Aminoglutethimide

De novo cholesterogenic activity was measured as described previously^{47 (link),48 (link)}. Y-1 cells were treated with siAd4BP/SF-1 (#1635) or siControl for 48 h, and then incubated for 1 h at 37 °C in a serum-free medium containing [1,2-¹⁴C]acetate (PerkinElmer, Boston, MA, USA), 50 μM aminoglutethimide (Sigma-Aldrich) and 2 μg/ml 58-035 (ACAT2; acyl-CoA cholesterol acyltransferase inhibitor) (Sigma-Aldrich). Total lipid fraction containing cholesterol was extracted with chloroform/methanol (2:1, v/v), and then separated by thin-layer chromatography on a silica gel using benzene-ethylacetate (2:3, v/v) as a solvent. The radioactivities of the spots for free and esterified cholesterol, visualized with iodine vapor, were determined by liquid scintillation counting. [1,2-¹⁴C]acetate incorporation into cholesterol was expressed as cpm/mg protein/h.

Sertoli cells (5 × 10⁵) at P21 were cultured for 3 days. The lactate concentration of 30 μl of the medium was determined at 6 and 24 h after medium change using the Lactate Assay Kit (BioVision, Milpitas, CA, USA). To determine cholesterol synthetic activity, Sertoli cells (1 × 10⁶) were incubated in a serum-free medium containing [1,2–¹⁴C]-acetate (PerkinElmer, Inc., Boston, MA USA), 50 μM aminoglutethimide (Sigma) and 2 μg/ml 58–035 (ACAT2; acyl-CoA cholesterol acyltransferase inhibitor; Sigma) for 2.5 or 3 h at 34 °C. All lipids, including cholesterol, were extracted using chloroform/methanol (2:1, v/v), then separated by thin-layer chromatography on silica gel with benzene-ethylacetate (2:3, v/v) as a solvent. The radioactivity of a spot containing free and esterified cholesterol visualized with iodine vapor was determined by liquid scintillation counting. The extent of [1,2-¹⁴C]-acetate incorporation into the cholesterol was expressed as cpm/μmol acetate/h.

Aminoglutethimide (Sigma, St. Louis, MO) was dissolved in 0.1M hydrochloric acid (pH 1.0) and diluted with RPMI medium in in vitro studies. The final concentration of hydrochloric acid was less than 0.05%. Aminoglutethimide was added at different doses based on our previous reports [8 (link), 20 (link)].