Figure 4.

Cholesterol ester synthesis and neutral lipid accumulation in SV-589 cells expressing wild-type and SCD-associated UBIAD1. A and B, SV589(ΔUBIAD1)/pMyc-UBIAD1 (WT) and SV589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 as described in the legend to Fig. 2. On day 1, the cells were refed medium A containing 10% DFCS. On day 2, the cells were refed the identical medium supplemented with 0.5 μCi/ml [³H]oleate and incubated for the indicated periods of time at 37 °C. Following incubations, the cells were harvested for preparation of lysates from which lipids were extracted and analyzed by TLC as described under “Experimental procedures.” Incorporation of [³H]oleate into [³H]cholesterol esters was determined by scintillation counting. The values were corrected for background as described in the legend to Fig. 2. Each value is the mean of triplicate incubations (± standard error). C, SV589(ΔUBIAD1)/pMyc-UBIAD1 (WT) and SV589(ΔUBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 at 3 × 10⁵ cells/60-mm dish with glass coverslips in medium A containing 5% FCS and 1 mm mevalonate. On day 1, the medium was switched to medium A supplemented with 10% FCS. On day 2, the cells were fixed and double-stained with 4′6-diamino-phenylindole for nuclei (blue) and with oil red O for neutral lipids (red) as described under “Experimental procedures.” D, quantification of the oil red O signal was performed using ImageJ. The values are the average of 10 images (± standard error). Scale bar, 20 μm. The p values were calculated using the Student's t test: ***, p ≤ 0.001.