p97-UBXD8 suppresses peroxisome flux through autophagy. (A) Schematic for pexophagy flux reporter eGFP-mCherry-PEX26. (B) Representative images of WT and UBXD8 KO cells transfected with eGFP-mCherry-PEX26. (C) WT and UBXD8 KO cells were transfected with the flux reporter and treated with 150 nM Torin1 for 18 h. Quantification showing the ratio of eGFP to (eGFP⁺mCherry⁺) in HeLa WT and UBXD8 KO cells. 50–100 cells were analyzed in N = 3 independent experiments. The violin plot shows median and 95% confidence intervals. **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA with Šidák’s multiple comparisons test. (D) Representative images of HeLa control or p97 siRNAs and eGFP-mCherry-PEX26. (E) Quantification showing the ratio of eGFP to (eGFP⁺mCherry⁺) in HeLa control, or p97-depleted cells. 50–100 cells were analyzed in N = 3 independent experiments. The violin plot shows median and 95% confidence intervals. *P < 0.05, **P < 0.001, **** P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. (F) Immunoblot showing p97 depletion. (G) Quantification showing the ratio of eGFP to (eGFP⁺mCherry⁺) in HeLa untreated, or CB5083 (10 μM for 2 h)-treated cells. 50–100 cells were analyzed in N = 3 independent experiments. The violin plot shows median and 95% confidence intervals. **P < 0.001, unpaired t test. The scale bar is 10 μM and 5 μM. Source data are available for this figure: SourceData F7.