( B ) The characteristic ubiquitylation ladder was observed for wild - type MITOL , but was absent in the K268A and K54A / K268A mutants . Black arrowhead indicates 3Flag - MITOL .

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

(B) The characteristic ubiquitylation ladder was observed for wild-type MITOL, but was absent in the K268A and K54A/K268A mutants. Black arrowhead indicates 3Flag-MITOL.


( C ) The MITOL K268A and K268R mutants were targeted to mitochondria under steady - state conditions , whereas peroxisomal localization following CCCP treatment was considerably disrupted . HeLa cells stably expressing HA - Parkin were transfected with 3Flag - MITOL wild - type , K268A , or K268R mutants , treated with 15 µM CCCP for 3 hours , and then subjected to immuno - staining with anti - Flag and anti - catalase antibodies . Scale bars , 10 µm .

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

(C) The MITOL K268A and K268R mutants were targeted to mitochondria under steady-state conditions, whereas peroxisomal localization following CCCP treatment was considerably disrupted. HeLa cells stably expressing HA-Parkin were transfected with 3Flag-MITOL wild-type, K268A, or K268R mutants, treated with 15 µM CCCP for 3 hours, and then subjected to immuno-staining with anti-Flag and anti-catalase antibodies. Scale bars, 10 µm.


( D ) The double K54 / K268 mutation did not change the MITOL localization pattern . The subcellular localization of the MITOL K268A or K268R mutants was not drastically changed by inclusion of the K54A or K54R mutations . Scale bars , 10 µm .

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

(D) The double K54/K268 mutation did not change the MITOL localization pattern. The subcellular localization of the MITOL K268A or K268R mutants was not drastically changed by inclusion of the K54A or K54R mutations. Scale bars, 10 µm.


( E ) Correlation statistics for the localization of 3Flag - MITOL wild - type , K268A , or K268R mutants with catalase . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

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

(E) Correlation statistics for the localization of 3Flag-MITOL wild-type, K268A, or K268R mutants with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( F ) The fold change in ubiquitylation of MITOL K268 , K40 , and K54 in valinomycin - treated samples versus untreated samples . After 3 hours of valinomycin treatment , PEX19 - / - HCT116 cells stably expressing HA - Parkin and 3Flag - MITOL were immunoprecipitated with anti - Flag magnetic beads , and then subjected to LC - MS / MS analysis for label - free quantification of ubiquitylated peptides . Error bars represent the mean ± s . e . m in three biological replicates .

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

(F) The fold change in ubiquitylation of MITOL K268, K40, and K54 in valinomycin-treated samples versus untreated samples. After 3 hours of valinomycin treatment, PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were immunoprecipitated with anti-Flag magnetic beads, and then subjected to LC-MS/MS analysis for label-free quantification of ubiquitylated peptides. Error bars represent the mean ± s.e.m in three biological replicates.


( A ) To generate MITOL - 3Flag knock - in ( KI ) HCT116 cell lines , 3xFlag gene cassettes were inserted upstream of the MITOL stop codon using CRISPR / Cas9 - based gene editing . Insertion of the 3Flag - tag was verified by immunoblotting with an anti - Flag antibody . Asterisk indicates a cross - reacting band .

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

(A) To generate MITOL-3Flag knock-in (KI) HCT116 cell lines, 3xFlag gene cassettes were inserted upstream of the MITOL stop codon using CRISPR/Cas9-based gene editing. Insertion of the 3Flag-tag was verified by immunoblotting with an anti-Flag antibody. Asterisk indicates a cross-reacting band.


( B ) The MITOL subcellular localization was observed in MITOL - 3Flag KI HCT116 cells stably expressing HA - Parkin with anti - Flag , anti - catalase , and anti - Hsp60 antibodies . Endogenous MITOL ( detectable with an anti - Flag antibody ) overlapped with Hsp60 under steady - state conditions , whereas 3 hours of valinomycin ( 10 µM ) treatment induced translocation of endogenous MITOL from mitochondria to peroxisomes . Higher magnification images of the boxed regions are shown in the bottom panel . Scale bars , 10 µm .

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

(B) The MITOL subcellular localization was observed in MITOL-3Flag KI HCT116 cells stably expressing HA-Parkin with anti-Flag, anti-catalase, and anti-Hsp60 antibodies. Endogenous MITOL (detectable with an anti-Flag antibody) overlapped with Hsp60 under steady-state conditions, whereas 3 hours of valinomycin (10 µM) treatment induced translocation of endogenous MITOL from mitochondria to peroxisomes. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 µm.


( C ) Correlation statistics for the localization of endogenous MITOL - 3Flag with catalase . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines show the medians , box limits indicate the 25th and 75th percentiles as determined by the R software package , and whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and the X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of endogenous MITOL-3Flag with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( D ) The extraction of endogenous MITOL from depolarized mitochondria is blocked by the p97 / VCP inhibitor NMS - 873 . Higher magnification images of the boxed regions are shown in the lower panel . Scale bars , 10 µm .

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

(D) The extraction of endogenous MITOL from depolarized mitochondria is blocked by the p97/VCP inhibitor NMS-873. Higher magnification images of the boxed regions are shown in the lower panel. Scale bars, 10 µm.


( E ) Correlation statistics for the localization of endogenous MITOL and PMP70 in the presence of NMS - 873 . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines show the medians , box limits indicate the 25th and 75th percentiles as determined by the R software package , and whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and the X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of endogenous MITOL and PMP70 in the presence of NMS-873. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( F ) Endogenous MITOL is ubiquitylated following valinomycin treatment . After MITOL - 3Flag KI HCT116 cells stably expressing HA - Parkin were treated with valinomycin for 3 hours , the collected cell lysates were immunoprecipitated with anti - Flag magnetic beads . The immunoprecipitates were blotted using anti - Flag and anti - ubiquitin antibodies . Red bars indicate ubiquitylation ; the black arrowhead indicates MITOL - 3Flag .

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

(F) Endogenous MITOL is ubiquitylated following valinomycin treatment. After MITOL-3Flag KI HCT116 cells stably expressing HA-Parkin were treated with valinomycin for 3 hours, the collected cell lysates were immunoprecipitated with anti-Flag magnetic beads. The immunoprecipitates were blotted using anti-Flag and anti-ubiquitin antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates MITOL-3Flag.


( A ) Distribution of exogenous 3Flag - MITOL in the mitochondria ‐ rich or peroxisome - rich fraction following cellular fractionation . HCT116 cells stably expressing HA - Parkin and 3Flag - MITOL were subjected to fractionation and detected using anti ‐ Tom20 , anti ‐ PMP70 , and anti ‐ Flag antibodies . Tom20 and PMP70 were used as mitochondrial and peroxisomal markers , respectively . The 3 , 000 g and 100 , 000 g pellets represent the mitochondria ‐ rich and peroxisomes - rich fractions .

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

(A) Distribution of exogenous 3Flag-MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were subjected to fractionation and detected using anti‐Tom20, anti‐PMP70, and anti‐Flag antibodies. Tom20 and PMP70 were used as mitochondrial and peroxisomal markers, respectively. The 3,000 g and 100,000 g pellets represent the mitochondria‐rich and peroxisomes-rich fractions.


( B ) The recovery ratio of 3Flag - MITOL between the mitochondria - enriched ( 3 , 000 g pellet ) and peroxisome - enriched fraction ( 100 , 000 g pellet ) following valinomycin treatment for the indicated times . The ratio of peroxisome - localized 3Flag - MITOL to mitochondria - localized 3Flag - MITOL increased with valinomycin treatment for 3 hours . Graphic data represent results of two biological replicates . In scatter plot , dots indicate individual data points . Black dots indicate the ratio of 3Flag - MITOL collected in the mitochondria - enriched fractions , and red dots are the ratio of 3Flag - MITOL collected in the peroxisome - enriched fractions . Mean values are also shown .

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

(B) The recovery ratio of 3Flag-MITOL between the mitochondria-enriched (3,000 g pellet) and peroxisome-enriched fraction (100,000 g pellet) following valinomycin treatment for the indicated times. The ratio of peroxisome-localized 3Flag-MITOL to mitochondria-localized 3Flag-MITOL increased with valinomycin treatment for 3 hours. Graphic data represent results of two biological replicates. In scatter plot, dots indicate individual data points. Black dots indicate the ratio of 3Flag-MITOL collected in the mitochondria-enriched fractions, and red dots are the ratio of 3Flag-MITOL collected in the peroxisome-enriched fractions. Mean values are also shown.


Distribution of endogenous MITOL in the mitochondria ‐ rich or peroxisome - rich fraction following cellular fractionation . The distribution of endogenous MITOL was examined using MITOL - 3Flag knock - in HCT116 cells . Treatment of cells with valinomycin for 3 hours reduced the amount of endogenous MITOL in the mitochondria - enriched fraction , but concomitantly increased endogenous MITOL in the peroxisomes - enriched fraction .

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

Distribution of endogenous MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. The distribution of endogenous MITOL was examined using MITOL-3Flag knock-in HCT116 cells. Treatment of cells with valinomycin for 3 hours reduced the amount of endogenous MITOL in the mitochondria-enriched fraction, but concomitantly increased endogenous MITOL in the peroxisomes-enriched fraction.


Distribution of endogenous MITOL in the mitochondria ‐ rich or peroxisome - rich fraction following cellular fractionation . The distribution of endogenous MITOL was examined using MITOL - 3Flag knock - in HCT116 cells . Graphic data represent results of two biological replicates . In scatter plot , dots indicate individual data points . Black dots indicate the ratio of MITOL - 3Flag collected in mitochondria - enriched fractions , and red dots are the ratio of MITOL - 3Flag collected in the peroxisome - enriched fractions . Mean values are also shown .

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

Distribution of endogenous MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. The distribution of endogenous MITOL was examined using MITOL-3Flag knock-in HCT116 cells. Graphic data represent results of two biological replicates. In scatter plot, dots indicate individual data points. Black dots indicate the ratio of MITOL-3Flag collected in mitochondria-enriched fractions, and red dots are the ratio of MITOL-3Flag collected in the peroxisome-enriched fractions. Mean values are also shown.


( A ) HeLa cells expressing HA - Parkin and wild type 3Flag - MITOL or 3Flag - MITOL lacking C - terminal 8 amino acids ( ∆ C8 ) were treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - Flag and anti - PMP70 antibodies . Expanded peroxisomes were observed in MITOL ∆ C8 - expressing cells . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm .

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

(A) HeLa cells expressing HA-Parkin and wild type 3Flag-MITOL or 3Flag-MITOL lacking C-terminal 8 amino acids (∆C8) were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-PMP70 antibodies. Expanded peroxisomes were observed in MITOL∆C8-expressing cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


( B ) 3Flag - MITOL ∆ C8 with the E3 - inactive Cys65Ser / Cys68Ser ( CS ) and H43W mutations were transfected into HeLa cells stably expressing HA - Parkin , treated with 15 µM CCCP , and then subjected to immunocytochemistry with anti - Flag and anti - PMP70 antibodies . After 3 hours of CCCP treatment , both the CS and H43W mutants localized on peroxisomes but expansion was not observed . Scale bars , 10 µm .

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

(B) 3Flag-MITOL∆C8 with the E3-inactive Cys65Ser/Cys68Ser (CS) and H43W mutations were transfected into HeLa cells stably expressing HA-Parkin, treated with 15 µM CCCP, and then subjected to immunocytochemistry with anti-Flag and anti-PMP70 antibodies. After 3 hours of CCCP treatment, both the CS and H43W mutants localized on peroxisomes but expansion was not observed. Scale bars, 10 µm.


( C ) HeLa cells expressing HA - Parkin and MITOL wild - type or ∆ C8 were treated with 15 µM CCCP for 3 hours , and the number of peroxisomes was counted as PMP70 - positive dots per 100 μm2 . In the box - plots , dots indicate individual data points , the center lines show the medians , box limits indicate the 25th and 75th percentiles as determined by the R software package , and whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and the X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test . The abundance of peroxisomes in cells expressing MITOL ∆ C8 was significantly decreased as compared with cells expressing wild - type MITOL .

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

(C) HeLa cells expressing HA-Parkin and MITOL wild-type or ∆C8 were treated with 15 µM CCCP for 3 hours, and the number of peroxisomes was counted as PMP70-positive dots per 100 μm2. In the box-plots, dots indicate individual data points, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. The abundance of peroxisomes in cells expressing MITOL∆C8 was significantly decreased as compared with cells expressing wild-type MITOL.


( D ) MITOL ∆ C8 causes a drastic expansion of peroxisomes following CCCP treatment . HeLa cells expressing HA - Parkin and wild - type or ∆ C8 MITOL were treated with 15 µM CCCP for 3 hours , and the approximate size of peroxisomes was determined as the number of pixels occupied by one peroxisome . The PMP70 - positive pixels per 100 µm2 were divided by the number of peroxisomes in the same area . In the box - plots , dots indicate individual data points , the center lines show the medians , box limits indicate the 25th and 75th percentiles as determined by the R software package , and whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and the X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

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

(D) MITOL∆C8 causes a drastic expansion of peroxisomes following CCCP treatment. HeLa cells expressing HA-Parkin and wild-type or ∆C8 MITOL were treated with 15 µM CCCP for 3 hours, and the approximate size of peroxisomes was determined as the number of pixels occupied by one peroxisome. The PMP70-positive pixels per 100 µm2 were divided by the number of peroxisomes in the same area. In the box-plots, dots indicate individual data points, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( A and B ) HeLa cells expressing HA - Parkin and MITOL ∆ C8 ( A ) or MITOL ∆ C8 lacking E3 activity ( B ) were treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - Flag and anti - ubiquitin antibodies . Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm .

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

(A and B) HeLa cells expressing HA-Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


T cell interface actin ( phalloidin ) re - organization during synapse symmetry breaking , imaged using SIM . Quantification of the shape elongation ( AR ; B , n = 44 for 5 ' , 39 for 20 ' ) , or relative fluorescence signal of the indicated proteins in the SIM images ( C , D ) normalized to the mean of values at 5 ' ; points represent values for individual cells . Arrowheads in ( C ) indicate foci . p values are : * * * P < 0 . 0001 for AR , * * P = 0 . 003 for foci , * * P = 0 . 008 for talin , and p = 0 . 36 for total F - actin using Mann - Whitney two - tailed test between populations of cells within the same experiment . Relationship between AR and foci at individual cell level , each point in the scatter plot represents value obtained from a single cell .

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

T cell interface actin (phalloidin) re-organization during synapse symmetry breaking, imaged using SIM. Quantification of the shape elongation (AR; B, n= 44 for 5', 39 for 20'), or relative fluorescence signal of the indicated proteins in the SIM images (C, D) normalized to the mean of values at 5'; points represent values for individual cells. Arrowheads in (C) indicate foci. p values are: ***P< 0.0001 for AR, **P =0.003 for foci, **P= 0.008 for talin, and p =0.36 for total F-actin using Mann-Whitney two-tailed test between populations of cells within the same experiment. Relationship between AR and foci at individual cell level, each point in the scatter plot represents value obtained from a single cell.


Integrin augmentation does not rescue synapse symmetry breaking . Cells were allowed to adhere to the APS for 5 ' or 10 ' , and were then treated with vehicle control , 0 . 5mM MnCl2 or 100nM A286982 for subsequent 10 ' . Cells were fixed , stained with phalloidin and anti - Talin antibody , and imaged using SIM ( F ) and analyzed for AR ( G ) as well as talin , total F - actin and actin foci at the synapse ( H ) . In ( E - H ) , n for 5 ' = 40 , for 20 ' = 62 , for MnCl2 = 59 , for A286982 = 53 . The values in the plots represent the intensity values normalized to the mean of 5 ' in each set . p values in the graph , n . s . > 0 . 05 ; * * * P < 0 . 001 ; * P = 0 . 01 ; * * P < 0 . 009 using Mann - Whitney two - tailed test between populations of cells within the same experiment .

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

Integrin augmentation does not rescue synapse symmetry breaking. Cells were allowed to adhere to the APS for 5' or 10', and were then treated with vehicle control, 0.5mM MnCl2 or 100nM A286982 for subsequent 10'. Cells were fixed, stained with phalloidin and anti-Talin antibody, and imaged using SIM (F) and analyzed for AR (G) as well as talin, total F-actin and actin foci at the synapse (H). In (E-H), n for 5'= 40, for 20'= 62, for MnCl2= 59, for A286982= 53. The values in the plots represent the intensity values normalized to the mean of 5' in each set. p values in the graph, n.s. > 0.05; ***P <0.001; *P= 0.01; **P<0.009 using Mann-Whitney two-tailed test between populations of cells within the same experiment.


Time - lapse imaging of LifeAct - GFP expressing T cell using Lattice lightsheet microscopy ( LLSM ) during synapse polarization . Shown are snapshots at selected timepoints ( left ) and a kymograph of actin foci ( denoted by arrows in the first image panel ) over time ( right ) for a representative T cell . Quantification of changes in actin foci and cellular aspect ratio in LLSM images during symmetry breaking . The values in the plot are the raw values normalized to their mean in each case .

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

Time-lapse imaging of LifeAct-GFP expressing T cell using Lattice lightsheet microscopy (LLSM) during synapse polarization. Shown are snapshots at selected timepoints (left) and a kymograph of actin foci (denoted by arrows in the first image panel) over time (right) for a representative T cell. Quantification of changes in actin foci and cellular aspect ratio in LLSM images during symmetry breaking. The values in the plot are the raw values normalized to their mean in each case.


T cells incubated with APS for the indicated time periods were processed for phalloidin - or immune - staining ( C , D ) Graph in ( D ) shows relative levels of total and active WASP ( WASP - phosphorylated at Y293 , pWASP ) , normalized to the mean levels at 5 ' ; for pWASP n in 5 ' = 49 , in 20 ' = 44 ; for WASP , n in 5 ' = 56 , 20 ' = 56 . P values , P * * = 0 . 0027 for pWASP and P * * = 0 . 0035 for WASP using Mann - Whitney two - tailed test . The scatterplot in ( D ) shows the relationship between active WASP and foci on a per cell basis .

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

T cells incubated with APS for the indicated time periods were processed for phalloidin- or immune-staining (C, D) Graph in (D) shows relative levels of total and active WASP (WASP-phosphorylated at Y293, pWASP), normalized to the mean levels at 5'; for pWASP n in 5'= 49, in 20'= 44; for WASP, n in 5'= 56, 20'= 56. P values, P**= 0.0027 for pWASP and P**= 0.0035 for WASP using Mann-Whitney two-tailed test. The scatterplot in (D) shows the relationship between active WASP and foci on a per cell basis.


T cells incubated with APS for the indicated time periods were processed for for western blotting ( E ) . Numbers in the graph in ( E ) indicate WASP band intensity divided by the actin band intensity , and resulting ratios normalized to the values at the 0 ' time point ( non - activated cells ) , from three independent experiments ( n = 3 ) . P value * = 0 . 025 , using paired two tailed t - test .

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

T cells incubated with APS for the indicated time periods were processed for for western blotting (E). Numbers in the graph in (E) indicate WASP band intensity divided by the actin band intensity, and resulting ratios normalized to the values at the 0' time point (non-activated cells), from three independent experiments (n=3). P value *= 0.025, using paired two tailed t-test.


Actin dynamics in mature synapse of LifeAct - GFP expressing T cell using TIRFM . The images show the foci ( pseudocolored red ) extracted from the total synaptic F - actin ( F - G ) , and kymograph shows the time course of foci and lamellar activity over a period of 210 sec . Histograms of foci and lamellar protrusion and retraction ( fluctuations ) dynamics in WT T cell ( H ) ; Lifetime of individual foci and lamellar events ( graph on the bottom right , each dot represents a single foci / lamellar protrusion - retraction event n = 126 for foci and n = 77 for lamellar protrusions ) . * * * P < 0 . 0001 using Mann - Whitney two - tailed test between populations of cells within the same experiment .

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

Actin dynamics in mature synapse of LifeAct-GFP expressing T cell using TIRFM. The images show the foci (pseudocolored red) extracted from the total synaptic F-actin (F-G), and kymograph shows the time course of foci and lamellar activity over a period of 210 sec. Histograms of foci and lamellar protrusion and retraction (fluctuations) dynamics in WT T cell (H); Lifetime of individual foci and lamellar events (graph on the bottom right, each dot represents a single foci/lamellar protrusion-retraction event n= 126 for foci and n= 77 for lamellar protrusions). ***P<0.0001 using Mann-Whitney two-tailed test between populations of cells within the same experiment.


Kymograph of the lamellar activity of WASP - deficient cells that lack actin foci .

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

Kymograph of the lamellar activity of WASP-deficient cells that lack actin foci.


Foci - deficient cells display poor traction forces in their synapse . WT or WASP - / - T cells were incubated on polyacrylamide substrates covalently functionalized with anti - CD3 and ICAM1 , and traction force measurements were carried out as described in ' Methods ' . The images in the right show traction force maps without ( left panels ) or with ( right panels ) force vectors . p value , * * P < 0 . 005 as determined by using Mann - Whitney two - tailed test between populations of cells within the same experiment , n = 9 and 11 .

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

Foci -deficient cells display poor traction forces in their synapse. WT or WASP-/- T cells were incubated on polyacrylamide substrates covalently functionalized with anti-CD3 and ICAM1, and traction force measurements were carried out as described in 'Methods'. The images in the right show traction force maps without (left panels) or with (right panels) force vectors. p value, **P<0.005 as determined by using Mann-Whitney two-tailed test between populations of cells within the same experiment, n = 9 and 11.


SIM imaging of WT or WASP - / - T cells activated by APS for 5 ' . The graphs show quantification of actin foci ( derived from phalloidin staining ) , pCasL and talin levels in the synapse normalized to the ' WT ' mean value in each case ( Middle graph ; in the case of foci , n = 63 for WT and 86 for WASP - / - ; in the case of pCasL n = 63 for WT and 78 for WASP - / - ; in the case of Talin , n = 59 for WT and 73 for WASP - / - ; in the case of AR , n = 46 for WT and 42 for WASP - / - ) ; right graph shows AR measurement . * * * P < 0 . 0001 ; p value for talin * P = 0 . 07 , as determined by using Mann - Whitney two - tailed test between populations of cells within the same experiment .

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

SIM imaging of WT or WASP-/- T cells activated by APS for 5'. The graphs show quantification of actin foci (derived from phalloidin staining), pCasL and talin levels in the synapse normalized to the 'WT' mean value in each case (Middle graph; in the case of foci, n= 63 for WT and 86 for WASP-/-; in the case of pCasL n= 63 for WT and 78 for WASP-/-; in the case of Talin, n= 59 for WT and 73 for WASP-/-; in the case of AR, n= 46 for WT and 42 for WASP-/-); right graph shows AR measurement. ***P <0.0001; p value for talin *P=0.07, as determined by using Mann-Whitney two-tailed test between populations of cells within the same experiment.


Human WAS patient CD4 + T cells - APC conjugates display synapse symmetry defects similar to those observed in mouse WASP - / - CD4 + T cells . CD4 + T cells purified from healthy controls or WAS patients were incubated with superantigen loaded HUVEC cells for 5 ' ( APC ; see ' Methods ' ) and imaged using confocal microscopy . Each image represents a maximum intensity projection of the synaptic area . Graph in the middle shows values normalized to the mean value of the ' Healthy ' case . In the case of ' actin ' and ' actin foci ' , n = 87 for healthy and n = 63 for WAS ; in the case of pCasL , n = 56 for healthy and n = 21 for WAS ; for ' AR ' graph , n = 88 for healthy and n = 63 for WAS case . P values ; * * * P < 0 . 001 ; n . s . P = 0 . 09 , as determined by using Mann - Whitney two - tailed test between populations of cells within the same experiment .

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

Human WAS patient CD4+T cells- APC conjugates display synapse symmetry defects similar to those observed in mouse WASP-/- CD4+ T cells. CD4+T cells purified from healthy controls or WAS patients were incubated with superantigen loaded HUVEC cells for 5' (APC; see 'Methods') and imaged using confocal microscopy. Each image represents a maximum intensity projection of the synaptic area. Graph in the middle shows values normalized to the mean value of the 'Healthy' case. In the case of 'actin' and 'actin foci', n= 87 for healthy and n= 63 for WAS; in the case of pCasL, n=56 for healthy and n= 21 for WAS; for 'AR' graph, n= 88 for healthy and n= 63 for WAS case. P values; ***P <0.001; n.s. P=0.09, as determined by using Mann-Whitney two-tailed test between populations of cells within the same experiment.


Simulation of T cell IS F - actin behavior and resultant mechanical tension incorporating the differential dynamics and positioning of foci and lamella across the synaptic interface , using Brownian dynamics equation with no inertia ( see ' Methods ' ) . The images show simulation snapshots at the beginning ( 0 min ; soon after the attachment and spreading of T cells on the substrates ) and the end of the simulations for the three IS cases - with persistent foci ( WT , left most panels ) , with no foci ( WASP - / - , middle right panels ) , and with WASP and consequently foci downregulated after a period of synapse maturation ( WT transitional ) . The colors in the images indicate myosin II motor ( pseudocolored teal ) , F - actin crosslinking proteins ( pseudocolored yellow ) , and the F - actin filaments ( pseudocolored blue for the low tension and the red for thigh tension actin filaments ) ; middle left image panels show the distribution of the aforementioned proteins at individual foci site at a higher magnification .

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

Simulation of T cell IS F-actin behavior and resultant mechanical tension incorporating the differential dynamics and positioning of foci and lamella across the synaptic interface, using Brownian dynamics equation with no inertia (see 'Methods'). The images show simulation snapshots at the beginning (0 min; soon after the attachment and spreading of T cells on the substrates) and the end of the simulations for the three IS cases - with persistent foci (WT, left most panels), with no foci (WASP-/-, middle right panels), and with WASP and consequently foci downregulated after a period of synapse maturation (WT transitional). The colors in the images indicate myosin II motor (pseudocolored teal), F-actin crosslinking proteins (pseudocolored yellow), and the F-actin filaments (pseudocolored blue for the low tension and the red for thigh tension actin filaments); middle left image panels show the distribution of the aforementioned proteins at individual foci site at a higher magnification.


Predicted stress ( tension normalized to the area ) profiles within the cytoskeletal architecture as the synaptic contact progresses in time , corresponding to the three cases shown in ( D ) .

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

Predicted stress (tension normalized to the area) profiles within the cytoskeletal architecture as the synaptic contact progresses in time, corresponding to the three cases shown in (D).


Live imaging of a T cell using SIM shows the inter - foci connections ( arrows in the inset ) as predicted in the simulations ; and their loss concomitant with loss of actin foci , as the T cell polarizes to initiate motility ( compare insets between 0 and 120 sec ) . ' 0 sec ' refers to the beginning of the observation of the cell , after it has attached to the substrate , spread and maintained synapse for 12 min . Scale bar , 5µm in main images and 1 . 5μm in the magnified insets .

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

Live imaging of a T cell using SIM shows the inter-foci connections (arrows in the inset) as predicted in the simulations; and their loss concomitant with loss of actin foci, as the T cell polarizes to initiate motility (compare insets between 0 and 120 sec). '0 sec' refers to the beginning of the observation of the cell, after it has attached to the substrate, spread and maintained synapse for 12 min. Scale bar, 5µm in main images and 1.5μm in the magnified insets.


Ultrastructural visualization of detergent extracted WT and WASP - / - T cell synaptic cytoskeleton using platinum replica electron microscopy ( top images ) . Each image shows individual synapse , dark zone in the middle of the synapse represents cell nucleus . Scale bar , 2 . 5μm . The bottom left micrograph is the magnified inset from WT synapse and shows actin foci ( identified as globular cluster of short filaments in the inset , marked with arrows ) - dependent interconnected cytoskeletal architecture that is associated with synapse symmetry as predicted in the model , and is lacking in the WASP - / - cell synapse ( bottom right micrograph - magnified inset from WASP - / - synapse ) . Scale bars , 1μm . This experiment was repeated at least twice with similar results .

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

Ultrastructural visualization of detergent extracted WT and WASP-/- T cell synaptic cytoskeleton using platinum replica electron microscopy (top images). Each image shows individual synapse, dark zone in the middle of the synapse represents cell nucleus. Scale bar, 2.5μm. The bottom left micrograph is the magnified inset from WT synapse and shows actin foci (identified as globular cluster of short filaments in the inset, marked with arrows)-dependent interconnected cytoskeletal architecture that is associated with synapse symmetry as predicted in the model, and is lacking in the WASP-/- cell synapse (bottom right micrograph- magnified inset from WASP-/- synapse). Scale bars, 1μm.This experiment was repeated at least twice with similar results.


Schematic of the cytoskeletal inhibitors and their targets used in the experiments . SIM imaging of mouse T cells activated for 5 ' on APS , then treated with the indicated inhibitors or vehicle control . Shown are the representative SIM images ( B ) , and quantification of actin foci ( from the phalloidin images ) , talin , or pCasL in the contact interface ( normalized to the mean value in the control case , C ) , and measured cell morphology aspect ratios ( D , E ) . Since CK666 treatment reduces F - actin intensity Kumari et al . , 2015 ( ) , the ' actin ' image of the CK666 treated cell in ( B ) was contrasted differently than the ' control ' and ' Blebb . ' cases for a clearer visualization of F - actin . p values , * P = 0 . 02 , * * P = 0 . 001 , n . s . > 0 . 05 . For the rest of the comparisons between the control and treatment cases , p < 0 . 0001 . For the bar graph in ( B ) , n for foci groups are 214 , 83 , 92 , 98 ; for pCasL groups are 100 , 68 , 91 , 92 ; and for Talin groups are 102 , 70 , 89 , 93 respectively . p value for the n . s . cases is > 0 . 05 , * P = 0 . 02 . In the foci , pCasL and talin groups , when not indicated , the p values are < 0 . 0001 using Mann - Whitney two - tailed test . Central values in the graphs represent Mean and error bars represent ± SEM . These experiments were repeated at least thrice with similar results . Scale bar , 5µm .

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

Schematic of the cytoskeletal inhibitors and their targets used in the experiments. SIM imaging of mouse T cells activated for 5' on APS, then treated with the indicated inhibitors or vehicle control. Shown are the representative SIM images (B), and quantification of actin foci (from the phalloidin images), talin, or pCasL in the contact interface (normalized to the mean value in the control case, C), and measured cell morphology aspect ratios (D, E). Since CK666 treatment reduces F-actin intensity Kumari et al., 2015(), the 'actin' image of the CK666 treated cell in (B) was contrasted differently than the 'control' and 'Blebb.' cases for a clearer visualization of F-actin. p values, *P =0.02, **P =0.001, n.s. >0.05. For the rest of the comparisons between the control and treatment cases, p <0.0001. For the bar graph in (B), n for foci groups are 214, 83, 92, 98; for pCasL groups are 100, 68, 91, 92; and for Talin groups are 102, 70, 89, 93 respectively. p value for the n.s. cases is > 0.05, *P =0.02. In the foci, pCasL and talin groups, when not indicated, the p values are <0.0001 using Mann-Whitney two-tailed test. Central values in the graphs represent Mean and error bars represent ± SEM. These experiments were repeated at least thrice with similar results. Scale bar, 5µm.


Simulation of synaptic F - actin network following localized myosin inactivation and inactivated myosin - mediated F - actin cross - linking within the area marked with dotted line in the left image . The graph shows the predicted alteration in mechanical stress profile , as myosin is inactivated crosslinked in the defined region ( marked by an arrow ) , ( G ) .

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

Simulation of synaptic F-actin network following localized myosin inactivation and inactivated myosin-mediated F-actin cross-linking within the area marked with dotted line in the left image. The graph shows the predicted alteration in mechanical stress profile, as myosin is inactivated crosslinked in the defined region (marked by an arrow), (G).


Experimental confirmation of localized actomyosin crosslinking and symmetry breaking . LifeAct - GFP expressing T cells were incubated with activating substrates , treated with vehicle control or 1µM Azido - blebb and their contact interface imaged using low excitation laser power ( 0 . 5 mW ) , using a spinning disc confocal microscope ( H ) . The montage shows the shape change and movement that synapse undergoes after it is exposed to a brief flash of blue light crosslinking Azido - Blebb and actomyosin network within the illuminated area ( shown with a box in the image ) . The deflection in center of mass ( COM ) within a minute was calculated and plotted for at least 10 cells in each case ( graphs in I ) . The left graphs in ( I ) show individual COM trajectory , and the graph on the right shows average COM movement of cells within a minute post photo - inactivation of myosinII . This experiment was repeated twice with similar results . Scale bar , 5µm .

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

Experimental confirmation of localized actomyosin crosslinking and symmetry breaking. LifeAct-GFP expressing T cells were incubated with activating substrates, treated with vehicle control or 1µM Azido-blebb and their contact interface imaged using low excitation laser power (0.5 mW), using a spinning disc confocal microscope (H). The montage shows the shape change and movement that synapse undergoes after it is exposed to a brief flash of blue light crosslinking Azido-Blebb and actomyosin network within the illuminated area (shown with a box in the image). The deflection in center of mass (COM) within a minute was calculated and plotted for at least 10 cells in each case (graphs in I). The left graphs in (I) show individual COM trajectory, and the graph on the right shows average COM movement of cells within a minute post photo-inactivation of myosinII. This experiment was repeated twice with similar results. Scale bar, 5µm.


T cells were activated using Alexa568 - 2C11 and ICAM1 reconstituted lipid bilayers for 5 min , fixed , stained with phalloidin and imaged using SIM . In the graph , n = 50 for WT and 61 for WASP - / - , P value , P * * * < 0 . 0001 , obtained using Mann - Whitney two - tailed test . The graphs in ( B ) show synaptic levels of indicated proteins , normalized to the mean levels of ' + WASP ' in each case . For the left graph , n = 48 , 46 , 52 , 46 respectively , and p value , P * = 0 . 03 . For the right graph , n = 50 for WT and n = 61 for WASP - / - in ' Actin ' , ' Foci ' , as well as ' pCasL ' cases . P values P * * * < 0 . 0001 , obtained using Mann - Whitney two - tailed test .

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

T cells were activated using Alexa568-2C11 and ICAM1 reconstituted lipid bilayers for 5 min, fixed, stained with phalloidin and imaged using SIM. In the graph, n= 50 for WT and 61 for WASP-/-, P value, P***<0.0001, obtained using Mann-Whitney two-tailed test. The graphs in (B) show synaptic levels of indicated proteins, normalized to the mean levels of '+WASP' in each case. For the left graph, n= 48, 46, 52, 46 respectively, and p value, P *= 0.03. For the right graph, n= 50 for WT and n= 61 for WASP-/- in 'Actin', 'Foci', as well as 'pCasL' cases. P values P*** <0.0001, obtained using Mann-Whitney two-tailed test.


Synapse breaking behavior of T cells on hydrogels of indicated stiffness , covalently conjugated with 2C11 and ICAM1 . These experiments were repeated at least thrice with similar results . In the left graph , n for WT = 14 , for WASP - / - , n = 19 ; P * * = 0 . 0015 ; in the graph on the right , n for WT = 40 , n for WASP - / - = 36 , P * * * = 0 . 0006 , as assessed using Mann - Whitney two - tailed test .

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

Synapse breaking behavior of T cells on hydrogels of indicated stiffness, covalently conjugated with 2C11 and ICAM1. These experiments were repeated at least thrice with similar results. In the left graph, n for WT= 14, for WASP-/-, n= 19; P**= 0.0015; in the graph on the right, n for WT= 40, n for WASP-/-= 36, P***= 0.0006, as assessed using Mann-Whitney two-tailed test.


IncuCyte cell proliferation curves of PC3 cells treated with the indicated concentrations of ASNase . Data information : For IncuCyte proliferation assays , images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well . All results were calculated from three replicates and presented as mean ± SD , unless otherwise stated . The p - value was calculated by two - tailed unpaired t test by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves of PC3 cells treated with the indicated concentrations of ASNase. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


IncuCyte cell proliferation curves for ASNS knockout ( sgASNS ) and control ( sgNon - targeting ) PC3 cells in the absence and presence of ASNase . Data information : For IncuCyte proliferation assays , images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well . All results were calculated from three replicates and presented as mean ± SD , unless otherwise stated . The p - value was calculated by two - tailed unpaired t test by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves for ASNS knockout (sgASNS) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


Volcano plots for the MAGeCK pipeline analysis of the sgRNA abundance from the screen . Green dots indicate positive controls and red dots indicate candidates with a fold discovery rate ( FDR ) < 0 . 003 .

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

Volcano plots for the MAGeCK pipeline analysis of the sgRNA abundance from the screen. Green dots indicate positive controls and red dots indicate candidates with a fold discovery rate (FDR) <0.003.


IncuCyte cell proliferation curves of SLC1A3 knockout ( sgSLC1A3 ) and control ( sgNon - targeting ) PC3 cells in the absence and presence of ASNase treatment . # 3 and # 4 represent 2 different sgRNAs targeting SLC1A3 . Data information : For IncuCyte proliferation assays , images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well . All results were calculated from three replicates and presented as mean ± SD , unless otherwise stated . The p - value was calculated by two - tailed unpaired t test by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase treatment. #3 and #4 represent 2 different sgRNAs targeting SLC1A3. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


Radioactive labeled aspartate and glutamate uptake measurement in control ( sgNon - targeting ) and SLC1A3 knockout ( sgSLC1A3 ) PC3 cells . # 3 and # 4 represent two different sgRNAs targeting SLC1A3 . Radioactive labeled leucine uptake was used as a control . Data was normalized to the reads of control PC3 cells .

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

Radioactive labeled aspartate and glutamate uptake measurement in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells. #3 and #4 represent two different sgRNAs targeting SLC1A3. Radioactive labeled leucine uptake was used as a control. Data was normalized to the reads of control PC3 cells.


Endogenous levels of aspartate , asparagine , glutamate and glutamine in control ( sgNon - targeting ) and SLC1A3 knockout ( sgSLC1A3 ) PC3 cells with or without ASNase for 3 days . Median peak intensity was used for the read normalization .

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

Endogenous levels of aspartate, asparagine, glutamate and glutamine in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells with or without ASNase for 3 days. Median peak intensity was used for the read normalization.


IncuCyte cell proliferation curves of SLC1A3 knockout ( sgSLC1A3 # 3 ) PC3 cells treated with ASNase and supplemented with either esterified - aspartate ( 6mM ) or esterified - glutamate ( 6mM ) , and esterified - leucine ( 6mM ) as a control . Data information : For IncuCyte proliferation assays , images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well . All results were calculated from three replicates and presented as mean ± SD , unless otherwise stated . The p - value was calculated by two - tailed unpaired t test by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3#3) PC3 cells treated with ASNase and supplemented with either esterified-aspartate (6mM) or esterified-glutamate (6mM), and esterified-leucine (6mM) as a control. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


RT - qPCR analysis was used to determine the relative SLC1A3 mRNA expression ( to GAPDH ) in different prostate and breast cancer cell lines , as indicated . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

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

RT-qPCR analysis was used to determine the relative SLC1A3 mRNA expression (to GAPDH) in different prostate and breast cancer cell lines, as indicated. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


cell lines were transduced with either control ( sgNon - targeting ) or sgSLC1A3 . Aspartate uptake levels were determined and compared between control and SLC1A3 KO in these cell lines . Leucine uptake level was used for normalization . The numbers above the control column denote the basal aspartate uptake capacity . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

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

cell lines were transduced with either control (sgNon-targeting) or sgSLC1A3. Aspartate uptake levels were determined and compared between control and SLC1A3 KO in these cell lines. Leucine uptake level was used for normalization. The numbers above the control column denote the basal aspartate uptake capacity. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


RT - qPCR was used to determine the relative mRNA levels ( to GAPDH ) of aspartate / glutamate transporter genes ( SLC1A1 , SLC1A2 , SLC1A3 , SLC1A6 and SLC1A7 ) in LNCaP , BT549 and SUM159PT cells . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 used to determine the relative mRNA levels (to GAPDH) of aspartate/glutamate transporter genes (SLC1A1, SLC1A2, SLC1A3, SLC1A6 and SLC1A7) in LNCaP, BT549 and SUM159PT cells. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


batch of cancer cells subjected to IncuCyte cell proliferation assays in the absence or presence of ASNase at indicated concentrations . ' ns ' indicates no significant difference . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

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

batch of cancer cells subjected to IncuCyte cell proliferation assays in the absence or presence of ASNase at indicated concentrations. 'ns' indicates no significant difference. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


MCF7 and DU145 cells were transduced with either lentiviral empty vector ( control ) or lentiviral vector containing a V5 - tagged SLC1A3 coding sequence ( V5 - SLC1A3 ) . Relative SLC1A3 mRNA levels ( to GAPDH ) were determined by RT - qPCR .

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

MCF7 and DU145 cells were transduced with either lentiviral empty vector (control) or lentiviral vector containing a V5-tagged SLC1A3 coding sequence (V5-SLC1A3). Relative SLC1A3 mRNA levels (to GAPDH) were determined by RT-qPCR.


Immunofluorescence staining of the V5 - tagged SLC1A3 in MCF7 and DU145 cells using anti - V5 antibody . Green staining indicates the plasma membrane localization of V5 - SLC1A3 and blue DAPI staining marks the nuclei . Scale bar stands for 5 μm .

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

Immunofluorescence staining of the V5-tagged SLC1A3 in MCF7 and DU145 cells using anti-V5 antibody. Green staining indicates the plasma membrane localization of V5-SLC1A3 and blue DAPI staining marks the nuclei. Scale bar stands for 5 μm.


Relative aspartate uptake levels in control and V5 - SLC1A3 - expressed MCF7 and DU145 cells . Leucine uptake level was used for normalization .

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

Relative aspartate uptake levels in control and V5-SLC1A3-expressed MCF7 and DU145 cells. Leucine uptake level was used for normalization.


Control and V5 - SLC1A3 expressed MCF7 and DU145 cells were subjected to IncuCyte cell proliferation assays with or without ASNase at indicated concentrations .

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

Control and V5-SLC1A3 expressed MCF7 and DU145 cells were subjected to IncuCyte cell proliferation assays with or without ASNase at indicated concentrations.


DU145 cells were supplemented with cell - permeable aspartate ( 6mM , esterified ) or glutamate ( 3mM , esterified ) following ASNase treatment , with esterified leucine ( 6mM or 3mM ) as control .

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

DU145 cells were supplemented with cell-permeable aspartate (6mM, esterified) or glutamate (3mM, esterified) following ASNase treatment, with esterified leucine (6mM or 3mM) as control.


PC3 , DU145 and V5 - SLC1A3 - DU145 cells were subjected to ASNase and TFB - TBOA treatment at indicated concentrations and cell proliferation was measured by IncuCyte assay .

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

PC3, DU145 and V5-SLC1A3-DU145 cells were subjected to ASNase and TFB-TBOA treatment at indicated concentrations and cell proliferation was measured by IncuCyte assay.


PC3 cells were treated under indicated conditions for 9 days and subjected to BrdU assays to determine cell cycle distributions . ASNase ( 0 . 3 U / ml ) , TFB - TBOA ( 5 μM ) .

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

PC3 cells were treated under indicated conditions for 9 days and subjected to BrdU assays to determine cell cycle distributions. ASNase (0.3 U/ml), TFB-TBOA (5 μM).


DU145 and V5 - SLC1A3 - DU145 cells were treated under indicated conditions with ASNase ( 0 . 2 U / ml ) or TFB - TBOA ( 20μM ) or both , and subjected to IncuCyte analysis for apoptotic cell counts .

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

DU145 and V5-SLC1A3-DU145 cells were treated under indicated conditions with ASNase (0.2 U/ml) or TFB-TBOA (20μM) or both, and subjected to IncuCyte analysis for apoptotic cell counts.


PC3 cells were treated under ASNase ( 0 . 3 U / ml ) , or TFB - TBOA ( 5 μM ) conditions for 3 days and cell lysates were extracted and intracellular contents of aspartate , asparagine , glutamate and glutamine were determined by liquid - chromatography mass spectrometry ( LC - MS ) . Data information : Median peak intensity was used for raw data normalization Results were calculated based on three replicates and presented as mean ± SD ( unless otherwise stated ) . The p - value was calculated by two - tailed unpaired t test from Prism7 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

PC3 cells were treated under ASNase (0.3 U/ml), or TFB-TBOA (5 μM) conditions for 3 days and cell lysates were extracted and intracellular contents of aspartate, asparagine, glutamate and glutamine were determined by liquid-chromatography mass spectrometry (LC-MS). Data information: Median peak intensity was used for raw data normalization Results were calculated based on three replicates and presented as mean ± SD (unless otherwise stated). The p-value was calculated by two-tailed unpaired t test from Prism7. *p<0.05, **p<0.01, ***p<0.001.


key metabolites involved in urea cycle , pyrimidine synthesis , TCA cycle , oxidation , glycolysis and carnitines metabolism were determined . The NAD + / NADH ratio of the indicated conditions was calculated and normalized to control ( mean ± SEM ) . Dash line indicates indirect effect . TCA cycle : tricarboxylic acid cycle ; OAA : oxaloacetic acid ; UMP : uridine monophosphate ; CMP : cytidine monophosphate ; PEP : phosphoenolpyruvate ; NADH : nicotinamide adenine dinucleotide ( reduced form ) ; NAD + : nicotinamide adenine dinucleotide ( oxidized form ) ; NADPH : nicotinamide adenine dinucleotide phosphate ( reduced form ) ; NADP + : nicotinamide adenine dinucleotide phosphate ( oxidized form ) ; FAD : flavin adenine dinucleotide ; GSSG : glutathione disulfide ; HMG - CoA : 3 - hydroxy - 3 - methylglutaryl - CoA . Data information : Median peak intensity was used for raw data normalization Results were calculated based on three replicates and presented as mean ± SD ( unless otherwise stated ) . The p - value was calculated by two - tailed unpaired t test from Prism7 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

key metabolites involved in urea cycle, pyrimidine synthesis, TCA cycle, oxidation, glycolysis and carnitines metabolism were determined. The NAD+/NADH ratio of the indicated conditions was calculated and normalized to control (mean± SEM). Dash line indicates indirect effect. TCA cycle: tricarboxylic acid cycle; OAA: oxaloacetic acid; UMP: uridine monophosphate; CMP: cytidine monophosphate; PEP: phosphoenolpyruvate; NADH: nicotinamide adenine dinucleotide (reduced form); NAD+: nicotinamide adenine dinucleotide (oxidized form); NADPH: nicotinamide adenine dinucleotide phosphate (reduced form); NADP+: nicotinamide adenine dinucleotide phosphate (oxidized form); FAD: flavin adenine dinucleotide; GSSG: glutathione disulfide; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA. Data information: Median peak intensity was used for raw data normalization Results were calculated based on three replicates and presented as mean ± SD (unless otherwise stated). The p-value was calculated by two-tailed unpaired t test from Prism7. *p<0.05, **p<0.01, ***p<0.001.


A - C . PC3 ( A ) , DU145 ( B ) and DU145 - V5 - SLC1A3 ( C ) cells were treated with ASNase ( 0 . 3 U / ml in A ; 0 . 2 U / ml in B and C ) , TFB - TBOA ( 5 μM ) for 3 days as indicated and subjected to transcriptome analysis . Bioinformatics pathway or gene ontology ( GO ) biological process analysis was performed on the sets of genes that were upregulated or downregulated when PC3 cells were treated with ASNase and TFB - TBOA compared to mock . Transcriptome analysis was based on one biological replicate for each cell line and validated by real - time PCR experiments in Figures EV4A - C . Heatmap presents row scaled normalized read counts and the biological signaling pathway enrichment analysis was performed by ToppGene online

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

A-C. PC3 (A), DU145 (B) and DU145-V5-SLC1A3 (C) cells were treated with ASNase (0.3 U/ml in A; 0.2 U/ml in B and C), TFB-TBOA (5 μM) for 3 days as indicated and subjected to transcriptome analysis. Bioinformatics pathway or gene ontology (GO) biological process analysis was performed on the sets of genes that were upregulated or downregulated when PC3 cells were treated with ASNase and TFB-TBOA compared to mock. Transcriptome analysis was based on one biological replicate for each cell line and validated by real-time PCR experiments in Figures EV4A-C. Heatmap presents row scaled normalized read counts and the biological signaling pathway enrichment analysis was performed by ToppGene online program


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 .

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

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

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 .

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

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

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 .

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

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

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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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