Figure 5.

Mitophagy induction in response to the mitochondrial recruitment of ATG16L1[100-250] or ULK1 is dependent on PIK3C3 activity. (A) ARPE-19 mito-QC cells were transduced to express the indicated proteins for 48 h in the presence of VPS34-IN1 (1 μM) or DMSO (0.01%) for the entire 48 h. Cells were analyzed using widefield microscopy. Left: Representative images. Scale bar: 5 µm. Right: Quantification of mitophagy showing the mean number of mitolysosomes per cell from at least 3 independent experiments with a minimum of 33 cells being analyzed per condition in each experiment. Statistics: Two-way ANOVA + Tukey’s multiple comparisons test. (B) ARPE-19 cells stably expressing mitochondrially localized mCherry-FIS1[101-152] were transduced with the indicated proteins for 48 h in the presence of VPS34-IN1 (1 μM) for the entire 48 h or BafA1 (50 nM) for the last 24 h or were treated with DMSO as a control. Cells were lysed and samples were analyzed by immunoblotting. Left: Representative immunoblot. Right: Quantification of mitophagy showing the levels of the mitochondrial protein HSPD1 normalized to VCL from 4 independent experiments. Statistics: Two-way ANOVA + Tukey’s multiple comparisons test. (C) ARPE-19 cells stably expressing mitochondrially localized mCherry-FIS1[101-152] were transduced with the indicated proteins or were not transduced (CTRL) for 24 h in the presence of VPS34-IN1 (1 μM) or DMSO (0.01%) for the entire 24 h before immunofluorescence staining using an anti-WIPI2 antibody and confocal microscopy analysis. Left: Representative images. Scale bar: 10 µm. Right: Quantification of the mean number of WIPI2 puncta on mCherry-positive mitochondria from 3 independent experiments with a minimum of 34 cells being analyzed per condition in each experimental replicate. Statistics: wo-way ANOVA + Tukey’s multiple comparisons test.