Fig. 5
PINK1 accumulation is unaltered in VPS35D620N cells as a result of depolarization via antimycin A and oligomycin. a Representative immunoblots of protein extracts from WT and VPS35D620N (clone 1 and clone 2) cells treated with DMSO, 1 μM antimycin A, 1 μM oligomycin, or a combination of both drugs for 24 h. Blots were stained with PINK1, Parkin and β-actin (total protein loading control) antibodies. b Quantification of PINK1 and Parkin levels from immunoblot analysis in (a). Shown are relative PINK1 and Parkin levels after 1 μM oligomycin or 1 μM antimycin A and 1 μm oligomycin stimulation for 24 h compared to the WT cells. Each red dot depicts a separate experiment. Statistical analyses were performed by one-way ANOVA followed by Tukey’s post-hoc test. c Representative fluorescence images of WT or VPS35D620N (clone 1 and 2) cells treated with 1 μM antimycin A and 1 μM oligomycin. From top to bottom: endogenous TOM20 staining; endogenous PINK1 staining; overlay image of TOM20 (magenta), PINK1 (green) and DAPI staining for nuclei (blue); zoom in of highlighted area in overlay image. Scale bar, 10 μm, or 4 μm for zoomed images. d Quantification of co-localization of endogenous TOM20 and PINK1 in WT and VPS35D620N (clones 1 and 2) cells treated with 1 μM antimycin A and 1 μM oligomycin, as shown in (c). Each dot represents the Pearson coefficient calculated for one cell. Red lines show the median Pearson coefficient per cell line. Statistical analysis was performed using a Kruskal-Wallis test followed by a pairwise Mann-Whitney U-test with Benjamini-Hochberg multiple testing correction. e Representative fluorescence images of WT (left panels) or VPS35D620N (right panels) cells that stably express COX8-EGFP-mCherry treated with 1 μM antimycin A and 1 μM oligomycin for 24 h. The signal from the yellow particles originates from EGFP and mCherry and highlights cytoplasmic mitochondria. The red particles show quenched EGFP signal and normal mCherry signal, reflecting mitochondria transported into an acidic compartment (mitophagolysosome). Scale bar, 10 μm. f Quantification of yellow and red particles in WT and VPS35 D620N cells in (e). Each dot represents the proportion of total yellow and red particles for one cell. Red lines show the median proportion per cell line per condition. Statistical analysis was performed using a beta regression. n.s. non-significant, *P < 0.05, **P < 0.01