Supplementary Figure 3: Lysosomal localization of a subset of OSBP in cells.
a, A pool of OSBP is localized to LAMP2-positive lysosomes. (left) HEK-293A cells stably expressing wild type or the indicated variants of FLAG-GFP-OSBP were subjected to immunofluorescence for endogenous LAMP2. Scale bar, 10 µm. (middle) Quantitation of the fraction of LAMP2-positive lysosomes that are positive for OSBP (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 7 fields of view per genotype; n represents cell number: WT (n = 13), ΔELSK (n = 11), ΔPH (n =13), ANOVA with Dunnett’s multiple comparison test. ****Adjusted P = 0.0001 vs. WT cells). (right) Expression levels of the wild type FLAG-GFP-OSBP in relative to those of the endogenous OSBP in HEK-293A cells. Cells were harvested and subjected to immunoblot analysis. Experiment repeated two times. b, A pool of OSBP is localized to LAMP2-positive lysosomes where mTOR and p18 are present. (left) HEK-293A cells stably expressing either wild type or PH domain-deleted FLAG-GFP-OSBP were subjected to immunofluorescence for endogenous LAMP2, mTOR and p18, respectively. Scale bar, 10 µm. (right) Quantitation of the fraction of LAMP2-, mTOR-, p18-labeled vesicles that are positive for OSBP (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 7 fields of view per genotype; n represents cell number: LAMP2 for WT (n = 9), ΔPH (n = 12), two-tailed, unpaired t-test. ****P = 1.07095 x 10-6 vs. WT; mTOR for WT (n = 10), ΔPH (n = 12), two-tailed, unpaired t-test. ****P = 3.89303 x 10-7 vs. WT; p18 for WT (n = 11), ΔPH (n = 9), two-tailed, unpaired t-test. ****P = 1.54976 x 10-7 vs. WT). c, OSW-1 treatment triggers clustering of OSBP on lysosomes. (left) HEK-293A cells stably expressing FLAG-GFP-OSBP were treated with either DMSO or with the OSBP inhibitor OSW-1 (10 nM) for 2h and subjected to immunofluorescence for endogenous LAMP2. Scale bar, 10 µm. (right) Quantitation of the fraction of LAMP2-positive lysosomes that are positive for OSBP (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 7 fields of view per treatment; n represents cell number: DMSO (n = 11), OSW-1 (n = 10), two-tailed, unpaired t-test. ****P = 3.47655 x 10-8 vs. DMSO). d, Depletion of VAPA and VAPB increases the lysosomal localization of OSBP. (left) Cells expressing the indicated shRNAs were subjected to immunofluorescence for endogenous OSBP and LAMP2. Scale bar, 10 µm. (right) Quantitation of the fraction of LAMP2-positive lysosomes that are positive for OSBP (box plots showing the min, 1st quartile, median, 3rd quartile, and max, 10 fields of view per genotype; n represents cell number: shLuc (n = 18), shVAPA/B (n = 17), two-tailed, unpaired t-test. ****P = 9.24193 x 10-11 vs. shLuc). (bottom) knockdown of VAPA/B was validated by immunoblotting. Experiments repeated two times. Statistics source data are provided in Supplementary Table 2.