Fig. 6

Syntaxin 17 (STX17) initiates mitophagy in the hierarchical autophagic pathway. a The schematic model illustrating the genetic hierarchy and temporal relationship of autophagy proteins. b, c Wild-type (WT) or mitochondrial fission 1 protein knockout (Fis1 KO) HeLa cells were transfected with the indicated plasmids for 24 h. Cells were fixed and imaged by fluorescence microscopy. Hoechst, blue. Scale bar, 10 µm. Arrowheads indicate mitophagic cells. d, g Fis1 KO cells were transfected with respective small interfering RNA (siRNA) to silence the indicated genes for 24 h. Then, green fluorescent protein (GFP)-tagged vector or STX17 (green) was transfected into cells for further 24 h. Mitochondrial Tim23 (red) and LC3 (cyan) signals are shown by confocal microscopy. Hoechst, blue. Scale bar, 10 µm. Arrowheads indicate mitophagic cells. e, h Quantification of mitophagy percentage of d or g. Error bar represents SD. ***P < 0.001 (two-tailed unpaired Student’s t test from three independent experiments, n = 150). f, i Fis1 KO HeLa cells were transfected with the indicated siRNA for 24 h and then treated with respective siRNA again plus GFP-tagged vector or STX17 for further 6 h. Hereafter, cells were cultured in medium with or without chloroquine (CQ) for another 66 h. Cell lysates were immunoblotted for outer mitochondrial membrane (OMM), intermembrane space (IMS), inner membrane mitochondrial (IMM), and matrix proteins. j A schematic summary of the recruitment of canonical autophagy proteins for STX17 overexpression-induced mitophagy of Fis1 KO cells