Fig. 1.
Generation of ROS in the mitochondrial inner membrane induces Parkin translocation and mitophagy, A. Menadione induces rapid production of both mitochondrial superoxide and hydrogen peroxide. PC6 cells stained with mitochondrial superoxide indicator MitoSOX™ Red (0.5 μM, left panel) or transfected with genetically encoded mitochondrial (middle panel) or cytosolic (right panel) H2O2 Hyper7 sensors were treated with 20 μM menadione and exposed for 385 nM LED Light Source for 1s at the end of the experiment to induce a massive ROS generation (positive control). Curves are depicted as mean ± SEM. Insets show subcellular localization of the indicator or sensor before the treatment. B. Menadione induces Parkin translocation. Representative superresolution Airyscan images of PC6 cells transfected with Parkin-EYFP (green) and mitochondria-targeted Kate2 (red) after treatment with DMSO or 20 μM menadione for 4 h. C. Quantification of Parkin-EYFP translocation to the mitochondria. The percentage of PC6 cells with Parkin-EYFP translocated to the mitochondria increases after menadione treatment in a concentration-dependent manner. *P < 0.05, ***P < 0.001 and ****P < 0.0001, n = 6 dishes, 20 fields per dish, Welch's ANOVA followed by Dunnett's T3 multiple comparison test. D. Co-treatment with ROS scavenger glutathione inhibits the menadione-induced Parkin translocation. The percentage of PC6 cells with Parkin-EYFP translocated to mitochondria in the presence of 20 μM menadione or/and 500 μM GSH-MEE. ****P < 0.0001, n = 3–6 dishes, 20 fields per dish, one-way ANOVA followed by Sidak's multiple comparison test. E. Menadione-induced ROS trigger mitophagy. Representative superresolution Airyscan images of cortical neurons transfected with a mix of EGFP-LC3B and GFP-LC3C (green) and mitochondria-targeted Kate2 (red) after treatment with DMSO or 20 μM menadione for 4 h. F. Quantification of the number of mitochondria co-localised with the autophagosome marker in neurons. *P < 0.05, **P < 0.01 and ***P < 0.001, n = 5 dishes, 5–10 cells per dish, one-way ANOVA followed by Dunnett's T3 multiple comparison test. G. Co-treatment with ROS scavenger glutathione inhibits the menadione-induced mitophagy in neurons. The number of mitochondrial colocalizations with EGFP-LC3B and GFP-LC3C in the presence of 20 μM menadione or/and 500 μM GSH-MEE. **P < 0.01 and ****P < 0.0001, n = 6 dishes, 8 cells per dish, one-way ANOVA followed by Sidak's multiple comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)