blot from 10 experiments is shown. NT- non-transfected; p-RelA - phospho-RelA; p-IκBα - phospho-IκBα; cl - cleaved caspases or PARP-1. GAPDH or vinculin served as loading controls.


Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in ( A ) . Data are means of 10 ( phospho - Rel and cleaved caspase 7 ) , 9 ( p52 , caspase 8 and cleaved caspase 9 ) , 7 ( phosphorylated and total IκB ) or 5 ( cleaved caspase 3 ) independent experiments . Error bars are SEM . Statistical significance was assessed using the following tests : one - sample t - test ( caspase 8 , cleaved caspases 9 , 7 and 3 , total and phospho - IκBα ) or Wilcoxon signed rank test ( phospho - RelA and p52 ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in (A). Data are means of 10 (phospho-Rel and cleaved caspase 7), 9 (p52, caspase 8 and cleaved caspase 9), 7 (phosphorylated and total IκB) or 5 (cleaved caspase 3) independent experiments. Error bars are SEM. Statistical significance was assessed using the following tests: one-sample t-test (caspase 8, cleaved caspases 9, 7 and 3, total and phospho-IκBα) or Wilcoxon signed rank test (phospho-RelA and p52); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


Immunoblot showing induction of the NF - κB pathway ( p52 ) and apoptosis ( cleaved caspase 7 ) in xenograft samples described in Fig 3C . cl - cleaved caspase . Vinculin was used as a loading control .

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Immunoblot showing induction of the NF-κB pathway (p52) and apoptosis (cleaved caspase 7) in xenograft samples described in Fig 3C. cl - cleaved caspase. Vinculin was used as a loading control.


Analysis of the impact of RIPK1 inhibitor ( necrostatin - 1 ) or pan - caspase inhibitor ( Q - VD - Oph ) on cell viability of HCT116 VPS4B - / - cells transfected with siRNA ( non - targeting siCTRL # 1 or different siVPS4A duplexes : # 2 , # 4 or # 5 ) . Cell viability was assessed 72 h after siRNA transfection . Necrostatin - 1 ( 50 µM ) , Q - VP - Oph ( 20 µM ) or vehicle were added to the medium 48 h before viability assessment . Data are means of 5 independent experiments ± SEM . All values were normalized , viability of siCTRL - transfected and vehicle - treated cells was set as 100 % . Two - tailed unpaired t - test ; * * p < 0 . 01 .

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Analysis of the impact of RIPK1 inhibitor (necrostatin-1) or pan-caspase inhibitor (Q-VD-Oph) on cell viability of HCT116 VPS4B-/- cells transfected with siRNA (non-targeting siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Cell viability was assessed 72 h after siRNA transfection. Necrostatin-1 (50 µM), Q-VP-Oph (20 µM) or vehicle were added to the medium 48 h before viability assessment. Data are means of 5 independent experiments ±SEM. All values were normalized, viability of siCTRL-transfected and vehicle-treated cells was set as 100%. Two-tailed unpaired t-test; **p<0.01.


Measurement of ATP released to the cell medium by HCT116 VPS4B - / - cells non - transfected ( NT ) or transfected with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A duplexes : # 2 , # 4 or # 5 ) . Cell culture media were exchanged 16 h after transfection , and fresh media were conditioned for the next 52 - 58 h . For non - transfected cells ( NT ) , the same treatment protocol was used but without the transfection mixture .

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Measurement of ATP released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.


Measurement of HMGB1 released to the cell medium by HCT116 VPS4B - / - cells non - transfected ( NT ) or transfected with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A duplexes : # 2 , # 4 or # 5 ) . Cell culture media were exchanged 16 h after transfection , and fresh media were conditioned for the next 52 - 58 h . For non - transfected cells ( NT ) , the same treatment protocol was used but without the transfection mixture .

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Measurement of HMGB1 released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.


Microscopy images presenting cell surface calreticulin ( green ) in HCT116 VPS4B - / - cells 48 h after transfection with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A # 2 ) . As a positive control for detection of cell surface calreticulin , non - transfected cells ( NT ) were treated with 2 µM mitoxantrone for 24 h . In blue , DAPI staining . Scale bar , 15 µm .

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Microscopy images presenting cell surface calreticulin (green) in HCT116 VPS4B-/- cells 48 h after transfection with siRNA (non-targeting siCTRL#1 or targeting siVPS4A#2). As a positive control for detection of cell surface calreticulin, non-transfected cells (NT) were treated with 2 µM mitoxantrone for 24 h. In blue, DAPI staining. Scale bar, 15 µm.


Flow cytometric analysis of calreticulin exposed on the cell surface of VPS4A - depleted HCT116 VPS4B - / - cells 66 h after siRNA transfection ( siVPS4A duplexes : # 2 , # 4 or # 5 were used ) . Non - transfected ( NT ) or siCTRL # 1 - transfected cells served as negative controls . Left panel , percentage of cells positive for calreticulin in the population of live ( DAPI - negative ) cells , data are means of 4 independent experiments ± SEM . The Mann - Whitney U test ; * * p < 0 . 01 . Right panel , representative dot plot diagrams of flow cytometric analysis of cell surface - exposed calreticulin . Primary rabbit anti - calreticulin and control isotype IgG antibodies were used for staining , followed by secondary AlexaFluor 647 - conjugated antibody .

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Flow cytometric analysis of calreticulin exposed on the cell surface of VPS4A-depleted HCT116 VPS4B-/- cells 66 h after siRNA transfection (siVPS4A duplexes: #2, #4 or #5 were used). Non-transfected (NT) or siCTRL#1-transfected cells served as negative controls. Left panel, percentage of cells positive for calreticulin in the population of live (DAPI-negative) cells, data are means of 4 independent experiments ±SEM. The Mann-Whitney U test; **p<0.01. Right panel, representative dot plot diagrams of flow cytometric analysis of cell surface-exposed calreticulin. Primary rabbit anti-calreticulin and control isotype IgG antibodies were used for staining, followed by secondary AlexaFluor 647-conjugated antibody.


qPCR analysis of M1 ( pro - inflammatory ) and M2 ( anti - inflammatory ) macrophage polarization markers in mouse bone marrow - derived macrophages ( BMDMs ) incubated for 24 h in conditioned media ( CM ) collected from control ( siCTRL # 1 ) , Vps4a - and / or Vps4b - depleted CT - 26 cells . For double Vps4a + b depletion various combinations of siVps4a # 2 or # 5 and siVps4B # 3 or # 4 duplexes were used . Data were normalized and are presented as the fold change of expression of a given M1 or M2 marker in BMDMs treated with CM from Vps4 - depleted cells compared to its expression in BMDMs treated with CM from siCTRL # 1 - transfected cells ( set as 1 ) . Data are means of 4 independent experiments ± SEM . One - sample t - test ; ns - non - significant ( p ≥ 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 .

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qPCR analysis of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage polarization markers in mouse bone marrow-derived macrophages (BMDMs) incubated for 24 h in conditioned media (CM) collected from control (siCTRL#1), Vps4a- and/or Vps4b-depleted CT-26 cells. For double Vps4a+b depletion various combinations of siVps4a#2 or #5 and siVps4B#3 or #4 duplexes were used. Data were normalized and are presented as the fold change of expression of a given M1 or M2 marker in BMDMs treated with CM from Vps4-depleted cells compared to its expression in BMDMs treated with CM from siCTRL#1-transfected cells (set as 1). Data are means of 4 independent experiments ±SEM. One-sample t-test; ns - non-significant (p≥0.05), *p<0.05, **p<0.01.


( A ) Stable RNAi screening of cancer - related genes was conducted to identify gene knockdowns that enhance reprogramming of MEFs into iPSCs . The graph shows the 10 shRNAs that were most strongly , positively selected in iPSCs compared to non - reprogrammed cells on day 14 of reprogramming ( n = 3 biological replicates ) .

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(A) Stable RNAi screening of cancer-related genes was conducted to identify gene knockdowns that enhance reprogramming of MEFs into iPSCs. The graph shows the 10 shRNAs that were most strongly, positively selected in iPSCs compared to non-reprogrammed cells on day 14 of reprogramming (n=3 biological replicates).


( B - E ) Mouse embryonic fibroblasts ( MEFs ) were co - infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors . The co - transduced cells were sorted and were reprogrammed for 14 days : ( B ) Representative images and ( C ) quantification of alkaline - phosphatase - positive ( AP + ) iPSC colonies on day 14 of reprogramming by transfection of 4 reprogramming factors ( = 4F : Oct4 , Sox2 , Klf4 , c - Myc = OSKM ) or 3 factors ( = 3F : OSK ) . [ n = 3 biological replicates per group ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by one - sided t - test analyzed by one - sided t - test with Holm - Sidak correction for multiple testing ] .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-E) Mouse embryonic fibroblasts (MEFs) were co-infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors. The co-transduced cells were sorted and were reprogrammed for 14 days: (B) Representative images and (C) quantification of alkaline-phosphatase-positive (AP+) iPSC colonies on day 14 of reprogramming by transfection of 4 reprogramming factors (=4F: Oct4, Sox2, Klf4, c-Myc = OSKM) or 3 factors (=3F: OSK). [n=3 biological replicates per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test analyzed by one-sided t-test with Holm-Sidak correction for multiple testing].


Mouse embryonic fibroblasts ( MEFs ) were co - infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors . The co - transduced cells were sorted and were reprogrammed for 14 days : ( D ) Representative FACS

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mouse embryonic fibroblasts (MEFs) were co-infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors. The co-transduced cells were sorted and were reprogrammed for 14 days: (D) Representative FACS profiles


( E ) percentage of Oct4 - GFP + iPSCs obtained after 14 days of reprogramming of MEFs from Oct4 - eGFP mice using 4F - transduction on day zero that were infected with shRNAs targeting Tnfaip2 or a scrambled shRNA control [ n = 5 biological replicates ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by one - sided t - test with Holm - Sidak correction for multiple testing ] .

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(E) percentage of Oct4-GFP+ iPSCs obtained after 14 days of reprogramming of MEFs from Oct4-eGFP mice using 4F-transduction on day zero that were infected with shRNAs targeting Tnfaip2 or a scrambled shRNA control [n=5 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test with Holm-Sidak correction for multiple testing].


] . ( F ) mRNA of Tnfaip2 was measured by RT - qPCR at the indicated days of reprogramming by 4F - transduction on day zero [ n = 3 biological replicates ; log - transformed data were not normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) , P value for the upregulation of Tnfaip2 on consecutive test days - 9 , - 12 and - 14 was calculated starting with the probability of maximum rank - based difference , i . e . having two groups of three data points perfectly separating , p1 , b = 0 . 1 ( bidirectional ) and p1 , u = 0 . 05 ( unidirectional ) ; the probability of finding a triple series of max . difference starting at some timepoint is Ptriple , tp = p1 , b · p1 , u · p1 , u , and finding such a triple somewhere across the 5 - step time series is Ptriple = Ptriple , tp + 2 · ( 1 - p1 , u ) · Ptriple , tp = 0 . 0007 .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

]. (F) mRNA of Tnfaip2 was measured by RT-qPCR at the indicated days of reprogramming by 4F-transduction on day zero [n=3 biological replicates; log-transformed data were not normally distributed (P>0.05 as per Shapiro-Wilk test), P value for the upregulation of Tnfaip2 on consecutive test days-9,-12 and -14 was calculated starting with the probability of maximum rank-based difference, i.e. having two groups of three data points perfectly separating, p1,b = 0.1 (bidirectional) and p1,u = 0.05 (unidirectional); the probability of finding a triple series of max. difference starting at some timepoint is Ptriple,tp = p1,b · p1,u · p1,u, and finding such a triple somewhere across the 5-step time series is Ptriple = Ptriple,tp + 2 · (1- p1,u) · Ptriple,tp = 0.0007.


( C ) RT - qPCR analysis of Smed - exoc3 mRNA expression of total RNA ( whole body - derived ) from exoc3 ( RNAi ) - treated planarians compared to control - injected planarians on day - 38 of the injection scheme [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) ; data were analyzed by one - sided t - test ] .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) RT-qPCR analysis of Smed-exoc3 mRNA expression of total RNA (whole body-derived) from exoc3(RNAi)-treated planarians compared to control-injected planarians on day-38 of the injection scheme [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test); data were analyzed by one-sided t-test].


( D ) Representative photographs of exoc3 ( RNAi ) - treated planarians vs . control planarians at day - 38 of the injection protocol as shown in panel B ( n = 4 experimental replicates with 6 - 10 biological replicates per experiment , scale bar : 0 . 5 mm ) . Number ratios in the photographs depict the total number of animals showing the indicated phenotype .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Representative photographs of exoc3(RNAi)-treated planarians vs. control planarians at day-38 of the injection protocol as shown in panel B (n=4 experimental replicates with 6-10 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs depict the total number of animals showing the indicated phenotype.


Planarians were injected with Smed - exoc3 dsRNA = exoc3 ( RNAi ) or with control gfp ( RNAi ) injections . ( A ) The representative photographs show staining for phosphorylation of 10th serine residue of Histone 3 ( H3S10p , a stem cell marker in planarians ) at days - 38 of the injection protocol ( n = 3 experimental replicates with 8 - 10 biological replicates per experiment , scale bar : 0 . 5 mm ) .

O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Planarians were injected with Smed-exoc3 dsRNA = exoc3(RNAi) or with control gfp(RNAi) injections. (A) The representative photographs show staining for phosphorylation of 10th serine residue of Histone 3 (H3S10p, a stem cell marker in planarians) at days-38 of the injection protocol (n=3 experimental replicates with 8-10 biological replicates per experiment, scale bar: 0.5 mm).


exoc3 ( RNAi ) - treated planarians versus gfp ( RNAi ) - treated controls were analyzed at the indicated time - points of the dsRNA injection protocol : ( B ) Quantification of H3S10p + cells per mm2 in planarians at day - 30 , - 34 and - 38 of the injection protocol [ n = 3 experimental replicates with 5 - 7 biological replicates per experiment ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by multiple t - tests with Holm - Sidak correction for multiple comparisons ] .

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exoc3(RNAi)-treated planarians versus gfp(RNAi)-treated controls were analyzed at the indicated time-points of the dsRNA injection protocol: (B) Quantification of H3S10p+ cells per mm2 in planarians at day-30, -34 and -38 of the injection protocol [n=3 experimental replicates with 5-7 biological replicates per experiment; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].


FACS - based time - course analysis on changes in the fraction of somatic stem cells ( X1 - population ) at indicated time points . Cells were obtained from whole body - trypsinized planarians of gfp ( RNAi ) - injected controls and exoc3 ( RNAi ) - treated planarians and stained with the cytoplasmic dye Calcein - AM and the nuclear ( DNA ) dye Hoechst 33342 : ( C ) Representative FACS

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FACS-based time-course analysis on changes in the fraction of somatic stem cells (X1-population) at indicated time points. Cells were obtained from whole body-trypsinized planarians of gfp(RNAi)-injected controls and exoc3(RNAi)-treated planarians and stained with the cytoplasmic dye Calcein-AM and the nuclear (DNA) dye Hoechst 33342: (C) Representative FACS profiles


( D ) quantification of the relative number of X1 cells [ n = 3 biological replicates ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by multiple t - tests with Holm - Sidak correction for multiple comparisons ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) quantification of the relative number of X1 cells [n=3 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].


RT - qPCR analysis for makers of differentiating progenitor cells from the epidermal lineage on total ( whole body derived ) RNA from exoc3 ( RNAi ) - treated planarians ( E ) Expression analysis of differentiation marker prog - 2 on day - 38 of the injection scheme [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test and analyzed by one - sided t - test ] ,

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-qPCR analysis for makers of differentiating progenitor cells from the epidermal lineage on total (whole body derived) RNA from exoc3(RNAi)-treated planarians (E) Expression analysis of differentiation marker prog-2 on day-38 of the injection scheme [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test and analyzed by one-sided t-test],


( F ) Expression analysis of marker genes of differentiated cells ( prog - 1 and Smed - odc - 1 ; on day - 30 , - 34 and - 38 of the injection scheme [ n = 3 biological replicates per group and time point except for prog - 1 expression at day 38 , which includes 6 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by multiple t - tests with Holm - Sidak correction for multiple comparisons ] . For Smed - odc - 1 log - transformed mean centered group data were normally distributed and and analyzed by multiple t - tests with Holm - Sidak correction for multiple comparisons ] .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Expression analysis of marker genes of differentiated cells (prog-1 and Smed-odc-1; on day-30, -34 and -38 of the injection scheme [n= 3 biological replicates per group and time point except for prog-1 expression at day 38, which includes 6 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons]. For Smed-odc-1 log-transformed mean centered group data were normally distributed and and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].


Planarians were injected with exoc3 ( RNAi ) or with control gfp ( RNAi ) injections . Organ phenotypes were analyzed on day - 38 of the injection protocol . Organ homeostasis was analyzed by staining against specific markers : ( A ) The structure of the brain / central nervous system ( CNS ) was analyzed by staining against synapsin ( 3C11 ) . The cartoon represents the phenotypic change ( upper panel ) . Representative photographs are shown in the lower panel ( 3 experimental replicates with 2 - 3 biological replicates per experiment , scale bar : 0 . 5 mm ) . Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype . There was a significant increase in the occurrence of atrophic cephalic ganglia and sensory neuron loss in exoc3 ( RNAi ) - treated planarians vs . controls ( cg : cephalic ganglia ; sn : sensory neurons ) .

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Planarians were injected with exoc3(RNAi) or with control gfp(RNAi) injections. Organ phenotypes were analyzed on day-38 of the injection protocol. Organ homeostasis was analyzed by staining against specific markers: (A) The structure of the brain/central nervous system (CNS) was analyzed by staining against synapsin (3C11). The cartoon represents the phenotypic change (upper panel). Representative photographs are shown in the lower panel (3 experimental replicates with 2-3 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype. There was a significant increase in the occurrence of atrophic cephalic ganglia and sensory neuron loss in exoc3(RNAi)-treated planarians vs. controls (cg: cephalic ganglia; sn: sensory neurons).


( B ) Planarian visual system was analyzed by staining against VC1 . Schematic cartoon ( upper panel ) and representative images ( lower panel ) are shown ( 3 experimental replicates with 3 biological replicates per experiment , scale bar : 0 . 5 mm ) . Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype . Arrows point to photosensitive cells ( ps ) ; pigmentary cells ( pc ) and to the optic chiasm ( oc ) .

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(B) Planarian visual system was analyzed by staining against VC1. Schematic cartoon (upper panel) and representative images (lower panel) are shown (3 experimental replicates with 3 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype. Arrows point to photosensitive cells (ps); pigmentary cells (pc) and to the optic chiasm (oc).


RNA - seq was conducted on freshly isolated X1 cells from exoc3 ( RNAi ) - treated compared to X1 cells from gfp ( RNAi ) - treated planarians on day - 38 of the injection protocol . The histograms depict the overlap ( blue ) of DEGs in X1 cells of Smed - exoc3 - depleted planarians versus controls ( n = 3 biological replicates ; adjusted P < 0 . 05 ) with genes expressed in the indicated sample sets of ( A ) neoblast stem

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA-seq was conducted on freshly isolated X1 cells from exoc3(RNAi)-treated compared to X1 cells from gfp(RNAi)-treated planarians on day-38 of the injection protocol. The histograms depict the overlap (blue) of DEGs in X1 cells of Smed-exoc3-depleted planarians versus controls (n=3 biological replicates; adjusted P< 0.05) with genes expressed in the indicated sample sets of (A) neoblast stem cells


( B - E ) The heat maps depict the intersection of DEGs with strong expression signals ( log2 fold change > 0 . 5 or < - 0 . 5 ) discovered in X1 cells of exoc3 - depleted planarians versus controls ( n = 3 biological replicates ) with the indicated , previously published data sets on gene expression profiles in ( B - D ) neoblast cells or ( E ) differentiating progenitor cells of epidermal lineage . The colour scale represents the gene - wise z - score calculated from normalized gene expression levels while purple and green indicate upregulated and downregulated genes in X1 cells of exoc3 - depleted planarians versus controls , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(B-E) The heat maps depict the intersection of DEGs with strong expression signals (log2 fold change > 0.5 or < -0.5) discovered in X1 cells of exoc3-depleted planarians versus controls (n= 3 biological replicates) with the indicated, previously published data sets on gene expression profiles in (B-D) neoblast cells or (E) differentiating progenitor cells of epidermal lineage. The colour scale represents the gene-wise z-score calculated from normalized gene expression levels while purple and green indicate upregulated and downregulated genes in X1 cells of exoc3-depleted planarians versus controls, respectively.


RNA - seq was conducted on freshly isolated X1 cells from exoc3 ( RNAi ) - treated compared to X1 cells from gfp ( RNAi ) - treated planarians on day - 38 of the injection protocol . The histograms depict the overlap ( blue ) of DEGs in X1 cells of Smed - exoc3 - depleted planarians versus controls ( n = 3 biological replicates ; adjusted P < 0 . 05 ) with genes expressed in the indicated sample sets of ( F ) differentiating progenitor cells of epidermal lineage from the indicated publications while the sum of overlapping ( blue ) and set - exclusive ( grey ) genes represents the total number of genes of a cluster that were analyzed in this study .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA-seq was conducted on freshly isolated X1 cells from exoc3(RNAi)-treated compared to X1 cells from gfp(RNAi)-treated planarians on day-38 of the injection protocol. The histograms depict the overlap (blue) of DEGs in X1 cells of Smed-exoc3-depleted planarians versus controls (n=3 biological replicates; adjusted P< 0.05) with genes expressed in the indicated sample sets of (F) differentiating progenitor cells of epidermal lineage from the indicated publications while the sum of overlapping (blue) and set-exclusive (grey) genes represents the total number of genes of a cluster that were analyzed in this study.


Planarians ( S . mediterranea ) were injected with exoc3 ( RNAi ) or with control gfp ( RNAi ) . ( B ) Representative photographs of morphology of exoc3 knockdown planarian vs . control planarian on days - 29 , - 31 and - 37 of the protocol Number ratios in the photographs indicate the photographically represented phenotypes .

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Planarians (S. mediterranea) were injected with exoc3(RNAi) or with control gfp(RNAi). (B) Representative photographs of morphology of exoc3 knockdown planarian vs. control planarian on days-29, -31 and -37 of the protocol Number ratios in the photographs indicate the photographically represented phenotypes.


Organ regeneration was analyzed by immunostaining of different organ compartments in regenerating exoc3 ( RNAi ) - treated planarians vs . gfp ( RNAi ) - treated control on day 37 of the protocol ( C ) Eyes were analyzed by staining against VC1 . Representative images are shown ( 3 experimental replicates with 4 - 5 biological replicates per experiment ) . Arrows point to photosensitive cells ( ps ) ; pigmentary cells ( pc ) and to the optic chiasm ( oc ) . Number ratios in the photographs refer to planarians with regenerative defects for the indicated phenotype ( scale bar : 0 . 5 mm ) .

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Organ regeneration was analyzed by immunostaining of different organ compartments in regenerating exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated control on day 37 of the protocol (C) Eyes were analyzed by staining against VC1. Representative images are shown (3 experimental replicates with 4-5 biological replicates per experiment). Arrows point to photosensitive cells (ps); pigmentary cells (pc) and to the optic chiasm (oc). Number ratios in the photographs refer to planarians with regenerative defects for the indicated phenotype (scale bar: 0.5 mm).


Organ regeneration was analyzed by immunostaining of different organ compartments in regenerating exoc3 ( RNAi ) - treated planarians vs . gfp ( RNAi ) - treated control on day 37 of the protocol ( D ) Representative photographs of staining with planarian pan - neural marker 3C11 ( alias Synapsin ) marking cephalic ganglia and sensory neurons Number ratios in the photographs refers to planarians with regenerative exhibiting the photographically represented phenotype , characterized by underdeveloped bilobed cephalic ganglia and atrophy of sensory neurons in exoc3 ( RNAi ) - treated planarians versus controls ( scale bar : 0 . 5 mm , cg : cephalic ganglia ; sn : sensory neurons ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Organ regeneration was analyzed by immunostaining of different organ compartments in regenerating exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated control on day 37 of the protocol (D) Representative photographs of staining with planarian pan-neural marker 3C11 (alias Synapsin) marking cephalic ganglia and sensory neurons Number ratios in the photographs refers to planarians with regenerative exhibiting the photographically represented phenotype, characterized by underdeveloped bilobed cephalic ganglia and atrophy of sensory neurons in exoc3(RNAi)-treated planarians versus controls (scale bar: 0.5 mm, cg: cephalic ganglia; sn: sensory neurons).


One thousand ESCs of the indicated genotypes were placed into hanging - drop cultures to form embryoid bodies ( EBs ) . After 3 days ( referred to as day - 0 ) , differentiation was induced by exposure to differentiation medium ( days - 1 to days - 3 after medium change ) . ( A ) Representative photographs of EBs of the indicated genotypes at day - 0 and day - 3 after differentiation induction ( experiments were conducted in 3 biological replicates ; n = 5 - 42 EBs per biological replicate , scale bar : 0 . 02 mm ) .

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One thousand ESCs of the indicated genotypes were placed into hanging-drop cultures to form embryoid bodies (EBs). After 3 days (referred to as day-0), differentiation was induced by exposure to differentiation medium (days-1 to days-3 after medium change). (A) Representative photographs of EBs of the indicated genotypes at day-0 and day-3 after differentiation induction (experiments were conducted in 3 biological replicates; n=5-42 EBs per biological replicate, scale bar: 0.02 mm).


RNA - seq analysis was conducted on Tnfaip2 - / - EBs vs . WT EBs on day - 3 of differentiation induction of EBs ( n = 3 biological replicates ) . The heat maps depict the intersection of DEGs with the following gene sets ( B ) " stem cell maintenance : positive and negative regulators " ,

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA-seq analysis was conducted on Tnfaip2-/- EBs vs. WT EBs on day-3 of differentiation induction of EBs (n=3 biological replicates). The heat maps depict the intersection of DEGs with the following gene sets (B) "stem cell maintenance: positive and negative regulators",


RNA - seq analysis was conducted on Tnfaip2 - / - EBs vs . WT EBs on day - 3 of differentiation induction of EBs ( n = 3 biological replicates ) . The heat maps depict the intersection of DEGs with the following gene sets ; , ( C ) " mesoderm development "

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA-seq analysis was conducted on Tnfaip2-/- EBs vs. WT EBs on day-3 of differentiation induction of EBs (n=3 biological replicates). The heat maps depict the intersection of DEGs with the following gene sets ;, (C) "mesoderm development"


RNA - seq analysis was conducted on Tnfaip2 - / - EBs vs . WT EBs on day - 3 of differentiation induction of EBs ( n = 3 biological replicates ) . The heat maps depict the intersection of DEGs with the following gene ( D ) " ectoderm development " Tnfaip2 and 5 other stem cell - related genes ( Tfcp2l1 , Dppa3 , Fbxo15 , Zfp42 and Klf2 ) were additionally incorporated based on literature searches . The color scale represents the gene - wise z - score calculated from normalized gene expression levels . The asterisk refers to the gene identified in this study .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA-seq analysis was conducted on Tnfaip2-/- EBs vs. WT EBs on day-3 of differentiation induction of EBs (n=3 biological replicates). The heat maps depict the intersection of DEGs with the following gene (D) "ectoderm development" Tnfaip2 and 5 other stem cell-related genes (Tfcp2l1, Dppa3, Fbxo15, Zfp42 and Klf2) were additionally incorporated based on literature searches. The color scale represents the gene-wise z-score calculated from normalized gene expression levels. The asterisk refers to the gene identified in this study.


( E ) Representative image of immunofluorescence staining against the ectodermal marker ( SOX1 ) and the mesodermal marker ( T alias Brachyury ) in WT and Tnfaip2 - / - EBs on day - 3 of differentiation induction . ( 3 repeat experiments were conducted on a total number of 9 - 10 EBs per genotype , scale bar : 20µm ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Representative image of immunofluorescence staining against the ectodermal marker (SOX1) and the mesodermal marker (T alias Brachyury) in WT and Tnfaip2-/- EBs on day-3 of differentiation induction. (3 repeat experiments were conducted on a total number of 9-10 EBs per genotype, scale bar: 20µm).


( F ) mRNA expression of Tnfaip2 measured by RT - qPCR in ESCs on the indicated days of differentiation induction of WT EBs [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) , the P value for the upregulation of Tnfaip2 on consecutive time points ( day - 0 , - 2 and - 3 ) was calculated starting with the probability of maximum rank - based difference , i . e . having two groups of three data points perfectly separating , p1 , b = 0 . 1 ( bidirectional ) and P1 , u = 0 . 05 ( unidirectional ) ; the probability of finding a triple series of max . difference starting at some timepoint is Ptriple , tp = p1 , b · p1 , u · p1 , u , and finding such a triple somewhere across the 4 - step time series is Ptriple = Ptriple , tp + ( 1 - p1 , u ) · Ptriple , tp = 0 . 0005 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) mRNA expression of Tnfaip2 measured by RT-qPCR in ESCs on the indicated days of differentiation induction of WT EBs [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test), the P value for the upregulation of Tnfaip2 on consecutive time points (day-0, -2 and -3) was calculated starting with the probability of maximum rank-based difference, i.e. having two groups of three data points perfectly separating, p1,b = 0.1 (bidirectional) and P1,u = 0.05 (unidirectional); the probability of finding a triple series of max. difference starting at some timepoint is Ptriple,tp = p1,b · p1,u · p1,u, and finding such a triple somewhere across the 4-step time series is Ptriple = Ptriple,tp + (1- p1,u) · Ptriple,tp = 0.0005.


( B ) The Volcano plots show the differentially expressed proteins identified from the proteome analysis of Tnfaip2 - / - EBs versus WT EBs at the indicated days after induction of differentiation . Vim ( marked in blue asterisk ) was the only protein that featured among top 100 differentially expressed proteins at all time points by implementing selection criteria of an average log2ratio > + / - 1 . 2 and a - log10Qvalue < 2 . Vim expression was reduced at all timepoints of in vitro differentiation in Tnfaip2 - / - EBs vs . WT EBs ( n = 5 biological replicates , see also Dataset EV8 for top 100 regulated proteins ) . Sphere size illustrates the magnitude of log2fold changes as does color coding : blue - downregulated proteins , red - upregulated proteins ; log2fold marking by color intensity : strong intensity > 2 , medium intensity > 1 . 5 , faded color > 1 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) The Volcano plots show the differentially expressed proteins identified from the proteome analysis of Tnfaip2-/- EBs versus WT EBs at the indicated days after induction of differentiation. Vim (marked in blue asterisk) was the only protein that featured among top 100 differentially expressed proteins at all time points by implementing selection criteria of an average log2ratio > +/-1.2 and a -log10Qvalue <2. Vim expression was reduced at all timepoints of in vitro differentiation in Tnfaip2-/- EBs vs. WT EBs (n=5 biological replicates, see also Dataset EV8 for top 100 regulated proteins). Sphere size illustrates the magnitude of log2fold changes as does color coding: blue-downregulated proteins, red-upregulated proteins; log2fold marking by color intensity: strong intensity >2, medium intensity > 1.5, faded color >1.


( C ) mRNA expression of Vim on indicated days of WT EB and Tnfaip2 - / - EB differentiation ( n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by multiple t - tests with Holm - Sidak correction for multiple testing ) .

O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) mRNA expression of Vim on indicated days of WT EB and Tnfaip2-/- EB differentiation (n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple testing).


FACS analysis of lipid droplet ( LD ) content of WT ( blue ) and Tnfaip2 - / - ( red ) EBs by BODIPY493 / 503 staining at the indicated days after differentiation induction : ( D ) Representative FACS blot of BODIPY staining . Unstained WT EBs served as a negative control ( grey ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FACS analysis of lipid droplet (LD) content of WT (blue) and Tnfaip2-/- (red) EBs by BODIPY493/503 staining at the indicated days after differentiation induction: (D) Representative FACS blot of BODIPY staining. Unstained WT EBs served as a negative control (grey).


FACS analysis of lipid droplet ( LD ) content of WT ( blue ) and Tnfaip2 - / - ( red ) EBs by BODIPY493 / 503 staining at the indicated days after differentiation induction : ( E ) Quantification of staining intensity [ n = 3 independent cultures per genotype ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by multiple t - tests with Holm - Sidak correction for multiple comparisons ] .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FACS analysis of lipid droplet (LD) content of WT (blue) and Tnfaip2-/- (red) EBs by BODIPY493/503 staining at the indicated days after differentiation induction: (E) Quantification of staining intensity [n=3 independent cultures per genotype; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].


Transmission Electron Microscopy ( TEM ) was used to determine the number of LDs in EB cultures derived from WT and Tnfaip2 - / - ES cells on day - 1 after differentiation induction . ( n = 5 independent cultures per genotype , n = 100 images per replicate ) . ( F ) Representative micrographs of TEM analysis of EB cells of the indicated genotype . Yellow arrows point to LDs , scale bars : 1 µm .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Transmission Electron Microscopy (TEM) was used to determine the number of LDs in EB cultures derived from WT and Tnfaip2-/- ES cells on day-1 after differentiation induction. (n=5 independent cultures per genotype, n=100 images per replicate). (F) Representative micrographs of TEM analysis of EB cells of the indicated genotype. Yellow arrows point to LDs, scale bars: 1 µm.


Electron Microscopy ( TEM ) was used to determine the number of LDs in EB cultures derived from WT and Tnfaip2 - / - ES cells on day - 1 after differentiation induction . ( n = 5 independent cultures per genotype , n = 100 images per replicate ) . ( G ) Quantification of LDs per TEM field . Data were not normally distributed ( P < 0 . 05 as per Shapiro - Wilk test ) and analyzed by Mann - Whitney test .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Electron Microscopy (TEM) was used to determine the number of LDs in EB cultures derived from WT and Tnfaip2-/- ES cells on day-1 after differentiation induction. (n=5 independent cultures per genotype, n=100 images per replicate). (G) Quantification of LDs per TEM field. Data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and analyzed by Mann-Whitney test.


( H ) UPLC - MS / MS analysis revealed a reduction in triacylglycerol ( TAG ) content in Tnfaip2 - / - EBs compared to WT EBs at the indicated time points after differentiation induction [ n = 3 independent cultures of EBs per time point ; data of EBs on day - 0 to day - 3 of differentiation were statistically analyzed ; mean centered group data were normally distributed and ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by one - sided t - test with Holm Sidak correction for multiple testing ] .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) UPLC-MS/MS analysis revealed a reduction in triacylglycerol (TAG) content in Tnfaip2-/- EBs compared to WT EBs at the indicated time points after differentiation induction [n=3 independent cultures of EBs per time point; data of EBs on day-0 to day-3 of differentiation were statistically analyzed; mean centered group data were normally distributed and (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test with Holm Sidak correction for multiple testing].


( A ) Heatmap of CPT1A protein expression as measured in proteome analysis of Tnfaip2 - / - and WT ESCs and during differentiation induction of EB cultures ( n = 5 biological replicates ) . The colour scale represents average log2ratios .

O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Heatmap of CPT1A protein expression as measured in proteome analysis of Tnfaip2-/- and WT ESCs and during differentiation induction of EB cultures (n=5 biological replicates). The colour scale represents average log2ratios.


( B ) Expression of Cpt1a mRNA as determined by qRT - PCR in WT and Tnfaip2 - / - EBs at the indicated days of differentiation induction [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by multiple t - tests with Holm - Sidak correction for multiple comparisons ] .

O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Expression of Cpt1a mRNA as determined by qRT-PCR in WT and Tnfaip2-/- EBs at the indicated days of differentiation induction [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].


Differentiation - induced WT EBs and Tnfaip2 - / - EBs were treated with palmitic acid ( PA ) and palmitoyl - L - carnitine ( PC ) ( 8μM each ) or a vehicle control ( C , D ) The diameters of the rosette structure of differentiated WT EBs and Tnfaip2 - / - EBs were measured on day - 3 after differentiation induction . ( n = 3 repeat experiments with 7 - 62 EBs per experiment per group ) : ( C ) Representative photographs ( scale bar : 0 . 02 mm ) and ( D ) quantification of the diameter of the rosette structure of the indicated groups [ data were not normally distributed ( P < 0 . 05 as per Shapiro - Wilk test ) and thus analyzed by Mann - Whitney U test with Holm Sidak correction for multiple comparisons ] .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Differentiation-induced WT EBs and Tnfaip2-/- EBs were treated with palmitic acid (PA) and palmitoyl-L-carnitine (PC) (8μM each) or a vehicle control (C,D) The diameters of the rosette structure of differentiated WT EBs and Tnfaip2-/- EBs were measured on day-3 after differentiation induction. (n=3 repeat experiments with 7-62 EBs per experiment per group): (C) Representative photographs (scale bar: 0.02 mm) and (D) quantification of the diameter of the rosette structure of the indicated groups [data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and thus analyzed by Mann-Whitney U test with Holm Sidak correction for multiple comparisons].


RNA - seq was conducted on day - 3 of differentiation induction of Tnfaip2 - / - EBs that were either treated with PA / PC or with a vehicle control ( n = 3 independent pools of 7 - 62 EBs per group ) . The heat maps depict the intersection of DEGs with the following gene sets ( E ) " stem cell maintenance : positive and negative regulators " ,

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets (E) "stem cell maintenance: positive and negative regulators",


RNA - seq was conducted on day - 3 of differentiation induction of Tnfaip2 - / - EBs that were either treated with PA / PC or with a vehicle control ( n = 3 independent pools of 7 - 62 EBs per group ) . The heat maps depict the intersection of DEGs with the following gene sets ; , ( F ) " mesoderm development "

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets ;, (F) "mesoderm development"


RNA - seq was conducted on day - 3 of differentiation induction of Tnfaip2 - / - EBs that were either treated with PA / PC or with a vehicle control ( n = 3 independent pools of 7 - 62 EBs per group ) . The heat maps depict the intersection of DEGs with the following gene sets ( G ) " ectoderm development " . Five other stem cell - related genes ( Tfcp2l1 , Dppa3 , Fbxo15 , Zfp42 and Klf2 ) were additionally incorporated based on literature searches . The asterisks indicate the reverted changes in gene expression that were seen in Tnfaip2 - / - EBs versus WT EBs The color scale represents the gene - wise z - score calculated from normalized gene expression levels .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets (G) "ectoderm development". Five other stem cell-related genes (Tfcp2l1, Dppa3, Fbxo15, Zfp42 and Klf2) were additionally incorporated based on literature searches. The asterisks indicate the reverted changes in gene expression that were seen in Tnfaip2-/- EBs versus WT EBs The color scale represents the gene-wise z-score calculated from normalized gene expression levels.


( H ) Representative image of immunofluorescence staining against ectodermal marker ( SOX1 ) and the mesodermal marker ( T alias Brachyury ) on day - 3 of differentiation induction of Tnfaip2 - / - EBs + / - co - treatment with PA / PC . Three repeat experiments were conducted with a total number of 9 EBs per

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative image of immunofluorescence staining against ectodermal marker (SOX1) and the mesodermal marker (T alias Brachyury) on day-3 of differentiation induction of Tnfaip2-/- EBs +/- co-treatment with PA/PC. Three repeat experiments were conducted with a total number of 9 EBs per group


Oct4 - eGFP reporter MEFs or WT MEFs were infected with shRNAs Cells were co - infected with 4 reprogramming factors ( OSKM ) ( A ) Representative images of AP staining of iPSC

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

Oct4-eGFP reporter MEFs or WT MEFs were infected with shRNAs Cells were co-infected with 4 reprogramming factors (OSKM) (A) Representative images of AP staining of iPSC colonies


Oct4 - eGFP reporter MEFs or WT MEFs were infected with shRNAs Cells were co - infected with 4 reprogramming factors ( OSKM ) and double - infected cells were FACS sorted and grown for 14 days , when reprogramming efficiencies were determined by FACS . ( B ) representative FACS profiles of Oct4 - positive iPSCs on day 14 , ( C ) quantification of the percentage of Oct4 - GFP + iPSCs in MEF cultures that were infected with 2 different shRNAs targeting Vim or a scrambled shRNA control [ n = 3 biological replicates per group ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by t - test with Holm - Sidak correction multiple comparisons ] .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Oct4-eGFP reporter MEFs or WT MEFs were infected with shRNAs Cells were co-infected with 4 reprogramming factors (OSKM) and double-infected cells were FACS sorted and grown for 14 days, when reprogramming efficiencies were determined by FACS. (B) representative FACS profiles of Oct4-positive iPSCs on day 14, (C) quantification of the percentage of Oct4-GFP+ iPSCs in MEF cultures that were infected with 2 different shRNAs targeting Vim or a scrambled shRNA control [n=3 biological replicates per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak correction multiple comparisons].


Oct4 - eGFP reporter MEFs or WT MEFs were infected with combinations of shRNAs . ( D ) Representative FACS profiles of mouse - specific pluripotency cell surface marker SSEA1 + iPSCs on day 14 of reprogramming and ( E ) quantification of the percentage of SSEA1 + iPSCs in MEF cultures that were infected with the indicated shRNAs targeting Vim and / or Tnfaip2 [ n = 5 biological replicates ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) but showed unequal variance ; data were analyzed by Brown - Forsythe and Welch ANOVA and Dunnett ' s multiple comparisons test ] .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Oct4-eGFP reporter MEFs or WT MEFs were infected with combinations of shRNAs. (D) Representative FACS profiles of mouse-specific pluripotency cell surface marker SSEA1+ iPSCs on day 14 of reprogramming and (E) quantification of the percentage of SSEA1+ iPSCs in MEF cultures that were infected with the indicated shRNAs targeting Vim and/or Tnfaip2 [n=5 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) but showed unequal variance; data were analyzed by Brown-Forsythe and Welch ANOVA and Dunnett's multiple comparisons test].


combinations of shRNAs . RT - qPCR was performed to determine ( F ) Vim mRNA expression in scrambled - shRNA infected MEFs versus shRNA - Tnfaip2 - infected MEFs or Infected cells were purified by BFP - sorting on day - 4 after infection [ n = 3 biological replicates ; log transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by t - test with Holm - Sidak for multiple comparisons ] .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

combinations of shRNAs. RT-qPCR was performed to determine (F) Vim mRNA expression in scrambled-shRNA infected MEFs versus shRNA-Tnfaip2-infected MEFs or Infected cells were purified by BFP-sorting on day-4 after infection [n=3 biological replicates; log transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak for multiple comparisons].


RT - qPCR was performed to determine ( G ) Tnfaip2 mRNA expression in scrambled - shRNA infected MEFs versus shRNA - Vim - infected MEFs . G ) Infected cells were purified by BFP - sorting on day - 4 after infection [ n = 3 biological replicates ; log transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by t - test with Holm - Sidak for multiple comparisons ] .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-qPCR was performed to determine (G) Tnfaip2 mRNA expression in scrambled-shRNA infected MEFs versus shRNA-Vim-infected MEFs. G) Infected cells were purified by BFP-sorting on day-4 after infection [n=3 biological replicates; log transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak for multiple comparisons].


Oct4 - eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA ( OE indicates overexpression ) , Cells were co - infected with 4 reprogramming factors ( 4F = Oct4 , Sox2 , Klf4 , c - myc = OSKM ) and double - infected cells were FACS sorted for analysis . ( A ) RT - qPCR analysis on day - 4 after infection to determine RNA expression of Tnfaip2 or Vim in Tnfaip2 - depleted cells with ectopic expression of Vim ( left ) or in Vim - depleted cells with ectopic expression of Tnfaip2 ( right ) . Scramble shRNA ( shScr ) - infected MEFs served as a control [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by t - test ] .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Oct4-eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), Cells were co-infected with 4 reprogramming factors (4F= Oct4, Sox2, Klf4, c-myc = OSKM) and double-infected cells were FACS sorted for analysis. (A) RT-qPCR analysis on day-4 after infection to determine RNA expression of Tnfaip2 or Vim in Tnfaip2-depleted cells with ectopic expression of Vim (left) or in Vim-depleted cells with ectopic expression of Tnfaip2 (right). Scramble shRNA (shScr)-infected MEFs served as a control [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test].


Oct4 - eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA ( OE indicates overexpression ) , ( B ) RT - qPCR analysis on day - 4 after infection to determine RNA expression of Vim or Tnfaip2 in Tnfaip2 - depleted cells with ectopic expression of Vim ( left ) or in Vim - depleted cells with ectopic expression of Tnfaip2 ( right ) . Scramble shRNA ( shScr ) - infected MEFs served as a control [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by t - test ] .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Oct4-eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), (B) RT-qPCR analysis on day-4 after infection to determine RNA expression of Vim or Tnfaip2 in Tnfaip2-depleted cells with ectopic expression of Vim (left) or in Vim-depleted cells with ectopic expression of Tnfaip2 (right). Scramble shRNA (shScr)-infected MEFs served as a control [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test].


Oct4 - eGFP reporter MEFs or WT MEFs were ( A - D ) infected with a vector expressing the indicated shRNA in combination with the indicated cDNA ( OE indicates overexpression ) , ] . ( C ) Representative images of FACS profiles and ( D ) quantification of SSEA1 + iPSCs on day 14 after infection of MEF cultures with the indicated combination of shRNAs plus cDNAs ( OE ) or scrambled shRNA control [ n = 3 independent cultures per group ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by one - way ANOVA and Dunnett ' s multiple comparison test ]

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Oct4-eGFP reporter MEFs or WT MEFs were (A-D) infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), ]. (C) Representative images of FACS profiles and (D) quantification of SSEA1+ iPSCs on day 14 after infection of MEF cultures with the indicated combination of shRNAs plus cDNAs (OE) or scrambled shRNA control [n=3 independent cultures per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-way ANOVA and Dunnett's multiple comparison test ]


Oct4 - eGFP reporter MEFs or WT MEFs were ( E , F ) infected with the indicated shRNA with or without combined treatment with Etomoxir - a chemical inhibitor of CPT1A . ( E ) Representative FACS profiles and ( F ) quantification of the percentage of SSEA1 + iPSCs in MEF cultures on day - 18 after infection with shRNAs targeting Tnfaip2 or a scrambled shRNA control with or without continuous treatment with Etomoxir [ n = 5 biological replicates ; data were not normally distributed ( P < 0 . 05 as per Shapiro - Wilk test ) and analyzed by Mann - Whitney U test with Holm Sidak correction for multiple comparisons ] .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Oct4-eGFP reporter MEFs or WT MEFs were (E,F) infected with the indicated shRNA with or without combined treatment with Etomoxir - a chemical inhibitor of CPT1A. (E) Representative FACS profiles and (F) quantification of the percentage of SSEA1+ iPSCs in MEF cultures on day-18 after infection with shRNAs targeting Tnfaip2 or a scrambled shRNA control with or without continuous treatment with Etomoxir [n=5 biological replicates; data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and analyzed by Mann-Whitney U test with Holm Sidak correction for multiple comparisons].


( A ) TAG levels in exoc3 ( RNAi ) - treated planarians vs . gfp ( RNAi ) - treated controls as determined by UPLC - MS / MS , [ n = 4 experimental replicates on pools of 25 planarians per group per experiment ; data were normally

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) TAG levels in exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated controls as determined by UPLC-MS/MS, [n=4 experimental replicates on pools of 25 planarians per group per experiment; data were normally distributed


( C ) RT - qPCR analysis of exoc3 mRNA expression in planarians of the indicated genotypes and treatment

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

(C) RT-qPCR analysis of exoc3 mRNA expression in planarians of the indicated genotypes and treatment schedule


( D ) Representative images of regenerating planarians that were injected with gfp - RNAi , Smed - β - catenin - 1 - RNAi or Smed - β - catenin - 1 - RNAi in combination with PA / PC injection Heads and tails were cut on day - 10 , images were taken on day - 17 after amputation . Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Representative images of regenerating planarians that were injected with gfp-RNAi, Smed-β-catenin-1-RNAi or Smed-β-catenin-1-RNAi in combination with PA/PC injection Heads and tails were cut on day-10, images were taken on day-17 after amputation. Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype.


( E ) Images of body plan maintenance in non - injured planarians that were injected with gfp - RNAi , exoc3 - RNAi or exoc3 - RNAi in combination with PA / PC injection Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Images of body plan maintenance in non-injured planarians that were injected with gfp-RNAi, exoc3-RNAi or exoc3-RNAi in combination with PA/PC injection Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype.


( F ) RT - qPCR analysis of marker genes of early stem cell differentiation ( prog - 1 and prog - 2 ) and late stem cell differentiation ( Smed - odc1 ) in planarians exposed to the indicated treatment regiments on day - 38 of the experiment as indicated in panel - B [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by one - way ANOVA and Dunnett ' s multiple comparison test ] .

O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) RT-qPCR analysis of marker genes of early stem cell differentiation (prog-1 and prog-2) and late stem cell differentiation (Smed-odc1) in planarians exposed to the indicated treatment regiments on day-38 of the experiment as indicated in panel-B [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-way ANOVA and Dunnett's multiple comparison test].


( a ) Silver - stained gel image of the affinity - purified complexes of PSPC1 , SFPQ and NONO . The respective proteins are marked along with AcTEV protease used for the elution of purified proteins .

O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(a) Silver-stained gel image of the affinity-purified complexes of PSPC1, SFPQ and NONO. The respective proteins are marked along with AcTEV protease used for the elution of purified proteins.


( f ) Western blotting of affinity purified complexes of PSPC1 , SFPQ and NONO with SWI / SNF antibodies . cBAF , PBAF , ncBAF specific , as well as common subunits , are marked .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(f) Western blotting of affinity purified complexes of PSPC1, SFPQ and NONO with SWI/SNF antibodies. cBAF, PBAF, ncBAF specific, as well as common subunits, are marked.


( a ) Silver - stained gel image of the affinity - purified complexes of ARID1A and ARID1B . The bait and a few other SWI / SNF subunits are marked as per their molecular weight .

O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(a) Silver-stained gel image of the affinity-purified complexes of ARID1A and ARID1B. The bait and a few other SWI/SNF subunits are marked as per their molecular weight.


Heat maps showing the enriched proteins ( dNSAFavg * 1000 ) normalized to the bait identified in MudPIT analysis focusing on various ( c ) SWI / SNF

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (c) SWI/SNF subunits


Heat maps showing the enriched proteins ( dNSAFavg * 1000 ) normalized to the bait identified in MudPIT analysis focusing on various ( e ) PSPs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (e) PSPs.


( f ) Western blotting of affinity purified complexes of ARID1A and ARID1B with bonafide paraspeckle components . SMARCA4 was used as a positive control for purification .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O

(f) Western blotting of affinity purified complexes of ARID1A and ARID1B with bonafide paraspeckle components. SMARCA4 was used as a positive control for purification.


( a ) Halo purification was performed from nuclear extracts of scramble and various knockdown HEK293FRT cells expressing Halo - FLAG - tagged ARID1B . Input and eluted samples were resolved on a gel followed by western blotting with various antibodies . Western with SWI / SNF subunits - SMARCA4 and SMARCD1 were used as the control for purification , while beta - actin was used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(a) Halo purification was performed from nuclear extracts of scramble and various knockdown HEK293FRT cells expressing Halo-FLAG-tagged ARID1B. Input and eluted samples were resolved on a gel followed by western blotting with various antibodies. Western with SWI/SNF subunits- SMARCA4 and SMARCD1 were used as the control for purification, while beta-actin was used as a loading control.


( e , f ) ( e ) RIP qPCR and ( f ) PCR analysis performed using an antibody against endogenous ARID1B . for monitoring NEAT1 _ 2 levels . RIP with NONO and IgG was used as a positive and negative control respectively . GAPDH levels were also assessed in the assay . Data are expressed as a percent of Input .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e, f) (e) RIP qPCR and (f) PCR analysis performed using an antibody against endogenous ARID1B. for monitoring NEAT1_2 levels. RIP with NONO and IgG was used as a positive and negative control respectively. GAPDH levels were also assessed in the assay. Data are expressed as a percent of Input.


( c ) smFISH analysis was performed to detect NEAT1 signal in the nucleus . The scale bar corresponds to 10 µm . Graph showing the FISH signal quantification across the knockdowns in comparison to scramble control . The number of nuclei ( n ) used for quantification is mentioned .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) smFISH analysis was performed to detect NEAT1 signal in the nucleus. The scale bar corresponds to 10 µm. Graph showing the FISH signal quantification across the knockdowns in comparison to scramble control. The number of nuclei (n) used for quantification is mentioned.


Western blots showing changing protein levels on the path to senescence in IMR90 and HUVECs . Passage 6 cells represent the proliferating state , and passages 21 and 34 the senescent state for HUVECs and IMR90 , respectively .

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Western blots showing changing protein levels on the path to senescence in IMR90 and HUVECs. Passage 6 cells represent the proliferating state, and passages 21 and 34 the senescent state for HUVECs and IMR90, respectively.


Super - resolution ( gSTED ) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained with DAPI . Bar : 2μm .

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Super-resolution (gSTED) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained with DAPI. Bar: 2μm.


Representative immunofluorescence images of IMR90 and HUVECs ( left ) showing reduced HMGB1 levels in senescent nuclei ; bean plots quantify this reduction ( right ; N is the number of cells analyzed per each condition / cell type ) . Bars : 5 μm . * : P < 0 . 01 ; Wilcoxon - Mann - Whitney test .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative immunofluorescence images of IMR90 and HUVECs (left) showing reduced HMGB1 levels in senescent nuclei; bean plots quantify this reduction (right; N is the number of cells analyzed per each condition/cell type). Bars: 5 μm. *: P<0.01; Wilcoxon-Mann-Whitney test.


Genome browser view of HMGB1 ChIP - seq ( normalized to input ; mean from two replicates ) from proliferating HUVEC ( red ) or proliferating / senescent IMR90 ( grey / green ) in the TMEM92 locus .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Genome browser view of HMGB1 ChIP-seq (normalized to input; mean from two replicates) from proliferating HUVEC (red) or proliferating/senescent IMR90 (grey/green) in the TMEM92 locus.


Heatmap showing protein ( SASP , from http : / / www . saspatlas . com / ; log fold ratio ) and gene expression levels ( log2FC ) of IMR90 SASP - related genes embedded in high - HMGB1 ( red ) TADs like those in panel C . * * : genes bound by HMGB1 in ChIP - seq data are more than expected by chance ; P > 0 . 001 , hypergeometric test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heatmap showing protein (SASP, from http://www.saspatlas.com/; log fold ratio)and gene expression levels (log2FC) of IMR90 SASP-related genes embedded in high-HMGB1 (red) TADs like those in panel C. **: genes bound by HMGB1 in ChIP-seq data are more than expected by chance; P>0.001, hypergeometric test.


Immunofluorescence ( top ) , western blot ( middle ) and RT - qPCR analyses ( bottom ; mean fold - change ± S . D . from two biological replicates ) confirm HMGB1 knockdown in IMR90 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

Immunofluorescence (top), western blot (middle) and RT-qPCR analyses (bottom; mean fold-change ±S.D. from two biological replicates) confirm HMGB1 knockdown in IMR90.


Representative images of IMR90 overexpressing HMGB1 - GFP , immunostained for p21 and CTCF , and counterstained with DAPI . IMR90 transfected with empty vectors provide a control . Bar : 5 μm .

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Representative images of IMR90 overexpressing HMGB1-GFP, immunostained for p21 and CTCF, and counterstained with DAPI. IMR90 transfected with empty vectors provide a control. Bar: 5 μm.


Genome browser views showing HMGB1 sCLIP data ( black ) along the ASH1L , CCNL2 and TET2 loci ; input tracks ( grey ) provide background levels . * : significantly - enriched peaks . The consensus motif for HMGB1 binding on RNA is also shown ( bottom right ) .

O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

Genome browser views showing HMGB1 sCLIP data (black) along the ASH1L, CCNL2 and TET2 loci; input tracks (grey) provide background levels. *: significantly-enriched peaks. The consensus motif for HMGB1 binding on RNA is also shown (bottom right).


Bar graphs showing mean fold - change of selected mRNAs ( over ± S . D . from two biological replicates ) from ILF3 - ( purple ) , RBMX - ( white ) or PNN - knockdown experiments ( blue ) in proliferating IMR90 . * : significantly different to siRNA controls ; P < 0 . 01 , unpaired two - tailed Student ' s t - test . Dashed line indicates no change in expression .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graphs showing mean fold-change of selected mRNAs (over ±S.D. from two biological replicates) from ILF3- (purple), RBMX- (white) or PNN-knockdown experiments (blue) in proliferating IMR90. *: significantly different to siRNA controls; P<0.01, unpaired two-tailed Student's t-test. Dashed line indicates no change in expression.