Nicotinamide

Human adrenal NCI‐H295R cells were maintained under normal growth conditions (growth medium, GM) in DMEM:F‐12 medium (HyClone Corporation) with 0.1% Insulin‐Transferrin‐Selenium Supplement (Gibco), 2.5% Nu‐Serum I (Becton Dickinson) and 100 U/mL penicillin and streptomycin (HyClone Corporation). The serum‐free medium (starvation medium, SM) only contained DMEM:F‐12 and 100 U/mL penicillin and streptomycin. Cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C and the cells were divided once a week. Nicotinamide (Beyotime Biotechnology) was dissolved in distilled water and used at a final concentration of 1–25 mmol/L. Cells were subcultured in 12‐well plates at a density of 5 × 10⁵ cells/well, in a volume of 1.0 mL medium. When the cells were at 60%–70% confluence, the medium was replaced with starvation medium supplemented with 1, 5, or 25 mmol/L (n = 3) of NAM. The untreated control group was treated with the starvation medium supplemented with the same volume of distilled water. After 24 hours, the cell culture medium was removed and stored at −80°C until hormone analysis. The remaining cells were lysed in RIPA buffer for subsequent protein quantification. Cells treated with 25 mmol/L as the highest concentration were used for RNA isolation and RNA‐sequencing.