Canonical autophagy receptors are dispensable for peroxisome maintenance and OPTN-mediated pexophagy in HeLa cells. (A–D) OPTN-mediated pexophagy occurs independently of the canonical receptors NBR1 and SQSTM1. CT and ΔSQSTM1ΔNBR1 double knockout (DKO) HeLa cells were transfected with untagged PEX14 on day 1 and infected with lentiviral particles encoding either GFP or OPTN-GFP on day 2. On day 4, cells were harvested for IB using antibodies against GFP, PEX14, ABCD3, and GAPDH. (A) Representative blots, with molecular mass markers (in kDa) indicated on the left. (B–D) Densitometry analysis of the relative levels of (B) PEX14 (14), (C) ABCD3, and (D) OPTN-GFP. Signal intensities were normalized to the mean value of the PEX14/GFP-expressing CT cells (set to 100%). Bars represent the mean ± SD of three biological replicates. (E–H) Peroxisome abundance is maintained in HeLa cells lacking classical autophagy receptors. Control (CT) and pentaKO HeLa cells were analyzed by IB and IF. (E) Immunoblot validation of pentaKO HeLa cells. Lysates from control (CT) and pentaKO (KO) cells were subjected to IB using antibodies against the indicated proteins. Molecular mass markers (in kDa) are shown on the left. (F) Representative IF images showing peroxisomes in CT and pentaKO cells. Nuclei were counterstained with DAPI. Scale bar, 10 µm. (G) Quantification of relative peroxisome number. Each dot represents a single cell. To ensure consistency across cell lines, images were acquired at the nuclear focal plane to minimize potential bias from imaging depth. (H) Densitometric analysis of PEX14 protein levels in CT and pentaKO cells. Signal intensities were normalized to the mean value of CT cells. Data represent the mean ± SD from three biological replicates. Statistical comparisons were performed between relevant conditions (ns, not statistically significant; **, P < 0.01). Source data are available for this figure: SourceData F9.