Fig. 7: MMEL promotes mitochondrial ROS production under hypoxia.

a–c Control or STX17 knockdown (shSTX17) HeLa cells expressing mito-GFP were cultured in normoxia or hypoxia for 24 h and then stained with mitoSOX (a). The relative mitoSOX fluorescence intensity (ratio to mito-GFP fluorescence intensity) of the fragmented, tubular, or large spherical mitochondria (megamitochondria,) in cells described in (a) was further analyzed. 300 mitochondria from 30 cells under normoxia or hypoxia were quantified (b). c The relative level of the mitoSOX fluorescence intensity was displayed. d, e HeLa cells treating with DMSO or N-acetyl-L-cysteine (NAC) were cultured in or hypoxia for 0 h (normoxia), 24 h, or 48 h. Cell lysates were analyzed by Western blotting using the indicated antibodies (d). The relative protein levels were evaluated by densitometry analysis (e). Error bars indicate the mean ± SD of the experiments, n = 3 independent experiments, statistical significance was assessed by two-tailed t-test. f–h Control or STX17 knockdown (shSTX17) HeLa cells stably expressing mitoGFP (mitochondria) treating with DMSO (control) or N-acetyl-L-cysteine (NAC) were cultured in or hypoxia for 0 h (normoxia), 24 h, and then immunostained with anti-LAMP1 antibodies, and analyzed by 3D imaging with confocal microscopy with Airyscan. Mitochondria (green) and lysosome (red) were displayed using 3D surface reconstructions (f). The events of mitochondria-lysosome contacts and megamitochondria engulfing lysosome (MMEL) from 10 cells were quantified in each experiment, and the number of M-L contacts and MMEL per 100 lysosomes was displayed (g). The number of megamitochondria described in “f” were also quantified (h) according to the criteria detailed in “Methods”. i Under normoxia (a), mitochondria-lysosome contacts contribute to mitochondrial fission. Under hypoxia (b), many normal mitochondria contact (mitochondria-mitochondria contact, M-M contact) and fuse to form megamitochondria, which then contact and engulf lysosomes with the help of the STX17-SNAP29-VAMP7 complex. Then lysosomes release some hydrolases to degrade mitochondrial proteins and other molecules. In addition, mitochondrial proteases (MP) are activated in hypoxia, which cooperates with MMEL to mediate the inverted mitophagy. Bars of (b, c, g and h) represent mean ± SEM, n = 3 independent experiments, statistical significance was assessed by a two-way ANOVA. P values are indicated in the figure. Source data are provided as a Source Data file.