Fig. 6.

Effect of U18666A on the synthesis of 27-HC and HMGR activity. A: 27-HC biosynthesis. Four flasks were incubated overnight in medium A. Pairs of flasks were then incubated for 2 h at 37° C in medium A containing either 0.2% ethanol (black bar) or 0.2% ethanol + 4.5 μM U18666A (gray bar). To increase cell cholesterol, the medium in each flask was replaced with 4 ml PBS, a pulse of 18 mg HPCD bearing 0.43 mg cholesterol added, and the flasks incubated for 7 min at 37°C. The buffer was replaced with medium A containing 0.2% ethanol 4.5 μM U18666A and the flasks incubated at 37°C for an additional 3 h. 27-HC was then determined in triplicate on each of the duplicate flasks, a zero-time background of 4.6 ng 27-HC/mg protein subtracted and the data plotted as means ± SEM relative to the control. B: HMGR activity. The experiment was as in A except that single flasks were used, HMGR activity was determined in duplicate, and the data expressed as the mean ± average deviation relative to controls. Data are from one of two similar experiments.