Fig. 3.

Oxidative stress causes extensive mitochondrial ubiquitination but mitophagy occurs independent of ubiquitin-binding autophagy receptors. HeLa cells were treated with 500 μM CoCl₂ along with 25 μM Z-VAD-FMK for 24 h. A. The cells were either treated with CoCl₂ alone or in combination with NAC. The isolated mitochondrial fractions were analyzed for the extent of total ubiquitin, K48 and K63-linked polyubiquitin by immunoblotting. B. The cells were stained with Mitotracker Red followed by immunostaining with LC-3B and Ubiquitin antibodies. Panels on the right display co-localization between mitochondria, ubiquitin and LC-3B by fluorescence intensity line measurement (yellow line). Scale bar: 10 μm. C. The whole cell extracts of CoCl₂ treated or control HeLa WT and 5KO cells were analyzed for HIF-1α, MFN2 and tubulin (loading control) by immunoblotting. Alternatively, isolated mitochondrial fractions from these cells were subjected to western blotting for detection of total ubiquitination and HSP60 (loading control). D. Control or CoCl₂ treated WT and 5KO HeLa cells stably expressing Mitokeima were analyzed by FACS for mitophagy quantification (ns: not significant). Data is shown as mean + SEM of three experiments. E. Control, CoCl₂ and CCCP (10 μM for 6 h) treated HeLa cells overexpressing Parkin were immunostained with TOM20 and OPTN or NDP52 antibodies. Scale bar: 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)