Figure 5.

Effect of brefeldin A on sterol-regulated acyl-CoA:cholesterol acyltransferase activity. On day 0, CHO-7 cells were set up at 2.5 × 10⁵ cells per 60-mm dish in medium A supplemented with 5% FCS. On day 2, cells were switched to medium A containing 5% newborn calf lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.2% ethanol in the absence or presence (+ sterols) of 1 μg/ml 25-hydroxycholesterol plus 10 μg/ml cholesterol as indicated. After incubation at 37°C for 11 hr, the cells were incubated for an additional 5 hr with 0.1% methanol in the absence or presence of 10 μg/ml brefeldin A as indicated. During the final 2 hr, each monolayer was pulse-labeled with 0.2 mM sodium [¹⁴C]oleate (7,600 dpm/pmol), after which the cells were harvested for measurement of the rate of [¹⁴C]oleate incorporation into cholesteryl [¹⁴C]oleate as described previously (23). Error bars denote the SD of triplicate values.