2.2 Cell culture and treatment
The human adrenal cortical adenocarcinoma cell line
(NCI-H295R) were plated into cell
culture flasks with DMEM medium with 10% fetal bovine serum, 1%
streptomycin, and penicillin in a 5% CO2 humidified
incubator at 37°C. Cells were treated with dexamethasone (0, 20, 100,
500 nM) or cortisol (300, 150, 75 nM) for 24 h, and then harvested for
further analysis. Chemically synthesized GRα plasmid, miR-370-3p mimics,
Sirt3 siRNA oligo was obtained from Suzhou GenePharma Co., Ltd. (Suzhou,
China). IGF1 (791-MG) was purchased from Abcam. NCI-H295R cells were
seeded into six-well plates at 1 × 105 cells/ well one
day before GRα plasmid, miR-370-3p mimics, Sirt3 siRNA oligo
transfection to reach the 50% confluency, respectively. After the
transfection procedures were performed using Lipofectamine 3000
(Invitrogen, Thermo Fisher Scientific; L3000150) for 24 h according to
the manufacturer’s instructions, the transfection mixture medium was
replaced by the medium with or without cortisol (300 nM) or
dexamethasone (500 nM) for 24 h. The cells were then lysed and total RNA
was extracted using the TRIzol reagent (Invitrogen Co.; 15596018), or
the cells were lysed using RIPA buffer for protein extraction.