Role of autophagy in UBXD8 regulation of peroxisome numbers. (A) HeLa WT and UBXD8 KO cells were treated with Torin1 (1 mM for 16 h) stained for peroxisomes using catalase. (B) Quantification of peroxisomes per cell. 100–150 cells were analyzed in N = 3 independent experiments. The violin plot shows median and 95% confidence intervals. **P < 0.01, ****P < 0.0001, two-way ANOVA with Dunnett’s multiple comparisons test. (C) HeLa WT and UBXD8 KO cells were depleted of ATG5 using siRNA. Cells were stained for peroxisomes using catalase. (D) Quantification of peroxisome abundance from A. 100–150 cells were analyzed in N = 3 independent experiments. The violin plot shows median and 95% confidence intervals. *P < 0.05, ****P < 0.0001, two-way ANOVA with Dunnett’s multiple comparisons test. (E) Immunoblot showing ATG5 depletion. (F) Immunoblot showing HeLa WT and UBXD8 KO cells treated with Torin1 (1 mM for 4 h) and bafilomycin A (50 nM for 4 h) indicating UBXD8 stabilization. Scale bars are 5 μM (A) and 10 μM (C). Source data are available for this figure: SourceData FS4.