The pathogenic Spastin^K388R mutant disrupts LD–peroxisome contact formation, leading to peroxidated lipid accumulation in LDs. (A) Schematic diagram representing lipid peroxidation assay using BODIPY-C11 to monitor LD peroxidation. (B) Colocalization of BODIPY-C11– and BODIPY-665/676–labeled LDs in HeLa cells. Representative confocal MIP images are shown. (C) Relative lipid peroxidation in LDs in control and in Halo-M1 Spastin– or Halo-M1 Spastin^K388R–overexpressing HeLa cells in the absence or presence of cumyl-OOH. Means ± SEM are shown (32–56 cells from at least three independent experiments). **, P < 0.01; ***, P < 0.001. (D) Distribution of NBD-C12 in Halo-M1 Spastin^K388R and mCherry-SKL–overexpressing HeLa cells following pulse-chase described in Fig. 7 D. Representative confocal images are shown. (E) Relative NBC-C12 intensity in peroxisomes as described in D. Means ± SEM are shown (15 cells from three independent experiments). Gray dashed line is a replica from Fig. 7 F. (F and G) Association between LDs and peroxisomes in HeLa cells coexpressing mApple-M1 Spastin and mTagBFP2-M1 Spastin or mTagBFP2-M1 Spastin^K388R (F), or coexpressing mApple-M1 Spastin and mTagBFP2-M87 Spastin or mTagBFP2-M87 Spastin^K388R (G). Representative confocal MIP images are shown. (H) Fraction of LD overlapping with peroxisome as described in F and G. Means ± SEM are shown (13–22 cells from two independent experiments). ***, P < 0.001.