Fig. 2.

Dependence of the biosynthesis of 27-HC on cholesterol load in wild-type and NPC1-deficient cells. Replicate flasks were preincubated overnight in medium A. The cells were layered with 4 ml PBS, pulses of HPCD ± cholesterol added, and the flasks incubated for 7 min at 37°C. For depletion, the pulses contained from 10 to 32 mg HPCD; for enrichment, the pulses contained mixtures ranging from 6.6 mg HPCD + 0.16 mg cholesterol to 19.5 mg HPCD + 0.47 mg cholesterol. This buffer was replaced with medium A, and the flasks incubated at 37°C for 4 h. Then, total 27-HC, cholesterol, and cell protein values were determined. (O) Wild-type fibroblasts (2 experiments); (Δ) NPC1-deficient fibroblasts (2 experiments). An average zero-time background value of 4.6 ng 27-HC/mg cell protein was subtracted from each data point. 27-HC and cholesterol values were normalized to those obtained for the cells treated with 10 mg HPCD (the 1.0/1.0 point). The average values for the 1.0/1.0 points for the two wild-type cells at 4 h were 35 ± 3 μg cholesterol/mg protein and 13 ± 1 ng 27-HC synthesized/mg protein. The corresponding average values for the two NPC1 flasks were 62 ± 1 μg cholesterol/mg protein and 18 ± 0.5 ng 27-HC synthesized/mg protein. The two lines are linear least square fits to the data below and above the 1.0/1.0 point, excluding the NPC1 outlier.