Fig. 2. Effect of cholesterol depletion or enhancement on the cholesterol content of AML12 cells.

The cholesterol (chol.) level in AML12 cells was reduced (cholesterol depletion; CD) by treatment with lovastatin and mevalonate (50 μM each) in medium containing 10% LPDS (for control samples, 10% FCS), or elevated (cholesterol enrichment; CE) by incubation with cholesterol-MβCD complex (5 mM MβCD, 300 μg/ml cholesterol) in complete growth medium for 16 h, followed by 30 min serum starvation under the same conditions (see “Methods”). The level of free (non-esterified) and total cholesterol was determined using the Abcam cholesterol assay kit (“Methods”). In each experiment, the values obtained in untreated (control) cells were taken as 100%. a Cholesterol depletion. Data are mean ± SEM of four independent experiments. b Cholesterol enrichment. Bars are mean ± SEM of six independent experiments. The results show similar levels of reduction (a) or elevation (b) in total and free cholesterol. c Effect of statin-mediated CD on free cholesterol level at the plasma membrane. After the CD treatment, the membrane cholesterol was extracted by short incubation with a high level of MβCD (30 mM, 30 min, 37 °C), and the free cholesterol level was assayed. Data are mean ± SEM of four independent experiments. d Effect of CE on the free plasma membrane cholesterol. The CE treatment was followed by extraction of the membrane cholesterol by a short exposure to MβCD as described in (c). Data are mean ± SEM of three independent experiments. Asterisks indicate significant differences between the pairs indicated by the brackets. ns not significant. In (a, b), significance was calculated using Student’s two-tailed t test. In (c, d), significance was evaluated using one-way ANOVA followed by Bonferroni post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001).