Figure 6.

Complex I inhibition severely impairs mevalonate pathway expression in liver carcinoma cells.A, dose-dependent inhibition of oxygen consumption by complex I inhibition. The effect of increasing concentrations of rotenone on basal respiration in the presence of 5 mM glucose was studied. Data are expressed as percentage of baseline oxygen consumption as described in Figure 1B. Shown is the average ±standard deviation from five replicates. B, apoptosis in HepG2 cells caused by increasing concentrations of rotenone. Staurosporine (100 nM) was included as a positive control. Apoptosis was measured as described for Figure 1C after 2 days in culture in the presence of increasing concentrations of rotenone. Bar indicates mean four independent experiments ±standard deviation. Individual measurements shown (black circles). C and D, expression of SREBP2 precursor and cleaved form, SQLE (E), and HMGCR (F) in HepG2 cells was followed by Western blotting. HepG2 cells were treated with either dimethyl sulfoxide (0.1%) or rotenone (100 nM) in either normal media or lipid-deficient media for 2 days as described for Figure 5A. The GAPDH signal was used as a loading control. D, HepG2 cells were grown as in C. For the final 2 h, cholesterol was acutely added or removed using cholesterol (50 μM) loaded or empty cyclodextrin respectively. G, RNA expression of ACAT2, SQLE, HMGCR, and HMGCS1 in HepG2 cells grown under conditions as described for panel C. Expression changes are given as fold change compared to cells grown in normal medium without rotenone. Shown are the results from three independent experiments (black circles) ± standard deviation. Significant differences from control were calculated by t tests followed by Benjamini–Hochberg multiple testing correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HMGCR, 3-Hydroxy-3-Methylglutaryl-CoA reductase; SQLE, squalene epoxidase; SREBP2, sterol regulatory element–binding protein 2.