Fig. 2

ORP1L is involved in endosomal cholesterol transport and signaling. a Guide RNA targeting sites for ORP1L and ORP1S. b Western blotting analysis of HeLa, ORP1L KO, and ORP1L/ORP1S DKO cells. c U18666A-mediated LEL cholesterol release assay in HeLa, ORP1L KO, and ORP1L/1S DKO cells (scale bar = 10 μm). d Quantitation of cytosolic filipin intensities of cells (6 h) in c. e Cholesterol esterification assay in HeLa, ORP1L KO, and ORP1L/1S DKO cells. TAG, triacylglycerol. f Quantitation of cholesteryl [¹⁴C]-esters formation in e by densitometry. g Western blotting analysis of HeLa, ORP1L KO, and ORP1L/ORP1S DKO cells starved in EBSS and then chased with FBS. h Densitometry of p-S6K/S6K in g. All values are means ± s.d. (error bar) (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant; n = 3–10) Statistical analyses use one-way ANOVA. i qRT-PCR analysis of PI3KC2β knockdown in ORP1L KO and HeLa control cells. j Cholesterol esterification assay in ORP1L KO and HeLa control cells with or without siPI3KC2β knockdown. k Quantitation of cholesteryl [¹⁴C]-esters formation in j by densitometry