Figure 1Western blot verification of knockout of FASN , ACC1 , and / or ACC2 in MDA - MB - 231 cells . Cell proliferation rate of MDA - MB - 231 and HeLa cells with knockout of FASN , ACC1 , and / or ACC2 . Colony formation ability of MDA - MB - 231 cells with knockout of FASN , ACC1 , and / or ACC2 . Invasion ability of MDA - MB - 231 cells with knockout of FASN , ACC1 , and / or ACC2 . The right panel shows the pictures of invasive cells , and the cell numbers are quantified in the left panel . Tumor formation ability in nude mice of MDA - MB - 231 and HeLa cells with gene knockout as indicated .

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Figure 1Western blot verification of knockout of FASN, ACC1, and/or ACC2 in MDA-MB-231 cells.Cell proliferation rate of MDA-MB-231 and HeLa cells with knockout of FASN, ACC1, and/or ACC2.Colony formation ability of MDA-MB-231 cells with knockout of FASN, ACC1, and/or ACC2.Invasion ability of MDA-MB-231 cells with knockout of FASN, ACC1, and/or ACC2. The right panel shows the pictures of invasive cells, and the cell numbers are quantified in the left panel.Tumor formation ability in nude mice of MDA-MB-231 and HeLa cells with gene knockout as indicated.


Figure 4Western blot verification of knockout of KAR or TECR in MDA - MB - 231 cells . B , C . Cell proliferation rate and colony formation ability of MDA - MB - 231 cells with knockout of KAR or TECR . Error bars represent mean ± SD . Data are from triplicate experiments , and all experimental data were verified in at least two independent experiments . * * P < 0 . 01 ( t - test ) ; asterisk shows comparison with wild type group . Tumor formation ability in nude mice of MDA - MB - 231 cells with gene knockout , as indicated . Relative cellular levels of fatty acids in MDA - MB - 231 cells with knockout of KAR or TECR . Each colored datum shows the mean from three independent cultures . G . Relative cellular levels of lipid - containing fatty acids in MDA - MB - 231 cells with knockout of KAR or TECR . The top curve plot indicates the fraction of the corresponding fatty acid . Each colored datum shows the mean from three independent cultures . H . Mass isotopomer analysis of fatty acids in MDA - MB - 231 cells with knockout of KAR or TECR cultured with 10 mM of 13C6 - glucose for 48 h . Error bars represent mean ± SD . Data are from three independent cultures .

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Figure 4Western blot verification of knockout of KAR or TECR in MDA-MB-231 cells.B,C. Cell proliferation rate and colony formation ability of MDA-MB-231 cells with knockout of KAR or TECR. Error bars represent mean ± SD. Data are from triplicate experiments, and all experimental data were verified in at least two independent experiments. **P < 0.01 (t-test); asterisk shows comparison with wild type group.Tumor formation ability in nude mice of MDA-MB-231 cells with gene knockout, as indicated.Relative cellular levels of fatty acids in MDA-MB-231 cells with knockout of KAR or TECR. Each colored datum shows the mean from three independent cultures.G. Relative cellular levels of lipid-containing fatty acids in MDA-MB-231 cells with knockout of KAR or TECR. The top curve plot indicates the fraction of the corresponding fatty acid. Each colored datum shows the mean from three independent cultures.H. Mass isotopomer analysis of fatty acids in MDA-MB-231 cells with knockout of KAR or TECR cultured with 10 mM of 13C6-glucose for 48 h. Error bars represent mean ± SD. Data are from three independent cultures.


Figure 6MDA - MB - 231 Wt cells or ACC - DKO cells were pretreated for 24 h and then cultured in FBS medium or DFBS medium with the indicated treatment for 5 days . Treatments were as follows : z - VAD - FMK ( z - VAD , 10 μM ) , necrostain - 1 ( Nec - 1 , 2 μM ) , Disulfiram ( 0 . 4 μM ) , ferrostain - 1 ( Ferr - 1 , 10 μM ) , Trolox ( 100 μM ) , N - Acetyl - L - cysteine ( NAC , 3 mM ) , and deferoxamine mesylate ( DFOM , 10 μM ) . Data are presented relative to the values obtained for the control cells cultured in FBS medium . Dotted line shows the survival level of cells in DFBS medium without treatment ( control ) . Relative cellular levels of ROS in MDA - MB - 231 / ACC - DKO and Wt cells cultured in FBS medium or DFBS medium for 5 days with or without exogenous VLCFA supplement . The inhibitory effects of H2O2 and PMS on MDA - MB - 231 ACC - DKO or Wt cells cultured in FBS medium for 24 h with indicated concentrations . The protein levels of PARP1 and caspase - 3 in MDA - MB - 231 / ACC - DKO and Wt cultured in FBS or DFBS medium . Annexin - V & Dead cell ( 7 - AAD ) flow cytometry analysis of MDA - MB - 231 / ACC - DKO and Wt cultured in DFBS medium . The right panel shows the quantification of annexin V - positive cells in the left panels . The relative cell survival of MDA - MB - 231 / ACC - DKO or Wt with control vector or Bcl - 2 or Bcl - xL overexpression cultured in FBS medium or DFBS medium for 5 days . The left panels show the cell survival , and the right panels verify the overexpression of Bcl - 2 and Bcl - xL . The transmembrane potential in mitochondria of MDA - MB - 231 / ACC - DKO and Wt cells cultured in FBS medium or DFBS medium was detected by FACS analysis . The bar graphs show the percentage of JC - 1 monomer . MDA - MB - 231 / ACC - DKO and Wt cells were stained for Mito Tracker to visualize mitochondria . Scale bar , 10 μm . Mitochondrial morphology was scored in the cell types indicated . Data represent three independent experiments , and at least 100 cells were counted per cell type .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6MDA-MB-231 Wt cells or ACC-DKO cells were pretreated for 24 h and then cultured in FBS medium or DFBS medium with the indicated treatment for 5 days. Treatments were as follows: z-VAD-FMK (z-VAD, 10 μM), necrostain-1 (Nec-1, 2 μM), Disulfiram (0.4 μM), ferrostain-1 (Ferr-1, 10 μM), Trolox (100 μM), N-Acetyl-L-cysteine (NAC, 3 mM), and deferoxamine mesylate (DFOM, 10 μM). Data are presented relative to the values obtained for the control cells cultured in FBS medium. Dotted line shows the survival level of cells in DFBS medium without treatment (control).Relative cellular levels of ROS in MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium for 5 days with or without exogenous VLCFA supplement.The inhibitory effects of H2O2 and PMS on MDA-MB-231 ACC-DKO or Wt cells cultured in FBS medium for 24 h with indicated concentrations.The protein levels of PARP1 and caspase-3 in MDA-MB-231/ACC-DKO and Wt cultured in FBS or DFBS medium.Annexin-V & Dead cell (7-AAD) flow cytometry analysis of MDA-MB-231/ACC-DKO and Wt cultured in DFBS medium. The right panel shows the quantification of annexin V-positive cells in the left panels.The relative cell survival of MDA-MB-231/ACC-DKO or Wt with control vector or Bcl-2 or Bcl-xL overexpression cultured in FBS medium or DFBS medium for 5 days. The left panels show the cell survival, and the right panels verify the overexpression of Bcl-2 and Bcl-xL.The transmembrane potential in mitochondria of MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium was detected by FACS analysis. The bar graphs show the percentage of JC-1 monomer.MDA-MB-231/ACC-DKO and Wt cells were stained for Mito Tracker to visualize mitochondria. Scale bar, 10 μm. Mitochondrial morphology was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type.


Figure 7MDA - MB - 231 / ACC - DKO cells were in FBS medium with or without exogenous LCFA or VLCFA supplementation , and were stained for MitoTracker to visualize mitochondria . Scale bar , 10 μm . Mitochondrial morphology in ( D ) was scored in the cell types indicated . Data represent three independent experiments , and at least 100 cells were counted per cell type . Error bars represent mean ± SD , and data are from triplicate experiments .

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Figure 7MDA-MB-231/ACC-DKO cells were in FBS medium with or without exogenous LCFA or VLCFA supplementation, and were stained for MitoTracker to visualize mitochondria. Scale bar, 10 μm.   Mitochondrial morphology in (D) was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type. Error bars represent mean ± SD, and data are from triplicate experiments.


Figure 1Left panel , a scheme of chromosome 18 copy number alterations depicting the distal long arm loss across TCGA Pan - Cancer dataset . Vertical red line indicates the localization of VPS4B . Right panel , enlarged fragment of chromosome 18 showing frequent deletions of VPSB locus in cancer samples . Deletions are marked in blue , amplified regions are marked in red . Analysis of VPS4B copy number alterations in TCGA Pan - Cancer dataset . Cancer types were sorted according to the mean VPS4B copy number after removing germline values . Scatter plot , analysis of VPS4B mRNA expression ( number of transcripts per million ) plotted against VPS4B copy number from TCGA CRC patient samples ( n = 376 ) ; plot generated using cBioPortal Gao et al , 2013 ( ) . Pie chart , summary of all types of VPS4B copy number alterations based on the analysis of data from 615 CRC samples deposited in the Colorectal Adenocarcinoma ( TCGA , Provisional ) qPCR analysis of VPS4B mRNA abundance in normal colon , adenoma and CRC samples . Adenocarcinoma ( n = 26 ) ; adenoma ( n = 42 ) ; normal colon ( n = 24 ) . Green horizontal bars indicate means and red whiskers indicate SD . Examples of immunohistochemical staining of VPS4B in normal colon and matched CRC samples as an illustration of the scoring system used for the evaluation presented in ( F ) . 3 + - very intensive staining , 2 + - medium - intensive staining , 1 + - weak staining , 0 - no staining . Scale bar , 50 µm .

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Figure 1Left panel, a scheme of chromosome 18 copy number alterations depicting the distal long arm loss across TCGA Pan-Cancer dataset. Vertical red line indicates the localization of VPS4B. Right panel, enlarged fragment of chromosome 18 showing frequent deletions of VPSB locus in cancer samples. Deletions are marked in blue, amplified regions are marked in red. Analysis of VPS4B copy number alterations in TCGA Pan-Cancer dataset. Cancer types were sorted according to the mean VPS4B copy number after removing germline values.Scatter plot, analysis of VPS4B mRNA expression (number of transcripts per million) plotted against VPS4B copy number from TCGA CRC patient samples (n=376); plot generated using cBioPortal Gao et al, 2013(). Pie chart, summary of all types of VPS4B copy number alterations based on the analysis of data from 615 CRC samples deposited in the Colorectal Adenocarcinoma (TCGA, Provisional)qPCR analysis of VPS4B mRNA abundance in normal colon, adenoma and CRC samples. Adenocarcinoma (n=26); adenoma (n=42); normal colon (n=24). Green horizontal bars indicate means and red whiskers indicate SD.Examples of immunohistochemical staining of VPS4B in normal colon and matched CRC samples as an illustration of the scoring system used for the evaluation presented in (F). 3+ - very intensive staining, 2+ - medium-intensive staining, 1+ - weak staining, 0 - no staining. Scale bar, 50 µm.


Figure 2Analysis of viability of HCT116 cells assessed 96 h after transfection with independent non - targeting siRNA ( two different duplexes used , siCTRL # 1 or # 2 ) or targeting VPS4A ( duplexes # 1 or # 2 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 3 independent experiments ± SEM . All values were normalized , averaged ( avrg ) viability of siCTRL # 1 and # 2 - transfected cells was set as 100 % , the Kruskal - Wallis test followed by Dunn ' s multiple comparison post hoc test ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 . Left panel , analysis of clonogenic growth of HCT116 cells assessed 15 days after transfection with non - targeting ( siCTRL # 1 ) , VPS4A - or / and VPS4B - targeting siRNAs used in various duplex combinations as indicated . Right panel , images of HCT116 clones taken at the day of clonogenic growth assessment . Data are means of 3 independent experiments ± SEM . NT - non - transfected . All values were normalized , clonogenic growth of siCTRL # 1 - transfected cells was set as 1 . One - sample t - test ; ns - non - significant ( p ≥ 0 . 05 ) , * * * * p < 0 . 0001 . Viability of RKO and SW480 cells assessed as in ( A ) , growth of DLD - 1 cells assessed in BrdU incorporation assay 96 h after siRNA transfection . Various independent non - targeting ( siCTRL # 1 or # 2 ) and VPS4A - or B - targeting siRNA duplexes were used ( # 1 , # 2 , # 5 or # 1 , # 2 , respectively ) . All values were normalized , averaged ( avrg ) viability of siCTRL # 1 and # 2 - transfected cells was set as 100 % . Data are means of 3 independent experiments ± SEM . The Kruskal - Wallis test followed by Dunn ' s multiple comparison post hoc ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 , * * * p < 0 . 001 . VPS4B copy number and dependency scores of selected cancer cell lines obtained in CRISPR / Cas9 and RNAi screens deposited in the DepMap portal ( https : / / depmap . org / portal / ) . VPS4B copy number status estimated across different cell lines using TaqMan assay . The whiskers represent the minimal and maximal copy number in a given triplicate readout . The BCL2 gene localized on 18q in the close vicinity to VPS4B was analyzed as a control . VPS4B and VPS4A protein abundance in selected normal and cancer cell lines analyzed by immunoblotting . Vinculin and GAPDH were used as loading controls . Analysis of viability of HOP62 lung cancer cells assessed 144 h after transfection with independent non - targeting siRNA ( two different duplexes used , siCTRL # 1 or # 2 ) or targeting VPS4A ( duplexes # 2 or # 5 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 4 independent experiments ± SEM . All values were normalized , cell viability of averaged ( avrg ) siCTRL # 1 - and # 2 - transfected cells was set as 100 % . Two - tailed unpaired t - test ; * * * * p < 0 . 0001 . Top panel , analysis of clonogenic growth of HOP62 cells assessed 14 days after transfection with various siRNA duplexes as indicated . Bottom panel , images of HOP62 clones taken at the day of clonogenic growth assessment . Data are means of 4 independent experiments ± SEM . All values were normalized , colony area of averaged ( avrg ) siCTRL # 1 and # 2 - transfected cells was set as 1 . The Mann - Whitney U test ; * * p < 0 . 01 . Analysis of viability of SNU410 pancreatic cancer cells assessed 168 h after transfection with independent non - targeting siRNA ( three different duplexes used , siCTRL # 1 , # 2 or # 3 ) or targeting VPS4A ( duplexes # 2 , # 4 or # 5 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 3 independent experiments ± SEM . All values were normalized , cell viability of averaged ( avrg ) siCTRL # 1 - , # 2 - and # 3 - transfected cells was set as 100 % . Two - tailed unpaired t - test ; * * * * p < 0 . 0001 .

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Figure 2Analysis of viability of HCT116 cells assessed 96 h after transfection with independent non-targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #1 or #2), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, averaged (avrg) viability of siCTRL#1 and #2-transfected cells was set as 100%, the Kruskal-Wallis test followed by Dunn's multiple comparison post hoc test; ns - non-significant (p≥0.05), **p<0.01.Left panel, analysis of clonogenic growth of HCT116 cells assessed 15 days after transfection with non-targeting (siCTRL#1), VPS4A- or/and VPS4B-targeting siRNAs used in various duplex combinations as indicated. Right panel, images of HCT116 clones taken at the day of clonogenic growth assessment. Data are means of 3 independent experiments ±SEM. NT - non-transfected. All values were normalized, clonogenic growth of siCTRL#1-transfected cells was set as 1. One-sample t-test; ns - non-significant (p≥0.05), ****p<0.0001.Viability of RKO and SW480 cells assessed as in (A), growth of DLD-1 cells assessed in BrdU incorporation assay 96 h after siRNA transfection. Various independent non-targeting (siCTRL#1 or #2) and VPS4A- or B-targeting siRNA duplexes were used (#1, #2, #5 or #1, #2, respectively). All values were normalized, averaged (avrg) viability of siCTRL#1 and #2-transfected cells was set as 100%. Data are means of 3 independent experiments ±SEM. The Kruskal-Wallis test followed by Dunn's multiple comparison post hoc; ns - non-significant (p≥0.05), **p<0.01, ***p<0.001.VPS4B copy number and dependency scores of selected cancer cell lines obtained in CRISPR/Cas9 and RNAi screens deposited in the DepMap portal (https://depmap.org/portal/).VPS4B copy number status estimated across different cell lines using TaqMan assay. The whiskers represent the minimal and maximal copy number in a given triplicate readout. The BCL2 gene localized on 18q in the close vicinity to VPS4B was analyzed as a control.VPS4B and VPS4A protein abundance in selected normal and cancer cell lines analyzed by immunoblotting. Vinculin and GAPDH were used as loading controls.Analysis of viability of HOP62 lung cancer cells assessed 144 h after transfection with independent non-targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #2 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 4 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1- and #2-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001.Top panel, analysis of clonogenic growth of HOP62 cells assessed 14 days after transfection with various siRNA duplexes as indicated. Bottom panel, images of HOP62 clones taken at the day of clonogenic growth assessment. Data are means of 4 independent experiments ±SEM. All values were normalized, colony area of averaged (avrg) siCTRL#1 and #2-transfected cells was set as 1. The Mann-Whitney U test; **p<0.01.Analysis of viability of SNU410 pancreatic cancer cells assessed 168 h after transfection with independent non-targeting siRNA (three different duplexes used, siCTRL#1, #2 or #3) or targeting VPS4A (duplexes #2, #4 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1-, #2- and #3-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001.


Figure 3Left panel , growth of HCT116 VPS4B - / - shVPS4A cells as xenografts in mice in the presence or absence of doxycycline . Day 1 indicates the first day of doxycycline administration . n = 9 for each group , each mouse bearing one tumor , ± SEM . Two - tailed unpaired t - test ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 . Right panel , scatter plot representing end - point volumes of single xenografts . Bars represent means ± SEM . Immunoblotting analysis of VPS4A abundance in xenograft samples from untreated and doxycycline - treated mice ( 6 separate xenograft samples for each group ) . Lysates of HCT116 VPS4B - / - cells transfected with control or VPS4A - targeting siRNA marked VPS4A protein detection . GAPDH served as a loading control . Growth of HCT116 VPS4B - / - shCTRL # 1 cells as xenografts in mice in the presence or absence of doxycycline . The arrow indicates the first day of doxycycline ( Dox ) administration . n = 2 mice for each group , each mouse bearing two tumors , ± SEM .

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Figure 3Left panel, growth of HCT116 VPS4B-/- shVPS4A cells as xenografts in mice in the presence or absence of doxycycline. Day 1 indicates the first day of doxycycline administration. n=9 for each group, each mouse bearing one tumor, ±SEM. Two-tailed unpaired t-test; ns - non-significant (p≥0.05), **p<0.01. Right panel, scatter plot representing end-point volumes of single xenografts. Bars represent means ±SEM.Immunoblotting analysis of VPS4A abundance in xenograft samples from untreated and doxycycline-treated mice (6 separate xenograft samples for each group). Lysates of HCT116 VPS4B-/- cells transfected with control or VPS4A-targeting siRNA marked VPS4A protein detection. GAPDH served as a loading control.Growth of HCT116 VPS4B-/- shCTRL#1 cells as xenografts in mice in the presence or absence of doxycycline. The arrow indicates the first day of doxycycline (Dox) administration. n=2 mice for each group, each mouse bearing two tumors, ±SEM.


Figure 4Heatmap visualizing expression of genes related to inflammatory response ( left panel ) and positive regulation of cell death ( right panel ) generated from the GO analysis of biological processes across different transfection conditions with at least 3 biological replicates .

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Figure 4Heatmap visualizing expression of genes related to inflammatory response (left panel) and positive regulation of cell death (right panel) generated from the GO analysis of biological processes across different transfection conditions with at least 3 biological replicates.


Figure 5Immunoblotting analysis of the canonical and noncanonical branches of the NF - κB pathway and mediators of caspase - dependent cell death . Lysates of HCT116 VPS4B - / - cells were collected 66 h after transfection with siRNA ( siCTRL # 1 or different siVPS4A duplexes : # 2 , # 4 or # 5 ) . Lysates of HCT116 VPS4B + / + and non - transfected HCT116 VPS4B - / - cells were used to monitor the basal pathway activity . Representative blot from 10 experiments is shown . NT - non - transfected ; p - RelA - phospho - RelA ; p - IκBα - phospho - IκBα ; cl - cleaved caspases or PARP - 1 . GAPDH or vinculin served as loading controls . Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in ( A ) . Data are means of 10 ( phospho - Rel and cleaved caspase 7 ) , 9 ( p52 , caspase 8 and cleaved caspase 9 ) , 7 ( phosphorylated and total IκB ) or 5 ( cleaved caspase 3 ) independent experiments . Error bars are SEM . Statistical significance was assessed using the following tests : one - sample t - test ( caspase 8 , cleaved caspases 9 , 7 and 3 , total and phospho - IκBα ) or Wilcoxon signed rank test ( phospho - RelA and p52 ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Immunoblot showing induction of the NF - κB pathway ( p52 ) and apoptosis ( cleaved caspase 7 ) in xenograft samples described in Fig 3C . cl - cleaved caspase . Vinculin was used as a loading control . Analysis of the impact of RIPK1 inhibitor ( necrostatin - 1 ) or pan - caspase inhibitor ( Q - VD - Oph ) on cell viability of HCT116 VPS4B - / - cells transfected with siRNA ( non - targeting siCTRL # 1 or different siVPS4A duplexes : # 2 , # 4 or # 5 ) . Cell viability was assessed 72 h after siRNA transfection . Necrostatin - 1 ( 50 µM ) , Q - VP - Oph ( 20 µM ) or vehicle were added to the medium 48 h before viability assessment . Data are means of 5 independent experiments ± SEM . All values were normalized , viability of siCTRL - transfected and vehicle - treated cells was set as 100 % . Two - tailed unpaired t - test ; * * p < 0 . 01 .

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Figure 5Immunoblotting analysis of the canonical and noncanonical branches of the NF-κB pathway and mediators of caspase-dependent cell death. Lysates of HCT116 VPS4B-/- cells were collected 66 h after transfection with siRNA (siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Lysates of HCT116 VPS4B+/+ and non-transfected HCT116 VPS4B-/- cells were used to monitor the basal pathway activity. Representative blot from 10 experiments is shown. NT- non-transfected; p-RelA - phospho-RelA; p-IκBα - phospho-IκBα; cl - cleaved caspases or PARP-1. GAPDH or vinculin served as loading controls.Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in (A). Data are means of 10 (phospho-Rel and cleaved caspase 7), 9 (p52, caspase 8 and cleaved caspase 9), 7 (phosphorylated and total IκB) or 5 (cleaved caspase 3) independent experiments. Error bars are SEM. Statistical significance was assessed using the following tests: one-sample t-test (caspase 8, cleaved caspases 9, 7 and 3, total and phospho-IκBα) or Wilcoxon signed rank test (phospho-RelA and p52); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Immunoblot showing induction of the NF-κB pathway (p52) and apoptosis (cleaved caspase 7) in xenograft samples described in Fig 3C. cl - cleaved caspase. Vinculin was used as a loading control.Analysis of the impact of RIPK1 inhibitor (necrostatin-1) or pan-caspase inhibitor (Q-VD-Oph) on cell viability of HCT116 VPS4B-/- cells transfected with siRNA (non-targeting siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Cell viability was assessed 72 h after siRNA transfection. Necrostatin-1 (50 µM), Q-VP-Oph (20 µM) or vehicle were added to the medium 48 h before viability assessment. Data are means of 5 independent experiments ±SEM. All values were normalized, viability of siCTRL-transfected and vehicle-treated cells was set as 100%. Two-tailed unpaired t-test; **p<0.01.


Figure 6Measurement of ATP released to the cell medium by HCT116 VPS4B - / - cells non - transfected ( NT ) or transfected with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A duplexes : # 2 , # 4 or # 5 ) . Cell culture media were exchanged 16 h after transfection , and fresh media were conditioned for the next 52 - 58 h . For non - transfected cells ( NT ) , the same treatment protocol was used but without the transfection mixture . Measurement of HMGB1 released to the cell medium by HCT116 VPS4B - / - cells non - transfected ( NT ) or transfected with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A duplexes : # 2 , # 4 or # 5 ) . Cell culture media were exchanged 16 h after transfection , and fresh media were conditioned for the next 52 - 58 h . For non - transfected cells ( NT ) , the same treatment protocol was used but without the transfection mixture . Microscopy images presenting cell surface calreticulin ( green ) in HCT116 VPS4B - / - cells 48 h after transfection with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A # 2 ) . As a positive control for detection of cell surface calreticulin , non - transfected cells ( NT ) were treated with 2 µM mitoxantrone for 24 h . In blue , DAPI staining . Scale bar , 15 µm . Flow cytometric analysis of calreticulin exposed on the cell surface of VPS4A - depleted HCT116 VPS4B - / - cells 66 h after siRNA transfection ( siVPS4A duplexes : # 2 , # 4 or # 5 were used ) . Non - transfected ( NT ) or siCTRL # 1 - transfected cells served as negative controls . Left panel , percentage of cells positive for calreticulin in the population of live ( DAPI - negative ) cells , data are means of 4 independent experiments ± SEM . The Mann - Whitney U test ; * * p < 0 . 01 . Right panel , representative dot plot diagrams of flow cytometric analysis of cell surface - exposed calreticulin . Primary rabbit anti - calreticulin and control isotype IgG antibodies were used for staining , followed by secondary AlexaFluor 647 - conjugated antibody . qPCR analysis of M1 ( pro - inflammatory ) and M2 ( anti - inflammatory ) macrophage polarization markers in mouse bone marrow - derived macrophages ( BMDMs ) incubated for 24 h in conditioned media ( CM ) collected from control ( siCTRL # 1 ) , Vps4a - and / or Vps4b - depleted CT - 26 cells . For double Vps4a + b depletion various combinations of siVps4a # 2 or # 5 and siVps4B # 3 or # 4 duplexes were used . Data were normalized and are presented as the fold change of expression of a given M1 or M2 marker in BMDMs treated with CM from Vps4 - depleted cells compared to its expression in BMDMs treated with CM from siCTRL # 1 - transfected cells ( set as 1 ) . Data are means of 4 independent experiments ± SEM . One - sample t - test ; ns - non - significant ( p ≥ 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 .

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Figure 6Measurement of ATP released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.Measurement of HMGB1 released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.Microscopy images presenting cell surface calreticulin (green) in HCT116 VPS4B-/- cells 48 h after transfection with siRNA (non-targeting siCTRL#1 or targeting siVPS4A#2). As a positive control for detection of cell surface calreticulin, non-transfected cells (NT) were treated with 2 µM mitoxantrone for 24 h. In blue, DAPI staining. Scale bar, 15 µm.Flow cytometric analysis of calreticulin exposed on the cell surface of VPS4A-depleted HCT116 VPS4B-/- cells 66 h after siRNA transfection (siVPS4A duplexes: #2, #4 or #5 were used). Non-transfected (NT) or siCTRL#1-transfected cells served as negative controls. Left panel, percentage of cells positive for calreticulin in the population of live (DAPI-negative) cells, data are means of 4 independent experiments ±SEM. The Mann-Whitney U test; **p<0.01. Right panel, representative dot plot diagrams of flow cytometric analysis of cell surface-exposed calreticulin. Primary rabbit anti-calreticulin and control isotype IgG antibodies were used for staining, followed by secondary AlexaFluor 647-conjugated antibody.qPCR analysis of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage polarization markers in mouse bone marrow-derived macrophages (BMDMs) incubated for 24 h in conditioned media (CM) collected from control (siCTRL#1), Vps4a- and/or Vps4b-depleted CT-26 cells. For double Vps4a+b depletion various combinations of siVps4a#2 or #5 and siVps4B#3 or #4 duplexes were used. Data were normalized and are presented as the fold change of expression of a given M1 or M2 marker in BMDMs treated with CM from Vps4-depleted cells compared to its expression in BMDMs treated with CM from siCTRL#1-transfected cells (set as 1). Data are means of 4 independent experiments ±SEM. One-sample t-test; ns - non-significant (p≥0.05), *p<0.05, **p<0.01.


Fig 1A ) Time - dependent GSK - 3 inhibition of VP2 . 51 at 1 and 2 μM . B ) Kinetic GSK - 3 inhibition data determined for VP2 . 51 . ATP concentrations in the reaction mixture varied from 1 to 50 μM . Compound concentrations used are depicted in the plot , and the concentration of GS - 2 was kept constant at 12 . 5 μM . Each point is the mean of two different experiments , each one analyzed in duplicate . C ) Kinase profiling of VP2 . 51 on human recombinant kinases . The percentage ( % ) of kinase activity after the treatment with a fixed concentration ( 10 μM ) of the compound is shown .

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Fig 1A) Time-dependent GSK-3 inhibition of VP2.51 at 1 and 2 μM.B) Kinetic GSK-3 inhibition data determined for VP2.51. ATP concentrations in the reaction mixture varied from 1 to 50 μM. Compound concentrations used are depicted in the plot, and the concentration of GS-2 was kept constant at 12.5 μM. Each point is the mean of two different experiments, each one analyzed in duplicate.C) Kinase profiling of VP2.51 on human recombinant kinases. The percentage (%) of kinase activity after the treatment with a fixed concentration (10 μM) of the compound is shown.


Fig 2A ) Evolution of rat weight of different treatment groups ( 12 animals in each group ) : control , L - BMAA ( 300 mg / kg / day during 5 consecutive days treated at weaning ) , VP2 . 51 ( 2 , 5 mg / kg during 15 days ) with a group treated at PT1 and another at PT30 , L - BMAA ( with the same treatment as the group of L - BMAA ) + VP2 . 51 ( with the same treatment groups VP2 . 51 ) with a group treated at PT1 and another at PT30 . B ) . Evolution of neurological evaluation in the treatment assay with VP2 . 51 ( 12 animals in each group ) . The control group and the groups treated exclusively with VP2 . 51 ( at PT1 and PT30 ) are not shown because its assessment coincides with the horizontal axis . The time when the inhibitor is added is indicated with arrows .

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Fig 2A) Evolution of rat weight of different treatment groups (12 animals in each group): control, L-BMAA (300 mg/kg/day during 5 consecutive days treated at weaning), VP2.51 (2,5 mg/kg during 15 days) with a group treated at PT1 and another at PT30, L-BMAA (with the same treatment as the group of L-BMAA) + VP2.51 (with the same treatment groups VP2.51) with a group treated at PT1 and another at PT30.B). Evolution of neurological evaluation in the treatment assay with VP2.51 (12 animals in each group). The control group and the groups treated exclusively with VP2.51 (at PT1 and PT30) are not shown because its assessment coincides with the horizontal axis. The time when the inhibitor is added is indicated with arrows.


Fig 3A ) Analysis of LC3 - II and P - mTOR by western - blot in the motor cortex of control and treated animals . Proteins were detected by Western blotting using β - tubulin for calibration of sample loading . The protein bands were quantified by Image Multigauge v3 . 0 software . Immunoblots are representative of one of three experiments with similar results . These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM . * , * * Indicate differences of p - value < 0 . 05 < 0 . 01 with the control group , respectively . # , # # # indicate differences of p - value < 0 . 05 < 0 . 001 with the group of animals treated with L - BMAA , respectively . B ) Analysis of LC3 - II and P - mTOR by western - blot in the lumbar spinal cord of control and treated animals . Proteins were detected by Western blotting using β - tubulin for calibration of sample loading . The protein bands were quantified by Image Multigauge v3 . 0 software . Immunoblots are representative of one of three experiments with similar results . These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM . * , * * * Indicate differences of p - value < 0 . 05 < 0 . 001 with the control group , respectively . # # Indicate differences of p - value < 0 . 01 with the group of animals treated with L - BMAA .

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Fig 3A) Analysis of LC3-II and P-mTOR by western-blot in the motor cortex of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, ** Indicate differences of p-value <0.05 <0.01 with the control group, respectively. #, ### indicate differences of p-value <0.05 <0.001 with the group of animals treated with L-BMAA, respectively.B) Analysis of LC3-II and P-mTOR by western-blot in the lumbar spinal cord of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, *** Indicate differences of p-value <0.05 <0.001 with the control group, respectively. ## Indicate differences of p-value <0.01 with the group of animals treated with L-BMAA.


Fig 4B ) Cell viability measured in the experiment with or without inhibitors .

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Fig 4B) Cell viability measured in the experiment with or without inhibitors.


Figure 1Representative images of combined IF for H3K27me3 or H2AK119ub ( green ) with RNA FISH for Xist ( red ) in Xist - TetOP lines ( for clone 1 of each mutant type ) upon D2 in DOX conditions ; Blue - DAPI staining ; Scale bar : 10 µm . Graph represents the mean % + S . E . M . of Xist - coated X chromosomes enriched for H3K27me3 or H2AK119ub in the different Xist - TetOP mutants ( for clone 1 of each mutant type ) from 2 - to - 4 independent experiments ; A minimum of 50 Xist - coated X chromosomes were counted per experiment ; Only p - values corresponding to significant differences from unpaired Student ' s t - test comparing mutants to Xist FL are indicated as * ( p - value < 0 . 05 ) .

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Figure 1Representative images of combined IF for H3K27me3 or H2AK119ub (green) with RNA FISH for Xist (red) in Xist-TetOP lines (for clone 1 of each mutant type) upon D2 in DOX conditions; Blue - DAPI staining; Scale bar: 10 µm.Graph represents the mean % + S.E.M. of Xist-coated X chromosomes enriched for H3K27me3 or H2AK119ub in the different Xist-TetOP mutants (for clone 1 of each mutant type) from 2-to-4 independent experiments; A minimum of 50 Xist-coated X chromosomes were counted per experiment; Only p-values corresponding to significant differences from unpaired Student's t-test comparing mutants to Xist FL are indicated as * (p-value < 0.05).


Figure 2Top 20 protein hits from the ChIRP - MS of Xist FL ; The ranking was based on fold - enrichment of Xist FL DOX versus Xist FL noDOX ; Weakly annotated protein isoforms with an Annotation score in UniprotKB < 3 ( out of 5 ) were excluded ; Fold - enrichment for Xist ΔB + C is also displayed for comparison ; Light green boxes correspond to proteins as Xist interactors [ 13 ] ; protein in red ( RNF2 / RING1B ) represents a protein not found in the Xist ΔB + C interactome ; Protein in light brown ( TRIM71 ) is less enriched in Xist ΔB + C than in Xist FL . Scatter plot displaying the differences in peptide counts between Xist FL and Xist ΔB + C for the 74 out of 81 Xist - interactors with a minimum of fold - change of 2 . 5 in Xist FL or Xist ΔB + C ; Shown is the log2 fold change of peptide counts of each mutant in DOX conditions compared with the Xist FL in noDOX conditions ; proteins retrieved by both Xist FL and Xist ΔB + C ChIRPs with a proposed role in XCI such as SPEN , RBM15 , WTAP , YTHDC1 and hnRNPU are indicated ; light brown dots mark proteins more represented in Xist FL than in Xist ΔB + C ChIRPs , while red dots display proteins which are only retrieved by Xist FL ChIRP .

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Figure 2Top 20 protein hits from the ChIRP-MS of Xist FL; The ranking was based on fold-enrichment of Xist FL DOX versus Xist FL noDOX; Weakly annotated protein isoforms with an Annotation score in UniprotKB < 3 (out of 5) were excluded; Fold-enrichment for Xist ΔB+C is also displayed for comparison; Light green boxes correspond to proteins as Xist interactors [13]; protein in red (RNF2/RING1B) represents a protein not found in the Xist ΔB+C interactome; Protein in light brown (TRIM71) is less enriched in Xist ΔB+C than in Xist FL.Scatter plot displaying the differences in peptide counts between Xist FL and Xist ΔB+C for the 74 out of 81 Xist-interactors with a minimum of fold-change of 2.5 in Xist FL or Xist ΔB+C; Shown is the log2 fold change of peptide counts of each mutant in DOX conditions compared with the Xist FL in noDOX conditions; proteins retrieved by both Xist FL and Xist ΔB+C ChIRPs with a proposed role in XCI such as SPEN, RBM15, WTAP, YTHDC1 and hnRNPU are indicated; light brown dots mark proteins more represented in Xist FL than in Xist ΔB+C ChIRPs, while red dots display proteins which are only retrieved by Xist FL ChIRP.


Figure 3Plots showing H3K27me3 and H2AK119ub accumulation over the X chromosome in Xist FL and Xist ΔB + C with upon DOX induction at day 2 ( D2 ) of differentiation ; Each dot represents a single 10Kb window and its enrichment relative to noDOX condition ; Black line is a loess regression on all windows ; Xist locus is represented by a blue long line , active genes by green lines . Violin plots quantifying H3K27me3 and H2AK119ub enrichment over intergenic regions , active promoters and active gene bodies in the X chromosome in Xist FL and Xist ΔB + C cell lines at D2 upon DOX induction ; Shown is the log2 fold change of DOX vs noDOX conditions ; violin plots represent the distribution of the values , the horizontal band is the median , the lower and upper hinges correspond to the 25th and 75th percentiles ; n = indicates the number of regions / genes analyzed ; p - values were calculated using paired Wilcoxon test , comparing Xist FL and Xist ΔB + C cell lines . Average plots showing the mean enrichment of H3K27me3 ( top ) and H2AK119ub ( bottom ) over all X - linked initially active transcriptional start sites ( TSS ) ; Shown is the mean of normalized log2 enrichment of DOX vs noDOX in both Xist FL and Xist ΔB + C cell lines . Genome browser plots showing H3K27me3 ( top ) and H2AK119ub ( bottom ) enrichments in a region encompassing the inactive Atp1b4 and the initially active Lamp2 genes within the XqA3 . 3 region and the initially active Rlim / Rnf12 gene at the XqD region ; Region around the promoter of Rlim / Rnf12 is highlighted in yellow .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Plots showing H3K27me3 and H2AK119ub accumulation over the X chromosome in Xist FL and Xist ΔB+C with upon DOX induction at day 2 (D2) of differentiation; Each dot represents a single 10Kb window and its enrichment relative to noDOX condition; Black line is a loess regression on all windows; Xist locus is represented by a blue long line, active genes by green lines.Violin plots quantifying H3K27me3 and H2AK119ub enrichment over intergenic regions, active promoters and active gene bodies in the X chromosome in Xist FL and Xist ΔB+C cell lines at D2 upon DOX induction; Shown is the log2 fold change of DOX vs noDOX conditions; violin plots represent the distribution of the values, the horizontal band is the median, the lower and upper hinges correspond to the 25th and 75th percentiles; n = indicates the number of regions/genes analyzed; p-values were calculated using paired Wilcoxon test, comparing Xist FL and Xist ΔB+C cell lines.Average plots showing the mean enrichment of H3K27me3 (top) and H2AK119ub (bottom) over all X-linked initially active transcriptional start sites (TSS); Shown is the mean of normalized log2 enrichment of DOX vs noDOX in both Xist FL and Xist ΔB+C cell lines.Genome browser plots showing H3K27me3 (top) and H2AK119ub (bottom) enrichments in a region encompassing the inactive Atp1b4 and the initially active Lamp2 genes within the XqA3.3 region and the initially active Rlim/Rnf12 gene at the XqD region; Region around the promoter of Rlim/Rnf12 is highlighted in yellow.


Figure 4Graph represents the mean % + S . E . M . of Xist - coated chromosomes presenting an active Pgk1 or Lamp2 gene as determined by RNA FISH ( as represented in B ) at D2 in the presence of DOX ( Xist FL was also used in noDOX conditions ) in the different Xist - TetOP mutants ; each bar represents the mean from to 2 - to - 4 independent experiments ; A minimum of 59 Xist - coated chromosomes were counted per experiment ; For Xist FL noDOX a minimum of 100 cells ( which do not have Xist - coated chromosome ) were counted ; Only p - values corresponding to significant differences comparing mutants ( or Xist FL noDOX ) to Xist FL DOX are indicated as * ( p - value < 0 . 05 ) or * * * ( p - value < 0 . 01 ) , unpaired Student ' s t - test ; dashed line marks the mean percentage of silencing for the Lamp2 gene in Xist FL DOX . Representative RNA FISH images for Xist ( red ) and nascent - transcript of Pgk1 ( green ) in Xist - TetOP lines at day 4 of differentiation in the presence of DOX ( Xist FL is also shown in noDOX conditions ) ; DNA stained in blue by DAPI ; Numbers represent % of Xist - coated X - chromosomes ± S . E . M . with active Pgk1 gene ( except for Xist FL noDOX , where numbers represent % of cells with Pgk1 active gene ) ; The values represent 2 - to - 4 independent experiments , where a minimum of 50 Xist - coated chromosomes were counted per experiment ; Significant differences compared with Xist FL ( DOX ) are indicated as * ( p - value < 0 . 05 ) or * * * ( p - value < 0 . 01 ) , unpaired Student ' s t - test . Clustering analysis of the normalized RNA - seq counts on the X chromosome ( chrX ) for all the duplicates of Xist FL , Xist ΔA and Xist ΔB + C in DOX and noDOX conditions . Plots displays the log2 ( fold - change ) in the expression of X - linked genes along the chrX comparing DOX versus noDOX samples for Xist FL , Xist ΔA and Xist ΔB + C at day 2 of differentiation ; red dots correspond to genes which are differently expressed in DOX vs noDOX ( p < 0 . 05 , Limma t - test ) , while black dots represent genes which are not differentially expressed between the two conditions ( p ≥ 0 . 05 ) . Violin plots displaying the average log2 ( fold - change ) in gene expression between DOX and noDOX conditions on the chrX in Xist FL , Xist ΔA and Xist ΔB + C at day 2 of differentiation ; violin plots represent the distribution of the values , the horizontal band is the median , the lower and upper hinges correspond to the 25th and 75th percentiles ; p - values were calculated using paired Wilcoxon test ; n = indicates the number of genes analyzed . Box plots displaying the log2 ( DOX / noDOX ) fold - change in expression of X - linked genes in Xist FL and Xist ΔB + C categorized according to the enrichment of H3K27me3 and H2AK119ub marks at promoters in Xist ΔB + C upon DOX induction ( with no or little accumulation vs accumulation ) ; the horizontal band of the boxplot is the median of the values , the lower and upper hinges correspond to the 25th and 75th percentiles , the upper whisker extends from the hinge to the largest value not further than 1 . 5 interquartile range from the hinge , the lower whisker extends from the hinge to the smallest value at most 1 . 5 interquartile range of the hinge ; p - values between samples were calculated using paired Wilcoxon test ; n = indicates the number of genes analyzed . Genome browser plots showing RNA - seq reads , H3K27me3 and H2AK119ub nChIP reads around the Abcb7 gene for Xist FL ( left ) and Xist ΔB + C ( right ) at day 2 of differentiation in both DOX and noDOX conditions .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Graph represents the mean % + S.E.M. of Xist-coated chromosomes presenting an active Pgk1 or Lamp2 gene as determined by RNA FISH (as represented in B) at D2 in the presence of DOX (Xist FL was also used in noDOX conditions) in the different Xist-TetOP mutants; each bar represents the mean from to 2-to-4 independent experiments; A minimum of 59 Xist-coated chromosomes were counted per experiment; For Xist FL noDOX a minimum of 100 cells (which do not have Xist-coated chromosome) were counted; Only p-values corresponding to significant differences comparing mutants (or Xist FL noDOX) to Xist FL DOX are indicated as * (p-value < 0.05) or *** (p-value < 0.01), unpaired Student's t-test; dashed line marks the mean percentage of silencing for the Lamp2 gene in Xist FL DOX.Representative RNA FISH images for Xist (red) and nascent-transcript of Pgk1 (green) in Xist-TetOP lines at day 4 of differentiation in the presence of DOX (Xist FL is also shown in noDOX conditions); DNA stained in blue by DAPI; Numbers represent % of Xist-coated X-chromosomes ± S.E.M. with active Pgk1 gene (except for Xist FL noDOX, where numbers represent % of cells with Pgk1 active gene); The values represent 2-to-4 independent experiments, where a minimum of 50 Xist-coated chromosomes were counted per experiment; Significant differences compared with Xist FL (DOX) are indicated as * (p-value < 0.05) or *** (p-value < 0.01), unpaired Student's t-test.Clustering analysis of the normalized RNA-seq counts on the X chromosome (chrX) for all the duplicates of Xist FL, Xist ΔA and Xist ΔB+C in DOX and noDOX conditions.Plots displays the log2(fold-change) in the expression of X-linked genes along the chrX comparing DOX versus noDOX samples for Xist FL, Xist ΔA and Xist ΔB+C at day 2 of differentiation; red dots correspond to genes which are differently expressed in DOX vs noDOX (p < 0.05, Limma t-test), while black dots represent genes which are not differentially expressed between the two conditions (p ≥ 0.05).Violin plots displaying the average log2(fold-change) in gene expression between DOX and noDOX conditions on the chrX in Xist FL, Xist ΔA and Xist ΔB+C at day 2 of differentiation; violin plots represent the distribution of the values, the horizontal band is the median, the lower and upper hinges correspond to the 25th and 75th percentiles; p-values were calculated using paired Wilcoxon test; n = indicates the number of genes analyzed.Box plots displaying the log2(DOX/noDOX) fold-change in expression of X-linked genes in Xist FL and Xist ΔB+C categorized according to the enrichment of H3K27me3 and H2AK119ub marks at promoters in Xist ΔB+C upon DOX induction (with no or little accumulation vs accumulation); the horizontal band of the boxplot is the median of the values, the lower and upper hinges correspond to the 25th and 75th percentiles, the upper whisker extends from the hinge to the largest value not further than 1.5 interquartile range from the hinge, the lower whisker extends from the hinge to the smallest value at most 1.5 interquartile range of the hinge; p-values between samples were calculated using paired Wilcoxon test; n = indicates the number of genes analyzed.Genome browser plots showing RNA-seq reads, H3K27me3 and H2AK119ub nChIP reads around the Abcb7 gene for Xist FL (left) and Xist ΔB+C (right) at day 2 of differentiation in both DOX and noDOX conditions.


Figure 1LGG - 1 : : GFP - positive puncta labeling autophagic membranes [ 22 ] were counted in wild - type or in food - limited animals . ( A ) Micrographs of eat - 2 ( ad1116 ) L3 larvae expressing GFP - tagged lgg - 1 / LC3 . Arrow indicates autophagic focus . Magnification is indicated . ( B ) Average number of LGG - 1 : : GFP - containing puncta in eat - 2 ( ad1116 ) mutants and N2 wild - type animals ( WT ) , p < 0 . 0001 . ( C ) Average number of LGG - 1 : : GFP - containing puncta in N2 wild - type , food - restricted animals grown in liquid media ( WT , food limited ) and N2 wild - type animals grown in liquid with a higher concentration of bacteria ( WT , fully fed ) , p < 0 . 0001 ; see Methods . Between three and ten seam cells were counted in each of 20 - 40 animals using high - power microscopy and averaged . n , total number of seam cells observed . Error bars : ± SEM . p - Values were calculated as unpaired , two - tailed t - test . Animals were raised at 20 ° C . Please see Table S1 for quantification of all data .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1LGG-1::GFP-positive puncta labeling autophagic membranes [22] were counted in wild-type or in food-limited animals.(A) Micrographs of eat-2(ad1116) L3 larvae expressing GFP-tagged lgg-1/LC3. Arrow indicates autophagic focus. Magnification is indicated.(B) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) mutants and N2 wild-type animals (WT), p < 0.0001.(C) Average number of LGG-1::GFP-containing puncta in N2 wild-type, food-restricted animals grown in liquid media (WT, food limited) and N2 wild-type animals grown in liquid with a higher concentration of bacteria (WT, fully fed), p < 0.0001; see Methods.Between three and ten seam cells were counted in each of 20-40 animals using high-power microscopy and averaged. n, total number of seam cells observed. Error bars: ±SEM. p-Values were calculated as unpaired, two-tailed t-test. Animals were raised at 20 °C. Please see Table S1 for quantification of all data.


Figure 2 ( A ) Survival curves of eat - 2 ( ad1116 ) animals fed either control bacteria or bacteria expressing bec - 1 dsRNA during adulthood at 20 ° C . Mean lifespan was 23 . 7 d for control and 19 . 6 d for bec - 1 RNAi , p < 0 . 0001 , Log - rank ( Mantel - Cox ) test . This experiment was performed a total of six times , and bec - 1 RNAi shortened the lifespan of eat - 2 animals ∼ 15 % - 30 % . Please see Table 1 for additional data . ( B ) Survival curves of N2 wild - type animals ( WT ) fed either control bacteria or bacteria expressing bec - 1 dsRNA during adulthood at 20 ° C . These assays were performed concurrently with the eat - 2 mutant lifespan analysis in Figure 2A . Mean lifespan was 17 . 3 d for control and 18 . 9 d for bec - 1 RNAi . p = 0 . 045 , Log - rank ( Mantel - Cox ) test . Depletion of bec - 1 did not significantly change the lifespan of N2 or sterile fer - 15 ( b26 ) ; fem - 1 ( hc17 ) animals in any of six experiments . Please see Table 1 for additional data . ( C ) Survival curves of eat - 2 ( ad1116 ) animals fed either control bacteria or bacteria expressing vps - 34 dsRNA during adulthood at 20 ° C . Mean lifespan was 27 . 6 d for control and 22 . 8 d for vps - 34 RNAi , p = 0 . 0003 , Log - rank ( Mantel - Cox ) test . This experiment was performed a total of four times . Please see Table 1 for additional data . ( D ) Survival curves of sterile fer - 15 ( b26 ) ; fem - 1 ( hc17 ) animals ( WT ) fed either control bacteria or bacteria expressing vps - 34 dsRNA during adulthood at 20 ° C . These assays were performed at the same time as the eat - 2 lifespan analysis shown in Figure 2C . Mean lifespan was 21 . 5 d for control and 23 . 3 d for bec - 1 RNAi . p = 0 . 14 , Log - rank ( Mantel - Cox ) test . Depletion of vps - 34 did not significantly change the lifespan of N2 or sterile fer - 15 ( b26 ) ; fem - 1 ( hc17 ) animals in each of six different experiments . Please see Table 1 for additional data .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Survival curves of eat-2(ad1116) animals fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. Mean lifespan was 23.7 d for control and 19.6 d for bec-1 RNAi, p < 0.0001, Log-rank (Mantel-Cox) test. This experiment was performed a total of six times, and bec-1 RNAi shortened the lifespan of eat-2 animals ∼15%-30%. Please see Table 1 for additional data.(B) Survival curves of N2 wild-type animals (WT) fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. These assays were performed concurrently with the eat-2 mutant lifespan analysis in Figure 2A. Mean lifespan was 17.3 d for control and 18.9 d for bec-1 RNAi. p = 0.045, Log-rank (Mantel-Cox) test. Depletion of bec-1 did not significantly change the lifespan of N2 or sterile fer-15(b26); fem-1(hc17) animals in any of six experiments. Please see Table 1 for additional data.(C) Survival curves of eat-2(ad1116) animals fed either control bacteria or bacteria expressing vps-34 dsRNA during adulthood at 20 °C. Mean lifespan was 27.6 d for control and 22.8 d for vps-34 RNAi, p = 0.0003, Log-rank (Mantel-Cox) test. This experiment was performed a total of four times. Please see Table 1 for additional data.(D) Survival curves of sterile fer-15(b26); fem-1(hc17) animals (WT) fed either control bacteria or bacteria expressing vps-34 dsRNA during adulthood at 20 °C. These assays were performed at the same time as the eat-2 lifespan analysis shown in Figure 2C. Mean lifespan was 21.5 d for control and 23.3 d for bec-1 RNAi. p = 0.14, Log-rank (Mantel-Cox) test. Depletion of vps-34 did not significantly change the lifespan of N2 or sterile fer-15(b26); fem-1(hc17) animals in each of six different experiments. Please see Table 1 for additional data.


Figure 3 ( A ) Average number of LGG - 1 : : GFP - containing puncta in eat - 2 ( ad1116 ) progeny of animals fed either control bacteria or bacteria expressing bec - 1 , vps - 34 or pha - 4 dsRNA their entire lives . p ≤ 0 . 0001 for bec - 1 , vps - 34 and pha - 4 RNAi treatments compared to control , respectively , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . ( B ) Average number of LGG - 1 : : GFP - containing puncta in rab - 10 ( ok1494 ) progeny of animals fed either control bacteria or bacteria expressing pha - 4 dsRNA their entire lives ; p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . Please see Figure 1 for details and Table S1 for quantification of data . Feeding mutants for several generations with pha - 4 dsRNA , inhibited development and sharply decreased the number of eggs laid ( [ 46 ] , data not shown ) . However , the loss of many embryos was unlikely to bias our findings , since many progeny of daf - 2 mutants treated with pha - 4 RNAi also died but those that did not exhibited a robust autophagy phenotype ( Figure 6 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34 or pha-4 dsRNA their entire lives. p ≤ 0.0001 for bec-1, vps-34 and pha-4 RNAi treatments compared to control, respectively, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details.(B) Average number of LGG-1::GFP-containing puncta in rab-10(ok1494) progeny of animals fed either control bacteria or bacteria expressing pha-4 dsRNA their entire lives; p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. Please see Figure 1 for details and Table S1 for quantification of data. Feeding mutants for several generations with pha-4 dsRNA, inhibited development and sharply decreased the number of eggs laid ([46], data not shown). However, the loss of many embryos was unlikely to bias our findings, since many progeny of daf-2 mutants treated with pha-4 RNAi also died but those that did not exhibited a robust autophagy phenotype (Figure 6).


Figure 4 ( A ) Average number of LGG - 1 : : GFP - containing puncta in let - 363 / TOR RNAi - arrested animals compared to N2 wild - type animals ( WT ) grown on control ( vector - only ) bacteria , p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . We were not able to detect increased LGG - 1 : : GFP puncta in long - lived let - 363 ( RNAi ) adults ; however , one generation of daf - 2 RNAi , our positive control , did not significantly increase the number of foci in adults either ( data not shown ) . Please see Table S1 for quantification of all data . ( B ) Average number of LGG - 1 : : GFP - containing puncta in daf - 15 ( m81 ) / unc - 24 ( e138 ) heterozygotes ( daf - 15 / + ) compared to N2 wild - type animals ( WT ) , p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . Please see Table S1 for quantification of all data . daf - 15 encodes the TOR - binding partner Raptor . ( C ) Survival curves of daf - 15 ( m81 ) / unc - 24 ( e138 ) heterozygotes ( daf - 15 / + , strain DR412 ) fed either control bacteria or bacteria expressing bec - 1 dsRNA during adulthood at 20 ° C . Mean lifespan : daf - 15 / + animals grown on control RNAi - bacteria : 25 . 1 d , daf - 15 / + animals on bec - 1 RNAi : 20 . 8 d , p = 0 . 0008 , Log - rank ( Mantel - Cox ) test . The lifespan of daf - 15 / + animals grown on bec - 1 RNAi - bacteria during adulthood was measured again , yielding similar results ; the lifespan of daf - 15 / + animals was also measured three times following whole - life RNAi exposure . In these experiments , bec - 1 RNAi generally shortened the mean lifespan of daf - 15 / + animals to a greater extent than it shortened the lifespan of wild type . ( We also attempted to perform double - RNAi experiments , in which animals were cultured on a 50 : 50 mixture of let - 363 / TOR and bec - 1 [ or control ] RNAi bacteria . Although the trends we saw were in the expected direction , the effects produced by half - strength RNAi were small and not statistically significant [ data not shown ] . ) Please see Table 2 for additional data . ( D ) Survival curves of wild - type animals derived from strain DR412 ( WT ) fed either control bacteria or bacteria expressing bec - 1 dsRNA throughout their whole life at 20 ° C . These assays were performed concurrently with the daf - 15 / + lifespan analysis shown in Figure 4C . WT grown on control RNAi - bacteria : 21 . 4 d , WT on bec - 1 RNAi - bacteria : 21 . 1 d ( p = 0 . 34 ) , p between daf - 15 / + and WT grown on control RNAi - bacteria , p < 0 . 0001 , Log - rank ( Mantel - Cox ) test . Please see Table 2 for additional data .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Average number of LGG-1::GFP-containing puncta in let-363/TOR RNAi-arrested animals compared to N2 wild-type animals (WT) grown on control (vector-only) bacteria, p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. We were not able to detect increased LGG-1::GFP puncta in long-lived let-363(RNAi) adults; however, one generation of daf-2 RNAi, our positive control, did not significantly increase the number of foci in adults either (data not shown). Please see Table S1 for quantification of all data.(B) Average number of LGG-1::GFP-containing puncta in daf-15(m81)/unc-24(e138) heterozygotes (daf-15/+) compared to N2 wild-type animals (WT), p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Please see Table S1 for quantification of all data. daf-15 encodes the TOR-binding partner Raptor.(C) Survival curves of daf-15(m81)/unc-24(e138) heterozygotes (daf-15/+, strain DR412) fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. Mean lifespan: daf-15/+ animals grown on control RNAi-bacteria: 25.1 d, daf-15/+animals on bec-1 RNAi: 20.8 d, p = 0.0008, Log-rank (Mantel-Cox) test. The lifespan of daf-15/+ animals grown on bec-1 RNAi-bacteria during adulthood was measured again, yielding similar results; the lifespan of daf-15/+ animals was also measured three times following whole-life RNAi exposure. In these experiments, bec-1 RNAi generally shortened the mean lifespan of daf-15/+ animals to a greater extent than it shortened the lifespan of wild type. (We also attempted to perform double-RNAi experiments, in which animals were cultured on a 50:50 mixture of let-363/TOR and bec-1 [or control] RNAi bacteria. Although the trends we saw were in the expected direction, the effects produced by half-strength RNAi were small and not statistically significant [data not shown].) Please see Table 2 for additional data.(D) Survival curves of wild-type animals derived from strain DR412 (WT) fed either control bacteria or bacteria expressing bec-1 dsRNA throughout their whole life at 20 °C. These assays were performed concurrently with the daf-15/+ lifespan analysis shown in Figure 4C. WT grown on control RNAi-bacteria: 21.4 d, WT on bec-1 RNAi-bacteria: 21.1 d (p = 0.34), p between daf-15/+ and WT grown on control RNAi-bacteria, p < 0.0001, Log-rank (Mantel-Cox) test. Please see Table 2 for additional data.


Figure 5 ( A ) Average number of LGG - 1 : : GFP - containing puncta in N2 wild - type animals ( WT ) fed either control bacteria or bacteria expressing daf - 2 ( as a control ) or rab - 10 dsRNA for two generations . p < 0 . 0001 for either daf - 2 or rab - 10 RNAi treatment compared to control RNAi treatment , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . Please see Table S1 for quantification of all data . ( B ) Average number of LGG - 1 : : GFP - containing puncta in rab - 10 ( ok1494 ) mutants compared to N2 wild - type animals ( WT ) , p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . Please see Figure 1 for details . Please see Table S1 for quantification of all data . ( C ) Survival curves of rab - 10 ( ok1494 ) animals fed either control bacteria or bacteria expressing bec - 1 or vps - 34 dsRNA during adulthood at 20 ° C . Mean lifespan was 27 . 9 d for control , 19 . 9 d for bec - 1 RNAi , and 22 . 1 d for vps - 34 RNAi , all pair - wise comparisons to control , p < 0 . 0001 , Log - rank ( Mantel - Cox ) test . This experiment was performed two times . Please see Table 3 for additional data . ( D ) Survival curves of N2 wild - type animals ( WT ) fed either control bacteria or bacteria expressing bec - 1 or vps - 34 dsRNA during adulthood at 20 ° C . These assays were performed at the same time as the rab - 10 lifespan analysis shown in Figure 5C . Mean lifespan was 22 . 9 d for control , 23 . 6 d for bec - 1 RNAi , and 24 . 5 d for vps - 34 RNAi , pair - wise comparison to control , p = 0 . 057 and p = 0 . 16 , respectively , Log - rank ( Mantel - Cox ) test . Please see Table 1 for additional data .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Average number of LGG-1::GFP-containing puncta in N2 wild-type animals (WT) fed either control bacteria or bacteria expressing daf-2 (as a control) or rab-10 dsRNA for two generations. p < 0.0001 for either daf-2 or rab-10 RNAi treatment compared to control RNAi treatment, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Please see Table S1 for quantification of all data.(B) Average number of LGG-1::GFP-containing puncta in rab-10(ok1494) mutants compared to N2 wild-type animals (WT), p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. Please see Figure 1 for details. Please see Table S1 for quantification of all data.(C) Survival curves of rab-10(ok1494) animals fed either control bacteria or bacteria expressing bec-1 or vps-34 dsRNA during adulthood at 20 °C. Mean lifespan was 27.9 d for control, 19.9 d for bec-1 RNAi, and 22.1 d for vps-34 RNAi, all pair-wise comparisons to control, p < 0.0001, Log-rank (Mantel-Cox) test. This experiment was performed two times. Please see Table 3 for additional data.(D) Survival curves of N2 wild-type animals (WT) fed either control bacteria or bacteria expressing bec-1 or vps-34 dsRNA during adulthood at 20 °C. These assays were performed at the same time as the rab-10 lifespan analysis shown in Figure 5C. Mean lifespan was 22.9 d for control, 23.6 d for bec-1 RNAi, and 24.5 d for vps-34 RNAi, pair-wise comparison to control, p = 0.057 and p = 0.16, respectively, Log-rank (Mantel-Cox) test. Please see Table 1 for additional data.


Figure 6 ( A ) Average number of LGG - 1 : : GFP - containing puncta in daf - 2 ( e1370 ) progeny of animals fed either control bacteria or bacteria expressing bec - 1 , vps - 34 , or pha - 4 dsRNA for their entire lives . p < 0 . 0001 for bec - 1 and vps - 34 RNAi - bacteria compared to control RNAi - bacteria , respectively , p = 0 . 17 for pha - 4 RNAi - bacteria compared to control RNAi - bacteria , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . Feeding daf - 2 mutants for several generations with pha - 4 dsRNA sharply decreased the number of eggs laid ( data not shown ) . The mean lifespan of daf - 2 ( e1370 ) animals was shortened 12 . 5 % by pha - 4 RNAi ( [ 28 ] and data not shown ) , and we measured an 11 % decrease in puncta in daf - 2 ( e1370 ) animals fed pha - 4 RNAi . Even though this decrease was not statistically significant it remains possible that it relates to the small difference seen in lifespan . ( B ) Average number of LGG - 1 : : GFP - containing puncta in daf - 16 ( mu86 ) ; daf - 2 ( e1370 ) double mutants compared to daf - 2 ( e1370 ) animals ; p = 0 . 50 , unpaired , two - tailed t - test . N2 wild - type animals ( WT ) are shown for comparison . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . The double mutant expressing the LGG - 1 reporter had a mean lifespan similar to non - transgenic daf - 16 ; daf - 2 double mutants ( data not shown , [ 65 ] ) . Please see Table S1 for quantification of all data .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Average number of LGG-1::GFP-containing puncta in daf-2(e1370) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34, or pha-4 dsRNA for their entire lives. p < 0.0001 for bec-1 and vps-34 RNAi-bacteria compared to control RNAi-bacteria, respectively, p = 0.17 for pha-4 RNAi-bacteria compared to control RNAi-bacteria, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Feeding daf-2 mutants for several generations with pha-4 dsRNA sharply decreased the number of eggs laid (data not shown). The mean lifespan of daf-2(e1370) animals was shortened 12.5% by pha-4 RNAi ([28] and data not shown), and we measured an 11% decrease in puncta in daf-2(e1370) animals fed pha-4 RNAi. Even though this decrease was not statistically significant it remains possible that it relates to the small difference seen in lifespan.(B) Average number of LGG-1::GFP-containing puncta in daf-16(mu86); daf-2(e1370) double mutants compared to daf-2(e1370) animals; p = 0.50, unpaired, two-tailed t-test. N2 wild-type animals (WT) are shown for comparison. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. The double mutant expressing the LGG-1 reporter had a mean lifespan similar to non-transgenic daf-16; daf-2 double mutants (data not shown, [65]). Please see Table S1 for quantification of all data.


Figure 172h after stimulation with anti - CD3 and anti - CD28 , the cells were re - stimulated with P + I for 2h , and the mRNA was subjected to qRT - PCR using the indicated primers for the lineage specifying transcription factors ( B ) or cytokines ( C ) . The graphs represent an average of at least 4 independent experiments . In each graph , the expression level in the control was set as 100 % . P - value was calculated by t - test . * p < 0 . 05 , * * * p < 0 .

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Figure 172h after stimulation with anti-CD3 and anti-CD28, the cells were re-stimulated with P+I for 2h, and the mRNA was subjected to qRT-PCR using the indicated primers for the lineage specifying transcription factors (B) or cytokines (C). The graphs represent an average of at least 4 independent experiments. In each graph, the expression level in the control was set as 100%. P-value was calculated by t-test. * p<0.05, ***p<0.005


Figure 2EVs were extracted from the schistosomal - growing medium and were subjected to cryo - TEM analysis : images were taken as described previously [ 90 ] . The black arrows indicate EVs at the size of ~ 100nm . EVs were extracted from the schistosomal - growing medium and were subjected AFM analysis : images were taken from the schistosomal - growing medium ( I ) or from 1 / 10 dilution of this medium ( II ) . The small images magnify the arrow - indicated EVs . ( III ) Image of the extract deriving from the unused schistosomal - medium . EVs were extracted from the schistosomal - growing medium and were subjected NTA size fractionation of particles analysis ; the graph represents average of 5 different measurements .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2EVs were extracted from the schistosomal-growing medium and were subjected to cryo-TEM analysis: images were taken as described previously [90]. The black arrows indicate EVs at the size of ~100nm. EVs were extracted from the schistosomal-growing medium and were subjected AFM analysis: images were taken from the schistosomal-growing medium (I) or from 1/10 dilution of this medium (II). The small images magnify the arrow-indicated EVs. (III) Image of the extract deriving from the unused schistosomal-medium. EVs were extracted from the schistosomal-growing medium and were subjected NTA size fractionation of particles analysis; the graph represents average of 5 different measurements.


Figure 3EVs were purified from either schistosomal - growing medium ( supernatant ) or unused schistosomal - medium as control . Both pellets were stained using Thiazole Orange . Purified labeled ~ 5 × 106 EVs were incubated with 5 × 105 freshly purified Th cells that were stimulated with anti - CD3 and anti - CD28 antibodies for 10 minutes . Images from the same slide were taken by inverted confocal microscopy at the indicated time points . The arrows in the enlarged images point toward cells labeled with EVs . The scale bars in all images represent 20μm . The percentage of labeled cells was calculated by dividing the number of labeled cells by the total numbers of cells in the same image . The mean + / - SEM was calculated from 3 independent

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3EVs were purified from either schistosomal-growing medium (supernatant) or unused schistosomal-medium as control. Both pellets were stained using Thiazole Orange. Purified labeled ~5 × 106 EVs were incubated with 5 × 105 freshly purified Th cells that were stimulated with anti-CD3 and anti-CD28 antibodies for 10 minutes. Images from the same slide were taken by inverted confocal microscopy at the indicated time points. The arrows in the enlarged images point toward cells labeled with EVs. The scale bars in all images represent 20μm. The percentage of labeled cells was calculated by dividing the number of labeled cells by the total numbers of cells in the same image. The mean +/- SEM was calculated from 3 independent images


Figure 4Schistosomal - miRNA was extracted from either EVs that were isolated from schistosomal - growing medium or control pellets that were isolated from unused schistosomal - medium as described in the Materials and Methods ( Schistosomal - EVs purification section ) . Equal aliquots were subjected to qRT - PCR with specific primers to the schistosomal - miR - 10 , Bantam and miR - 125 ; the expression is presented as ∆ Ct from background Ct that was set as 40 cycles .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Schistosomal- miRNA was extracted from either EVs that were isolated from schistosomal-growing medium or control pellets that were isolated from unused schistosomal-medium as described in the Materials and Methods (Schistosomal-EVs purification section). Equal aliquots were subjected to qRT-PCR with specific primers to the schistosomal-miR-10, Bantam and miR-125; the expression is presented as ∆Ct from background Ct that was set as 40 cycles.


Figure 5The trans - well system was performed as in Fig . 1 . Schistosome - exposed Th cells or control Th cells were subjected to qRT - PCR with specific schistosomal - Taqman primers to ( A ) miR - 10 - 5p , ( B ) Bantam , and ( C ) miR - 125b . The trans - well system was performed as in Fig . 1 . The Control wells contained only unused schistosomal - medium ; Supernatant wells contained 0 . 1 μm of the filtered schistosomal - growing medium ; Th wells contained sorted CD4 + CD11c - from spleens ; Th + APCs contained sorted CD4 + CD11c - and CD4 - CD11c + in 5 : 1 ratio , respectively . In Data information : In ( A - E ) the expression is presented as arbitrary units calculated as 2 ∆ ∆ CT of miRNA relative to snRNA U6 and in ( F ) mRNA relative to β2 microglobulin . The mean + / - SEM was calculated from at least 3 independent experiments ( except in F of 2 independent experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5The trans-well system was performed as in Fig. 1. Schistosome-exposed Th cells or control Th cells were subjected to qRT-PCR with specific schistosomal-Taqman primers to (A) miR-10-5p, (B) Bantam, and (C) miR-125b.The trans-well system was performed as in Fig. 1. The Control wells contained only unused schistosomal-medium; Supernatant wells contained 0.1 μm of the filtered schistosomal-growing medium; Th wells contained sorted CD4+CD11c- from spleens; Th+APCs contained sorted CD4+CD11c- and CD4-CD11c+ in 5:1 ratio, respectively. In Data information: In (A-E) the expression is presented as arbitrary units calculated as 2∆∆CT of miRNA relative to snRNA U6 and in (F) mRNA relative to β2 microglobulin. The mean +/- SEM was calculated from at least 3 independent experiments (except in F of 2 independent experiments).


Figure 6Image of a schistosome - infected mouse . The lymph nodes , from which the Th cells were isolated , are indicated . Th cells were isolated from the indicated lymph nodes and spleen . In the case of uninfected mice , the Th cells from different lymph nodes and the spleen were mixed . qRT - PCR was performed with specific Taqman primers to either schistosomal - miR - 10 or - Bantam . The presence of the miRNAs is presented as arbitrary units calculated as 2 ∆ ∆ CT of the miRNA divided by snRNA U6 . The mean + / - SEM was calculated from 4 independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Image of a schistosome-infected mouse. The lymph nodes, from which the Th cells were isolated, are indicated.Th cells were isolated from the indicated lymph nodes and spleen. In the case of uninfected mice, the Th cells from different lymph nodes and the spleen were mixed. qRT-PCR was performed with specific Taqman primers to either schistosomal-miR-10 or -Bantam. The presence of the miRNAs is presented as arbitrary units calculated as 2∆∆CT of the miRNA divided by snRNA U6. The mean +/- SEM was calculated from 4 independent experiments.