Supplementary Figure 1: OSBP-delivered cholesterol accumulates on the surface of NPC1-null lysosomes. | Nature Cell Biology

Supplementary Figure 1: OSBP-delivered cholesterol accumulates on the surface of NPC1-null lysosomes.

From: ER–lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann–Pick type C

Supplementary Figure 1

a, Coomassie blue staining of recombinant GST-tagged D4H*-mCherry. b, NPC1-deleted HEK-293T cells were treated with 1 μM LysoSensor Green DND-189 for 30 min, fixed, permeabilized with liquid nitrogen (LN2) where indicated, and subjected to cholesterol labeling by GST-D4H*-mCherry and filipin. Scale bar, 10 μm. c, Accumulation of D4H*-mCherry on the plasma membrane of non-permeabilized cells. Control HEK-293T cells were subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, and stained for endogenous LAMP2. Scale bar, 10 μm. d, NPC1 knockout MEFs display cholesterol accumulation at the lysosomal membrane, in addition to the lysosomal lumen. Wild type and NPC1 knockout MEFs were fixed, breached with LN2, subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, and stained for endogenous LAMP1. Scale bar, 10 µm. e, NPC1 inhibition via U18666A treatment induces cholesterol deposition at the limiting membrane as well as in the lumen of lysosomes. Control human fibroblasts were treated with U18666A (5 µg/ml) for 6h and subjected to cholesterol labeling and LAMP2 staining. Scale bar, 10 µm. f, Quantitation of co-localization of D4H*-mCherry with filipin-labeled cholesterol deposits in control human fibroblasts treated with either DMSO or U18666A (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), U18666A (n = 10), two-tailed, unpaired t-test. ****P = 5.35648 x 10-13 vs. U18666A). g, Depletion of OSBP significantly suppresses the accumulation of lysosomal membrane cholesterol in human NPC1 patient-derived fibroblasts. Human NPC1 patient-derived fibroblasts depleted of ORPs via shRNA were subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, then stained for endogenous LAMP2. Scale bar, 10 µm. See quantitation in Fig. 1f. Experiments in a–d, and g performed independently two times. Statistics source data are provided in Supplementary Table 2.

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