Effects of 5-Aza-2-DC on ABCB1 expression in HepG2 cells
First, the optimal concentration and administration time for 5-Aza-2-DC were determined (Figure 4A-C ). Then, based on findings in literature, 10 μM 5-Aza-2-DC was used to investigate the effects ofABCB1 methylation status on its expression in HepG2 cells. We found that after treatment with 10 μM 5-Aza-2-DC for 24, 48 and 72 h,ABCB1 mRNA and protein expression levels significantly increased at 72 h (Figure 4D-F ). Therefore, 10 μM 5-Aza-2-DC and 72 h were selected as the optimal concentration and treatment duration, respectively, for the determination of the effects of 5-Aza-2-DC on the methylation levels of the three ABCB1 CpG sites (cg12501229, cg00634941, and cg05496710).
Effects of 5-Aza-2-DC on ABCB1 methylation levels and tacrolimus metabolism in HepG2 cells
A pyrophosphorylation assay was performed to investigate the effects of 5-Aza-2-DC on ABCB1 methylation. As shown in Figure 5 , we found that the methylation levels of the ABCB1 sites in the 5-Aza-2-DC-treated group (10 μM) were significantly lower (P< 0.001) than those in the untreated group (0 μM). To determine the effects of 5-Aza-2-DC on tacrolimus metabolism, intracellular tacrolimus concentrations were determined by UHPLC-MS/MS. We found the tacrolimus contents of 5-Aza-2-DC-treated cells to be significantly lower than those of untreated cells at 0, 4, and 6 h following the removal of tacrolimus-containing medium (P< 0.05). At 12 h, although there was a slight increase in intracellular tacrolimus concentrations, its levels in the 5-Aza-2-DC-treated group were still significantly lower than those in the untreated group (0 μM), and this may be due to its intracellular metabolism in a dynamic equilibrium scenario (Figure 6 ).