Figure 4A Representative western blot of HeLa cells downregulated for G3BPs and overexpressing untagged CLUH . Asterisks indicates signal of previous incubation with anti - G3BP1 antibody . Pan - actin was used as loading control . B Confocal images of anti - CLUH staining in HeLa cells downregulated for G3BPs and overexpressing untagged CLUH . Insets show 2 . 6x enlarged area . Scale bar , 10 µm . C Quantification of cells with CLUH - granules from experiments shown in ( B ) ( n = 3 independent experiments , > 50 cells per experiment per condition ) . Data information data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . D Life imaging of WT and CLUH KO HeLa cells transfected with G3BP1 - GFP plasmid and incubated in HBSS medium with or without CHX . Cells were recorded for a maximum of 2 h . Insets show 2 . 5x enlarged areas . Scale bar , 10 µm . E Total number of cells analyzed by life imaging for the indicated experiments shown in ( D ) . " Positive " relates to a cell which formed G3BP1 granules at the end of the recording . F Confocal images of anti - G3BP1 staining in primary hepatocytes derived from Li - CluhWT and Li - CluhKO mice grown in indicated media . Arrows point to G3BP1 - granules . Scale bar , 10 µm . G Quantification of percentage of cells with G3BP1 - positive granules of experiment shown in F . Cells were analyzed in a blind fashion ( n = 3 mice per genotype ; number of cells in total : WT Basal , 449 ; KO Basal , 303 ; WT HBSS , 146 ; KO HBSS , 161 ) . Data information : , data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . H Western blot of primary WT and KO hepatocytes stained with indicated antibodies . Asterisks indicates unspecific signal . Pan - actin was used as loading control . I Quantification of western blot ( n = 3 per genotype per condition ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . J mRNA levels of G3bp1 in primary hepatocytes grown under indicated conditions . ( n = 4 per genotype per condition ) . Data information data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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

Figure 4A Representative western blot of HeLa cells downregulated for G3BPs and overexpressing untagged CLUH. Asterisks indicates signal of previous incubation with anti-G3BP1 antibody. Pan-actin was used as loading control.B Confocal images of anti-CLUH staining in HeLa cells downregulated for G3BPs and overexpressing untagged CLUH. Insets show 2.6x enlarged area. Scale bar, 10 µm. C Quantification of cells with CLUH-granules from experiments shown in (B) (n=3 independent experiments, >50 cells per experiment per condition). Data information data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test). D Life imaging of WT and CLUH KO HeLa cells transfected with G3BP1-GFP plasmid and incubated in HBSS medium with or without CHX. Cells were recorded for a maximum of 2 h. Insets show 2.5x enlarged areas. Scale bar, 10 µm. E Total number of cells analyzed by life imaging for the indicated experiments shown in (D). "Positive" relates to a cell which formed G3BP1 granules at the end of the recording. F Confocal images of anti-G3BP1 staining in primary hepatocytes derived from Li-CluhWT and Li-CluhKO mice grown in indicated media. Arrows point to G3BP1-granules. Scale bar, 10 µm. G Quantification of percentage of cells with G3BP1-positive granules of experiment shown in F. Cells were analyzed in a blind fashion (n=3 mice per genotype; number of cells in total: WT Basal, 449; KO Basal, 303; WT HBSS, 146; KO HBSS, 161). Data information: , data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test). H Western blot of primary WT and KO hepatocytes stained with indicated antibodies. Asterisks indicates unspecific signal. Pan-actin was used as loading control.I Quantification of western blot (n=3 per genotype per condition). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test). J mRNA levels of G3bp1 in primary hepatocytes grown under indicated conditions. (n=4 per genotype per condition). Data information data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


Figure 5A , B Volcano plots showing significantly changed proteins in KO hepatocytes relative to WT in basal ( A ) and HBSS ( B ) conditions . F Correlation of protein fold changes in KO in respect to WT hepatocytes under basal condition and upon HBSS ( 2 h ) . Only genes previously found in CLUH RIP experiments in HeLa cells ( Gao et al . , 2014 ) are plotted . Inset shows magnification of upper part of the graph .

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

Figure 5A, B Volcano plots showing significantly changed proteins in KO hepatocytes relative to WT in basal (A) and HBSS (B) conditions.F Correlation of protein fold changes in KO in respect to WT hepatocytes under basal condition and upon HBSS (2 h). Only genes previously found in CLUH RIP experiments in HeLa cells (Gao et al., 2014) are plotted. Inset shows magnification of upper part of the graph.


Figure 6A Representative western blot of primary hepatocytes starved in HBSS for the indicated time points and probed with indicated antibodies . Pan - actin was used as loading control . B , C Quantification of western blots as shown in ( A ) . Antibody signal was normalized to pan - actin signal and signal of phospho - protein was normalized to signal of total protein ( n = 4 independent experiments ) . Data information : In ( B , C ) , data shows the mean ± SEM . * * , P ≤ 0 . 01 for genotype and * * * , P ≤ 0 . 001 for time ( Two - way ANOVA ) . Error bars show minimum and maximum values . D Representative western blot of total protein extracts from livers of starved Li - CluhWT and Li - CluhKO mice probed with indicated antibodies . GAPDH was used as loading control . E , F Quantification of western blots as shown in ( D ) . Antibody signal was normalized to GAPDH signal and signal of phospho - protein was normalized to signal of the total protein ( n = 8 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ( Student ' s t - test ) . ( H , J ) * , P ≤ 0 . 05 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . G Confocal images of primary hepatocytes grown in HBSS for 16 h and stained with anti - cleaved caspase 3 antibody . Scale bar , 25 µm . H Quantification of cleaved caspase 3 positive hepatocytes treated with or without rapamycin . More than 100 cells per replicate were analyzed in a blind fashion ( n = 3 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . Error bars show minimum and maximum values . I IHC staining of cleaved caspase 3 in liver sections of starved Li - CluhWT and Li - CluhKO mice . Arrows point to positive apoptotic foci . Scale bar , 200 µm . J Quantification of positive apoptotic foci The number of positive spots was relativized to the area analyzed ( n = 5 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . K . Confocal images of primary hepatocytes grown in indicated media and stained with anti - G3BP1 and anti - mTOR . Scale bar , 10 µm . L Manders ' colocalization coefficient between G3BP1 and mTOR staining ( n > 50 cells isolated from 3 mice ) . Data information : In ( L ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ( Student ' s t - test ) . ( H , J ) * , P ≤ 0 . 05 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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

Figure 6A Representative western blot of primary hepatocytes starved in HBSS for the indicated time points and probed with indicated antibodies. Pan-actin was used as loading control. B, C Quantification of western blots as shown in (A). Antibody signal was normalized to pan-actin signal and signal of phospho-protein was normalized to signal of total protein (n=4 independent experiments). Data information: In (B, C), data shows the mean ± SEM. **, P≤0.01 for genotype and ***, P≤0.001 for time (Two-way ANOVA). Error bars show minimum and maximum values.D Representative western blot of total protein extracts from livers of starved Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. GAPDH was used as loading control. E, F Quantification of western blots as shown in (D). Antibody signal was normalized to GAPDH signal and signal of phospho-protein was normalized to signal of the total protein (n=8 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test). (H, J) *, P≤0.05; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).G Confocal images of primary hepatocytes grown in HBSS for 16 h and stained with anti-cleaved caspase 3 antibody. Scale bar, 25 µm.H Quantification of cleaved caspase 3 positive hepatocytes treated with or without rapamycin. More than 100 cells per replicate were analyzed in a blind fashion (n=3 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. Error bars show minimum and maximum values.I IHC staining of cleaved caspase 3 in liver sections of starved Li-CluhWT and Li-CluhKO mice. Arrows point to positive apoptotic foci. Scale bar, 200 µm.J Quantification of positive apoptotic foci The number of positive spots was relativized to the area analyzed (n=5 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM.K. Confocal images of primary hepatocytes grown in indicated media and stained with anti-G3BP1 and anti-mTOR. Scale bar, 10 µm. L Manders' colocalization coefficient between G3BP1 and mTOR staining (n >50 cells isolated from 3 mice). Data information: In (L), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test). (H, J) *, P≤0.05; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


Figure 7A Confocal images of liver cryosections from Li - CluhWT and Li - CluhKO - LC3 - GFP mice . Scale bar , 25 µm . B Quantification of LC3 signal morphology of images shown in ( A ) . Graphs show relative number of LC3 autophagosomes and the relative area occupied per nuclei . 4 images were quantified per animal and averaged . Nuclear staining was excluded for the analysis ( n = 4 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t - test ) . C Confocal images of primary hepatocytes derived from Li - CluhWT and Li - CluhKO - LC3 - GFP mice . Hepatocytes were starved for 2 h in HBSS . Scale bar , 10μm . D - E Quantification of LC3 puncta number ( D ) and area ( E ) per cell ( n ≥ 100 cells isolated from 3 mice ) . Data information : In ( D , E ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . F Western blot of total protein extracts from livers of Li - CluhWT and Li - CluhKO mice probed with LAMP1 . GAPDH was used as loading control . G Quantification of western blot shown in ( F ) . WT : n = 4 ; KO : n = 3 . Data information : In data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t - test ) . H Representative western blot of primary hepatocytes treated for 2 h with bafilomycin A in the indicated media and probed with indicated antibodies . Two different exposure times are shown for LC3 . I refers to total LC3 and II indicates lipidated form of LC3 . Asterisk indicates signal of previous incubation . I Quantification of LC3 levels in western blots as shown in ( H ) ( n = 4 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . J Quantification of autophagic flux of western blot as shown in ( H ) . The flux was calculated as the increase in LC3II upon bafilomycin A treatment in each condition . The flux in KO cells was normalized to flux in WT cells . ( n = 4 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t - test ) . Quantification of p62 levels in western blots ( n = 4 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . Quantification of CLUH levels in western blots ( n = 4 independent experiments ) . Data information data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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

Figure 7A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO-LC3-GFP mice. Scale bar, 25 µm. B Quantification of LC3 signal morphology of images shown in (A). Graphs show relative number of LC3 autophagosomes and the relative area occupied per nuclei. 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=4 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).C Confocal images of primary hepatocytes derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice. Hepatocytes were starved for 2 h in HBSS. Scale bar, 10μm. D-E Quantification of LC3 puncta number (D) and area (E) per cell (n ≥100 cells isolated from 3 mice). Data information: In (D, E), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).F Western blot of total protein extracts from livers of Li-CluhWT and Li-CluhKO mice probed with LAMP1. GAPDH was used as loading control. G Quantification of western blot shown in (F). WT: n=4; KO: n=3. Data information: In data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).H Representative western blot of primary hepatocytes treated for 2 h with bafilomycin A in the indicated media and probed with indicated antibodies. Two different exposure times are shown for LC3. I refers to total LC3 and II indicates lipidated form of LC3. Asterisk indicates signal of previous incubation. I Quantification of LC3 levels in western blots as shown in (H) (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).J Quantification of autophagic flux of western blot as shown in (H). The flux was calculated as the increase in LC3II upon bafilomycin A treatment in each condition. The flux in KO cells was normalized to flux in WT cells. (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).Quantification of p62 levels in western blots (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).Quantification of CLUH levels in western blots (n=4 independent experiments). Data information data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


Figure 8Western blot analysis of total protein extracts from livers of 8 weeks old Li - CluhWT and Li - CluhKO mice probed with indicated antibodies . GAPDH was used as loading control . B Quantification of western blot shown in ( A ) . WT : n = 4 ; KO : n = 3 . Data information data are presented as histograms showing the mean ± SEM . * * * , P ≤ 0 . 001 ; * , P ≤ 0 . 05 ( Student ' s t - test ) . C Western blot analysis of isolated mitochondria from Li - CluhWT and Li - CluhKO mice probed with indicated antibodies . PonceauS staining was used as a loading control . D Quantification of western blot ( n = 3 mice per genotype ) . Data information : , data are presented as histograms showing the mean ± SEM . * * * , P ≤ 0 . 001 ; * , P ≤ 0 . 05 ( Student ' s t - test ) . E Confocal images of liver cryosections derived from Li - CluhWT and Li - CluhKO - LC3 - GFP mice and stained with anti - TOM20 antibody . On the right side , indicated boxes were 3 . 5x magnified . Arrows point to colocalizing spots . Scale bar , 25 μm . F Manders ' colocalization coefficient between TOM20 and LC3 from experiments shown in ( E ) . 5 images were analyzed per animal to get an averaged value ( n = 5 mice per genotype ) . Data information , data are presented as histograms showing the mean ± SEM . * * * , P ≤ 0 . 001 ; * , P ≤ 0 . 05 ( Student ' s t - test ) . G Confocal images of primary hepatocytes derived from Li - CluhWT and Li - CluhKO - LC3 - GFP mice and stained with anti - TOM20 antibody . When indicated cells were treated with rapamycin ( 200 nM for 4h ) . Bottom panels show 2 . 3x magnified areas for each channel of indicated boxes . Arrows point to colocalizing spots . Scale bar , 10 μm . H Manders ´ colocalization coefficient from experiment shown in ( G ) ( n ≥ 50 cells isolated from 3 mice per genotype ) . Data information : In ( H ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . I Confocal images of WT and Cluh KO MEFs transfected with mCherry - GFP - FIS101 - 152 and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A / oligomycin . Arrows indicate mitophagosomes . Scale bar , 10μm . J Quantification of percentage of cells with mitophagosomes . Cells were considered positive when red signal was clearly recognizable ( n = 3 - 5 independent experiments , > 100 cells per experiment ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . K Confocal images of WT and Cluh KO MEFs transfected with mCherry - GFP - FIS101 - 152 , incubated with 200 nM rapamycin and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A / oligomycin . Arrows indicate mitophagosomes . Scale bar , 10 μm . L Quantification of percentage of cells with mitophagosomes . Cells were considered positive when red signal was clearly recognizable ( n = 3 - 5 independent experiments , > 100 cells per experiment ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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

Figure 8Western blot analysis of total protein extracts from livers of 8 weeks old Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. GAPDH was used as loading control. B Quantification of western blot shown in (A). WT: n=4; KO: n=3. Data information data are presented as histograms showing the mean ± SEM. ***, P≤0.001; *, P≤0.05 (Student's t-test).C Western blot analysis of isolated mitochondria from Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. PonceauS staining was used as a loading control.D Quantification of western blot (n=3 mice per genotype). Data information: , data are presented as histograms showing the mean ± SEM. ***, P≤0.001; *, P≤0.05 (Student's t-test).E Confocal images of liver cryosections derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice and stained with anti-TOM20 antibody. On the right side, indicated boxes were 3.5x magnified. Arrows point to colocalizing spots. Scale bar, 25 μm. F Manders' colocalization coefficient between TOM20 and LC3 from experiments shown in (E). 5 images were analyzed per animal to get an averaged value (n=5 mice per genotype). Data information , data are presented as histograms showing the mean ± SEM. ***, P≤0.001; *, P≤0.05 (Student's t-test).G Confocal images of primary hepatocytes derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice and stained with anti-TOM20 antibody. When indicated cells were treated with rapamycin (200 nM for 4h). Bottom panels show 2.3x magnified areas for each channel of indicated boxes. Arrows point to colocalizing spots. Scale bar, 10 μm. H Manders´ colocalization coefficient from experiment shown in (G) (n ≥ 50 cells isolated from 3 mice per genotype). Data information: In (H), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).I Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101-152 and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A/ oligomycin. Arrows indicate mitophagosomes. Scale bar, 10μm. J Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n= 3-5 independent experiments, >100 cells per experiment). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).K Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101-152, incubated with 200 nM rapamycin and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A/ oligomycin. Arrows indicate mitophagosomes. Scale bar, 10 μm. L Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n= 3-5 independent experiments, >100 cells per experiment). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


Figure 9A Confocal images of liver cryosections from Li - CluhWT and Li - CluhKO mice injected with rapamycin or mock solution and stained with anti - TOM20 antibody . Scale bar , 25 µm . B Quantification of morphological parameters of experiments shown in ( A ) . 4 images were quantified per animal and averaged . Nuclear staining was excluded for the analysis ( n = 3 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . * * , P ≤ 0 . 01 ; * , P ≤ 0 . 05 ( Student ' s t - test ) . C Confocal images of liver cryosections from Li - CluhWT and Li - CluhKO mice injected with rapamycin or mock solution and stained with anti - LAMP1 antibody . Scale bar , 25 µm . D Quantification of morphological parameters of experiments shown in ( C ) . 4 images were quantified per animal and averaged . Nuclear staining was excluded for the analysis ( n = 3 mice per genotype ) . Data information : , data are presented as histograms showing the mean ± SEM . * * , P ≤ 0 . 01 ; * , P ≤ 0 . 05 ( Student ' s t - test ) . E Confocal images of COS - 7 cells downregulated for the indicated genes and stained with anti - TOM20 antibody . Scale bar , 10 µm . F Representative western blot of COS - 7 cells downregulated for the indicated genes of experiments and probed with indicated antibodies . Asterisks indicate unspecific signal or signal of previous incubation . G Quantification of morphological parameters of experiments ( n > 100 cells from 3 independent experiments ) . Data information : In ( G ) , data presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . ( G ) * * * , P ≤ 0 . 001 ; * * , P ≤ 0 . 01 * , P ≤ 0 . 05 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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

Figure 9A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-TOM20 antibody. Scale bar, 25 µm. B Quantification of morphological parameters of experiments shown in (A). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype).Data information: data are presented as histograms showing the mean ± SEM. **, P≤0.01; *, P≤0.05 (Student's t-test).C Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-LAMP1 antibody. Scale bar, 25 µm. D Quantification of morphological parameters of experiments shown in (C). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype). Data information: , data are presented as histograms showing the mean ± SEM. **, P≤0.01; *, P≤0.05 (Student's t-test).E Confocal images of COS-7 cells downregulated for the indicated genes and stained with anti-TOM20 antibody. Scale bar, 10 µm.F Representative western blot of COS-7 cells downregulated for the indicated genes of experiments and probed with indicated antibodies. Asterisks indicate unspecific signal or signal of previous incubation.G Quantification of morphological parameters of experiments (n>100 cells from 3 independent experiments). Data information: In (G), data presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (G) ***, P≤0.001; **, P≤0.01 *, P≤0.05 (One-way ANOVA, Tukey's multiple comparisons test).


Figure 4D ) Proline biosynthesis protein levels in breast cancer subtypes ( Log2 ratios of light vs . SILAC standard ) . The upper and lower limits of the box show values from the first to the third quartile , with the horizontal line indicating the median . The whiskers extend 1 . 5 times the interquartile range from the edges of the box . Outlier values were plotted outside the whiskers . Significance was determined using paired Student ' s t - test and p values were corrected using permutation based FDR . * p < 0 . 05 and * * * p < 0 . 001 . E ) High abundance of PYCR1 in the cancer tissue was validated by immunohistochemistry on matched tissue specimens from patients in this study ( n = 3 ) ; Scale bar : 200 μm . F ) Quantification of IHC shows percentage of cells with 2 + and 3 + PYCR1 staining intensity . Data is expressed as mean ± SD of three patient samples . Paired samples were compared by a Student ' s t - test . * p < 0 . 05 and * * * p < 0 . 001 . G ) Kaplan Meier survival curve for PYCR1 levels in residual cancer in the current proteomics data . Cox univariate p value , risk table and hazard ratio with 95 % CI indicated .

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

Figure 4D) Proline biosynthesis protein levels in breast cancer subtypes (Log2 ratios of light vs. SILAC standard). The upper and lower limits of the box show values from the first to the third quartile, with the horizontal line indicating the median. The whiskers extend 1.5 times the interquartile range from the edges of the box. Outlier values were plotted outside the whiskers. Significance was determined using paired Student's t-test and p values were corrected using permutation based FDR . *p < 0.05 and *** p < 0.001.E) High abundance of PYCR1 in the cancer tissue was validated by immunohistochemistry on matched tissue specimens from patients in this study (n=3); Scale bar: 200 μm.F) Quantification of IHC shows percentage of cells with 2+ and 3+ PYCR1 staining intensity. Data is expressed as mean ± SD of three patient samples. Paired samples were compared by a Student's t-test. *p < 0.05 and *** p < 0.001.G) Kaplan Meier survival curve for PYCR1 levels in residual cancer in the current proteomics data. Cox univariate p value, risk table and hazard ratio with 95% CI indicated.


Figure 5A ) Seahorse experiment measuring oxygen consumption rate ( OCR ) in MCF7 cells upon addition of oligomycin , FCCP and a mixture of antimycin and rotenone . Arrows indicate the points of addition of each inhibitor . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Similar results were obtained in three biological experiments . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values were indicated as follows * * p < 0 . 01 and * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances . B ) Bar plot indicates average basal OCR and spare respiratory capacity in seahorse measurements . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 and * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances . C ) 13C5 - Glutamine derived carbon labeling patterns of TCA cycle intermediates fumarate , malate and citrate in MCF7 cells . Data are presented as mean ± SD of triplicate samples . Isotopologues are represented as M + n where M indicates the mass and n equals number of 13C incorporatedD ) Seahorse experiment measuring extracellular acidification rate ( ECAR ) in MCF7 cells upon addition of glucose , oligomycin and 2 - deoxyglucose ( 2DG ) . Arrows indicate the points of addition of each inhibitor . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * * p < 0 . 01 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances . E ) Bar plot indicates average glycolytic capacity and glycolytic reserve in seahorse measurements . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Similar results were obtained in three biological experiments . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * * p < 0 . 01 and * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances . F ) Normalized lactate concentration upon PYCR1 KO . Data represents mean ± SE of three biological experiments . Samples are compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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

Figure 5A) Seahorse experiment measuring oxygen consumption rate (OCR) in MCF7 cells upon addition of oligomycin, FCCP and a mixture of antimycin and rotenone. Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values were indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.B) Bar plot indicates average basal OCR and spare respiratory capacity in seahorse measurements. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.C) 13C5 -Glutamine derived carbon labeling patterns of TCA cycle intermediates fumarate, malate and citrate in MCF7 cells. Data are presented as mean ± SD of triplicate samples. Isotopologues are represented as M+n where M indicates the mass and n equals number of 13C incorporatedD) Seahorse experiment measuring extracellular acidification rate (ECAR) in MCF7 cells upon addition of glucose, oligomycin and 2-deoxyglucose (2DG). Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.E) Bar plot indicates average glycolytic capacity and glycolytic reserve in seahorse measurements. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments.Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.F) Normalized lactate concentration upon PYCR1 KO. Data represents mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


Figure 6A ) Representative pictures of transwell migration and invasion after PYCR1 knockout . Scale bar : 100 μm . B ) Bar plot represents mean ± SD of three biological replicates for transwell migration and invasion . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p - values are reported as follows * * * p < 0 . 001 . C ) Representative images of colony formation in soft agar upon PYCR1 KO . Scale bar : 100 μm . D ) Growth measurements of MCF7 wild type and PYCR1 KO cells over 96 hours . Data represents mean ± SE of three biological experiments . Samples were compared using paired Student ' s t - test . E - H ) Bar plots show percentage of viable cells after treatment with 0 . 75 mM H2O2 ( E ) , 0 . 06 µM paclitaxel ( F ) , 0 . 15 µM doxorubicin ( G ) , 2 . 5 mM cyclophosphamide ( H ) . Data represent mean ± SE of three biological experiments . Samples are compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 . I ) Tumor volume measurements for 26 days in MCF7 injected NSG mice . CRISPR control and PYCR1 KO tumors with ( n = 8 ) and without treatment ( n = 5 ) are shown . Data represents mean ± SE . Pax : Paclitaxel , Dox : Doxorubicin . Groups were compared by one way ANOVA followed by Tukey ' s multiple comparisons test for pairwise group comparisons . Corrected p - values are reported as follows * p < 0 . 05 , * * p < 0 . 01 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances . J ) Bar plot indicates mean ± SE of tumor volume measurements for day 26 . CRISPR control and PYCR1 KO tumors with ( n = 8 ) and without treatment ( n = 5 ) were compared by one way ANOVA followed by Tukey ' s multiple comparisons test for pairwise group comparisons . Corrected p - values are reported as follows * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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

Figure 6A) Representative pictures of transwell migration and invasion after PYCR1 knockout. Scale bar :100 μm. B) Bar plot represents mean ± SD of three biological replicates for transwell migration and invasion. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p-values are reported as follows *** p < 0.001. C) Representative images of colony formation in soft agar upon PYCR1 KO. Scale bar: 100 μm.D) Growth measurements of MCF7 wild type and PYCR1 KO cells over 96 hours. Data represents mean ± SE of three biological experiments. Samples were compared using paired Student's t-test.E-H) Bar plots show percentage of viable cells after treatment with 0.75 mM H2O2 (E), 0.06 µM paclitaxel (F), 0.15 µM doxorubicin (G), 2.5 mM cyclophosphamide (H). Data represent mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05.I) Tumor volume measurements for 26 days in MCF7 injected NSG mice. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) are shown. Data represents mean ± SE. Pax: Paclitaxel, Dox: Doxorubicin. Groups were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *p < 0.05, ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.J) Bar plot indicates mean ± SE of tumor volume measurements for day 26. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


Figure 1 . ( A ) HeLa cells expressing HA - Parkin were treated with CCCP or DMSO ( control ) and then immunostained with the indicated antibodies . ( B ) HeLa cells stably expressing HA - Parkin or intact SH - SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments . I , C , and M indicate input , cytosol - rich supernatant , and mitochondria - rich membrane pellet , respectively . ( D ) Polarized mitochondria stained with MitoTracker red ( red arrowheads ) were not labeled by Parkin . In contrast , damaged mitochondria marked by Parkin ( green arrowheads ) were not stained with MitoTracker red . ( E ) HeLa cells expressing HA - Parkin with various pathogenic mutations were treated with CCCP , followed by immunocytochemistry . ( A , D , and E ) Higher magnification views of the boxed areas are shown in the insets . ( F ) Parkin colocalization with mitochondria was analyzed in > 100 cells per mutation . Example figures indicative of robust colocalization ( counted as 1 ) , weak colocalization ( counted as 0 . 5 ) , and no colocalization ( counted as 0 ) are shown . Error bars represent the mean ± SD values of at least three experiments .

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

Figure 1.(A) HeLa cells expressing HA-Parkin were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies.(B) HeLa cells stably expressing HA-Parkin or intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments. I, C, and M indicate input, cytosol-rich supernatant, and mitochondria-rich membrane pellet, respectively.(D) Polarized mitochondria stained with MitoTracker red (red arrowheads) were not labeled by Parkin. In contrast, damaged mitochondria marked by Parkin (green arrowheads) were not stained with MitoTracker red.(E) HeLa cells expressing HA-Parkin with various pathogenic mutations were treated with CCCP, followed by immunocytochemistry. (A, D, and E) Higher magnification views of the boxed areas are shown in the insets. (F) Parkin colocalization with mitochondria was analyzed in >100 cells per mutation. Example figures indicative of robust colocalization (counted as 1), weak colocalization (counted as 0.5), and no colocalization (counted as 0) are shown. Error bars represent the mean ± SD values of at least three experiments.


Figure 2 . ( A ) HeLa cells expressing wild - type Parkin or E3 - inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies . When E3 - inactivating mutations were introduced into Parkin , the mitochondrial ubiquitylation signal disappeared . ( B and C ) HeLa cells expressing HA - Parkin ( B ) or expressing both Mt - GFP and HA - Parkin ( C ) were treated with CCCP or DMSO ( control ) and then immunostained with the indicated antibodies . ( D ) Localization of GFP - Parkin to the mitochondria after CCCP treatment . ( A - D ) Higher magnification views of the boxed areas are shown in the insets . ( E ) HeLa cell lysates expressing GFP - Parkin were immunoprecipitated by anti - GFP antibody , followed by immunoblotting with the indicated antibodies . ( F ) Straight immunoblotting of HA - and GFP - Parkin in the absence or presence of CCCP . Note the slower migrating ladders derived from ubiquitylation ( Ub ) in only the GFP - Parkin with CCCP lane . ( G ) GFP - Parkin - expressing HeLa cells with various pathogenic mutations ( Fig . 1 C ) were treated with CCCP and subjected to immunoblotting . Asterisks show ubiquitylation of GFP - Parkin . Vertical black lines indicate that intervening lanes have been spliced out . IB , immunoblot ; IP , immunoprecipitation .

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

Figure 2.(A) HeLa cells expressing wild-type Parkin or E3-inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies. When E3-inactivating mutations were introduced into Parkin, the mitochondrial ubiquitylation signal disappeared.(B and C) HeLa cells expressing HA-Parkin (B) or expressing both Mt-GFP and HA-Parkin (C) were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies.(D) Localization of GFP-Parkin to the mitochondria after CCCP treatment. (A-D) Higher magnification views of the boxed areas are shown in the insets.(E) HeLa cell lysates expressing GFP-Parkin were immunoprecipitated by anti-GFP antibody, followed by immunoblotting with the indicated antibodies.(F) Straight immunoblotting of HA- and GFP-Parkin in the absence or presence of CCCP. Note the slower migrating ladders derived from ubiquitylation (Ub) in only the GFP-Parkin with CCCP lane.(G) GFP-Parkin-expressing HeLa cells with various pathogenic mutations (Fig. 1 C) were treated with CCCP and subjected to immunoblotting. Asterisks show ubiquitylation of GFP-Parkin. Vertical black lines indicate that intervening lanes have been spliced out. IB, immunoblot; IP, immunoprecipitation.


Figure 3 . ( A ) The number of HeLa cells with N - terminal - or C - terminal - tagged PINK1 localized to the mitochondria was counted in > 100 cells . ( B and C ) Exogenous nontagged PINK1 ( B ) or endogenous PINK1 ( C ) in HeLa cells was immunostained with the indicated antibodies . ( D ) The number of HeLa cells with endogenous PINK1 localized to the mitochondria was counted as in A . ( A and D ) Error bars represent the mean ± SD values of least three experiments . ( E ) Endogenous PINK1 gradually accumulated after CCCP treatment . The first through the fifth lanes show endogenous PINK1 , and the sixth lane shows overexpressed untagged PINK1 . Note that the asterisk indicates a cross - reacting band because it was not affected by overproduction of untagged PINK1 . ( F ) Subcellular fractionation of endogenous PINK1 . Intact SH - SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments ( same sample as Fig . 1 B ) . I , C , and M indicate input , cytosol - rich supernatant , and the mitochondria - rich membrane pellet , respectively . ( G ) HeLa cells were treated with CCCP and cycloheximide as depicted , followed by immunoblotting with the indicated antibodies . LDH , lactate dehydrogenase . ( F and G ) Asterisks indicate a cross - reacting band . ( H ) N - terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP . The top panel shows control HeLa cells expressing only GFP . ( B , C , and H ) Higher magnification views of the boxed areas are shown in the insets . Bars , 10 µm .

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

Figure 3.(A) The number of HeLa cells with N-terminal- or C-terminal-tagged PINK1 localized to the mitochondria was counted in >100 cells.(B and C) Exogenous nontagged PINK1 (B) or endogenous PINK1 (C) in HeLa cells was immunostained with the indicated antibodies. (D) The number of HeLa cells with endogenous PINK1 localized to the mitochondria was counted as in A. (A and D) Error bars represent the mean ± SD values of least three experiments.(E) Endogenous PINK1 gradually accumulated after CCCP treatment. The first through the fifth lanes show endogenous PINK1, and the sixth lane shows overexpressed untagged PINK1. Note that the asterisk indicates a cross-reacting band because it was not affected by overproduction of untagged PINK1.(F) Subcellular fractionation of endogenous PINK1. Intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments (same sample as Fig. 1 B). I, C, and M indicate input, cytosol-rich supernatant, and the mitochondria-rich membrane pellet, respectively.(G) HeLa cells were treated with CCCP and cycloheximide as depicted, followed by immunoblotting with the indicated antibodies. LDH, lactate dehydrogenase. (F and G) Asterisks indicate a cross-reacting band.(H) N-terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP. The top panel shows control HeLa cells expressing only GFP. (B, C, and H) Higher magnification views of the boxed areas are shown in the insets. Bars, 10 µm.


Figure 4 . ( A ) PINK1 knockout ( PINK1 − / − ) or control ( PINK1 + / + ) MEFs were transfected with HA - Parkin , treated with CCCP , and subjected to immunocytochemistry with the indicated antibodies . Higher magnification views of the boxed areas are shown in the insets . ( B ) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig . 3 A . ( C and D ) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1 − / − MEFs . MEFs stably expressing GFP - Parkin were treated with CCCP for 4 h and subjected to immunoblotting ( C ) ( C and D ) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1 − / − MEFs . MEFs stably expressing GFP - Parkin were treated with CCCP for 24 h , followed by immunocytochemistry ( D ) . IB , immunoblot ; Ub , ubiquitylation . ( E ) The number of MEFs without anti - Tom20 antibody - detectable mitochondria was counted as in Fig . 3 A . In the example figure ( left ) , blue arrowheads indicate cells without anti - Tom20 antibody - detectable mitochondria , and red arrowheads indicate cells harboring anti - Tom20 antibody - detectable mitochondria .

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

Figure 4.(A) PINK1 knockout (PINK1−/−) or control (PINK1+/+) MEFs were transfected with HA-Parkin, treated with CCCP, and subjected to immunocytochemistry with the indicated antibodies. Higher magnification views of the boxed areas are shown in the insets. (B) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig. 3 A.(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 4 h and subjected to immunoblotting (C)(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 24 h, followed by immunocytochemistry (D). IB, immunoblot; Ub, ubiquitylation.(E) The number of MEFs without anti-Tom20 antibody-detectable mitochondria was counted as in Fig. 3 A. In the example figure (left), blue arrowheads indicate cells without anti-Tom20 antibody-detectable mitochondria, and red arrowheads indicate cells harboring anti-Tom20 antibody-detectable mitochondria.


Figure 5 . ( A ) Schematic depiction of pathogenic and deletion mutants of PINK1 used in this study . MTS , mitochondria - targeting sequence ; TMD , transmembrane domain . ( B ) Subcellular localization of Parkin in PINK1 − / − cells complemented by various pathogenic and deletion mutants of PINK1 - Flag . Cells were treated with CCCP . Higher magnification views of the boxed areas are shown in the insets . ( C ) The number of cells with Parkin - positive mitochondria was counted as in Fig . 3 A . Error bars represent the mean ± SD values of least three experiments . ( D ) PINK1 − / − MEFs complemented by various PINK1 mutants were treated with CCCP and subjected to immunoblotting using anti - Parkin or anti - Flag ( tag of PINK1 ) antibodies . IB , immunoblot ; Ub , ubiquitylation .

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

Figure 5.(A) Schematic depiction of pathogenic and deletion mutants of PINK1 used in this study. MTS, mitochondria-targeting sequence; TMD, transmembrane domain. (B) Subcellular localization of Parkin in PINK1−/− cells complemented by various pathogenic and deletion mutants of PINK1-Flag. Cells were treated with CCCP. Higher magnification views of the boxed areas are shown in the insets. (C) The number of cells with Parkin-positive mitochondria was counted as in Fig. 3 A. Error bars represent the mean ± SD values of least three experiments.(D) PINK1−/− MEFs complemented by various PINK1 mutants were treated with CCCP and subjected to immunoblotting using anti-Parkin or anti-Flag (tag of PINK1) antibodies. IB, immunoblot; Ub, ubiquitylation.


Figure 1 ( A ) Immunoblot analysis with anti - LC3 antibody shows that NO donors reduced LC3 - II levels in rat primary cortical neurons and decreased autophagosome synthesis in bafilomycin A1 - treated rat primary cortical neurons and HeLa cells . ( B - E ) Confocal microscopy images and analysis of autophagic flux by automated Cellomics microscopy show that NO donors reduced autolysosome numbers in mRFP - GFP - LC3 HeLa cells grown in full medium ( FM ) ( B and D ) or starved with HBSS ( C and E ) . Cells in HBSS had more autolysosomes compared to FM ( C ) . Autolysosome number in untreated cells in FM is set at 100 % . ( F ) NO donors increased EGFP - HDQ74 aggregates in a dose - dependent manner in EGFP - HDQ74 - transfected Atg5 + / + , but not in Atg5 − / − , MEFs . Atg5 − / − MEFs had more EGFP - HDQ74 aggregates than Atg5 + / + MEFs . ( G and H ) Confocal microscopy images ( H ) and immunofluorescence analysis with anti - Atg16 antibody ( G ) show that NO donors reduced the percentage of HeLa cells with Atg16 - positive structures ( arrows ) under starvation . Graphical data denote mean ± SEM .

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

Figure 1(A) Immunoblot analysis with anti-LC3 antibody shows that NO donors reduced LC3-II levels in rat primary cortical neurons and decreased autophagosome synthesis in bafilomycin A1-treated rat primary cortical neurons and HeLa cells.(B-E) Confocal microscopy images and analysis of autophagic flux by automated Cellomics microscopy show that NO donors reduced autolysosome numbers in mRFP-GFP-LC3 HeLa cells grown in full medium (FM) (B and D) or starved with HBSS (C and E). Cells in HBSS had more autolysosomes compared to FM (C). Autolysosome number in untreated cells in FM is set at 100%.(F) NO donors increased EGFP-HDQ74 aggregates in a dose-dependent manner in EGFP-HDQ74-transfected Atg5+/+, but not in Atg5−/−, MEFs. Atg5−/− MEFs had more EGFP-HDQ74 aggregates than Atg5+/+ MEFs.(G and H) Confocal microscopy images (H) and immunofluorescence analysis with anti-Atg16 antibody (G) show that NO donors reduced the percentage of HeLa cells with Atg16-positive structures (arrows) under starvation. Graphical data denote mean ± SEM.


Figure 2 ( A ) NO donors reduced JNK1 phosphorylation in HeLa cells , as analyzed by immunoprecipitation with anti - JNK1 agarose - conjugated beads and immunoblotting with anti - phospho - JNK antibody . ( B ) NO donors reduced Bcl - 2 phosphorylation in HeLa cells , as analyzed by immunoblotting with anti - phospho - Bcl - 2 antibody . C ) Immunoblot analysis with anti - phospho - Bcl - 2 antibody shows that DETA NONOate could not reduce phospho - Bcl - 2 in Flag - CA JNK1 - transfected HeLa cells , where phospho - Bcl - 2 was higher compared to mock - transfected cells . ( D and E ) Immunoprecipitation with anti - Flag M2 affinity agarose gel and immunoblotting with anti - Myc antibody shows that NO donors increased the interaction of Flag - Beclin 1 with WT Myc - Bcl - 2 ( D ) , but not with AAA Myc - Bcl - 2 ( E ) , in HeLa cells transfected with WT Myc - Bcl - 2 ( D ) or AAA Myc - Bcl - 2 ( E ) along with either empty Flag or Flag - Beclin 1 . Asterisk denotes IgG band ( E ) . ( F ) Immunoprecipitation with anti - Flag M2 affinity agarose gel and immunoblotting with anti - Vps34 antibody shows that NO donors decreased Flag - Beclin 1 - hVps34 interaction in HeLa cells transfected with hVps34 along with either empty Flag or Flag - Beclin 1 . ( G ) Immunoprecipitation with anti - Flag M2 affinity agarose gel and immunoblotting with anti - Vps34 antibody shows that AAA Myc - Bcl - 2 decreased Flag - Beclin 1 - hVps34 interaction in the presence ( right panel ) or absence ( left panel ) of DETA NONOate in HeLa cells transfected with hVps34 along with either empty Flag or Flag - Beclin 1 , and with pcDNA3 . 1 or AAA Myc - Bcl - 2 . Graphical data denote mean ± SEM .

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

Figure 2(A) NO donors reduced JNK1 phosphorylation in HeLa cells, as analyzed by immunoprecipitation with anti-JNK1 agarose-conjugated beads and immunoblotting with anti-phospho-JNK antibody.(B) NO donors reduced Bcl-2 phosphorylation in HeLa cells, as analyzed by immunoblotting with anti-phospho-Bcl-2 antibody.C) Immunoblot analysis with anti-phospho-Bcl-2 antibody shows that DETA NONOate could not reduce phospho-Bcl-2 in Flag-CA JNK1-transfected HeLa cells, where phospho-Bcl-2 was higher compared to mock-transfected cells.(D and E) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Myc antibody shows that NO donors increased the interaction of Flag-Beclin 1 with WT Myc-Bcl-2 (D), but not with AAA Myc-Bcl-2 (E), in HeLa cells transfected with WT Myc-Bcl-2 (D) or AAA Myc-Bcl-2 (E) along with either empty Flag or Flag-Beclin 1. Asterisk denotes IgG band (E).(F) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that NO donors decreased Flag-Beclin 1-hVps34 interaction in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1.(G) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that AAA Myc-Bcl-2 decreased Flag-Beclin 1-hVps34 interaction in the presence (right panel) or absence (left panel) of DETA NONOate in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1, and with pcDNA3.1 or AAA Myc-Bcl-2. Graphical data denote mean ± SEM.


Figure 3 ( A ) Immunoblot analysis with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated WT Bcl - 2 and AAA Bcl - 2 MEFs . ( B and C ) NO donors decreased EGFP - LC3 vesicles in EGFP - LC3 - transfected WT Bcl - 2 and AAA Bcl - 2 MEFs ( C ) . Images were acquired by a confocal microscope ( B ) . WT Bcl - 2 and AAA Bcl - 2 MEFs were analyzed separately . ( D ) NO donors increased EGFP - HDQ74 aggregates in EGFP - HDQ74 - transfected WT Bcl - 2 and AAA Bcl - 2 MEFs . WT Bcl - 2 and AAA Bcl - 2 MEFs were analyzed separately . ( E ) Immunoblot analysis with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated WT Jnk , Jnk1 − / − , and Jnk2 − / − MEFs . ( F ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in HeLa cells . ( G ) Confocal microscope images of immunofluorescence with anti - phospho - S6 antibody show that DETA NONOate increased S6 phosphorylation ; arrowhead shows a cell where this effect was not observed . ( H ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in WT Bcl - 2 and AAA Bcl - 2 MEFs . ( I ) Confocal microscope images of immunofluorescence with anti - phospho - mTOR and anti - LAMP1 antibodies in HeLa cells show that DETA NONOate increased phospho - mTOR but did not alter its distribution with lysosomes . Graphical data denote mean ± SEM .

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

Figure 3(A) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Bcl-2 and AAA Bcl-2 MEFs.(B and C) NO donors decreased EGFP-LC3 vesicles in EGFP-LC3-transfected WT Bcl-2 and AAA Bcl-2 MEFs (C). Images were acquired by a confocal microscope (B). WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately.(D) NO donors increased EGFP-HDQ74 aggregates in EGFP-HDQ74-transfected WT Bcl-2 and AAA Bcl-2 MEFs. WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately.(E) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Jnk, Jnk1−/−, and Jnk2−/− MEFs.(F) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in HeLa cells.(G) Confocal microscope images of immunofluorescence with anti-phospho-S6 antibody show that DETA NONOate increased S6 phosphorylation; arrowhead shows a cell where this effect was not observed.(H) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in WT Bcl-2 and AAA Bcl-2 MEFs.(I) Confocal microscope images of immunofluorescence with anti-phospho-mTOR and anti-LAMP1 antibodies in HeLa cells show that DETA NONOate increased phospho-mTOR but did not alter its distribution with lysosomes. Graphical data denote mean ± SEM.


Figure 4 ( A ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in Tsc2 + / + MEFs , but not in Tsc2 − / − , MEFs . ( B ) NO donors reduced phosphorylation of IKKα / β and AMPKα in MEFs , as analyzed by immunoblotting with anti - phospho - IKKα / β and anti - phospho - AMPKα antibodies , respectively . ( C ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in Ikkβ + / + MEFs , but not in Ikkβ − / − MEFs . ( D ) Immunoblot analysis with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated Tsc2 + / + and Tsc2 − / − MEFs . ( E ) DETA NONOate decreased EGFP - LC3 vesicles in Tsc2 + / + and Tsc2 − / − MEFs transfected with EGFP - LC3 , along with either pcDNA3 . 1 or AAA Myc - Bcl - 2 . Although it further reduced EGFP - LC3 vesicles in Tsc2 + / + MEFs expressing AAA Myc - Bcl - 2 compared to mock - transfected Tsc2 + / + MEFs , this effect was not seen in Tsc2 − / − MEFs . Tsc2 + / + and Tsc2 − / − MEFs were analyzed separately . Graphical data denote mean ± SEM .

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

Figure 4(A) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Tsc2+/+ MEFs, but not in Tsc2−/−, MEFs.(B) NO donors reduced phosphorylation of IKKα/β and AMPKα in MEFs, as analyzed by immunoblotting with anti-phospho-IKKα/β and anti-phospho-AMPKα antibodies, respectively.(C) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Ikkβ+/+ MEFs, but not in Ikkβ−/− MEFs.(D) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated Tsc2+/+ and Tsc2−/− MEFs.(E) DETA NONOate decreased EGFP-LC3 vesicles in Tsc2+/+ and Tsc2−/−MEFs transfected with EGFP-LC3, along with either pcDNA3.1 or AAA Myc-Bcl-2. Although it further reduced EGFP-LC3 vesicles in Tsc2+/+MEFs expressing AAA Myc-Bcl-2 compared to mock-transfected Tsc2+/+MEFs, this effect was not seen in Tsc2−/−MEFs. Tsc2+/+ and Tsc2−/−MEFs were analyzed separately. Graphical data denote mean ± SEM.


Figure 5 ( A - D ) Immunoblot analyses with anti - LC3 , along with anti - nNOS , anti - iNOS , and anti - eNOS antibodies , show decreased LC3 - II levels in stable HEK293 cell lines overexpressing nNOS ( A and D ) , iNOS - GFP ( B and D ) , or eNOS ( C and D ) compared to HEK293 control cells in the presence or absence of bafilomycin A1 . ( E and F ) mCherry - LC3 vesicles were reduced in mCherry - LC3 - transfected stable HEK293 NOS cell lines compared to HEK293 control cells cultured in HBSS , an effect that was restored in the NOS cells by L - NAME ( F ) . Images were acquired by a confocal microscope ( E ) . ( G and H ) Confocal microscopy images ( G ) and immunofluorescence analysis with anti - Atg16 antibody show that stable HEK293 NOS cell lines had fewer Atg16 - positive structures under starvation , compared to HEK293 control cells ( H ) . Graphical data denote mean ± SEM .

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

Figure 5(A-D) Immunoblot analyses with anti-LC3, along with anti-nNOS, anti-iNOS, and anti-eNOS antibodies, show decreased LC3-II levels in stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and D), or eNOS (C and D) compared to HEK293 control cells in the presence or absence of bafilomycin A1.(E and F) mCherry-LC3 vesicles were reduced in mCherry-LC3-transfected stable HEK293 NOS cell lines compared to HEK293 control cells cultured in HBSS, an effect that was restored in the NOS cells by L-NAME (F). Images were acquired by a confocal microscope (E).(G and H) Confocal microscopy images (G) and immunofluorescence analysis with anti-Atg16 antibody show that stable HEK293 NOS cell lines had fewer Atg16-positive structures under starvation, compared to HEK293 control cells (H). Graphical data denote mean ± SEM.


Figure 6 ( A - F ) Immunoblot analyses with anti - nNOS , anti - iNOS , anti - eNOS , anti - phospho - JNK1 , anti - phospho - Bcl - 2 , anti - phospho - S6K , and anti - phospho - S6 antibodies show that stable HEK293 cell lines overexpressing nNOS ( A and D ) , iNOS - GFP ( B and E ) , or eNOS ( C and F ) inhibited both the JNK1 - Bcl - 2 and mTORC1 pathways compared to the HEK293 control cells under basal ( FM ) ( A - C ) or starvation ( HBSS ) ( D - F ) conditions . Graphical data denote mean ± SEM .

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

Figure 6(A-F) Immunoblot analyses with anti-nNOS, anti-iNOS, anti-eNOS, anti-phospho-JNK1, anti-phospho-Bcl-2, anti-phospho-S6K, and anti-phospho-S6 antibodies show that stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and E), or eNOS (C and F) inhibited both the JNK1-Bcl-2 and mTORC1 pathways compared to the HEK293 control cells under basal (FM) (A-C) or starvation (HBSS) (D-F) conditions. Graphical data denote mean ± SEM.


Figure 7 ( A ) Immunoblot analyses with anti - LC3 antibody show that rapamycin ( 8 hr ) and L - NAME ( 24 hr ) increased autophagosome synthesis in bafilomycin A1 - treated mouse primary cortical neurons and HeLa cells . ( B and C ) Confocal microscopy images ( B ) and analysis of autophagic flux by automated Cellomics microscope ( C ) show that L - NAME increased autolysosomes in mRFP - GFP - LC3 HeLa cells . ( D ) L - NAME reduced EGFP - HDQ74 aggregates in EGFP - HDQ74 - transfected Atg5 + / + , but not in Atg5 − / − , MEFs . ( E ) Drosophila expressing mutant huntingtin exon 1 ( Q120 ) shows a significant decrease in neurodegeneration ( p < 0 . 001 , paired t test ) upon L - NAME treatment compared to DMSO . ( F ) Expression of an endogenous negative regulator of NOS activity ( NOS4 ) significantly attenuates neurodegeneration ( p < 0 . 05 , paired t test ) in Drosophila expressing mutant huntingtin exon 1 ( Q120 ) . ( G - I ) Images from the retina of transgenic HD zebrafish show mutant huntingtin aggregates ( arrows ) ( G ) . Treatment with rapamycin or L - NAME reduced the number of aggregates ( H ) . L - NAME did not reduce aggregates in the presence of NH4Cl , which increased aggregate count ( I ) .

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

Figure 7(A) Immunoblot analyses with anti-LC3 antibody show that rapamycin (8 hr) and L-NAME (24 hr) increased autophagosome synthesis in bafilomycin A1-treated mouse primary cortical neurons and HeLa cells.(B and C) Confocal microscopy images (B) and analysis of autophagic flux by automated Cellomics microscope (C) show that L-NAME increased autolysosomes in mRFP-GFP-LC3 HeLa cells.(D) L-NAME reduced EGFP-HDQ74 aggregates in EGFP-HDQ74-transfected Atg5+/+, but not in Atg5−/−, MEFs.(E) Drosophila expressing mutant huntingtin exon 1 (Q120) shows a significant decrease in neurodegeneration (p < 0.001, paired t test) upon L-NAME treatment compared to DMSO.(F) Expression of an endogenous negative regulator of NOS activity (NOS4) significantly attenuates neurodegeneration (p < 0.05, paired t test) in Drosophila expressing mutant huntingtin exon 1 (Q120).(G-I) Images from the retina of transgenic HD zebrafish show mutant huntingtin aggregates (arrows) (G). Treatment with rapamycin or L-NAME reduced the number of aggregates (H). L-NAME did not reduce aggregates in the presence of NH4Cl, which increased aggregate count (I).


Figure 1IFM images of KIF13A - YFP transfected control and Rab - knockdown HeLa cells . TN : average tubule number ( mean ± SEM , n = 3 ) . B , C Graphs represent the measurement of KIF13A - positive TN ( B ) and TL ( C ) in HeLa cells of Fig 1A ( mean ± SEM ) . n = 3 . nc : total number of cells . nt : total number of tubules . arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10 μm . IFM images of KIF13A - YFP and mCherry - Rab7A / 11A / 22A transfected HeLa cells arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10 μmLive cell imaging of GFP / mCherry - Rab22A with respect to mCherry - Rab11A or GFP - Rab7A in HeLa cells . Magnified view of insets ( at 0 , 20 , 40 seconds ) are shown separately ) , arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10 μmPull - down of different His - KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins . The bead bound His - KIF13A in each pull - down was shown as coomassie gel . * , non - specific bands .

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

Figure 1IFM images of KIF13A-YFP transfected control and Rab-knockdown HeLa cells. TN: average tubule number (mean±SEM, n=3). B, C Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 1A (mean± SEM). n=3. nc: total number of cells. nt: total number of tubules.arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm.IFM images of KIF13A-YFP and mCherry-Rab7A/11A/22A transfected HeLa cells arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μmLive cell imaging of GFP/mCherry-Rab22A with respect to mCherry-Rab11A or GFP-Rab7A in HeLa cells. Magnified view of insets (at 0, 20, 40 seconds) are shown separately ), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μmPull-down of different His-KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins. The bead bound His-KIF13A in each pull-down was shown as coomassie gel. *, non-specific bands.


Figure 2IFM images of KIF13A - YFP and mCherry - Rab22AWT / Q64L / S19N cotransfected HeLa cells . Arrowheads and arrows point to the KIF13A - positive tubular REs and E / SEs respectively . The colocalization coefficient ( r , in mean ± SEM ) between two markers was indicated separately . Scale bars : 10 μm Graphs represent the measurement of KIF13A - positive TN ( B ) and TL ( C ) in HeLa cells of Fig 2A . n = 3 . nc : total number of cells . nt : total number of tubulesSubcellular fractionation of HeLa cells to probe the localization of Rab22A ( red box ) with respect to other organelle specific proteins . * , non - specific bands .

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

Figure 2IFM images of KIF13A-YFP and mCherry-Rab22AWT/Q64L/S19N cotransfected HeLa cells. Arrowheads and arrows point to the KIF13A-positive tubular REs and E/SEs respectively. The colocalization coefficient (r, in mean±SEM) between two markers was indicated separately. Scale bars: 10 μm Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 2A. n=3. nc: total number of cells. nt: total number of tubulesSubcellular fractionation of HeLa cells to probe the localization of Rab22A (red box) with respect to other organelle specific proteins. *, non-specific bands.


Figure 3IFM images of KIF13A - YFP transfected control and Rab22A - , BLOC - 1Mu - , BLOC - 2HPS6 - knockdown HeLa cells . Cells were stained for AP - 1 ( γ ) or internalized with Tf - Alexa fluor 594 arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10 μmGraphs represent the measurement of KIF13A - positive TN ( B ) and TL ( C ) in HeLa cells of Fig 3A . n = 3 . nc : total number of cells . nt : total number of tubulesImmunoblotting analysis of proteins in control and Rab22A - , BLOC - 1 - , BLOC - 2 - depleted HeLa cells . * , non - specific bands . Protein band intensities were quantified and indicated on the gels . IFM images of mCherry - Rab22A and KIF13A - YFP cotransfected control and BLOC - 1Mu - , BLOC - 2HPS6 - knockdown HeLa cells ) , arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10

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

Figure 3IFM images of KIF13A-YFP transfected control and Rab22A-, BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells. Cells were stained for AP-1 (γ) or internalized with Tf-Alexa fluor 594 arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μmGraphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 3A. n=3. nc: total number of cells. nt: total number of tubulesImmunoblotting analysis of proteins in control and Rab22A-, BLOC-1-, BLOC-2-depleted HeLa cells. *, non-specific bands. Protein band intensities were quantified and indicated on the gels.IFM images of mCherry-Rab22A and KIF13A-YFP cotransfected control and BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells ), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


Figure 4BF and IFM images of control and Rab22A - depleted melanocytes . The colocalization coefficient ( r , in mean ± SEM ) between the two markers indicated separately . Nuclei are stained with Hoechst33258 ) , arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1 / GFP - Rab22A . Scale bars : 10 μmGraph represents the quantified melanin content in control sh , Rab22A sh and BLOC - 1 - melanocytes . n = 3 . The fold - change in melanin content ( mean ± SEM ) indicated separatelyImmunoblotting analysis of proteins in Control sh and Rab22A sh melanocytes ) , protein band intensities were quantified and indicated on the gelsEM of control and Rab22A - knockdown MNT - 1 melanocytes . Arrowheads indicate PMEL fibrils . Scale bars : 1 μm . I , II , III , IV : stages of melanosomes . M : mitochondria . Magnified view of insets are shown separately and they emphasize the stage I melanosomes in both conditions . Note : the presence of hybrid stage I / II melanosomes in Rab22A - knockdown cells . Insets ( b ) - ( d ) and ( g ) - ( h ) are from different cells of respective condition . Scale bars : 200 nm . BF and IFM images of GFP - Rab22A transfected BLOC - 1 - ( Mu ) melanocytes ) , arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1 / GFP - Rab22A . Scale bars : 10 μmImmunoblotting analysis of proteins in BLOC - 1 - and BLOC - 1R melanocytes protein band intensities were quantified and indicated on the

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

Figure 4BF and IFM images of control and Rab22A-depleted melanocytes. The colocalization coefficient (r, in mean±SEM) between the two markers indicated separately. Nuclei are stained with Hoechst33258 ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μmGraph represents the quantified melanin content in control sh, Rab22A sh and BLOC-1- melanocytes. n=3. The fold-change in melanin content (mean±SEM) indicated separatelyImmunoblotting analysis of proteins in Control sh and Rab22A sh melanocytes ), protein band intensities were quantified and indicated on the gelsEM of control and Rab22A-knockdown MNT-1 melanocytes. Arrowheads indicate PMEL fibrils. Scale bars: 1 μm. I, II, III, IV: stages of melanosomes. M: mitochondria. Magnified view of insets are shown separately and they emphasize the stage I melanosomes in both conditions. Note: the presence of hybrid stage I/II melanosomes in Rab22A-knockdown cells. Insets (b)-(d) and (g)-(h) are from different cells of respective condition. Scale bars: 200 nm.BF and IFM images of GFP-Rab22A transfected BLOC-1-(Mu) melanocytes ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μmImmunoblotting analysis of proteins in BLOC-1- and BLOC-1R melanocytes protein band intensities were quantified and indicated on the gels


Figure 5Subcellular membrane fractionation of control and Rab22A - , BLOC - 1 - , BLOC - 2 - knockdown HeLa cells and probed the fractions for pallidin , dysbindin and muted . Red colored box emphasizes BLOC - 1 membrane association in the respective cell types . Cytosol - membrane fractionation of homogenates from HeLa cells ( B as indicated . Protein band intensities were quantified and indicated the percentage membrane association on the gelsCytosol - membrane fractionation of homogenates fro melanocyte as indicated . Protein band intensities were quantified and indicated the percentage membrane association on the gelsPull - down of His - Rab22AWT using HeL lysate the bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel separatelPull - down of His - Rab22AWT usin melanocyte ( E ) lysate . In E , the beads were preloaded with GTPγS or GDP the bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel separatelPull - down of different His - KIF13A domains using HeLa cell lysate the bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel

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

Figure 5Subcellular membrane fractionation of control and Rab22A-, BLOC-1-, BLOC-2-knockdown HeLa cells and probed the fractions for pallidin, dysbindin and muted. Red colored box emphasizes BLOC-1 membrane association in the respective cell types.Cytosol-membrane fractionation of homogenates from HeLa cells (B as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gelsCytosol-membrane fractionation of homogenates fro melanocyte as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gelsPull-down of His-Rab22AWT using HeL lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatelPull-down of His-Rab22AWT usin melanocyte (E) lysate. In E, the beads were preloaded with GTPγS or GDP the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatelPull-down of different His-KIF13A domains using HeLa cell lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel


Figure 1C . Biotinylation of the centrosome and microtubules by V5 - BirA * - AURKA in different cell cycle stages . After 18 h biotin incubation , HEK293T : : V5 - BirA * - AURKA were fixed and stained for the fusion protein with V5 antibody , biotinylated proteins with the fluorescent streptavidin and the centrosome with gamma - tubulin antibody . DNA was stained with DAPI . Scale bar , 5 μm . D . Control and biotin - treated HEK293T cells stably expressing V5 - BirA * and V5 - BirA * - AURKA were lysed and biotinylated proteins were precipitated by streptavidin beads . The initial sample ( initial ) and captured biotinylated proteins ( pulldown ) were run on an SDS gel and immunoblotted with the fluorescent streptavidin and antibodies against V5 and vinculin ( loading control ) .

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

Figure 1C. Biotinylation of the centrosome and microtubules by V5-BirA*-AURKA in different cell cycle stages. After 18 h biotin incubation, HEK293T::V5-BirA*-AURKA were fixed and stained for the fusion protein with V5 antibody, biotinylated proteins with the fluorescent streptavidin and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 5 μm.D. Control and biotin-treated HEK293T cells stably expressing V5-BirA* and V5-BirA*-AURKA were lysed and biotinylated proteins were precipitated by streptavidin beads. The initial sample (initial) and captured biotinylated proteins (pulldown) were run on an SDS gel and immunoblotted with the fluorescent streptavidin and antibodies against V5 and vinculin (loading control).