Fig. 4.

The functional domains of mitochondrial fission 1 protein (Fis1) and syntaxin 17 (STX17) for mitophagy. a Schematic diagram of Fis1 truncations. The deleted regions are represented by dotted lines. b HEK293T Cells were transiently co-transfected with the indicated plasmids for 24 h. Cells were lysed and immunoprecipitated with anti-Flag antibody. Co-immunoprecipitated Myc-tagged STX17 was detected by immunoblotting. c Fis1 KO HeLa cells were co-transfected with green fluorescent protein (GFP)-tagged STX17 (green) and plasmids encoding mCherry-Fis1 truncations (red). Cells were fixed, stained with Tim23 antibody (cyan), and then imaged for fluorescence microscopy. Hoechst, blue. Scale bar, 10 µm. Arrowheads indicate mitophagic cells. d Quantification of c. ***P < 0.001, NS, not significant (two-tailed unpaired Student’s t test, n = 150 cells from three independent experiments). e Schematic representation of STX17 domain mutants. Dotted regions represent deleted domains. f HEK293T cells were co-transfected with the indicated plasmids. After 24 h, cells were extracted and co-immunoprecipitated with anti-Flag beads, followed by immunoblotting with anti-Myc and anti-Flag antibodies, respectively. g Fis1 KO cells were transfected with GFP-tagged vector or the indicated STX17 truncations for 24 h. Cells were imaged following immunostaining for Tim23 (red) and LC3 (cyan). Hoechst, blue. Scale bar, 10 µm. Arrowheads indicate mitophagic cells. h Quantitative analysis of mitophagy induced by STX17 full-length (FL) or truncations in Fis1 KO cells. ***P < 0.001, NS, not significant (two-tailed unpaired Student’s t test, n = 150 cells from three independent experiments). Data are mean ± SD (d, h)