Fig. 4: The role of STX17-SNAP29-VAMP7 complex in the mitochondria-lysosome contacts and MMEL.

a–c WT or ATG5 knockdown (shATG5) HeLa cells stably expressing GFP-STX17 (green) were exposed to normoxia or hypoxia for 24 h, and were immunostained with antibodies against TOMM20 (mitochondria, red), or LAMP1 (lysosome, blue), and were analyzed by confocal microscopy with Airyscan (a). The mode of localization of GFP-STX17 in “A” was drawn (b), green: GFP-STX17, red: TOMM20, blue: LAMP1 (lysosome). The number of GFP-STX17 puncta locating at megamitochondria-lysosome contact sites per cell were quantified (c), n = 10 cells for each experiment. d Control or ATG5 knockdown HeLa cells were transfected with or without Flag-STX17, cell lysates were immunoprecipitated (IP) with anti-Flag M2 affinity gel, followed by Western blotting using anti-SNAP29, anti-VAMP7, or anti-Flag antibodies. e,f Control, Syntaxin-17 knockdown (shSTX17) HeLa cells stably expressing mito-DsRed (mitochondria) were exposed to normoxia or hypoxia for 24 h, and were immunostained with an antibody against LAMP1 (lysosome), and were analyzed and imaged with confocal microscopy with Airyscan. Mitochondria (red) and lysosome (green) were displayed using 3D surface reconstructions overlaid upon original data with Imaris software (e). The bottom images are 3D surface reconstructions of the middle images (e). Mitochondria-lysosome contacts (contacts) and MMEL (engulfment) in control, STX17 knockdown (f) (10 cells in each experiment) were quantified, and the number of M-L contacts and MMEL per 100 lysosomes was displayed. All bars 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.