Supplementary Figure 6: OSBP regulates cholesterol-induced lysosomal recruitment and activation of mTORC1.

a, OSBP drives sterol-dependent lysosomal recruitment of mTORC1. HEK-293T cells expressing the indicated shRNAs were subjected to cholesterol depletion, followed by restimulation with MCD:cholesterol where indicated, and subjected to immunofluorescence for endogenous mTOR and LAMP2. Representative confocal microscopic images are shown. Scale bar, 10 μm. See quantitation in Fig. 2c. Experiments repeated two times. b, OSW-1 abolishes sterol-induced lysosomal recruitment of mTORC1. HEK-293T cells were subjected to cholesterol starvation and restimulation in the presence of DMSO or OSW-1 at the indicated concentrations and subjected to immunofluorescence for endogenous mTOR and LAMP2. Scale bar, 10 µm. c, Quantitation of co-localization between mTOR and LAMP2-positive lysosomes in cells subjected to the indicated treatments (mean ± s.d., 10 fields of view per treatment; n represents cell number: – chol (n = 150), + chol (n = 123), 1 nM OSW-1 + chol (n = 155), 10 nM OSW-1 + chol (n = 163), 20 nM OSW-1 + chol (n = 172), 50 nM OSW-1 + chol (n = 180), ANOVA with Dunnett’s multiple comparison test. **** Adjusted P = 0.0001 vs. + chol). d, Depletion of OSBP inhibits mTORC1 lysosomal localization in cells under full media conditions. HEK-293T cells depleted of OSBP were subjected to immunofluorescence for endogenous mTOR and LAMP2. Representative confocal microscopic images are shown. Scale bar, 10 μm. e, Quantitation of co-localization between mTOR and LAMP2-positive lysosomes in cells cultured in full media (mean ± s.d., 10 fields of view per genotype; n represents cell number: shLuc (n = 103), shOSBP (n = 118), two-tailed, unpaired t-test. ****P = 5.68202 x 10^-15 vs. shLuc). f, knockdown of OSBP impairs basal state mTORC1 signaling. Cells expressing the indicated shRNAs were harvested and analyzed by immunoblotting for the indicated proteins and phospho-proteins. Experiments repeated two times. g, Depletion of OSBP has a modest effect on mTORC1 activation by amino acids. HEK-293T cells expressing the indicated shRNAs were either starved of amino acids for 50 min, or starved and restimulated with amino acids for 10 min. Cell lysates were immunoblotted for the indicated proteins and phospho-proteins. Fold changes of the protein phosphorylation are indicated. Experiments repeated two times. h, Loss of OSBP does not affect lysosomal localization of the Ragulator-Rag GTPase complex. HEK-293T cells depleted of OSBP were subjected to cholesterol starvation and restimulation, followed by immunofluorescence for endogenous p18, RagC and LAMP2, respectively. Scale bar, 10 μm. See quantitation in Fig. 3a. Experiment performed once. Statistics source data are provided in Supplementary Table 2.