Fig. 2.

Promoter methylation is involved in regulating CTHRC1 expression in ESCC. a Promoter methylation of CTHRC1 in paired tissue specimens from 50 ESCC patients was detected by pyrosequencing assay. P < 0.0001, paired two-tailed Student’s t-test. b The number and percent of patients with higher or lower promoter methylation of CTHRC1 in ESCC tumour tissues compared with non-tumour tissues. T: tumour tissue; N: non-tumour tissue. c RT-PCR was performed to analyse the mRNA level of CTHRC1 in ESCC cells treated or not with the demethylating agent 5-aza-dC (10 μM) for 72 h. The significance of difference between groups was analysed using two-tailed Student’s t-test. Data are presented as the mean (n = 3) ± SD. *P < 0.05 and **P < 0.01. d Western blot was performed to detect the protein level of CTHRC1 in culture supernatants of cells with or without 5-aza-dC treatment. e Promoter methylation of CTHRC1 in ESCC cells with and without 5-aza-dC treatment was detected using a pyrosequencing assay