Journal: Redox Biology

Article Title: Ubiquitination and receptor-mediated mitophagy converge to eliminate oxidation-damaged mitochondria during hypoxia

doi: 10.1016/j.redox.2021.102047

Figure Lengend Snippet: Genuine hypoxia causes mitochondrial oxidative stress leading to their ubiquitination. HeLa cells were exposed to 1% Oxygen for 24 h (Hypoxia) or 21% Oxygen (Normoxia). A. The cell lysates corresponding to the indicated time points following hypoxia were analyzed for detection of HIF-1α and GAPDH by western blotting. B. The cells were immunostained with HSP60 antibody followed by image acquisition on a confocal microscope and the percent mitochondrial fragmentation was determined by evaluating a minimum of 100 cells across 10 different microscopic fields. C. The Oxygen Consumption Rate (OCR) was determined by Seahorse Extracellular Flux analyzer using the MitoStress test kit. D. The cells were stained with live cell imaging dyes Mitotracker Green and MitoSOX red and observed under a confocal microscope. The MitoSOX Red staining intensity was expressed in terms of fold change relative to the control. At least 100 cells across 10 different microscopic fields per condition were evaluated. E. The cells were exposed to normoxia and hypoxia alone or in combination with 1 μM Mitoquinone Q (MitoQ) or vehicle control (VC) and whole cell extracts were subjected to immunoblotting for the detection of HIF-1α, PHD2 and Actin (loading control). F. The cells were stained with MitoTracker Red followed by immunostaining with LC-3B antibody. G. The whole cell lysates of HeLa cells exposed to Normoxia (N) and Hypoxia (H) were subjected to immunoblotting for detection of the indicated mitochondrial proteins. H. The isolated mitochondrial fractions from control and CoCl 2 treated cells (left) and normoxic and hypoxic cells (right) were subjected to western blotting for evaluating the level of total ubiquitin and ATP5a (loading control). I. Wild type HeLa cells were stained with Mitotracker Red followed by immunostaining with Ubiquitin and LC-3B antibodies. The numbered circles mark their corresponding position in the individual channels and in the merged image to indicate the extent of their overlap. Scale bar for microscopy images: 10 μm. The blots are representative of three experiments. The data is expressed as mean + SEM of three experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: To identify the extent by which mitochondrial ROS contribute to HIF-1α stabilization in response to CoCl 2 , we treated cells with the mitochondria-targeted antioxidant Mitoquinone Q (MitoQ).

Techniques: Western Blot, Microscopy, Staining, Live Cell Imaging, Immunostaining, Isolation