Fig. 5.

KO of ERG28 results in reduced cholesterol synthesis. A and B: Wild-type or ERG28 KO cells were pretreated for 16 h in 10% FCLPDS DMEM:HG sterol-deficient media supplemented with 5 μM compactin with or without 10 μM 17-OHP and then radiolabeled with 1 μCi of [¹⁴C]-acetate for 4 h in sterol-deficient media with or without 10 μM 17-OHP. Cell lysates were normalized to total cellular protein concentration, and total nonsaponifiable sterols were harvested from the cells (A), dried, and separated on TLC Silica gel 60 F₂₅₄ plates in a heptane:ethyl acetate (2:1, v/v) mobile phase. Radiolabeled lipids were imaged after ∼ 1 week of exposure using the Typhoon FLA 9500 phosphor imager. Data were normalized to the wild-type and presented as mean ± SEM from n = 3 or 4 independent experiments. Statistical significance was determined using a paired two-tailed t test (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.005). B: Wild-type or ERG28 KO1 cells were radiolabeled in 10% FCS DMEM:HG with 2 μCi of [¹⁴C]-acetate for 4 h and lipids extracted as for A. TLC is representative of n = 2 independent experiments each conducted in triplicate. C–D: Huh7 wild-type cells and ERG28 KO1 cells were pretreated for 16 h in 10% FCLPDS DMEM:HG sterol-deficient media supplemented with 5 μM compactin with or without 10 μM 17-OHP and then radiolabeled with 1 μCi of [¹⁴C]-acetate for 2–24 h. Lipids were extracted as per Fig. 5A. Data were normalized to the wild-type 8 h time point and presented as mean ± SEM from n = 3 independent experiments. Statistical significance was determined using a paired two-tailed t-test, all time points were significant P < 0.05. E: After total nonsaponifiable sterols were harvested from the cells (Fig. 5A), the remaining lysate was acidified, and fatty acids were extracted, dried, and separated on TLC Silica gel 60 F₂₅₄ plates in a heptane:diethyl ether:acetic acid (90:30:1, v/v) mobile phase. F: Huh7 wild-type or ERG28 KO1 cells were transfected for 8 h with increasing amounts of CMV-ERG28-V5 as indicated, pretreated for 16 h in sterol-deficient media supplemented with 5 μM compactin, and then radiolabeled with 1 μCi of [¹⁴C]-acetate for 4 h in sterol-deficient media. Lipids were extracted as per Fig. 5A. Data expressed as levels of [¹⁴C]-sterol in KO compared with wild-type for each plasmid concentration to account for variations in transfection efficiency, normalized to sterol synthesis for the 1.0 μg condition for each cell line. Data for 0, 0.5, 0.75, and 1.0 μg are from n = 3 independent experiments and presented as mean ± SEM, data for 0.25 μg are from n = 2 independent experiments and presented as mean ± half range.