FIGURE 1.

Cellular cholesterol uptake mediated by different NPC1L1 variants. A, model of membrane topology of human NPC1L1. The 19 NS variants of NPC1L1 identified from low cholesterol absorbers are shown with yellow circles. The blue and red strips indicate the transmembrane domains of NPC1L1, and the red ones denote the sterol-sensing domain. B, cellular cholesterol uptake mediated by NPC1L1. Equal amounts of plasmids encoding human wild-type and the 19 NS variants of NPC1L1 were transfected into CRL-1601 cells, respectively. 24 h after transfection, cells were incubated in cholesterol-depleting medium to reduce cellular cholesterol, and then were refed with cholesterol-replenishing medium to deliver cholesterol. Then the cells were fixed, stained with filipin, and examined by confocal microscopy. Arrows indicate the cells without expression of the transfected plasmids. Arrowheads show the cells expressing the indicated NPC1L1 variants. Scale bar, 10 μm. C, quantification of cellular cholesterol uptake mediated by different NPC1L1 variants in B. For each variant, the filipin fluorescence intensity was measured by Image-Pro Plus 5.02 as described under “Experimental Procedures.” The dashed red line denotes the 1.5-fold of total cellular cholesterol of non-transfected control cells. Error bars represent standard deviations.