Figure 4

PE alters mitochondrial morphology basally and enhances mitophagosome formation to facilitate mitophagy under CCCP stress. (a) Left: Immunofluorescence images of TOMM20 in SY5Y cells treated with DMSO, 10 µM CCCP or 300 μg/ml PE for 16 h. Representative views of the different types of mitochondrial morphology are shown in the smaller inlets. Right: Quantification of mitochondrial interconnectivity (Top) and percentage of cells containing fragmented or “donut” mitochondrial morphology (Bottom). Mitochondrial interconnectivity is expressed as fold change relative to DMSO. (b) Electron micrographs depicting different mitochondrial morphology in SY5Y cells treated with DMSO or 300 μg/ml PE for 24 h. Donut- and C-shaped (partial donut) mitochondria are highlighted by yellow arrows. Autophagic vacuoles (red arrow) and lysosomes (green arrow) are found in close proximity to the donut/C-shaped mitochondria. (c) Left: Immunofluorescence images of TOMM20 and LC3 colocalization in SY5Y cells treated with DMSO, 300 µg/ml PE or PE with 1 µM vinblastine (Vb), in the absence (Basal/-CCCP) or presence of 10 µM CCCP for 16 h. Smaller inlets show close-up views of colocalization between TOMM20 and LC3. Right: Quantification of TOMM20 and LC3 colocalization per cell area, calculated as fold change against DMSO. Nuclei were stained with DAPI. 20 cells from random fields were analyzed for mitochondrial interconnectivity and TOMM20-LC3 colocalization. (d) Top: Immunoblot of TOMM20 in SY5Y cells treated with 10 µM cycloheximide (CHX), over a 12 h time-course treatment with 10 μM CCCP in the absence or presence of 300 μg/ml PE. Bottom: Quantification of TOMM20 levels at each time point, expressed as fold change against 0 h. Full-length blots are presented in Supplementary Figure S9c. (e) Top: Immunoblot of TOMM20 in SY5Y cells treated with 10 µM cycloheximide (CHX) and subjected to 10 μM CCCP alone or supplemented with 300 μg/ml PE in the absence or presence of lysosomal inhibition (NL with 1 µM Vb) for 12 h. Bottom: Quantification of CCCP-induced TOMM20 flux in the absence or presence of PE, expressed as TOMM20 levels under lysosomal inhibition against untreated condition. Full-length blots are presented in Supplementary Figure S9d. All values are mean + S.E.M (n = 3–7). Differences against DMSO or CCCP treatment only are significant at *p < 0.05, **p < 0.01, ***p < 0.005, ^#p < 0.05 and ^###p < 0.005. N.S = Not significant. Scale bar is 10 μm, unless otherwise stated.