Fig. 4.

Effect of methylation on POLD1 promoter activity and effect of 5-AzaC treatment on POLD1 and E2F1 expression. a The POLD1 promoter activity in HEK-293T cells transfected with the unmethylated and methylated pGL3 control and pGL3-POLD1 plasmids. Comparison of luciferase activity in HEK-293T cells cotransfected with a pRL-CMV vector containing β-galactosidase and a pGL3 vector containing a luciferase gene driven by a methylated or unmethylated POLD1 promoter fragment (− 1758 to + 49 bp). After 24 h, luciferase activity in cell lysates, related to β-galactosidase activity, was determined, and relative luciferase activity was normalized to the activity of an unmethylated control pGL3 vector. The results are presented as the ratio (%) of luciferase activity to the unmethylated control. Values represent the mean ± SD, with three independent experiments per group. b, c DNA methylation status of the POLD1 promoter in 48PD 2BS and 38PD WI-38 cells after treatment with 5 μM 5-AzaC by bisulfite DNA sequencing analysis. d, e The mRNA expression levels of POLD1 and E2F1 in 48PD 2BS cells treated with 5 μM 5-AzaC for 72 h. f, g The protein expression levels of POLD1 and E2F1 in 48PD 2BS cells treated with 5 μM 5-AzaC for 72 h. h, i The mRNA expression levels of POLD1 and E2F1 in 38PD WI-38 cells treated with 5 μM 5-AzaC for 72 h. j, k The protein expression levels of POLD1 and E2F1 in 38PD WI-38 cells treated with 5 μM 5-AzaC for 72 h. GAPDH was used for normalization of the data. The data were compared with the independent samples t-test, with three independent experiments in each group; *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001, vs. the untreated group