SUM159PT human breast cancer cells were orthotopically injected into the mammary glands of NSG mice . Once SUM159PT tumors reached 250mm3 volume , mice were treated with mock or ASNase ( 60U per day ) for 5 consecutive days ( n = 3 ) . Following treatment , mice were sacrificed , and blood , mammary glands and tumors were collected and subjected to mass spectrometry to determine the asparagine level . Essential amino acids were used for the raw data normalization . Data are presented as mean ± SD . Data information : The pretreatment started 2 days before the injection of cancer cells . And mice were either injected with 60 U ASNase or saline per day . The p - value was calculated by two - tailed unpaired t test in Prism7 , unless otherwise stated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

B-CELL_LINE B-ORGANISM B-DISEASE I-DISEASE O O O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O B-CELL_LINE O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SUM159PT human breast cancer cells were orthotopically injected into the mammary glands of NSG mice. Once SUM159PT tumors reached 250mm3 volume, mice were treated with mock or ASNase (60U per day) for 5 consecutive days (n=3). Following treatment, mice were sacrificed, and blood, mammary glands and tumors were collected and subjected to mass spectrometry to determine the asparagine level. Essential amino acids were used for the raw data normalization. Data are presented as mean ± SD. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.


The mouse breast cancer cell lines 4T1 and 4T1 - V5 - SLC1A3 were orthotopically implanted into the mammary glands of pretreated NSG mice . Presented is the volume measurements of arising tumors at day 9 . ( n = 13 mice for each group , except for 4T1 + ASNase , n = 12 ) . Data are presented as mean ± SEM . Data information : The pretreatment started 2 days before the injection of cancer cells . And mice were either injected with 60 U ASNase or saline per day . The p - value was calculated by two - tailed unpaired t test in Prism7 , unless otherwise stated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-ORGANISM B-DISEASE I-DISEASE O O B-CELL_LINE O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O 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-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The mouse breast cancer cell lines 4T1 and 4T1-V5-SLC1A3 were orthotopically implanted into the mammary glands of pretreated NSG mice. Presented is the volume measurements of arising tumors at day 9. (n=13 mice for each group, except for 4T1+ASNase, n=12). Data are presented as mean ± SEM. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.


tumors were surgically removed once reached a volume of ~ 500mm3 and collected and subjected to LC - MS to determine the levels of asparagine , aspartate , glutamine and glutamate . Leucine level was used as a control . Results are based on five tumor samples and presented as mean ± SEM . Data information : The pretreatment started 2 days before the injection of cancer cells . And mice were either injected with 60 U ASNase or saline per day . The p - value was calculated by two - tailed unpaired t test in Prism7 , unless otherwise stated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

B-DISEASE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-DISEASE 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-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

tumors were surgically removed once reached a volume of ~500mm3 and collected and subjected to LC-MS to determine the levels of asparagine, aspartate, glutamine and glutamate. Leucine level was used as a control. Results are based on five tumor samples and presented as mean ± SEM. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.


The human breast cancer cell lines MDA - MB - 231 and MDA - MB - 231 - V5 - SLC1A3 cells were intravenously injected into pretreated NSG mice . Once mice showed breathing problems , they were sacrificed , and lung and liver were collected and blindly scored for metastasis lesions . The p - value was calculated by one - tailed unpaired t test in Prism7 . Data are presented as mean ± SEM ( n = 8 ) . Data information : The pretreatment started 2 days before the injection of cancer cells . And mice were either injected with 60 U ASNase or saline per day . The p - value was calculated by two - tailed unpaired t test in Prism7 , unless otherwise stated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-ORGANISM B-DISEASE I-DISEASE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The human breast cancer cell lines MDA-MB-231 and MDA-MB-231-V5-SLC1A3 cells were intravenously injected into pretreated NSG mice. Once mice showed breathing problems, they were sacrificed, and lung and liver were collected and blindly scored for metastasis lesions. The p-value was calculated by one-tailed unpaired t test in Prism7. Data are presented as mean ± SEM (n=8). Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.


I Correlation between peptide - spectrum matches ( PSMs ) of identified proteins and their fold change calculated by SAINTexpress .

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I Correlation between peptide-spectrum matches (PSMs) of identified proteins and their fold change calculated by SAINTexpress.


A Pulldown and Western blot analysis validated the interaction between the viral protein ORF3a and its interactors , VPS11 and CLCC1 , and the binding of S protein to SPCS2 . HEK293T cells with SFB - tagged bait expression were collected and lysed . Cell lysates were subjected to pulldown assay using S - protein beads . Western blot analysis was conducted with the indicated antibodies . Cells transfected with vector or construct encoding control genes were included as controls in these experiments .

O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O 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 Pulldown and Western blot analysis validated the interaction between the viral protein ORF3a and its interactors, VPS11 and CLCC1, and the binding of S protein to SPCS2. HEK293T cells with SFB-tagged bait expression were collected and lysed. Cell lysates were subjected to pulldown assay using S-protein beads. Western blot analysis was conducted with the indicated antibodies. Cells transfected with vector or construct encoding control genes were included as controls in these experiments.


B Immunostaining analysis of protein localization . U2OS cells were transfected with construct encoding ORF3a . Cells were fixed and stained with the indicated antibodies . The green signal is CLCC1 or lysosome marker LAMP1 , the red signal is flag ( for SFB - ORF3a ) , and the blue signal indicates DAPI / nuclei . Scale bar : 10 µm .

O B-EXP_ASSAY O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O

B Immunostaining analysis of protein localization. U2OS cells were transfected with construct encoding ORF3a. Cells were fixed and stained with the indicated antibodies. The green signal is CLCC1 or lysosome marker LAMP1, the red signal is flag (for SFB-ORF3a), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.


B Comparison of SARS - CoV - 2 virus - host interaction to the genetic screening with CRISPR technique . Two different MOIs ( MOI 0 . 01 and MOI 0 . 3 ) were used in these screens and we compared our results with both of these datasets . Blue dots are the identified genes from the genetic screening . Orange dots are the highlighted overlapped genes between our virus - host interaction study and the results from the genetic screening . Top five overlapped genes are indicated .

O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

B Comparison of SARS-CoV-2 virus-host interaction to the genetic screening with CRISPR technique. Two different MOIs (MOI 0.01 and MOI 0.3) were used in these screens and we compared our results with both of these datasets. Blue dots are the identified genes from the genetic screening. Orange dots are the highlighted overlapped genes between our virus-host interaction study and the results from the genetic screening. Top five overlapped genes are indicated.


C Analyzing the number of HCIPs with RNA expression changes identified by single - cell RNA sequencing using cells from COVID - 19 patients .

O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE I-DISEASE I-DISEASE O O

C Analyzing the number of HCIPs with RNA expression changes identified by single-cell RNA sequencing using cells from COVID-19 patients.


D Pulldown and Western blot validation of the interaction between human coronavirus NSP1 proteins and the human proteins PYCR1 / PYCR2 . Cells transfected with vector or construct encoding GFP were included as controls in these experiments .

O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM B-GENEPROD O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O

D Pulldown and Western blot validation of the interaction between human coronavirus NSP1 proteins and the human proteins PYCR1/PYCR2. Cells transfected with vector or construct encoding GFP were included as controls in these experiments.


C Heat map of the N protein HCIPs identified by SFB - tandem affinity purification . N protein HCIPs were compared among seven human coronaviruses using the preys ' spectral counts . Three areas of the heat map are manually selected , enlarged and labeled as ( 1 ) , ( 2 ) , and ( 3 ) , which are enriched by all N proteins of the seven human coronaviruses . Functional characterization for each protein is shown with the red circles below the enlarged images .

O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O

C Heat map of the N protein HCIPs identified by SFB-tandem affinity purification. N protein HCIPs were compared among seven human coronaviruses using the preys' spectral counts. Three areas of the heat map are manually selected, enlarged and labeled as (1), (2), and (3), which are enriched by all N proteins of the seven human coronaviruses. Functional characterization for each protein is shown with the red circles below the enlarged images.


A Preys identified showing significant enrichment by N proteins specifically from one or more human coronaviruses . PSM , peptide - spectrum match .

O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM O O O O O O O O

A Preys identified showing significant enrichment by N proteins specifically from one or more human coronaviruses. PSM, peptide-spectrum match.


B Pulldown and Western blot validation of the interaction between N protein and G3BP1 / G3BP2 . Cells transfected with vector or construct encoding GFP or N protein from different human coronaviruses were compared in these experiments .

O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM I-ORGANISM O O O O O O

B Pulldown and Western blot validation of the interaction between N protein and G3BP1/G3BP2. Cells transfected with vector or construct encoding GFP or N protein from different human coronaviruses were compared in these experiments.


C , D Effect of SARS - CoV - 2 N protein on stress granule formation . A549 ( C ) and MCF10A ( D ) cells were transfected with pinducer20 - N protein of SARS - CoV - 2 . All the cells are treated with sodium arsenite but with or without Dox to induce N protein expression . Cells were fixed and stained with the indicated antibodies . The green signal is G3BP1 , the red signal is HA ( for N protein ) , and the blue signal indicates DAPI / nuclei . Scale bar : 10 µm .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O

C,D Effect of SARS-CoV-2 N protein on stress granule formation. A549 (C) and MCF10A (D) cells were transfected with pinducer20-N protein of SARS-CoV-2. All the cells are treated with sodium arsenite but with or without Dox to induce N protein expression. Cells were fixed and stained with the indicated antibodies. The green signal is G3BP1, the red signal is HA (for N protein), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.


E Quantification of the stress granule formation in A549 and MCF10A cells . Total of 30 cells were counted and a student ' s t test was used for the statistical analysis ( * * * , p < 0 . 001 ) . Box limits represent 25th percentile and 75th percentile ; horizontal line represents median . Whiskers display min . to max . values .

O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of the stress granule formation in A549 and MCF10A cells. Total of 30 cells were counted and a student's t test was used for the statistical analysis (***, p<0.001). Box limits represent 25th percentile and 75th percentile; horizontal line represents median. Whiskers display min. to max. values.


( 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 ) .

O O O O O O O B-DISEASE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(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 B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O B-GENEPROD O O 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-EXP_ASSAY

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 ] .

O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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) 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-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY 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 ) .

B-ORGANISM O O O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O 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 ] .

B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O 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-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-ORGANISM O B-EXP_ASSAY O O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY

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 ] ,

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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 ) .

B-ORGANISM O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O B-TISSUE I-TISSUE O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O B-ORGANISM O O O O B-TISSUE O B-TISSUE I-TISSUE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O

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 ) .

O O O B-ORGANISM O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O

(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

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O 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 ) .

O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O 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-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY 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 (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 B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE 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 ) .

O O B-CELL_TYPE O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O 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-TISSUE O O O O O O O O O O O O O

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 " ,

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-TISSUE O O O B-TISSUE O O O O O O O O B-TISSUE O 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-CELL_TYPE I-CELL_TYPE 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 "

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-TISSUE O O O B-TISSUE O O O O O O O O B-TISSUE O O O 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-TISSUE 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-TISSUE O O O B-TISSUE O O O O O O O O B-TISSUE 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-TISSUE O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O B-GENEPROD O O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O B-TISSUE O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-TISSUE O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-TISSUE O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O 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 ) .

B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-TISSUE 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 ] .

B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O B-TISSUE O O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O 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-EXP_ASSAY O O B-TISSUE O O O O O O O O O O B-SUBCELLULAR 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O B-TISSUE O O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O 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-SUBCELLULAR O B-EXP_ASSAY O O O O 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 B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-CELL_TYPE O O O O O B-TISSUE 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 B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE O B-GENEPROD O O O B-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-TISSUE O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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 " ,

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE 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 "

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-GENEPROD O O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE 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

B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE

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-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O B-GENEPROD O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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 ] .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O 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 ]

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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 ] .

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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

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(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

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(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 .

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(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 .

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(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 ] .

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(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 SHP1immunoprecipitation ( IP ) from Jurkat 1G4 ‐ CD8 cells . Upper panel of immunoblots shows expression levels in the input lysates , and isolated protein complexes are shown at the bottom . Rabbit IgGIP ( isotype ) is shown as control .

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A SHP1immunoprecipitation (IP) from Jurkat 1G4‐CD8 cells. Upper panel of immunoblots shows expression levels in the input lysates, and isolated protein complexes are shown at the bottom. Rabbit IgGIP (isotype) is shown as control.


B Lentiviral knock ‐ down / re ‐ expression constructs for THEMIS ‐ wt ‐ Strep and THEMIS ‐ dPRR1 ‐ Strep in 1G4 ‐ CD8 cells . Knock ‐ down / re ‐ expression constructs are based on shTHEMIS ‐ 128476 . Red arrowheads indicate the specific signal for endogenous THEMIS and re ‐ expressed THEMIS ‐ Strep . The higher molecular weight band observed in all lanes is a non ‐ specific signal .

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B Lentiviral knock‐down/re‐expression constructs for THEMIS‐wt‐Strep and THEMIS‐dPRR1‐Strep in 1G4‐CD8 cells. Knock‐down/re‐expression constructs are based on shTHEMIS‐128476. Red arrowheads indicate the specific signal for endogenous THEMIS and re‐expressed THEMIS‐Strep. The higher molecular weight band observed in all lanes is a non‐specific signal.


C Streptactinpull ‐ downs ( PDs ) of THEMIS ‐ Strep from 1G4 ‐ CD8 cells . Cells as described in ( B ) were stimulated with 6V tetramers , followed by Streptactin PDs of THEMIS and immunoblot analysis of isolated protein complexes . Separate experiments for SHP1 and SHP2 are shown .

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C Streptactinpull‐downs (PDs) of THEMIS‐Strep from 1G4‐CD8 cells. Cells as described in (B) were stimulated with 6V tetramers, followed by Streptactin PDs of THEMIS and immunoblot analysis of isolated protein complexes. Separate experiments for SHP1 and SHP2 are shown.


D SHP1IP in the presence and absence of SHP1 tail phosphorylation . SHP1 was immunoprecipitated from J . CaM1 . 6LCK ‐ Tet cells . Where indicated , LCK expression was induced with doxycycline prior to the experiment or Src ‐ kinase inhibitor PP2 was used to abolish residual SHP1 ‐ pY564 . The upper panel of immunoblots shows the effect of LCK activity on SHP1 ‐ pY564 levels . The red arrowhead indicates residual pY564 . Isolated protein complexes are shown in the lower panels .

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D SHP1IP in the presence and absence of SHP1 tail phosphorylation. SHP1 was immunoprecipitated from J.CaM1.6LCK‐Tet cells. Where indicated, LCK expression was induced with doxycycline prior to the experiment or Src‐kinase inhibitor PP2 was used to abolish residual SHP1‐pY564. The upper panel of immunoblots shows the effect of LCK activity on SHP1‐pY564 levels. The red arrowhead indicates residual pY564. Isolated protein complexes are shown in the lower panels.


E In vitro phosphatase treatment of the THEMIS : GRB2 : SHP1 complex . Bead ‐ bound THEMIS ‐ Strep complexes from 1G4 ‐ CD8 cells were incubated in alkaline phosphatase ( AP ) buffer in the absence ( lane 1 ) or presence of AP ( lane 2 ) , or left completely untreated ( lane 3 ) , prior to washing and elution .

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E In vitro phosphatase treatment of the THEMIS:GRB2:SHP1 complex. Bead‐bound THEMIS‐Strep complexes from 1G4‐CD8 cells were incubated in alkaline phosphatase (AP) buffer in the absence (lane 1) or presence of AP (lane 2), or left completely untreated (lane 3), prior to washing and elution.


F Anti ‐ HAIP from HEK293 cells transfected with HA ‐ SHP1 and GRB2 ‐ Myc constructs . GRB2 mutants used : W36K , N ‐ SH3 mutant ; W193K , C ‐ SH3 mutant . Upper panels show expression levels in the input ; isolated proteins complexes are shown at the bottom . Relative amounts of GRB2 ‐ Myc are normalized to the bait HA ‐ SHP1 .

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F Anti‐HAIP from HEK293 cells transfected with HA‐SHP1 and GRB2‐Myc constructs. GRB2 mutants used: W36K, N‐SH3 mutant; W193K, C‐SH3 mutant. Upper panels show expression levels in the input; isolated proteins complexes are shown at the bottom. Relative amounts of GRB2‐Myc are normalized to the bait HA‐SHP1.


G Far ‐ Western blot of GRB2 ‐ SH3 domains binding to full ‐ length SHP1 . HA IPs from empty vector or HA ‐ SHP1 ‐ transfected HEK293 cells were subjected to far ‐ Western blotting using recombinant GST ‐ tagged N ‐ or C ‐ SH3 domains of GRB2 . GST alone served as a background control . Blots were re ‐ probed for SHP1 loading for normalization of anti ‐ GST signals . Data shown are representative of three independent experiments .

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G Far‐Western blot of GRB2‐SH3 domains binding to full‐length SHP1. HA IPs from empty vector or HA‐SHP1‐transfected HEK293 cells were subjected to far‐Western blotting using recombinant GST‐tagged N ‐or C‐SH3 domains of GRB2. GST alone served as a background control. Blots were re‐probed for SHP1 loading for normalization of anti‐GST signals. Data shown are representative of three independent experiments.


A Lentiviral knock ‐ down ( KD ) of THEMIS expression in 1G4 ‐ CD8 cells . The efficiency of the THEMIS KD was assessed using immunoblotting .

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A Lentiviral knock‐down (KD) of THEMIS expression in 1G4‐CD8 cells. The efficiency of the THEMIS KD was assessed using immunoblotting.


B Phospho ‐ flow cytometry analysis of pERK responses in THEMIS KD cells . 1G4 ‐ CD8 control and THEMIS KD cells were stimulated with NY ‐ ESO ‐ 1 pMHC tetramers and analysed for ERK phosphorylation using flow cytometry . Data shown are representative of three independent experiments . C Quantification of pERK ‐ positive cells from ( B ) . Data from three independent experiments were used in the analysis . n = 3 , means ± SEM are shown ; two ‐ tailed unpaired Student ' s t ‐ test , * P 0 . 05 , * * P 0 . 01 .

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B Phospho‐flow cytometry analysis of pERK responses in THEMIS KD cells. 1G4‐CD8 control and THEMIS KD cells were stimulated with NY‐ESO‐1 pMHC tetramers and analysed for ERK phosphorylation using flow cytometry. Data shown are representative of three independent experiments.C Quantification of pERK‐positive cells from (B). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01.


D Phospho ‐ flow cytometry analysis of pERK responses against titrated concentrations of 9V tetramer . Time point was fixed at 60 s . Data shown are representative of three independent experiments . E Quantification of pERK ‐ positive cells from ( D ) . Data from three independent experiments were used in the analysis . n = 3 , means ± SEM are shown .

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D Phospho‐flow cytometry analysis of pERK responses against titrated concentrations of 9V tetramer. Time point was fixed at 60 s. Data shown are representative of three independent experiments.E Quantification of pERK‐positive cells from (D). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown.


A 1G4 ‐ CD8 control and THEMIS KD cells were stimulated with NY ‐ ESO ‐ 1 pMHC tetramers and analysed for pY142 ‐ CD3 ‐ ζ phosphorylation by flow cytometry . Data shown are representative of three independent experiments . B Quantification of pY142 ‐ CD3 ‐ ζ responses of cells from ( A ) . Data from three independent experiments were used in the analysis . Responses are expressed as percentage of the positive control sodium pervanadate . n = 3 , means ± SEM are shown ; two ‐ tailed unpaired Student ' s t ‐ test , * * P 0 . 01 .

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A 1G4‐CD8 control and THEMIS KD cells were stimulated with NY‐ESO‐1 pMHC tetramers and analysed for pY142‐CD3‐ζ phosphorylation by flow cytometry. Data shown are representative of three independent experiments.B Quantification of pY142‐CD3‐ζ responses of cells from (A). Data from three independent experiments were used in the analysis. Responses are expressed as percentage of the positive control sodium pervanadate. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, **P 0.01.


A Lentiviral knock ‐ down of THEMIS expression in primary humanCD4 + T cells . Following transduction , cells were expanded on CD3 / CD28 beads in the presence of exogenous IL ‐ 2 and analysed for THEMIS KD by immunoblotting on day 4 post ‐ transduction . Data shown are representative of three independent experiments .

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A Lentiviral knock‐down of THEMIS expression in primary humanCD4+T cells. Following transduction, cells were expanded on CD3/CD28 beads in the presence of exogenous IL‐2 and analysed for THEMIS KD by immunoblotting on day 4 post‐transduction. Data shown are representative of three independent experiments.


B pERK response in THEMIS KD humanCD4 + T cells . Transduced CD4 + cells were removed from beads , rested overnight and stimulated with CD3 mAb for the indicated time points . ERK phosphorylation was assessed by immunoblotting . Data shown are representative of three independent experiments .

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B pERK response in THEMIS KD humanCD4+T cells. Transduced CD4+ cells were removed from beads, rested overnight and stimulated with CD3 mAb for the indicated time points. ERK phosphorylation was assessed by immunoblotting. Data shown are representative of three independent experiments.


C Expression of CD25 and CD69 surface markers of THEMIS KD humanCD4 + T cells was assessed on day 4 post ‐ transduction by flow cytometry . Data shown are representative of three independent experiments .

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C Expression of CD25 and CD69 surface markers of THEMIS KD humanCD4+T cells was assessed on day 4 post‐transduction by flow cytometry. Data shown are representative of three independent experiments.


D CD69 up ‐ regulation on re ‐ stimulated THEMIS KD CD4 + T cells . CD3 / CD28 beads were removed on day 1 post ‐ transduction , and cells were cultured in IL ‐ 2 and IL ‐ 7 . After removal of cell debris at day 4 , cells were restimulated with CD3 / CD28 beads at the indicated bead to cell ratio . CD69 surface expression was assessed by flow cytometry 24 h later . Data shown are representative of two independent experiments . n = 3 , means ± SD are shown ; two ‐ tailed unpaired Student ' s t ‐ test , * P 0 . 05 , * * * P 0 . 001 .

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D CD69 up‐regulation on re‐stimulated THEMIS KD CD4+T cells. CD3/CD28 beads were removed on day 1 post‐transduction, and cells were cultured in IL‐2 and IL‐7. After removal of cell debris at day 4, cells were restimulated with CD3/CD28 beads at the indicated bead to cell ratio. CD69 surface expression was assessed by flow cytometry 24 h later. Data shown are representative of two independent experiments. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, *P 0.05, ***P 0.001.


A Donor ‐ derived humanCD4 + T cells transduced as in Fig A were analysed for apoptosis by Annexin ‐ V surface staining on day 4 post ‐ transduction . Data from three independent experiments were used in the analysis . n = 3 , means ± SD are shown ; two ‐ tailed unpaired Student ' s t ‐ test , * * P 0 . 01 .

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A Donor‐derived humanCD4+T cells transduced as in Fig A were analysed for apoptosis by Annexin‐V surface staining on day 4 post‐transduction. Data from three independent experiments were used in the analysis. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, **P 0.01.


B Annexin ‐ V staining of THEMIS KD 1G4 ‐ CD8 cells . THEMIS KD and control 1G4 ‐ CD8 cells were stimulated for 24 h with plate ‐ bound 6V tetramers . Annexin ‐ V surface expression was analysed by flow cytometry and is shown as fold ‐ increase over non ‐ stimulated . Data from three independent experiments were used in the analysis . n = 3 , means ± SD are shown ; two ‐ tailed unpaired Student ' s t ‐ test , * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; ns , not significant .

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B Annexin‐V staining of THEMIS KD 1G4‐CD8 cells. THEMIS KD and control 1G4‐CD8 cells were stimulated for 24 h with plate‐bound 6V tetramers. Annexin‐V surface expression was analysed by flow cytometry and is shown as fold‐increase over non‐stimulated. Data from three independent experiments were used in the analysis. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01, ***P 0.001; ns, not significant.