Fig. 4.

PS restores plasma membrane-to-ER transport of LDL-derived cholesterol in PTDSS1^−/− cells. (A) SREBP-2 processing in PTDSS1^−/− and NPC1^−/− CHO-K1 cells incubated with PS. On day 0, cells were set up in medium C with 5% FCS. On day 2, cells were switched to cholesterol-depletion medium C in the absence or presence of 20 μM PS. After 16 h, cells were switched to medium D containing CPN/MEV and 10% FCS in the absence or presence of 20 μM PS. After 6 h, cells were harvested for immunoblotting of SREBP-2 and H3K9Me3. (B) Cholesteryl ester synthesis in cells incubated with phospholipids. On day 0, cells were set up in medium C with 5% FCS. On day 2, cells were switched to cholesterol-depletion medium C, and the indicated liposomes. After a 12-h incubation, cells were switched to medium D containing CPN/MEV, 10% FCS, and the indicated liposomes. After 4 h, cells were pulse-labeled for 2 h with 0.1 mM sodium [¹⁴C]oleate (9495 dpm/nmol), after which the cellular content of cholesteryl [¹⁴C]oleate was measured. Each value represents an average of duplicate incubations. (C) Measurement of PM cholesterol by flow cytometry. On day 0, cells were set up in medium C with 5% FCS. On day 2, cells were switched to cholesterol-depletion medium C in the absence or presence of 20 µM of the indicated liposomes. After a 16-h incubation, cells were switched to medium D with CPN/MEV and supplemented with either 5% calf LPDS or 10% FCS as indicated and 20 µM of the indicated liposomes. After 6 h, cells were harvested for flow cytometry using AF-488-conjugated-PFO*. (D) Stimulation of cholesteryl ester synthesis by cholesterol-MCD in cells incubated with or without PS. On day 0, cells were set up in medium C with 5% FCS. On day 2, cells were refed with cholesterol-depletion medium C in the absence or presence of 10 µM PS liposomes. After a 12-h incubation, cells received medium D containing CPN/MEV and the indicated amount of cholesterol-MCD in the absence (Left) or presence (Right) of 10 µM PS liposomes. After 4 h, cells were pulse-labeled for 2 h with 0.1 mM sodium [¹⁴C]oleate (4588 dpm/nmol), after which the cellular content of cholesteryl [¹⁴C]oleate was measured. Each value represents the average of duplicate incubations, with individual values shown.