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.