Figure 2.

Reduced synthesis of cholesterol in SV-589 cells lacking UBIAD1. SV-589 and SV-589(ΔUBIAD1) cells were set up on day 0 at a density of 4 × 10⁵ cells/60-mm dish in medium A containing 10% FCS and 1 mm mevalonate. On day 1, the cells were switched to medium A containing 10% FCS. A, on day 2, the cells were refed the identical medium supplemented with 15 μCi/ml [¹⁴C]acetate; cold acetate was to obtain a final concentration of 0.5 mm. Following incubation for 4 h at 37 °C, the cells were harvested for preparation of lysates from which lipids were extracted and analyzed by TLC as described under “Experimental procedures.” Incorporation of [¹⁴C]acetate into [¹⁴C]cholesterol was determined by scintillation counting. A blank value, representing the amount of [¹⁴C]acetate incorporated into [¹⁴C]cholesterol in each cell line that was chilled to 4 °C, refed [¹⁴C]acetate-containing medium and immediately washed and extracted, was subtracted from each value. Each value is the mean of triplicate incubations (± standard error). B, on day 2, the cells were refed medium A supplemented with 10 μCi/ml [³H]mevalonolactone and either 10% FCS or LPDS. Following incubation for 4 h at 37 °C, the cells were harvested and lysed for lipid extraction; incorporation of [³H]mevalonolactone into [³H]cholesterol was determined as described in A. Each value is the mean of triplicate incubations (± standard error). The p values were calculated using Student's t test: NS, not significant; *, p ≤ 0.05; ***, p ≤ 0.001.