SNX10 modulates piecemeal mitophagy of OXPHOS components. (A) U2OS cells stably expressing the reporter pSu9-Halo-mGFP were reverse transfected with siCtrl or siSNX10 for 72 h. Cells were treated with TMR (100 nM) for 20 min, washed three times with PBS, and then treated with DFP (1 µM), DMOG (1 µM), or left untreated (control) for 24 h before lysis. (B and C) The relative Free Halo Tag expression was quantified using the formula (Free Halo/[Free Halo + Full Length]) normalized to actin. Data in B were log₂ transformed. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison tests to compare treatment groups to the control. Data represent mean ± SEM from three independent experiments. Data distribution was assumed to be normal but was not formally tested. (D) U2OS cells with inducible expression of SNX10-EGFP were treated with or without DFP (1 µM) for 24 h and stained with antibodies anti–COX-IV and anti-p62, followed by acquisition with a Nikon Ti2-E microscope with a Yokogawa CSU-W1 SoRa spinning disk 100×/1.45 NA oil immersion objective. Pixel intensity plot line graphs from control and DFP insets were generated with GraphPad Prism using two different y axis to enhance visualization. Scale bar: 10 µm. Insets: 4.08 × 4.08 µm. (E) U2OS cells subjected to reverse transfection with sip62 (20 nM) for 72 h were stained with an anti–COX-IV and anti-p62 antibody, prior to acquisition with a Nikon Ti2-E microscope with a Yokogawa CSU-W1 SoRa spinning disk 100×/1.45 NA oil immersion objective. 100×/1.45 NA oil immersion objective. Scale bar: 10 µm. (F) Quantification of COX-IV intensity from E represented as z-score from one experiment with individual data points corresponding to a single field of view (>30 cells per siRNA). Significance was determined by an unpaired two-tailed t test. Data distribution was assumed to be normal, but this was not formally tested. (G) Representative images of U2OS cells subjected to reverse transfection with siSNX10 (20 nM) for 72 h, followed by treatment with either IN1 or MRT for 24 h before fixation. After fixation, cells were stained with a COX-IV antibody, and images were captured using an ImageXpress Micro Confocal (Molecular Devices) at 20× magnification. (H and I) Quantification of COX-IV intensity from G represented as z-score from one independent experiment, with individual data points corresponding to a single field of view (>200 cells were analyzed for each condition). Significance was determined by one-way ANOVA followed by Šídák’s multiple comparisons test. Data distribution was assumed to be normal, but this was not formally tested. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001; nonsignificant differences are not depicted. Source data are available for this figure: SourceData F7.