Bafilomycin

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).

To mimic a diabetic environment in vitro, podocytes were grown in the presence of 100 nmol/l insulin (Tocris, Bristol, UK), 25 mmol/l glucose (Sigma), 1 ng/ml TNF-α and 1 ng/ml IL-6 (R&D systems, Abingdon, UK). d-Mannitol (Sigma) was used as a control for osmotic pressure in these assays. For initial chronic insulin exposure, podocytes were incubated with insulin at 10 nmol/l and 100 nmol/l for 10 days. Although supraphysiological (as physiological hyperinsulinaemia is typically within the range 1000–2000 pmol/l, occurring over an extended period of months or years), this is consistent with numerous in vitro studies of other cell types [25 (link)–30 (link)]. For short-term insulin stimulation, culture medium was replaced with serum- and insulin-free RPMI-1640 for 2–4 h, and podocytes were re-challenged with insulin at 10 or 100 nmol/l for 10 min. For inhibition of proteasomal and lysosomal degradation, podocytes were treated with 10 μmol/l MG-132 (Sigma) or 50 nmol/l bafilomycin (Tocris), respectively, for 8 h.