Figure 5.

Complex I inhibition affects posttranslational regulation of multiple cholesterol sensors on the ER.A, expression of SREBP2 precursor and cleaved SREBP2 after maturation in human primary fibroblasts following culture in the presence of DMSO (0.1%) or rotenone (100 nM) in either normal media or lipid-deficient media for 48 h as indicated. The lipid-deficient medium contained delipidated serum and atorvastatin (1 μM). Quantification of cleaved SREBP2 from three independent experiments is shown on the right. Individual values ±standard deviation is expressed as fold change compared to control cells in standard medium. Significance was calculated by ANOVA with Tukey HSD post-hoc test. B, gene expression of ACAT2, SQLE, and HMGCR measured by qPCR in human primary fibroblasts cultured and treated as in panel A. Individual measurements (black circles) ± standard deviation from three independent experiments are presented. Significance was calculated by ANOVA with Tukey HSD post-hoc test. C, protein expression of SREBP2 precursor and cleaved form in primary human fibroblasts following retention of cholesterol in endosomes. Cells were cultured in normal conditions, supplemented with either rotenone (100 nM), U18666a (2 μM), or both. Quantification of cleaved SREBP2 from three independent experiments is shown on the right. Individual values ±standard deviation are expressed as fold change compared to control cells in standard medium. Significance was calculated by ANOVA with Tukey HSD post-hoc test. D, representative Western blot of SREBP2 protein expression in human primary fibroblasts following acute manipulation of cellular cholesterol with methyl-β-cyclodextrins (MBCD). Fibroblasts were cultured in control medium for 2 days in the absence (0.1% DMSO) or presence of rotenone (100 nM). Two hours before protein extraction, cellular cholesterol was manipulated using vehicle, empty MBCD (0.1%), or cholesterol-loaded (50 μM) MBCD. Quantification of mature form of SREBP2. Individual measurements ±standard deviation compared to control untreated cells from four independent experiments are shown (right). Significance was calculated by ANOVA with Tukey HSD post-hoc test. E–G, protein expression of HMGCR (E), SQLE (F), and calnexin (G) in primary human fibroblasts (left). Cells were cultured as described for panel A. Quantification of HMGCR and SQLE Western blot signals (right) of four independent experiments ±standard deviation normalized to loading controls are shown. GAPDH or Vinculin protein signals were used as loading control. Significance was calculated between indicated samples by ANOVA with Tukey HSD post-hoc test. ns: not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 compared to DMSO control. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HMGCR, 3-Hydroxy-3-Methylglutaryl-CoA reductase; SQLE, squalene epoxidase; SREBP2, sterol regulatory element–binding protein 2.