FIGURE 6.

Overexpression of NPC1^I1061T rescues the cholesterol accumulation phenotype in npc1-null cells. A, npc1-deficient CHO cells were transiently transfected with either npc1^I1061T-GFP (top panels) or npc1^P692S-GFP (bottom panels) constructs and stained with filipin for unesterified cholesterol. Cells were examined by immunofluorescence for cholesterol (left panels) and GFP expression (right panels). npc1-null cells expressing npc1^I1061T-GFP (top panels, closed arrows) are filipin-negative, whereas non-transfected cells (top panels, open arrows) are filipin-positive indicative of NPC1 mutant phenotype. npc1-null cells expressing the non-functional mutant npc1^P692Sbottom panels, closed arrows) remain filipin-positive. Bar, 10 μm. B, NPC1-deficient human fibroblasts were transiently transfected with either GFP (left panel) or npc1^I1061T-GFP (right panel) constructs and tested for the ability to complement the NPC1 phenotype. Merged immunofluorescence images are shown for GFP (green) and cholesterol (blue) staining. In the right panel, npc1-null cells expressing npc1^I1061T-GFP (closed arrows) are complemented (low cholesterol staining), whereas in the left panel GFP expressing cells (closed arrows) are not complemented (high cholesterol staining). Non-transfected cells (open arrows) do not exhibit complementation. Bar, 50 μm.