Morphological characterization of OPTN-peroxisome clusters. (A) Distribution of OPTN-GFP and mCherry-PTS1 in HEK-293 cells in the presence or absence of a high-affinity plasma membrane–targeted anti-GFP nanobody. HEK-293 cells were co-transfected with plasmids encoding OPTN-GFP (OPTN) and mCherry-PTS1 (PTS1), with or without a plasmid coding for a high-affinity plasma membrane–targeted anti-GFP nanobody (GFP_Nb-PM). After 2 days, cells were treated with 200 nM Baf A1 for 2 h. Nuclei were counterstained with Hoechst 33258. Scale bars: 10 μm; insets: 2.5 µm. (B and C) Ectopic expression of OPTN-GFP induces peroxisome clustering and removal. HEK-293 cells were transfected with plasmids encoding either GFP or OPTN-GFP. After 2 days, the cells were processed for IF microscopy using anti-PEX14 antiserum. (B) Representative images of cells. Nuclei were counterstained with DAPI. Scale bar: 10 µm. (C) Quantification of relative peroxisome number. Each dot represents a single cell. To minimize potential bias due to imaging depth, images were acquired at the nuclear focal plane to ensure consistency across conditions. Statistical comparisons were made between transfected (+) and non-transfected (−) cells within each condition and between GFP- and OPTN-GFP–expressing cells (ns, nonsignificant; **, P < 0.01). Data are expressed as mean ± SD. (D) High-resolution imaging and 3D volume rendering of an mCherry-PTS1/OPTN-GFP aggregate in HEK-293 cells. Scale bar: 500 nm. (E and F) Maximum projection of an Airyscan confocal image stack of (E) HEK-293 cells and (F) MFF-deficient human fibroblasts (ΔMFF HuFs-T). The cells were pretreated with 100 nM Baf A1 for 6 h, immunostained for OPTN and PEX14, and analyzed by 3D Airyscan microscopy. Zoomed-in XY views highlight co-clusters of OPTN and PEX14, as indicated by the white color. Orthogonal XZ and YZ views show overlap across dimensions. Yellow dashed lines indicate positions of orthogonal sections.