Fig. 1.

A–C: HeLa cells were mock-transfected or transfected with anti-NPC1 siRNA for 72 h. When indicated in A, cells were treated for the last 24 h with 0.7 mM HPCD and fixed and stained with filipin (bar: 20 μm). In B, 36 h after transfection, cells were starved for 24 h in 10% LPDS and then labeled with [³H]OA for 12 h in 10% FCS. When indicated, 0.7 mM HPCD was added 12 h after starvation and maintained during OA incorporation. In C, cells were treated before the end of the 72 h with 0.7 mM HPCD, as indicated (all samples were treated equally with siRNAs, and the time of HPCD treatment was adjusted accordingly). Total mRNA was extracted, and LDLR mRNA was quantified by RT-PCR. D: HeLa NPC1 KO cells were treated for 24 h with 0.7 mM HPCD, and LDLR mRNA was quantified as in C. E: BHK cells were transfected as in A and treated or not for the last 24 h with 0.7 mM HPCD. A late endosomal fraction was prepared, and unesterified cholesterol was quantified (total cells and fractions) enzymatically. B–D: Data are expressed as a percentage of the mock control. UT, untreated.