Figure 3. Cholesterol and NEFA accumulation in Wt and NPC1 late endosomes and fibroblasts.

(A) The amount of cholesterol (CH) and NEFAs (FFA) in Wt and NPC1 endosomes was determined as described in the Materials and methods section. Cholesterol and NEFA levels in NPC1 endosomes were expressed as percentages of the amount in Wt endosomes, which was arbitrarily set at 100%. Values represent the average levels found in four independent preparations. (B) The amount of cholesterol (CH) in Wt and NPC1 crude PMS was quantified as described in the Materials and methods section. Values are expressed as above and represent the mean of four independent preparations. (C) Analysis of NEFA levels in Wt, NPC1-null and NPC1-I1061T human fibroblasts. An amount of lipid equivalent to 50 μg of cellular protein was extracted, separated by TLC and visualized as described in Figure 2. Wt1, GM5387 cells; Wt2, GM41B cells; Null, DMN98.16 cells; I1061T, GM3123 cells. (D) Analysis of cholesterol and NEFA levels in vesicles isolated from Wt, heterozygous (Het) and NPC1 mice. The amount of cholesterol in vesicles was determined using a diagnostic kit as described in the Materials and methods section. For NEFA analysis, an amount of lipid equivalent to 25 μg of vesicular protein was extracted, separated by TLC, visualized and quantified as described in Figure 2. Cholesterol and NEFA levels were expressed as percentages of the amount in Wt endosomes, which was arbitrarily set at 100%. Values represent the average levels found in three independent preparations from different mice.