22 r hydroxycholesterol 22r ohc

The MA-10 mouse Leydig cells were purchased from American Type Culture Collection (ATCC) and cultured in DMEM/F12 medium (Sigma-Aldrich; D8900) supplemented with 15% horse serum (Gibco), 1% penicillin–streptomycin (Gibco), 20 mM HEPES and 1.2 g/L of sodium bicarbonate. After pre-coating 0.1% gelatin on the dishes and plates for 4 h, the cells were maintained at 37℃ with 5% CO₂ in a humidified incubator. Palmitic acid (PA) and oleic acid (OA) were separately dissolved in pure ethanol (100 mM) and diluted to a working concentration with the culture medium containing 1% BSA to conjugate fatty acids. To stimulate steroidogenesis, the cells were treated with 50 μM 8-Br-cAMP (Tocris) for 4 h, and then the conditioned medium was collected for further measurements. We treated the cells with 10 µg/mL of 22(R)-hydroxycholesterol (22R-OHC; Sigma) to assay CYP11A1 activity and 1 µg/mL of pregnenolone (Sigma) to assay 3β-HSD activity for 4 h and measured the progesterone production. To modulate autophagic activities, the cells were treated with 50 nM bafilomycin (Tocris), 20 μM chloroquine (Sigma-Aldrich), and different concentrations of rapamycin (Tocris) for 24 h before analysis. For the knockdown experiments, SMARTpool specific siRNAs (Dharmacon) were reversely transfected using Lipofectamine RNAiMAX Transfection Reagent (Thermo).