Supplementary Figure 2: OSBP inactivation via OSW-1 treatment abrogates the accumulation of lysosomal membrane cholesterol in NPC1-deficient cells.
a, OSW-1 treatment in human NPC1 patient-derived fibroblasts abolishes cholesterol deposition on the lysosomal limiting membrane. Human patient cells were treated with either DMSO or 20 nM OSW-1 for 8h, fixed, permeabilized with LN2, subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, and stained for endogenous LAMP2. Scale bar, 10 μm. b, Quantitation of co-localization of D4H*-mCherry with filipin-labeled cholesterol deposits in human NPC1 patient-derived cells treated with either DMSO or OSW-1 (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 10 fields of view per treatment; n represents cell number: DMSO (n = 11), OSW-1 (n = 10), two-tailed, unpaired t-test. ****P = 1.07121 x 10-12 vs. DMSO). c, OSW-1 treatment in NPC1 knockout MEFs significantly reduces cholesterol levels on the lysosomal limiting membrane. NPC1 knockout MEFs were treated with either DMSO or 20 nM OSW-1 for 8h, fixed, permeabilized with LN2, subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, and stained for endogenous LAMP1. Scale bar, 10 μm. d, Quantitation of co-localization of D4H*-mCherry with filipin-labeled cholesterol deposits in NPC1 knockout MEFs treated with either DMSO or OSW-1 (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 10 fields of view per treatment; n represents cell number: DMSO (n = 30), OSW-1 (n = 34), two-tailed, unpaired t-test. ****P = 8.39052 x 10-15 vs. DMSO). e, OSW-1 treatment in NPC1-null cells significantly removes cholesterol levels on the lysosomal limiting membrane. NPC1-deleted HEK-293T cells expressing FLAG-GFP-Tmem192 were treated with either DMSO or 20 nM OSW-1 for 8h, fixed, permeabilized with LN2, and subjected to cholesterol labeling by GST-D4H*-mCherry and filipin. Scale bar, 10 μm. f, Quantitation of co-localization of D4H*-mCherry with filipin-labeled cholesterol deposits in NPC1-null cells treated with either DMSO or OSW-1 (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 10 fields of view per treatment; n represents cell number: DMSO (n = 66), OSW-1 (n = 73), two-tailed, unpaired t-test. ****P = 8.69018 x 10-11 vs. DMSO). Statistics source data are provided in Supplementary Table 2.