EDN3 promoter methylation in primary breast cancer. (a) Methylation-specific polymerase chain reaction (MSP) was performed on bisulphite-treated DNA from breast carcinomas (T) and matching normal breast tissue (N). Representative MSP results from eight patients are shown. MSP controls were commercially available poly-unmethylated (poly-U) or in vitro poly-methylated (poly-M) DNA. (b) Box plot demonstrating the significant association between EDN3 promoter methylation and EDN3 mRNA expression in these tissues. For 71 samples, EDN3 expression has been assessed in parallel to EDN3 promoter methylation. EDN3-methylated tumours show significant downregulation as compared with EDN3-unmethylated tumours (P = 0.005, Mann-Whitney U test). Horizontal lines indicate group medians, and boxes indicate 25% to 75% quartiles, range, peak and minimum. EDN3, endothelin-3; M, methylated; U, unmethylated.