Supplementary Figure 5: Exogenously supplied cholesterol is transported to the lysosome via an OSBP-dependent route.

a, HEK-293T cells stably expressing Tmem192-mRFP-3xHA (T192^-mRFP) were treated with DMSO or 20 nM OSW-1 for 2h, labeled with TopFluor-cholesterol (TF-Chol) complexed with MCD, chased at the indicated times, fixed, and imaged on a spinning disk confocal microscope. Scale bar, 10 µm. b, Quantitation of co-localization of TF-Chol with T192^-mRFP-labeled lysosomes in cells treated with DMSO or OSW-1 (box plots showing the min, 1^st quartile, median, 3^rd quartile, and max, data expressed as the fraction of T192^-mRFP-labeled lysosomes that are positive for TF-Chol, 10 fields of view for each treatment/time point; n represents cell number: DMSO_18 min (n = 106), DMSO_30 min (n = 85), DMSO_60 min (n = 78), DMSO_90 min (n = 77), DMSO_180 min (n = 87), OSW-1_18 min (n = 100), OSW-1_30 min (n = 82), OSW-1_60 min (n = 85), OSW-1_90 min (n = 83), and OSW-1_180 min (n = 107). Total peak area derived from the Area Under Curve (AUC) analysis: DMSO, 60.94 ± 3.111, OSW-1, 14.86 ± 1.849). Statistics source data are provided in Supplementary Table 2.