Fig. 3.

Syntaxin 17 (STX17)-induced mitophagy is PINK/Parkin-independent. a Wild-type (WT) or mitochondrial fission 1 protein knockout (Fis1 KO) HeLa cells were transfected with Flag-tagged vector or STX17 and green fluorescent protein (GFP)-tagged Parkin (green) for 24 h. Cells were analyzed by immunofluorescence microscopy using antibodies against Tom20 (red) and Flag (cyan). Hoechst, blue. Scale bar, 10 µm. White arrow indicates cell with STX17 overexpression-mediated mitophagy. b Bar chart shows the percentage of cells with Parkin translocation as indicated in a. Parkin translocation upon carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment at 10 µM for 4 h was quantified as positive control. Data are mean ± SD (n = 150 cells from three independent experiments). ***P < 0.001 (two-tailed unpaired Student’s t test). c WT or Fis1 KO HeLa cells were incubated with dimethyl sulfoxide (DMSO) or 10 µM CCCP for 16 h and then fixed and immunostained for Tim23 (red) and Fis1 (green). Cells transiently expressing GFP-tagged Parkin and treated with 10 µM CCCP for 16 h were used as a positive control for mitophagy. Hoechst, blue. Scale bar, 10 µm. d HeLa cells expressing GFP-tagged vector or STX17 were treated with 10 µM CCCP for 16 h and immunostained for Tim23 (red). Hoechst, blue. Scale bar, 10 µm. e WT or Fis1 KO HeLa cells were transfected with GFP-tagged vector or STX17 for 72 h. SH-SY5Y cell lysate was used as the positive control for the expression of endogenous Parkin. Cell lysates were immunoblotted for Parkin and mitochondrial proteins. f HeLa cells stably expressing GFP-Parkin (green) were transfected with small interfering RNA (siRNA) against control, Fis1 or STX17 for 48 h. Then, cells were treated with or without 10 µM CCCP for further 4 h. Cells were visualized by immunostaining for Tim23 (red) and LC3 (cyan). Hoechst, blue. Scale bar, 10 µm