Cell culture and treatments
The human hepatocellular carcinoma cell line, HepG2, which was purchased from the China Centre for Type Culture Collection (Wuhan, China), and confirmed by short tandem repeat analysis, was cultured in DMEM (HyClone/Thermo Fisher Scientific, Beijing, China) supplemented with 10% FBS (LONSA SCIENCE S.R.L., Montevideo, Uruguay). To determine the optimum concentration and administration period for 5-Aza-2-DC (Selleck, Shanghai, China) and tacrolimus (Selleck, Shanghai, China), HepG2 cells were seeded into 12-well plates at a density of 4×104 cells/well and cultured for 24 h at 37 °C in a 5% CO2 atmosphere. For treatment with 5-Aza-2-DC, the cells were exposed to 5-Aza-2-DC dissolved in DMSO (0.1% v/v) at a series of final concentrations (0, 0.1, 0.5, 1.0, 2.5, 5.0, 10, and 50 μM) for 24, 48, and 72 h. Then, cell viability was evaluated using the CCK-8 kit (Dojindo, Shanghai, China) according to the manufacturer’s instructions, and ABCB1 mRNA and protein expression levels were evaluated by RT-qPCR and Western blotting, respectively. For treatment with tacrolimus, the cells were exposed to tacrolimus dissolved in DMSO (0.1% v/v) at a series of final concentrations (0, 0.01, 0.1, 1.0, 10, 50, 100, and 200 μM) for 24, 48, and 72 h, and cell viability was evaluated using the CCK-8 kit. Based on the results of the optimum time determined by CCK-8 analysis, HepG2 cells were treated with 0, 40, 50, 60, 70, 80, 90, and 100 μM tacrolimus for 24 h, and cell viability was evaluated using the CCK-8 kit to determine the tacrolimus optimum concentration.