Fig. 7
From: STX17 dynamically regulated by Fis1 induces mitophagy via hierarchical macroautophagic mechanism
Transcription factor EB (TFEB) and Rab7 are involved in syntaxin 17 (STX17)-initiated mitophagy upon mitochondrial fission 1 protein (Fis1) loss. a Wild-type (WT) or Fis1 knockout (KO) HeLa cells were co-transfected with plasmids encoding green fluorescent protein (GFP)-Rab7 (green) and blue fluorescent protein (BFP)-STX17 (blue) for 24 h. Cells were fixed and immunostained for Tim23 (red) and Lamp2 (cyan). Cells were visualized as three-dimensional reconstruction images. b Fis1 KO HeLa cells were transfected with control-, TBC1D15-, or Rab7-small interfering RNA (siRNA) for 24 h. Cells were transiently transfected with GFP-tagged vector or STX17 (green) for further 24 h. Mitochondria and autophagosome were highlighted by Tim23 (red) and LC3 (cyan). Hoechst, blue. Scale bar, 10 µm. Arrowhead indicates mitophagic cell. c Quantification of mitophagic cells in b. ***P < 0.001 (two-tailed unpaired Student’s t test from three independent experiments, n = 150). d Fis1 KO cells were transfected with control- or Rab7-siRNA for 24 h. Cells were then again transfected with respective siRNA, plus GFP-tagged vector or STX17 for further 6 h. Hereafter, cells were cultured with or without chloroquine (CQ) for another 66 h. Cells were harvested and lysed, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel separation and immunoblotting against overall mitochondrial proteins. e WT or Fis1 KO HeLa cells were co-transfected with GFP-tagged vector or STX17 (green) and TFEB-Flag (cyan) for 24 h. Cells were fixed and immunostained against Tom20 (red) and Flag (cyan). Hoechst, blue. Scale bar, 10 µm. Arrowhead indicates mitophagic cell. f WT or Fis1 KO HeLa cells treated as in e were fractioned, lysed, and immunoblotted. Asterisk indicates non-specific bands. g Quantification of f. TFEB-Flag expression level was normalized to Tubulin (cytosol) or C23 (nuclear). Nuclear TFEB-Flag was indicated as the percentage of total TFEB-Flag. **P < 0.01, NS, not significant (two-tailed unpaired Student’s t test, n = 3). Error bars represent SD (c and g)
