LDH release was measured after cold stress for 24 h . FCCP ( 200 µM ) , antimycin A ( 50 µM ) , mitoQ and decylQ ( 500 nM ) were used as pretreatments for 30 min . Data are presented as mean ± SEM ; n = 4 ( G ) , biological replicates , * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , by one - way ANOVA followed by Dunnett ' s or Bonferroni ' s multiple comparison test . whose raw value were normalized by the C11 - BODIPY 581 / 591 without cold stress of non - treatment , are presented as mean ± SEM ; n = 5 , biological replicates . * * p < 0 . 01 , * p < 0 . 05 , by one - way ANOVA followed by Dunnett ' s multiple comparison test .

B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LDH release was measured after cold stress for 24 h. FCCP (200 µM), antimycin A (50 µM), mitoQ and decylQ (500 nM) were used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 4 (G) , biological replicates, ****p < 0.0001, ***p < 0.001, **p < 0.01, by one-way ANOVA followed by Dunnett's or Bonferroni's multiple comparison test. whose raw value were normalized by the C11-BODIPY 581/591 without cold stress of non-treatment, are presented as mean ± SEM; n = 5, biological replicates. **p < 0.01, *p < 0.05, by one-way ANOVA followed by Dunnett's multiple comparison test.


LDH release was measured after cold stress for 24 h . BAPTA - AM ( 10 µM ) was used as pretreatments for 30 min . Data are presented as mean ± SEM ; n = 3 , biological replicates , * * * * p < 0 . 0001 , by unpaired T - test .

B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LDH release was measured after cold stress for 24 h. BAPTA-AM (10 µM) was used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 3, biological replicates, ****p < 0.0001, by unpaired T-test.


LDH release was measured after cold stress for 16 h . Data are presented as mean ± SEM ; n = 5 , biological replicates , * p < 0 . 05 , by unpaired T - test .

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

LDH release was measured after cold stress for 16 h. Data are presented as mean ± SEM; n = 5, biological replicates, *p < 0.05, by unpaired T-test.


B : Silver nitrate staining of proteins pulled - down using the control peptide ( left ) or the FFAT peptide ( right ) , after SDS - PAGE . The two major differential bands are highlighted by

O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

B: Silver nitrate staining of proteins pulled-down using the control peptide (left) or the FFAT peptide (right), after SDS-PAGE. The two major differential bands are highlighted by arrows


C : Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction . Score : protein score based on the sum of the ion scores of all peptides identified ; Cov . ; percentage of the protein sequence covered by identified peptides ; Pep . : number of unique peptide sequences identified

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

C: Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction. Score: protein score based on the sum of the ion scores of all peptides identified; Cov.; percentage of the protein sequence covered by identified peptides; Pep.: number of unique peptide sequences identified


E : Western blot analysis of proteins pulled - down using the control ( Ctrl ) peptide ( left ) or the FFAT ( FFAT ) peptide ( right ) . The Input , FT ( flow - through ) , and Elution fractions correspond to HeLa cell total protein extract , unbound proteins and bound proteins , respectively . Representative illustration of at least two independent experiments

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

E: Western blot analysis of proteins pulled-down using the control (Ctrl) peptide (left) or the FFAT (FFAT) peptide (right). The Input, FT (flow-through), and Elution fractions correspond to HeLa cell total protein extract, unbound proteins and bound proteins, respectively. Representative illustration of at least two independent experiments


A : List of the 5 top scored proteins interacting with MOSPD2 and known to contain a FFAT motif ( see Appendix Table S1 for the full list ) . Proteins co - precipitated with GFP - MOSPD2 and GFP - MOSPD2 RD / LD proteins were identified by mass spectrometry ; proteins were filtered for their ability to bind MOSPD2 and not the RD / LD mutant . The sub - cellular localization of the proteins is indicated [ 53 , 65 - 69 ]

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

A: List of the 5 top scored proteins interacting with MOSPD2 and known to contain a FFAT motif (see Appendix Table S1 for the full list). Proteins co-precipitated with GFP-MOSPD2 and GFP-MOSPD2 RD/LD proteins were identified by mass spectrometry; proteins were filtered for their ability to bind MOSPD2 and not the RD/LD mutant. The sub-cellular localization of the proteins is indicated [53,65-69]


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagged STARD3 ( B ) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E ) anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F and anti - GFP ( B - F )

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

B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagged STARD3 (B) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E) anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge STARD3NL ( C ) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed usin anti - ORP1 ( C ) , and anti - GFP ( B - F )

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

B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge STARD3NL (C) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed usin anti-ORP1 (C), and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge ORP1L ( D Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E ) , anti - ORP1 ( D ) ) , anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F ) , and anti - GFP ( B - F )

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

B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge ORP1L (D Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E), anti-ORP1 (D) ), anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F ), and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge STARD11 ( E Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies Immunoprecipitated material was analyzed using anti - Flag ( B , D - F and anti - GFP ( B - F )

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

B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge STARD11 (E Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E anti-GFP (B-F), and anti-actin (B-F) antibodies Immunoprecipitated material was analyzed using anti-Flag (B, D-F and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) an HA - tagged PTPIP51 ( F ) ( WT and FFAT - deficient ) . Approximatively 5 µg of total protein extracts were analyzed by Western blot usin anti - HA ( F ) anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F ) , and anti - GFP ( B - F )

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

B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) an HA-tagged PTPIP51 (F) (WT and FFAT-deficient). Approximatively 5 µg of total protein extracts were analyzed by Western blot usin anti-HA (F) anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F ), and anti-GFP (B-F) antibodies


G : Immunoprecipitation ( anti - Flag ) experiment between Flag - tagged STARD3NL ( WT and FFAT - deficient ) and endogenous MOSPD2 . Proteins extracts and immunoprecipitated material were analyzed by Western blot using anti - MOSPD2 , anti - STARD3NL and anti - actin antibodies

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

G: Immunoprecipitation (anti-Flag) experiment between Flag-tagged STARD3NL (WT and FFAT-deficient) and endogenous MOSPD2. Proteins extracts and immunoprecipitated material were analyzed by Western blot using anti-MOSPD2, anti-STARD3NL and anti-actin antibodies


A : GFP - tagged MOSPD2 ( green ) expressing cells were labelled with an anti - MOSPD2 antibody ( magenta ) . The superposition of the green and magenta signals indicates that the anti - MOSPD2 antibody recognized efficiently MOSPD2 by immunofluorescence Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10

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

A: GFP-tagged MOSPD2 (green) expressing cells were labelled with an anti-MOSPD2 antibody (magenta). The superposition of the green and magenta signals indicates that the anti-MOSPD2 antibody recognized efficiently MOSPD2 by immunofluorescence Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm


B : Western blot analysis of MOSPD2 and VAP proteins in control HeLa cells ( WT ) and in HeLa cells expressing a control shRNA ( shCtrl ) and two individual shRNAs targeting MOSPD2 ( shMOSPD2 - α or shMOSPD2 - β ) . Quantification of MOSPD2 protein level is shown below . Means and error bars ( SD ) are shown . n : three independent experiments

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

B: Western blot analysis of MOSPD2 and VAP proteins in control HeLa cells (WT) and in HeLa cells expressing a control shRNA (shCtrl) and two individual shRNAs targeting MOSPD2 (shMOSPD2-α or shMOSPD2-β). Quantification of MOSPD2 protein level is shown below. Means and error bars (SD) are shown. n: three independent experiments


C : Endogenous MOSPD2 ( green ) was labelled in control HeLa cells ( WT ) , in cells expressing a control shRNA ( shCtrl ) , and in cells expressing two individual shRNAs targeting MOSPD2 ( shMOSPD2 - α or shMOSPD2 - β ) . Scale bar : 10 µm

O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

C: Endogenous MOSPD2 (green) was labelled in control HeLa cells (WT), in cells expressing a control shRNA (shCtrl), and in cells expressing two individual shRNAs targeting MOSPD2 (shMOSPD2-α or shMOSPD2-β). Scale bar: 10 µm


D : Endogenous MOSPD2 ( green ) staining in HeLa cells expressing the ER marker GFP - ER ( magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10

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

D: Endogenous MOSPD2 (green) staining in HeLa cells expressing the ER marker GFP-ER (magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm


E - F : Endogenous MOSPD2 ( green ) staining in HeLa cells expressing Flag - STARD3NL ( E ( anti - Flag ; magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay . Black rectangles indicate the positions of late endosomes ( E ) . Scale bars : 10 µm

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

E-F: Endogenous MOSPD2 (green) staining in HeLa cells expressing Flag-STARD3NL (E (anti-Flag; magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay. Black rectangles indicate the positions of late endosomes (E). Scale bars: 10 µm


E - F : Endogenous MOSPD2 ( green ) staining in HeLa cells expressin Flag - STARD3NL ∆ FFAT ( F ) ( anti - Flag ; magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10

O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

E-F: Endogenous MOSPD2 (green) staining in HeLa cells expressin Flag-STARD3NL ∆FFAT (F) (anti-Flag; magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm


G : Pearson correlation coefficients between endogenous MOSPD2 and Flag - STARD3NL ( WT or ΔFFAT ) staining are shown . Each dot represents a single cell ( number of cells : MOSPD2 - Flag - STARD3NL : 27 ; MOSPD2 - Flag - STARD3NL ΔFFAT : 26 ; from three independent experiments ) . Means and error bars ( SD ) are shown . Mann - Whitney test ( two - tailed P value ; * * * * : P < 0 , 0001 )

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

G: Pearson correlation coefficients between endogenous MOSPD2 and Flag-STARD3NL (WT or ΔFFAT) staining are shown. Each dot represents a single cell (number of cells: MOSPD2-Flag-STARD3NL: 27; MOSPD2-Flag-STARD3NL ΔFFAT: 26; from three independent experiments). Means and error bars (SD) are shown. Mann-Whitney test (two-tailed P value; ****: P< 0,0001)


A : Western blot analysis of MOSPD2 , VAP - A and VAP - B proteins in control HeLa cells ( WT ) and in HeLa cells transfected with a control siRNA ( siCtrl ) , and with siRNA targeting MOSPD2 ( siMOSPD2 ) , VAP - A and VAP - B ( siVAP - A / VAP - B ) , and MOSPD2 , VAP - A and VAP - B ( siMOSPD2 / VAP - A / VAP - B )

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O B-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

A: Western blot analysis of MOSPD2, VAP-A and VAP-B proteins in control HeLa cells (WT) and in HeLa cells transfected with a control siRNA (siCtrl), and with siRNA targeting MOSPD2 (siMOSPD2), VAP-A and VAP-B (siVAP-A/VAP-B), and MOSPD2, VAP-A and VAP-B (siMOSPD2/VAP-A/VAP-B)


B - F : TEM images of control HeLa cell ( B : HeLa - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

O O O O B-EXP_ASSAY O O O B-CELL_LINE O O O O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O

B-F: TEM images of control HeLa cell (B: HeLa -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images of control HeLa cell C : HeLa / siCtrl - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

O O O O B-EXP_ASSAY O O O B-CELL_LINE O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O

B-F: TEM images of control HeLa cell C: HeLa/siCtrl -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images o MOSPD2 ( D : siMOSPD2 - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

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

B-F: TEM images o MOSPD2 (D: siMOSPD2 -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images o VAP - A and VAP - B ( E : siVAP - A / VAP - B - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

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

B-F: TEM images o VAP-A and VAP-B (E: siVAP-A/VAP-B -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images o MOSPD2 , VAP - A and VAP - B ( F : siVAP - A / VAP - B / MOSPD2 ) - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

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

B-F: TEM images o MOSPD2, VAP-A and VAP-B (F: siVAP-A/VAP-B/MOSPD2) -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


G - I : Quantification by stereology of ER - mitochondria ( G contacts . The percentages of mitochondria perimeter in contact with the ER ( G are shown as means and error bars ( SEM ) . ( G ) 200 ( HeLa ) , 208 ( siCtrl ) , 199 ( siMOSPD2 ) , 202 ( siVAP - A / VAP - B ) , 199 ( siMOSPD2 / VAP - A / VAP - B ) mitochondria from 8 , 10 , 8 , 12 and 12 cells , respectively , were

O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O

G-I: Quantification by stereology of ER-mitochondria (G contacts. The percentages of mitochondria perimeter in contact with the ER (G are shown as means and error bars (SEM). (G) 200 (HeLa), 208 (siCtrl), 199 (siMOSPD2), 202 (siVAP-A/VAP-B), 199 (siMOSPD2/VAP-A/VAP-B) mitochondria from 8, 10, 8, 12 and 12 cells, respectively, were analyzed


G - I : Quantification by stereology o ER - endosome ( H contacts The percentages o ) , endosome perimeter in contact with the ER ( H are shown as means and error bars ( SEM ) ( H , I ) 202 ( HeLa ) , 189 ( siCtrl ) , 148 ( siMOSPD2 ) , 141 ( siVAP - A / VAP - B ) , 221 ( siMOSPD2 / VAP - A / VAP - B ) endosomes from 9 , 19 , 10 , 14 and 12 cells , respectively , were analyzed . Kruskal - Wallis with Dunn " s multiple comparison test ( * : P - values < 0 . 05 ; * * * : P < 0 , 001 ; * * * * : P < 0 . 0001 )

O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-I: Quantification by stereology o ER-endosome (H contacts The percentages o ), endosome perimeter in contact with the ER (H are shown as means and error bars (SEM) (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001)


G - I : Quantification by stereology o endosome - endosome ( I ) contacts . The percentages o endosome perimeter in contact with an endosome ( I ) are shown as means and error bars ( SEM ) ( H , I ) 202 ( HeLa ) , 189 ( siCtrl ) , 148 ( siMOSPD2 ) , 141 ( siVAP - A / VAP - B ) , 221 ( siMOSPD2 / VAP - A / VAP - B ) endosomes from 9 , 19 , 10 , 14 and 12 cells , respectively , were analyzed . Kruskal - Wallis with Dunn " s multiple comparison test ( * : P - values < 0 . 05 ; * * * : P < 0 , 001 ; * * * * : P < 0 . 0001 )

O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-I: Quantification by stereology o endosome-endosome (I) contacts. The percentages o endosome perimeter in contact with an endosome (I) are shown as means and error bars (SEM) (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001)


All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . A ) Immunohistochemistry analysis of small intestinal sections , using the indicated antibodies .

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

All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.A) Immunohistochemistry analysis of small intestinal sections, using the indicated antibodies.


All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . B ) Western blot analysis with protein extracts obtained from ex vivo - purified intestinal crypts and probed with the indicated antibodies .

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

All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.B) Western blot analysis with protein extracts obtained from ex vivo-purified intestinal crypts and probed with the indicated antibodies.


All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . C ) Haematoxylin and eosin staining of sections prepared from different intestinal tracts .

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

All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.C) Haematoxylin and eosin staining of sections prepared from different intestinal tracts.


All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . D ) Immunohistochemistry analysis of small intestinal sections , using specific antibodies for the proliferation marker KI67 .

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

All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.D) Immunohistochemistry analysis of small intestinal sections, using specific antibodies for the proliferation marker KI67.


All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . E ) Expression analysis by qRT - PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts . Graphs show mean ± SD for three replicates .

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

All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.E) Expression analysis by qRT-PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts. Graphs show mean ± SD for three replicates.


A ) PAS and ( B ) Alcian blue staining of sections prepared from the different intestinal tracts isolated from AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days .

O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O

A) PAS and (B) Alcian blue staining of sections prepared from the different intestinal tracts isolated from AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.


C ) Quantification ( mean ± SD ) of Goblet cells in crypts and villi from the same mice sown in A and B . More than 100 crypts and villi were scored for each condition after 15 days from the first β - naphthoflavone administration .

O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-TISSUE O O O B-ORGANISM O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O

C) Quantification (mean ± SD) of Goblet cells in crypts and villi from the same mice sown in A and B. More than 100 crypts and villi were scored for each condition after 15 days from the first β-naphthoflavone administration.


D ) Immunohistochemistry analyses in AhCre Eed + / + and AhCre Eedfl / fl mice injected with β - naphthoflavone and sacrificed after 15 days using a lysozyme - specific antibody ( upper panels : LYZ , a Paneth cell - specific marker ) . Histochemical assay probing for alkaline phosphatase activity is presented in the middle panels ( ALPI , an enterocyte - specific marker ) . Combined Ki67 immunohistochemistry and histochemical Alcian blue staining is presented in the lower panels ( Alcian / KI67 ) .

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

D) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice injected with β-naphthoflavone and sacrificed after 15 days using a lysozyme-specific antibody (upper panels: LYZ, a Paneth cell-specific marker). Histochemical assay probing for alkaline phosphatase activity is presented in the middle panels (ALPI, an enterocyte-specific marker). Combined Ki67 immunohistochemistry and histochemical Alcian blue staining is presented in the lower panels (Alcian/KI67).


E ) GFP expression from an Lgr5 - eGFPtransgene identifying LGR5 + ISCs in near - native agarose - embedded sections from Eed + / + and Eed - / - mice at 30 days from the first β - naphthoflavone administration . Cell nuclei were counterstained with DAPI .

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

E) GFP expression from an Lgr5-eGFPtransgene identifying LGR5+ ISCs in near-native agarose-embedded sections from Eed+/+ and Eed-/- mice at 30 days from the first β-naphthoflavone administration. Cell nuclei were counterstained with DAPI.


A ) Radiation - induced intestinal regeneration : AhCre Eed + / + and AhCre Eedfl / fl mice were injected with β - naphthoflavone , irradiated with 10 Gy 15 days later , and sacrificed 4 and 8 days after irradiation . Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin ( n = 6 ) .

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

A) Radiation-induced intestinal regeneration: AhCre Eed+/+ and AhCre Eedfl/fl mice were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 4 and 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (n=6).


B ) Immunohistochemistry analyses in AhCre Eed + / + and AhCre Eedfl / fl mice treated as in A using H3K27me3 and KI67 specific antibodies at 8 days post - irradiation . C ) Quantification ( mean ± SD ) of KI67 positive cells per crypt ( B ) . More than 100 crypts were scored for each condition .

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

B) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice treated as in A using H3K27me3 and KI67 specific antibodies at 8 days post-irradiation.C) Quantification (mean ± SD) of KI67 positive cells per crypt (B). More than 100 crypts were scored for each condition.


D ) In vitro organoids formation using crypts isolated from AhCre Eed + / + and AhCre Eed - / - mice 15 days after the first β - naphthoflavone injection . Organoids pictures were taken 5 days later at low and higher magnification ( insets ) .

O O O O O O O B-TISSUE O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

D) In vitro organoids formation using crypts isolated from AhCre Eed+/+ and AhCre Eed-/- mice 15 days after the first β-naphthoflavone injection. Organoids pictures were taken 5 days later at low and higher magnification (insets).


A ) Volcano plot showing the expression changes with a minimal fold change of 3 ( FC = 3 ) at genome - wide levels in AhCre Eed - / - crypts relative to AhCre Eed + / + crypts 15 days after β - naphthoflavone administration .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-TISSUE O O B-GENEPROD I-GENEPROD O O O B-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O

A) Volcano plot showing the expression changes with a minimal fold change of 3 (FC=3) at genome-wide levels in AhCre Eed-/- crypts relative to AhCre Eed+/+ crypts 15 days after β-naphthoflavone administration.


B ) Bar plot showing the percentage of H3K27me3 - positive promoters among the differentially regulated genes in Eed - / - crypts . All gene promoters and 5 independent random sampling of 167 gene promoters extracted the mouse genome are presented as specificity controls .

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

B) Bar plot showing the percentage of H3K27me3-positive promoters among the differentially regulated genes in Eed-/- crypts. All gene promoters and 5 independent random sampling of 167 gene promoters extracted the mouse genome are presented as specificity controls.


C ) Intensity profiles for H3K27me3 and SUZ12 occupancy at the promoters of up - regulated genes with respect to down - regulated genes in Eed - / - crypts .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O

C) Intensity profiles for H3K27me3 and SUZ12 occupancy at the promoters of up-regulated genes with respect to down-regulated genes in Eed-/- crypts.


D ) Genomic snapshots of the Cdkn2a locus showing SUZ12 occupancy and H3K27me3 deposition at the promoter regions of both the p16 and the ARF isoforms .

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

D) Genomic snapshots of the Cdkn2a locus showing SUZ12 occupancy and H3K27me3 deposition at the promoter regions of both the p16 and the ARF isoforms.


E ) Genomic snapshots of the RNAseq profile of the Cdkn2a locus in AhCre Eed + / + and AhCre Eed - / - crypts 15 days after β - naphthoflavone administration .

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

E) Genomic snapshots of the RNAseq profile of the Cdkn2a locus in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.


F ) Expression analysis by qRT - PCR ( mean ± SD ) of both Cdkn2a isoforms ( p16Ink4A and p19Arf ) in AhCre Eed + / + and AhCre Eed - / - crypts 15 days after β - naphthoflavone administration .

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

F) Expression analysis by qRT-PCR (mean ± SD) of both Cdkn2a isoforms (p16Ink4A and p19Arf) in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.


G ) Gene set enrichment analysis ( GSEA ) using specific secretory and enterocytic transcriptional signatures with respect to the list of up - regulated genes in Eed - / - crypts .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O

G) Gene set enrichment analysis (GSEA) using specific secretory and enterocytic transcriptional signatures with respect to the list of up-regulated genes in Eed-/- crypts.


A ) Haematoxylin and eosin ( top panels ) and PAS staining ( bottom panels ) of sections prepared from different intestinal tracts in AhCre Cdkn2a - / - Eed + / + and AhCre Cdkn2a - / - Eedfl / fl mice 15 days after the first β - naphthoflavone administration ( n = 5 ) .

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

A) Haematoxylin and eosin (top panels) and PAS staining (bottom panels) of sections prepared from different intestinal tracts in AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration (n=5).


B ) Quantification ( mean ± SD ) of Goblet cells along the crypt - to - villus axis from the staining shown in A . More than 100 crypts and villi were scored for each condition .

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

B) Quantification (mean ± SD) of Goblet cells along the crypt-to-villus axis from the staining shown in A. More than 100 crypts and villi were scored for each condition.


C ) Immunhistochemistry analysis using anti - KI67 antibody of intestinal sections prepared from AhCre Cdkn2a - / - Eed + / + and AhCre Cdkn2a - / - Eedfl / fl mice 15 days after the first β - naphthoflavone administration .

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

C) Immunhistochemistry analysis using anti-KI67 antibody of intestinal sections prepared from AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration.


D ) Radiation - induced intestinal regeneration : AhCre Cdkn2a - / - Eed + / + and AhCre Cdkn2a - / - Eedfl / fl mice ( n = 6 ) were injected with β - naphthoflavone , irradiated with 10 Gy 15 days later , and sacrificed 8 days after irradiation . Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin ( first panels ) and Alcian Blue ( last panels ) . Sections were also stained for immunohistochemistry analysis ( second and third panels ) using H3K27me3 and Ki67 - specific antibodies .

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

D) Radiation-induced intestinal regeneration: AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice (n=6) were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (first panels) and Alcian Blue (last panels). Sections were also stained for immunohistochemistry analysis (second and third panels) using H3K27me3 and Ki67-specific antibodies.


A ) Expression analysis by qRT - PCR ( mean ± SD ) of the indicated intestinal epithelial cell markers regulated by the Wnt and Notch signaling pathways that are involved in stem cell homeostasis , regionalization , and Goblet cell differentiation ( n = 3 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O 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) Expression analysis by qRT-PCR (mean ± SD) of the indicated intestinal epithelial cell markers regulated by the Wnt and Notch signaling pathways that are involved in stem cell homeostasis, regionalization, and Goblet cell differentiation (n=3).


B ) Genomic snapshots of the indicated genomic loci for SUZ12 occupancy and H3K27me3 deposition in intestinal crypts of wild type mice ( n = 3 ) .

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B) Genomic snapshots of the indicated genomic loci for SUZ12 occupancy and H3K27me3 deposition in intestinal crypts of wild type mice (n=3).


C ) Expression analysis by qRT - PCR ( mean ± SD ) of the Goblet cell markers ( Atoh1 , Gfi1 , Spdef ) , early secretory precursor cells ( Dll1 ) , NOTCH targets ( Hes1 , Olfm4 ) and intestinal stem cells ( Lgr5 , Rnf43 ) in wild type intestinal crypts purified 38hrs after treatment with the NOTCH inhibitor DBZ . DMSO served as vehicle negative control ( n = 4 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O

C) Expression analysis by qRT-PCR (mean ± SD) of the Goblet cell markers (Atoh1, Gfi1, Spdef), early secretory precursor cells (Dll1), NOTCH targets (Hes1, Olfm4) and intestinal stem cells (Lgr5, Rnf43) in wild type intestinal crypts purified 38hrs after treatment with the NOTCH inhibitor DBZ. DMSO served as vehicle negative control (n=4).


D ) ChIP analysis of Atoh1 and Gfi1 loci using H3K27me3 specific antibody . The same crypts described in C were used . H3K27me3 levels were normalized on Histone H3 density ( percentage of H3 signal ) . Graphs show mean ± SD .

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

D) ChIP analysis of Atoh1 and Gfi1 loci using H3K27me3 specific antibody. The same crypts described in C were used. H3K27me3 levels were normalized on Histone H3 density (percentage of H3 signal). Graphs show mean ± SD.


( a ) PPARγ mRNA expression in sperm from control and MSUS mice . Control n = 15 , MSUS n = 13 , two - tailed Student ' s t - test , P = 0 . 029 , t = 2 . 30 , df = 26 .

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

(a) PPARγ mRNA expression in sperm from control and MSUS mice. Control n = 15, MSUS n = 13, two-tailed Student's t-test, P = 0.029, t = 2.30, df = 26.


( c ) Relative luciferase luminescence in transfected GC - 1 cells exposed to serum from control or MSUS mice . Numbers correspond to serum from individual animals applied to different cell culture wells . Control n = 17 , MSUS n = 22 , two - tailed Student ' s t - test , P = 0 . 042 , t = 2 . 1 , df = 37 .

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

(c) Relative luciferase luminescence in transfected GC-1 cells exposed to serum from control or MSUS mice. Numbers correspond to serum from individual animals applied to different cell culture wells. Control n = 17, MSUS n = 22, two-tailed Student's t-test, P = 0.042, t = 2.1, df = 37.


( b ) Adult weight in the offspring of tesaglitazar - injected males ( Tesa - inj , n = 16 ) compared to the offspring of vehicle - injected males ( Vehicle - inj , n = 23 ) . Two - tailed Student ' s t - test , P = 0 . 019 , t = 2 . 44 , df = 37 .

O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Adult weight in the offspring of tesaglitazar-injected males (Tesa-inj, n = 16) compared to the offspring of vehicle-injected males (Vehicle-inj, n = 23). Two-tailed Student's t-test, P = 0.019, t = 2.44, df = 37.


( c ) Glucose level in the offspring of Tesa - inj ( n = 14 ) and Vehicle - inj ( n = 21 ) males during a glucose tolerance test . Repeated measures ANOVA , treatment effect P = 0 . 0083 , F ( 1 , 33 ) = 7 . 877 , time effect P < 0 . 0001 , F ( 4 , 132 ) = 347 . 7 , interaction P = 0 . 0776 , F ( 4 , 132 ) = 2 . 155 Conc . ; concentration .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Glucose level in the offspring of Tesa-inj (n = 14) and Vehicle-inj (n = 21) males during a glucose tolerance test. Repeated measures ANOVA, treatment effect P = 0.0083, F (1, 33) = 7.877, time effect P < 0.0001, F (4, 132) = 347.7, interaction P = 0.0776, F (4, 132) = 2.155 Conc.; concentration.


( a Differentially expressed transposable elements in sperm from Tesa - inj and MSUS males . Data represent genes with P < 0 . 05 and similar fold change . Fold change in heat map represents log2 ( fold change ) respective to the corresponding control group . Total overlap is presented in Extended Data Fig . 11 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a Differentially expressed transposable elements in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11.


b ) mRNAs / lincRNAs in sperm from Tesa - inj and MSUS males . Data represent genes with P < 0 . 05 and similar fold change . Fold change in heat map represents log2 ( fold change ) respective to the corresponding control group . Total overlap is presented in Extended Data Fig . 11 .

O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

b) mRNAs/lincRNAs in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11.


( c , d ) ( c ) Volcano plot and ( d ) heat map of differentially expressed ( FDR < 0 . 05 ) mRNA / lincRNA in sperm from Tesa - inj males . Dashed black lines in ( c ) represent y = 1 . 3 , equivalent to FDR = 0 . 05 , and x = ± 0 . 03 , equivalent to FC = 1 . 23 and 0 . 81 . Black dots represent top candidates , which all have FC > 2 or FC < 0 . 5 , and FDR < 0 . 05 . Teal dots represent non - significant genes .

O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,d) (c) Volcano plot and (d) heat map of differentially expressed (FDR < 0.05) mRNA/lincRNA in sperm from Tesa-inj males. Dashed black lines in (c) represent y = 1.3, equivalent to FDR = 0.05, and x = ±0.03, equivalent to FC = 1.23 and 0.81. Black dots represent top candidates, which all have FC > 2 or FC < 0.5, and FDR < 0.05. Teal dots represent non-significant genes.


( b ) Weight in adult male MSUS offspring and the offspring of males injected with MSUS serum compared to respective control groups . MSUS offspring n = 22 , Control offspring n = 24 , one - tailed Student ' s t - test ( data reproduced ) P = 0 . 045 , t = 1 . 734 , df = 44 . MSUS serum - injected offspring n = 30 , Control serum - injected offspring n = 31 , two - tailed Mann - Whitney U = 334 . 5 , P = 0 . 059 .

O O O B-EXP_ASSAY O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Weight in adult male MSUS offspring and the offspring of males injected with MSUS serum compared to respective control groups. MSUS offspring n = 22, Control offspring n = 24, one-tailed Student's t-test (data reproduced) P = 0.045, t = 1.734, df = 44. MSUS serum-injected offspring n = 30, Control serum-injected offspring n = 31, two-tailed Mann-Whitney U = 334.5, P = 0.059.


( c , d ) Blood glucose levels in MSUS offspring and the offspring of MSUS serum - injected males following a 30 - min restraint challenge . MSUS offspring n = 13 , Control offspring n = 12 , repeat measures ANOVA , treatment effect P = 0 . 016 , F ( 1 , 23 ) = 6 . 704 , time effect P < 0 . 0001 , F ( 3 , 69 ) = 53 . 13 , interaction P = 0 . 027 , F ( 3 , 69 ) = 3 . 25 , at 15 - min adjusted P = 0 . 0015 , t = 3 . 693 , df = 92 . MSUS serum - injected offspring n = 17 , and Control serum - injected offspring n = 14 , repeat measures ANOVA , treatment effect P = 0 . 023 , F ( 1 , 14 ) = 6 . 493 , time effect P < 0 . 0001 , F ( 3 , 42 ) = 48 . 4 , interaction P = 0 . 29 , F ( 3 , 42 ) = 1 . 29 . Conc . , concentration .

O O O O O B-TISSUE B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,d) Blood glucose levels in MSUS offspring and the offspring of MSUS serum-injected males following a 30-min restraint challenge. MSUS offspring n = 13, Control offspring n = 12, repeat measures ANOVA, treatment effect P = 0.016, F (1, 23) = 6.704, time effect P < 0.0001, F (3, 69) = 53.13, interaction P = 0.027, F (3, 69) = 3.25, at 15-min adjusted P = 0.0015, t = 3.693, df = 92. MSUS serum-injected offspring n = 17, and Control serum-injected offspring n = 14, repeat measures ANOVA, treatment effect P = 0.023, F (1,14) = 6.493, time effect P < 0.0001, F (3, 42) = 48.4, interaction P = 0.29, F (3, 42) = 1.29. Conc., concentration.


B , C , D . WT , ∆ cpt1 ∆ ept1 ( B ) , Δcho2 ( C ) or Δopi3 ( D ) cells expressing GFP - Atg8 were grown to logarithmic phase in SD - URA , and shifted to SD - N for 4 h of starvation . Cells were harvested at indicated time points and subjected to western blotting ( left panel ) . Pgk1 was used as a loading control . Middle panel - Autophagic activity during starvation was quantified by calculating the ratio of free GFP to total GFP ( GFP - Atg8 + free GFP ) . Statistical analysis was done by student ' s t - test ( paired , two tailed ) ( ns - not significant , * * * * , p ≤ 0 . 0001 ) , error bars represent SEM of at least 3 independent experiments . Right panel - Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount , during SD and SD - N .

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

B, C, D. WT, ∆cpt1∆ept1 (B), Δcho2 (C) or Δopi3 (D) cells expressing GFP-Atg8 were grown to logarithmic phase in SD-URA, and shifted to SD-N for 4 h of starvation. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was used as a loading control. Middle panel- Autophagic activity during starvation was quantified by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by student's t-test (paired, two tailed) (ns- not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount, during SD and SD-N.


E . WT , Δopi3 , and Δcho2 as well as Δatg1 ( negative autophagy control ) cells expressing chromosomally - tagged Fba1 - GFP were grown to log phase in SD , and starved in SD - N for 4 hours . Cells were harvested at indicated starvation time points and subjected to western blotting ( left panel ) . Pgk1 was monitored as a loading control . Right panel - Autophagic activity during starvation was quantified , by calculating the ratio of free GFP to total GFP ( Fba1 - GFP + free GFP ) .

O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O

E. WT, Δopi3, and Δcho2 as well as Δatg1 (negative autophagy control) cells expressing chromosomally-tagged Fba1-GFP were grown to log phase in SD, and starved in SD-N for 4 hours. Cells were harvested at indicated starvation time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Right panel- Autophagic activity during starvation was quantified, by calculating the ratio of free GFP to total GFP (Fba1-GFP + free GFP).


F . WT , Δopi3 , Δcho2 and Δatg1 cells on the background of pho8Δ60 pho13Δ strain , were grown to log phase in SD , and starved in SD - N for 4 hours . Pho8 activity was measured by the alkaline phosphatase assay .

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F. WT, Δopi3, Δcho2 and Δatg1 cells on the background of pho8Δ60 pho13Δ strain, were grown to log phase in SD, and starved in SD-N for 4 hours. Pho8 activity was measured by the alkaline phosphatase assay.


A . Representative images of GFP - Atg8 ( green ) and vacuole ( red , stained with FM4 - 64 ) . WT , Δopi3 and Δcho2 cells expressing GFP - Atg8 were grown to log phase in SD - URA medium and shifted to SD - N for 3 h . Cells were observed by confocal microscopy before ( SD ) and during nitrogen starvation ( SD - N ) ( left panel ) . Scale bar 1 µm . Right panel - Quantification of of cells with or without GFP inside vacuoles . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s , compared to WT ( * * * * , p ≤ 0 . 0001 ) ( ns - not significant ) , error bars represent SEM of at least 3 independent experiments . Number of cells analyzed for each strain and condition : SD ( WT ( n = 446 ) , Δcho2 ( n = 272 ) , Δopi3 ( n = 272 ) ) , SD - N ( WT ( n = 398 ) , Δcho2 ( n = 224 ) , Δopi3 ( n = 213 ) ) .

O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT, Δopi3 and Δcho2 cells expressing GFP-Atg8 were grown to log phase in SD-URA medium and shifted to SD-N for 3 h. Cells were observed by confocal microscopy before (SD) and during nitrogen starvation (SD-N) (left panel). Scale bar 1 µm. Right panel- Quantification of of cells with or without GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001) (ns- not significant), error bars represent SEM of at least 3 independent experiments. Number of cells analyzed for each strain and condition: SD (WT (n=446), Δcho2 (n=272), Δopi3 (n=272)), SD-N (WT (n=398), Δcho2 (n=224), Δopi3 (n=213)).


A , B . WT and Δopi3 cells expressing GFP - Atg8 were grown to log phase in SD - URA , and shifted to SD - N for 4 h , choline ( 1 mM ) was not supplemented ( - choline ) or supplemented to SD and SD - N ( + choline SD , SD - N ) or only to SD - N ( + choline , SD - N ) as indicated . Cells were harvested at indicated time points and subjected to western blotting ( left panel ) . Pgk1 was monitored as a loading control . Middle panel - Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP ( GFP - Atg8 + free GFP ) . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s , compared to WT ( ns - not significant , * * * * , p ≤ 0 . 0001 ) , error bars represent SEM of at least 3 independent experiments . Right panel - Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth ( SD ) or during starvation ( SD - N ) , with choline supplemented as indicated . Statistical analysis was done by Sidak ' s multiple comparisons test compared to WT ( ns - not significant , * , p ≤ 0 . 05 , * * , p ≤ 0 . 005 , * * * * , p ≤ 0 . 0001 ) , error bars represent SEM of at least 3 independent experiments .

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A, B. WT and Δopi3 cells expressing GFP-Atg8 were grown to log phase in SD-URA, and shifted to SD-N for 4 h, choline (1 mM) was not supplemented (-choline) or supplemented to SD and SD-N (+choline SD, SD-N) or only to SD-N (+choline, SD-N) as indicated. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Middle panel- Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (ns-not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth (SD) or during starvation (SD-N), with choline supplemented as indicated. Statistical analysis was done by Sidak's multiple comparisons test compared to WT (ns-not significant, *, p≤0.05, **, p≤0.005, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments.


C . Representative images of GFP - Atg8 ( green ) and vacuole ( red , stained with FM4 - 64 ) . WT and Δopi3 cells expressing GFP - Atg8 were grown in SD - URA and shifted to SD - N . Choline ( 1mM ) was either excluded ( - choline ) , added to growth and starvation medium ( + choline SD , SD - N ) or added only to starvation medium ( + choline , SD - N ) . Cells were observed using confocal microscopy during starvation ( left panel ) . Scale bar 1 µm . Right panel - Quantification of cells with GFP inside vacuoles . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s , compared to WT ( * * * * , p ≤ 0 . 0001 , ns - not significant ) , error bars represent SEM of 3 independent experiments . Number of cells analyzed for each strain condition ; - Choline : WT ( n = 487 ) , Δopi3 ( n = 316 ) , Choline SD , SD - N : WT ( n = 251 ) , Δopi3 ( n = 361 ) , Choline SD - N : WT ( n = 320 ) , Δopi3 ( n = 267 ) . D . PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP - Atg8 , with or without choline ( 1 mM ) supplementation to SD - N . Samples were taken after 4 h in SD - N medium .

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

C. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT and Δopi3 cells expressing GFP-Atg8 were grown in SD-URA and shifted to SD-N. Choline (1mM) was either excluded (-choline), added to growth and starvation medium (+choline SD, SD-N) or added only to starvation medium (+choline, SD-N). Cells were observed using confocal microscopy during starvation (left panel). Scale bar 1 µm. Right panel- Quantification of cells with GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001, ns- not significant), error bars represent SEM of 3 independent experiments. Number of cells analyzed for each strain condition; -Choline: WT (n=487), Δopi3 (n=316), Choline SD, SD-N: WT (n=251), Δopi3 (n=361), Choline SD-N: WT (n=320), Δopi3 (n=267). D. PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP-Atg8, with or without choline (1 mM) supplementation to SD-N. Samples were taken after 4 h in SD-N medium.


A . Representative Images of Atg5 - mNG with sfTq2 - Atg8 during SD - N . Δatg3 and Δatg3Δopi3 cells were grown to log phase in SD medium , shifted to SD - N for 4 h , and observed by Widefield microscopy ( left panel ) , scale bar 1 µm . Right panel - quantification of percentage of cells with Atg5 puncta during different time points . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s ns - not significant ) , error bars represent SEM of 2 independent experiments . Number of cells analyzed for each strain and time point ( 0 hour : Δatg3 ( n = 296 ) , Δatg3Δopi3 ( n = 111 ) , 1 hour : Δatg3 ( n = 325 ) , Δatg3Δopi3 ( n = 110 ) , 2 hours : Δatg3 ( n = 338 ) , Δatg3Δopi3 ( n = 135 ) , 4 hours : Δatg3 ( n = 309 ) , Δatg3Δopi3 ( n = 167 ) .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative Images of Atg5-mNG with sfTq2-Atg8 during SD-N. Δatg3 and Δatg3Δopi3 cells were grown to log phase in SD medium, shifted to SD-N for 4 h, and observed by Widefield microscopy (left panel), scale bar 1 µm. Right panel- quantification of percentage of cells with Atg5 puncta during different time points. Statistical analysis was done by Anova multiple comparisons test- Sidak's ns- not significant), error bars represent SEM of 2 independent experiments. Number of cells analyzed for each strain and time point (0 hour: Δatg3 (n=296), Δatg3Δopi3 (n=111), 1 hour: Δatg3 (n=325), Δatg3Δopi3 (n=110), 2 hours: Δatg3 (n=338), Δatg3Δopi3 (n=135), 4 hours: Δatg3 (n=309), Δatg3Δopi3 (n=167).


C . Representative images of WT and Δopi3 cells expressing GFP - Atg8 . Cells were grown to log phase in SD - URA medium , shifted to SD - N for 3 h and observed by Airyscan microscopy ( left panel ) . White dashes indicate cell boundaries , yellow dashes indicate phagophores . For each cell - Magnification of yellow dashed area ( top right ) , schematic representation ( bottom right ) . Scale bar 1 µm . Right panel - quantification of the percentage of cells with cup shaped GFP - Atg8 structures imaged by Airyscan microscopy . Statistical analysis was done by student ' s t - test ( unpaired ) ( * * p ≤ 0 . 05 ) , error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain , WT ( n = 45 ) , Δopi3 ( n = 110 ) .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of WT and Δopi3 cells expressing GFP-Atg8. Cells were grown to log phase in SD-URA medium, shifted to SD-N for 3 h and observed by Airyscan microscopy (left panel). White dashes indicate cell boundaries, yellow dashes indicate phagophores. For each cell - Magnification of yellow dashed area (top right), schematic representation (bottom right). Scale bar 1 µm. Right panel- quantification of the percentage of cells with cup shaped GFP-Atg8 structures imaged by Airyscan microscopy. Statistical analysis was done by student's t-test (unpaired) (** p≤0.05), error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain, WT (n=45), Δopi3 (n=110).


D . Representative CLEM images of WT and Δopi3 cells expressing GFP - Atg8 , grown to log phase in SD - URA medium and shifted to SD - N for 3h . Cells were harvested , deep frozen and processed for CLEM ( left panel ) , V - vacuole . For Δopi3 cell - Magnification of yellow dashed area of GFP - Atg8 positive phagophore ( top right ) , TEM image only ( middle right ) , schematic representation of a phagophore ( bottom right ) . Scale bar 1 µm . Right panel - quantification of the percentage of cells with cup shaped GFP - Atg8 positive cup shaped membrane structures imaged by CLEM . Statistical analysis was done by student ' s t - test ( unpaired ) ( * * * p ≤ 0 . 005 ) , error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain , WT ( n = 20 ) , Δopi3 ( n = 21 ) .

O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Representative CLEM images of WT and Δopi3 cells expressing GFP-Atg8, grown to log phase in SD-URA medium and shifted to SD-N for 3h. Cells were harvested, deep frozen and processed for CLEM (left panel), V-vacuole. For Δopi3 cell - Magnification of yellow dashed area of GFP-Atg8 positive phagophore (top right), TEM image only (middle right), schematic representation of a phagophore (bottom right). Scale bar 1 µm. Right panel- quantification of the percentage of cells with cup shaped GFP-Atg8 positive cup shaped membrane structures imaged by CLEM. Statistical analysis was done by student's t-test (unpaired) (*** p≤0.005), error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain, WT (n=20), Δopi3 (n=21).


A . Representative images of Δypt7 , Δopi3Δypt7 expressing GFP - Atg8 ( z - stack projection ) . Cells were grown to log phase in SD - URA medium and starved in SD - N , images were taken during starvation in indicated time points by widefield microscopy ( left panel ) . Scale bar 1 µm . Right panel - quantification of GFP - Atg8 puncta per cell in Δypt7 , Δopi3Δypt7 at indicated time points . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s ( * * * * , p ≤ 0 . 0001 , ns - not significant ) , compared to WT . Error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain and time point , 0 hours : Δypt7 ( n = 322 ) , Δopi3Δypt7 ( n = 194 ) , 1 . 5 hours : Δypt7 ( n = 220 ) , Δopi3Δypt7 ( n = 155 ) , 3 . 5 hours Δypt7 ( n = 216 ) , Δopi3Δypt7 ( n = 307 ) . B . Quantification of cup - shaped phagophores in Δypt7 and Δopi3Δypt7 cells expressing GFP - Atg8 during starvation ( 1 - 3h ) . Statistical analysis was done by students t - test ( * * * , p ≤ 0 . 001 ) , error bars represent SEM from at least 3 independent experiments . Number of cells analyzed for each strain , Δypt7 ( n = 347 ) , Δopi3Δypt7 ( n = 474 ) .

O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of Δypt7, Δopi3Δypt7 expressing GFP-Atg8 (z-stack projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, images were taken during starvation in indicated time points by widefield microscopy (left panel). Scale bar 1 µm. Right panel- quantification of GFP-Atg8 puncta per cell in Δypt7, Δopi3Δypt7 at indicated time points. Statistical analysis was done by Anova multiple comparisons test- Sidak's (****, p≤0.0001, ns- not significant), compared to WT. Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and time point, 0 hours: Δypt7 (n=322), Δopi3Δypt7 (n=194), 1.5 hours: Δypt7 (n=220), Δopi3Δypt7 (n=155), 3.5 hours Δypt7 (n=216), Δopi3Δypt7 (n=307). B. Quantification of cup-shaped phagophores in Δypt7 and Δopi3Δypt7 cells expressing GFP-Atg8 during starvation (1-3h). Statistical analysis was done by students t-test (***, p≤0.001), error bars represent SEM from at least 3 independent experiments. Number of cells analyzed for each strain, Δypt7 (n= 347), Δopi3Δypt7 (n= 474).


C . Representative images of mNG - tagged Atg8 colocalized with mScarletI - PXVam7 . WT , Δopi3 , Δatg1 and Δopi3Δatg1 cells on the background of Δymr1 , expressing mScarletI - PXVam7 under the CUP1 promoter from the knocked - out VPS38 locus , were grown to log phase in SD medium in the presence of 10 µM copper sulfate , and shifted to SD - N in the presence of 10 µM copper sulfate . Images were taken during SD - N ( 1 - 3 h ) by widefield microscopy . D . Quantification of autophagic structure diameters in WT and Δopi3 cells on the background of Δymr1Δvps38 ( C ) by ImageJ . Statistical analysis was done by student ' s t - test , unpaired , two sided ( ns - not significant ) , error bars represent SEM of at least three independent experiments . Number of cells analyzed for each strain , WT ( n = 51 ) , Δopi3 ( n = 28 ) .

O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of mNG-tagged Atg8 colocalized with mScarletI-PXVam7. WT, Δopi3, Δatg1 and Δopi3Δatg1 cells on the background of Δymr1, expressing mScarletI-PXVam7 under the CUP1 promoter from the knocked-out VPS38 locus, were grown to log phase in SD medium in the presence of 10 µM copper sulfate, and shifted to SD-N in the presence of 10 µM copper sulfate. Images were taken during SD-N (1-3 h) by widefield microscopy. D. Quantification of autophagic structure diameters in WT and Δopi3 cells on the background of Δymr1Δvps38 (C) by ImageJ. Statistical analysis was done by student's t-test, unpaired, two sided (ns- not significant), error bars represent SEM of at least three independent experiments. Number of cells analyzed for each strain, WT (n=51), Δopi3 (n=28).


E . Representative images of Δypt7 , Δopi3Δypt7 cells expressing GFP - Atg8 ( projection ) . Cells were grown to log phase in SD - URA medium and starved in SD - N , with supplementation of choline ( 1 mM ) to SD and SD - N as indicated . Images were taken during growth and starvation by confocal microscopy ( left panel ) . Scale bar 1 µm . Right panel - quantification of cell with more than two GFP - Atg8 puncta , in Δypt7 and Δopi3Δypt7 at growth ( SD ) or starvation ( SD - N ) in the presence ( + choline : SD , SD - N ) or absence ( - choline ) of choline ( 1 mM ) . Incidence were normalized to Δypt7 ( SD - N ) control . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s ( * * * , p ≤ 0 . 001 ) . Error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain and condition , SD : Δypt7 ( n = 209 ) , Δopi3Δypt7 ( n = 111 ) , SD - N : Δypt7 ( n = 118 ) , Δopi3Δypt7 ( n = 103 ) , SD + Choline : Δypt7 ( n = 107 ) , Δopi3Δypt7 ( n = 120 ) , Δypt7 ( n = 84 ) , Δopi3Δypt7 ( n = 74 ) .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative images of Δypt7, Δopi3Δypt7 cells expressing GFP-Atg8 (projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, with supplementation of choline (1 mM) to SD and SD-N as indicated. Images were taken during growth and starvation by confocal microscopy (left panel). Scale bar 1 µm. Right panel- quantification of cell with more than two GFP-Atg8 puncta, in Δypt7 and Δopi3Δypt7 at growth (SD) or starvation (SD-N) in the presence (+choline: SD, SD-N) or absence (-choline) of choline (1 mM). Incidence were normalized to Δypt7 (SD-N) control. Statistical analysis was done by Anova multiple comparisons test- Sidak's (***, p≤0.001). Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and condition, SD: Δypt7 (n=209), Δopi3Δypt7 (n=111), SD-N: Δypt7 (n=118), Δopi3Δypt7 (n=103), SD+Choline: Δypt7 (n=107), Δopi3Δypt7 (n=120), Δypt7 (n=84), Δopi3Δypt7 (n=74).


Overexpression of USP36 promotes SUMOylation in cells . Whole cell lysates ( WCL ) from H1299 ( A ) , U2OS and HeLa ( B ) cells transfected with control empty vector or Flag - USP36 were assayed by IB .

O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O B-CELL_LINE O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O

Overexpression of USP36 promotes SUMOylation in cells. Whole cell lysates (WCL) from H1299 (A), U2OS and HeLa (B) cells transfected with control empty vector or Flag-USP36 were assayed by IB.


H1299 cells transfected with WT USP36 or the indicated mutants and His - SUMO2 were subjected to Ni2 + - NTA agarose beads pull down ( PD ) followed by IB using anti - SUMO2 / 3 antibody .

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

H1299 cells transfected with WT USP36 or the indicated mutants and His-SUMO2 were subjected to Ni2+-NTA agarose beads pull down (PD) followed by IB using anti-SUMO2/3 antibody.


USP36 interacts with Ubc9 in cells . H1299 cell transfected with the indicated plasmids were subjected to co - IP with anti - Flag antibody followed by IB .

B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O

USP36 interacts with Ubc9 in cells. H1299 cell transfected with the indicated plasmids were subjected to co-IP with anti-Flag antibody followed by IB.


USP36 SUMOylates PARP1 in vitro . Recombinant T7 - PARP1 protein ( 0 . 1 μM ) was incubated with SUMO E1 ( 30 nM ) , Ubc9 ( 50 nM ) , SUMO2 ( 4 μM ) in the presence of USP361 - 800 ( 50 nM ) and / or ATP ( 2 . 5 mM ) at 30ºC for 5 hours and then assayed by IB .

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USP36 SUMOylates PARP1 in vitro. Recombinant T7-PARP1 protein (0.1 μM) was incubated with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the presence of USP361-800 (50 nM) and/or ATP (2.5 mM) at 30ºC for 5 hours and then assayed by IB.


USP36 SUMOylates Nhp2 and Nop58 in vitro . In vitro SUMOylation assays were performed by incubating recombinant Nhp2 ( F , G ) or Nop58 ( H ) ( 0 . 1 μM ) with SUMO E1 ( 30 nM ) , Ubc9 ( 50 nM ) , SUMO2 ( 4 μM ) in the absence or presence of USP361 - 800 ( 50 nM , 1x ; 100 nM , 2x ) and / or ATP ( 2 . 5 mM ) at 37ºC for two hours or the indicated times . The reactions were assayed by IB using anti - Nhp2 and anti - Nop58 antibodies , respectively .

B-GENEPROD B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O

USP36 SUMOylates Nhp2 and Nop58 in vitro. In vitro SUMOylation assays were performed by incubating recombinant Nhp2 (F, G) or Nop58 (H) (0.1 μM) with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the absence or presence of USP361-800 (50 nM, 1x; 100 nM, 2x) and/or ATP (2.5 mM) at 37ºC for two hours or the indicated times. The reactions were assayed by IB using anti-Nhp2 and anti-Nop58 antibodies, respectively.


USP36 does not change the levels of endogenous snoRNP proteins . HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB .

B-GENEPROD O O O O O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-ORGANISM O O O B-EXP_ASSAY O

USP36 does not change the levels of endogenous snoRNP proteins. HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB.


USP36 promotes the binding of Nhp2 to snoRNAs . H1299 cells transfected with Flag - Nhp2 and V5 - USP36 individually or together were assayed by RNA - IP with anti - Flag or control mouse IgG , followed by RT - qPCR detection of the indicated box H / ACA snoRNAs ( C ) . Shown is one representative experiment of three independent experiments . Data were presented as mean ± SD , n = 3 technical replicates . * P < 0 . 05 ; * * P < 0 . 01 , compared to Nhp2 alone ( Student ' s t - test ) .

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

USP36 promotes the binding of Nhp2 to snoRNAs. H1299 cells transfected with Flag-Nhp2 and V5-USP36 individually or together were assayed by RNA-IP with anti-Flag or control mouse IgG, followed by RT-qPCR detection of the indicated box H/ACA snoRNAs (C). Shown is one representative experiment of three independent experiments. Data were presented as mean ± SD, n=3 technical replicates. *P<0.05; **P<0.01, compared to Nhp2 alone (Student's t-test).


( B ) . USP36 is required for rRNA processing . HeLa cells transfected with scr or USP36 siRNA ( left panels ) or infected with scr or USP36 shRNA lentiviruses ( right panels ) were assayed for rRNA processing by Northern blot using 5 ' - ETS , ITS1 and ITS2 probes as indicated .

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

(B). USP36 is required for rRNA processing. HeLa cells transfected with scr or USP36 siRNA (left panels) or infected with scr or USP36 shRNA lentiviruses (right panels) were assayed for rRNA processing by Northern blot using 5'- ETS, ITS1 and ITS2 probes as indicated.


Knockdown of USP36 inhibits translation . HeLa cells infected with scr or USP36 shRNA lentiviruses were incubated with O - propargyl - puromycin ( OPP ) followed by Click - iT OPP protein synthesis assays . Representative images ( C ) and quantification from three independent experiments ( D ) . Data were presented as mean ± SD , n = 3 biological replicates . * * * P < 0 . 001 , compared to scr control ( Student ' s t - test ) . Scale bar = 50 μM .

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

Knockdown of USP36 inhibits translation. HeLa cells infected with scr or USP36 shRNA lentiviruses were incubated with O-propargyl-puromycin (OPP) followed by Click-iT OPP protein synthesis assays. Representative images (C) and quantification from three independent experiments (D). Data were presented as mean ± SD, n=3 biological replicates. ***P<0.001, compared to scr control (Student's t-test). Scale bar = 50 μM.


( A ) Cell proliferation for PC3 cells transfected with siSCR or siCLU monitored in real time for 60h with SP Dynamic Xcelligence system ( Voltage 2 . 5 K ) . Error bars represent mean ± SEM , n = 4 . Slopes compared by ANCOVA test , p < 0 . 0001 .

O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Cell proliferation for PC3 cells transfected with siSCR or siCLU monitored in real time for 60h with SP Dynamic Xcelligence system (Voltage 2.5 K). Error bars represent mean ± SEM, n=4. Slopes compared by ANCOVA test, p<0.0001.


( B ) Left panel : Cell cycle profile of PC3 cells transfected with siSCR or siCLU . Right panel : Percentage quantification of variation in G1 , S and G2 / M phases . Error bars represent mean ± SEM , n = 3 , * * p < 0 . 01 by paired Student ' s t - test . ( G2 / M population p = 0 . 0058 ; G1 p = 0 . 008 . ) . Inset : Western blot with CLU antibody in PC3 cells after CLU silencing . Vinculin was used as loading control .

O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O O O

(B) Left panel: Cell cycle profile of PC3 cells transfected with siSCR or siCLU. Right panel: Percentage quantification of variation in G1, S and G2/M phases. Error bars represent mean ± SEM, n=3, **p<0.01 by paired Student's t-test. (G2/M population p= 0.0058; G1 p=0.008.). Inset: Western blot with CLU antibody in PC3 cells after CLU silencing. Vinculin was used as loading control.


( C ) Left panel : RNA - Microarray analysis comparing PC3 cells transfected with siCLU versus siSCR and Kinexus phosphokinome microarray analysis comparing LNCaP - transfected with siCLU versus siSCR . Target genes expression is shown as fold change relative to siSCR , p ≤ 0 . 05 by an unpaired t - test for all the genes listed . Central panel : Venn diagram to graphically illustrate the overlap of mRNA and proteins involved in the regulation of mitosis in both transcriptome and phosphokinome analysis . The comparison of these 2 experiments shows modification of 4 mitosis regulators . Right panel : Western blot validating the expression levels of target proteins .

O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(C) Left panel: RNA-Microarray analysis comparing PC3 cells transfected with siCLU versus siSCR and Kinexus phosphokinome microarray analysis comparing LNCaP-transfected with siCLU versus siSCR. Target genes expression is shown as fold change relative to siSCR, p ≤0.05 by an unpaired t-test for all the genes listed. Central panel: Venn diagram to graphically illustrate the overlap of mRNA and proteins involved in the regulation of mitosis in both transcriptome and phosphokinome analysis. The comparison of these 2 experiments shows modification of 4 mitosis regulators. Right panel: Western blot validating the expression levels of target proteins.


( A ) Real - time PCR for CLU and Cdc25C in three different prostate cancer cell lines . Target genes expression was calculated relative to GAPDH and normalized to siSCR . Error bars represent mean ± SD , n = 3 .

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

(A) Real-time PCR for CLU and Cdc25C in three different prostate cancer cell lines. Target genes expression was calculated relative to GAPDH and normalized to siSCR. Error bars represent mean ± SD, n=3.


( B ) Western blot for CLU and Cdc25C after siSCR or si CLU transfection in the same prostate cancer cell lines as in A . Vinculin was used as loading control .

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

(B) Western blot for CLU and Cdc25C after siSCR or si CLU transfection in the same prostate cancer cell lines as in A. Vinculin was used as loading control.


( C ) Western blot for CLU and Cdc25C in PC3 cells after transfection with siSCR or siCLU followed by transfection with CLU or empty expression plasmid for additional 48h . Vinculin was used as loading

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O

(C) Western blot for CLU and Cdc25C in PC3 cells after transfection with siSCR or siCLU followed by transfection with CLU or empty expression plasmid for additional 48h. Vinculin was used as loading control


( A ) Upper panel : Western blot for CLU and Cdc25C from LNCaP xenografts harvested from mice treated with OGX - 011 ( n = 8 ) or SCR ASO control ( n = 6 ) . Vinculin was used as loading control . Lower panel : Quantification of the protein levels , relative to the loading control , reported as dot plots . * p < 0 . 05 by t - test followed by Welch ' s correction ( CLU p = 0 . 049 , Cdc25C p = 0 . 024 ) .

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

(A) Upper panel: Western blot for CLU and Cdc25C from LNCaP xenografts harvested from mice treated with OGX-011 (n=8) or SCR ASO control (n=6). Vinculin was used as loading control. Lower panel: Quantification of the protein levels, relative to the loading control, reported as dot plots. *p<0.05 by t-test followed by Welch's correction (CLU p= 0.049, Cdc25C p= 0.024).


( B ) Western blot with the correspondent quantification of protein levels ( left ) and real time PCR ( right ) from untreated LNCaP xenografts harvested at endpoint . The negative correlation between CLU - RNA and Cdc25C - RNA ΔΔCT values was calculated using a spearman test ( rho = - 0 . 86 ; p = 0 . 0107 ) .

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

(B) Western blot with the correspondent quantification of protein levels (left) and real time PCR (right) from untreated LNCaP xenografts harvested at endpoint. The negative correlation between CLU-RNA and Cdc25C-RNA ΔΔCT values was calculated using a spearman test (rho=-0.86; p=0.0107).