Figure 5

PE enhances the presence of PINK1 and Parkin on mitochondria to potentiate PINK1-Parkin dependent mitophagy under CCCP-induced stress. (a) Top: Immunofluorescence images of TOMM20 and PINK1 colocalization in SY5Y cells treated with DMSO, 10 µM CCCP or CCCP with 300 μg/ml PE for 16 h. Smaller inlets show close-up views of TOMM20 and PINK1 colocalization. Bottom: Quantification of TOMM20 and PINK1 colocalization per cell area, expressed as fold change against DMSO control. (b) Top: Immunofluorescence images of TOMM20 in mCherry-Parkin SY5Y stable cells treated with DMSO or 10 µM CCCP in the absence or presence of 300 μg/ml PE for 6 h. White arrows depict the colocalization between mitochondria aggregates and Parkin. Bottom: Quantification of percentage cells containing TOMM20 and Parkin colocalization. 20–30 cells from random fields were analyzed for the colocalization. Nuclei were stained with DAPI. (c,d) Top: Immunoblots of TOMM20 in WT (c) and GFP-Parkin (d) HeLa stable cells left untreated (UT), or treated with 10 µM CCCP or CCCP supplemented with 300 µg/ml PE for 2 h, 4 h, 6 h and 12 h. Bottom: Quantification of TOMM20 levels across the various treatment timepoints, expressed as fold change against 2 h UT condition. Full-length blots are presented in Supplementary Figure S10c and d. All values are mean + S.E.M (n = 3–7). Differences against DMSO, UT or CCCP are significant at *p < 0.05, **p < 0.01, ***p<0.005, ^#p < 0.05 and ^###p < 0.005. Scale bar is 10 μm, unless otherwise stated.