Figure S3.
SNX10 is dispensible for mitophagy of a matrix reporter and MDV formation.(A) U2OS cells stably expressing iMLS-GFP-mCherry were reverse transfected with Ctrl, SNX10, or ULK1 siRNAs (20 nM) for 72 h. DFP was added for the last 24 h and 50 nM BafA1 was added 16 h before fixation. Scale bar: 20 µm. The graph represents the mitolysosome area per cell from >1,000 cells based on images taken with ImageXpress Micro Confocal (Molecular devices) at 20× magnification. The bars show the means normalized to the control (siCtrl) cells ± SEM (n = 3). Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. (B) U2OS cells stably expressing MLS-GFP-mCherry and non-tagged Parkin were reverse transfected with Ctrl and SNX10 siRNAs (20 nM) for 72 h. CCCP was added for the last 24 h and 50 nM BafA1 was added 2 h before fixation. Scale bar: 20 µm. The graph represents the mitolysosome area per cell from >1,000 cells based on images taken with ImageXpress Micro Confocal (Molecular devices) at 20× magnification. The bars show the means normalized to the control (siCtrl) cells ± SEM (n = 3). Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. (C) Representative images of U2OS cells transfected with 20 nM siRNA: siCtrl (control) and two different siSNX10 oligoes (siSNX10 #1 and siSNX10 #2). Cells were stained with an anti-PDH and anti-TOMM20 antibody after fixation. Images were acquired using a Nikon CREST X-Light V3 spinning disk microscope utilizing a 60× oil objective. Scale bar: 10 µm. (D) Quantification of the data shown in C, performed using CellProfiler software. The graph displays the number of MDVs per cell, calculated as vesicles positive for TOMM20 only or PDH only (n = 3, >100 cells per condition in each replicate). Significance was assessed by two-way ANOVA followed by Dunnett’s multiple comparison test. Data distribution was assumed to be normal but was not formally tested. BafA1; bafilomycin A1 and CCCP; carbonyl cyanide m-chlorophenyl hydrazine.

SNX10 is dispensible for mitophagy of a matrix reporter and MDV formation. (A) U2OS cells stably expressing iMLS-GFP-mCherry were reverse transfected with Ctrl, SNX10, or ULK1 siRNAs (20 nM) for 72 h. DFP was added for the last 24 h and 50 nM BafA1 was added 16 h before fixation. Scale bar: 20 µm. The graph represents the mitolysosome area per cell from >1,000 cells based on images taken with ImageXpress Micro Confocal (Molecular devices) at 20× magnification. The bars show the means normalized to the control (siCtrl) cells ± SEM (n = 3). Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. (B) U2OS cells stably expressing MLS-GFP-mCherry and non-tagged Parkin were reverse transfected with Ctrl and SNX10 siRNAs (20 nM) for 72 h. CCCP was added for the last 24 h and 50 nM BafA1 was added 2 h before fixation. Scale bar: 20 µm. The graph represents the mitolysosome area per cell from >1,000 cells based on images taken with ImageXpress Micro Confocal (Molecular devices) at 20× magnification. The bars show the means normalized to the control (siCtrl) cells ± SEM (n = 3). Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. (C) Representative images of U2OS cells transfected with 20 nM siRNA: siCtrl (control) and two different siSNX10 oligoes (siSNX10 #1 and siSNX10 #2). Cells were stained with an anti-PDH and anti-TOMM20 antibody after fixation. Images were acquired using a Nikon CREST X-Light V3 spinning disk microscope utilizing a 60× oil objective. Scale bar: 10 µm. (D) Quantification of the data shown in C, performed using CellProfiler software. The graph displays the number of MDVs per cell, calculated as vesicles positive for TOMM20 only or PDH only (n = 3, >100 cells per condition in each replicate). Significance was assessed by two-way ANOVA followed by Dunnett’s multiple comparison test. Data distribution was assumed to be normal but was not formally tested. BafA1; bafilomycin A1 and CCCP; carbonyl cyanide m-chlorophenyl hydrazine.

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