OPTN-mediated pexophagy is cell type–dependent and correlates with endogenous OPTN and PEX14 levels. (A) Recruitment of OPTN to peroxisomes varies by cell type. Representative images of HCT116 and HeLa cells expressing OPTN-GFP and mCherry-PTS1, either alone (left panels) or together with a high-affinity plasma membrane–targeted anti-GFP nanobody (GFP_Nb-PM; right panels). Prior to analysis, cells were treated with 200 nM Baf A1 for 2 h. White arrowheads indicate colocalization of OPTN-GFP and mCherry-PTS1; yellow arrowheads mark peroxisomes recruited to the plasma membrane; and blue arrowheads indicate GFP_Nb-PM–induced foci, which may represent concentration-dependent condensates (López-Palacios and Andersen, 2023; Yang et al., 2024). (B and C) OPTN-GFP expression induces pexophagy in HCT116 but not in HeLa cells. HCT116 and HeLa cells stably expressing po-mKeima were transfected with a plasmid encoding either GFP or OPTN-GFP and processed for FACS analysis 2 days later, as described in the Materials and methods section. Pexophagy was quantified by gating GFP-positive cells based on reduced fluorescence in the neutral channel. (B) Representative flow cytometry plots (n = 3); color indicates cell density at a given position. (C) Quantification of the percentage of cells within the pexophagy gate. Data represent the mean ± SD from three independent biological replicates. Statistical comparisons were made relative to the GFP control (ns, nonsignificant; **, P < 0.01). (D–F) Relative expression levels of OPTN and PEX14 in HEK-293, HCT116, and HeLa cells. (D) Total lysates were analyzed by SDS-PAGE and IB using antibodies against OPTN, PEX14, and GAPDH. Representative blots are shown with molecular mass markers (kDa) indicated on the left. (E and F) Densitometric quantification of relative levels: (E) OPTN and (F) PEX14. Signal intensities were normalized to the mean value in HEK-293 cells, set at 100%. Bars represent mean ± SD from 3 biological replicates. Source data are available for this figure: SourceData FS1.