Fig. 4.

Effect of genistein and 5Aza-C on BTG3 methylation. (A). Representative sequencing results showing the effect of 5Aza-C (AZ) and genistein (G) on promoter methylation of the BTG3 gene. (B) Summarized results showing demethylation of CpG sites by 5Aza-C (Aza) and genistein (G) treatments in three RCC cell lines and a HK-2. (C) Summarized results showing promoter methylation status in 20 pairs of normal and tumor samples. Each pair was obtained from the same patient and microdissected by a certified pathologist into normal and tumor. The symbols open circle and asterisk indicate individual CpG sites in the CpG island. Bisulfite modification of the DNA was performed that changes the unmethylated cytosines to thymine thus represented by ‘TG’ in the sequence whereas the methylated cytosines remain unchanged and thus represented by ‘CG’ in the sequences. For cell lines, a single primer set (BSP_F2–R2) was used for BS-PCR whereas a nested PCR was performed for tissue samples using two primer sets BSP_F1–R1 and BSP_F2–R2.