Figure 1 Whole genome methylation microarray sequencing ofCYP3A5*3/*3 genotype donor liver. (A) Principal Component Analysis (PCA) diagram; (B) Distribution of methylation degree between the 5 kb upstream and downstream transcription start sites (TSS); (C) Heat map of differential methylation sites; (D) Scatter plot of different methylation sites. G1: low tacrolimus C0/D ratio group; G2: high tacrolimus C0/D ratio group; n = 15.
Figure 2 The methylation sites (red vertical bars) within theABCB1 methylation region located on chromosome 7 (A) and the methylation level at cg12501229 site (B) , cg00634941 site(C) , and cg05496710 site (D) of ABCB1 gene in donor livers carried with CYP3A5*3/*3 genotype and different tacrolimus C0/D ratio (High: n=12, Low: n=11) ; **P <0.01.
Figure 3 Correlation of ABCB1 mRNA expression with tacrolimus C0/D ratio in CYP3A5*3/*3 donor livers (n=23). (A) correlation analyses; (B , C) AnABCB1 mRNA expression level of 0.15 was used as the cut-off value to analyse the tacrolimus C0/D ratio (B ) and the tacrolimus C0/D ratio distribution (C ); *P <0.05.
Figure 4 Effects of 5-Aza-2-DC on cell viability, ABCB1expression in HepG2 cells. (A-C ) The effects of 5-Aza-2-DC on cell viability after treated at indicated concentrations of 5-Aza-2-DC for 24h (A ), 48h (B ), and 72h (C ), respectively; (D-F ) ABCB1 mRNA expression (D ) and protein expression (E , F ) after treated with 5-Aza-2-DC (10 μM) for 24 h, 48 h, 72 h treatment, respectively; *P <0.05,**P <0.01,***P <0.001vs 0μM 5-Aza-2-DC (DMSO; 0.1% v/v).
Figure 5 Effects of 5-Aza-2-DC (10 μM) on DNA methylation at three CpG sites located on ABCB1 promoter region in HepG2 cells; ***P <0.001 vs 0μM 5-Aza-2-DC (DMSO; 0.1% v/v).
Figure 6 Changes of tacrolimus concentration in HepG2 cells over time replaced with tacrolimus-free DMEM after treated with 5-Aza-2-DC (0 μM or 10 μM) 72h and then incubated with 60 μM tacrolimus for 48 h; *P <0.05, ***P <0.001vs 0μM 5-Aza-2-DC (DMSO; 0.1% v/v).