KO of MARCH5 blocks Torin1-induced PMP70 degradation and pexophagy. (A) WT and MARCH5 KO HeLa cells were treated with Torin1 (1 μM) or DMSO for 24 h and immunoblotted with indicated antibodies. (B) The protein levels in A were quantified with ImageJ, then normalized to β-actin. Values are mean ± SD, n = 3 independent experiments. **, P < 0.01; ***, P < 0.001; ns, P > 0.05 by two-tailed Student’s t test. (C) WT and MARCH5 KO HeLa cells were treated with Torin1 (1 μM) or DMSO for 24 h as indicated and immunostained for PMP70. Scale bars: 7 µm. (D) Quantification of peroxisome numbers (green puncta) in cells treated in C. Values are mean ± SD, n = 3 independent experiments, calculated using >50 cells. ****, P < 0.0001; n.s., not significant by one-way ANOVA and Tukey’s multiple comparisons test. (E) WT and MARCH5 KO HeLa cells stably expressing RFP-GFP-SKL were treated with Torin1 (1 μM for 24 h), folimycin (10 nM for 24 h), or a combination of the two. Cells were then analyzed by flow cytometry.