Mitophagy induced by Parkin. (a) HeLa cells stably expressing GFP-LC3 (green) transfected with mCherry-Parkin (not depicted) and treated with 10 μM CCCP for 1 h. Parkin-negative cells (left) display less overlap between autophagosomes and mitochondria (red) than Parkin-positive cells (right), as assessed by (b) counting the number of mitochondria encapsulated by LC3-positive autophagosomes in >30 cells per condition in at least three independent experiments. (c) HeLa cells stably expressing GFP-LC3 (green) and transiently transfected with mCherry-Parkin (white) were immunostained for cytochrome c (red) to reveal colocalization of LC3, Parkin, and mitochondria after 1 h exposure to CCCP. Arrows indicate mitochondria that colocalize with both mCherry-Parkin and GFP-LC3. Insets show an enlarged view of the boxed areas. (d) YFP-Parkin (green)-induced mitochondrial removal after 24 h of CCCP (10 μM) exposure observed in WT MEFs (left) failed to occur in ATG5−/− MEFs (right) quantified (e) in ≥150 cells in at least three experiments. Cells were stained for Tom20 (red). Outlines demarcate the edges of cells expressing YFP-Parkin. (f) 3-methyladenine (3MA) and bafilomycin blocked Parkin-induced mitophagy in HeLa cells quantified as in panel e. Error bars indicate standard deviation of at least three replicates. Bars: (c and d) 10 μm; (a and c, insets) 1 μm.