Co-overexpression of PEX14 and OPTN enhances OPTN recruitment and induces peroxisome clustering and pexophagy in HeLa cells. (A and B) Overexpression of PEX14 promotes the recruitment of OPTN to peroxisomes. HeLa cells were co-transfected with plasmids encoding (A) mCherry-PEX14 and OPTN-GFP, or (B) non-tagged PEX14, OPTN-GFP, and mCherry-PTS1. After 2 days, live-cell imaging was performed. White arrowheads indicate colocalization of OPTN-GFP with mCherry-PTS1. Notably, under pexophagy-inducing conditions, the mCherry reporter redistributes markedly to the cytosol. Scale bars, 10 µm; insets: 2.5 µm. (C–H) HeLa cells stably expressing po-mKeima were transfected with plasmids encoding the indicated proteins and cultured in DMEM. After 2 days, cells were processed for GFP-Trap and FACS analyses. Pexophagy was assessed by gating single-cell GFP-positive populations for reduce fluorescence intensity in the neutral channel. (C) Protein extracts (input) and the GFP-Trap were analyzed by SDS-PAGE and IB using antibodies against GFP, PEX14, and GAPDH. Representative blots are shown. Specific protein bands are marked by arrows. Molecular mass markers (in kDa) are indicated on the left. (D) Densitometric quantification of the relative ratio (RR) of PEX14/GFP and PEX14/OPTN-GFP fusion proteins in the GFP-Trap. Signal intensities for both GFP and PEX14 were normalized to 100%. Bars represent the mean ± SD of three biological replicates. (E and F) Representative flow cytometry plots of each group (n = 3), where colors indicate cell density. (G and H) Quantification of the percentage of cells within the gated area. Data represent the mean ± SD from three independent biological replicates. Statistical comparisons were performed between relevant conditions (ns, nonsignificant; ***, P < 0.001; ****, P < 0.0001). Source data are available for this figure: SourceData F8.