Figure 2.
RHCG promoter region is frequently hypermethylated in ESCCs. (A) Measurement of RHCG expression levels in a panel of ESCC cell lines compared with squamous epithelial cell line NE1 by Western blotting. (B) qRT-PCR analysis of RHCG expression after demethylation or histone acetylation treatment with 5-aza-dC or TSA in ESCC cell lines without RHCG expression. (C) High-resolution mapping of the methylation status of individual CpG site in the RHCG promoter by BGS in normal cell line NE1 and ESCC cell lines with or without 5-aza-dC treatment. A 270-bp region spanning the CpG island with 24 CpG sites was analyzed. Each CpG site analyzed is shown at the top row as a short vertical bar. The percentage of methylation was determined as percentage of methylated CpGs from 10 to 15 randomly sequenced clones and is displayed in the pie charts. (D-E) Representative MSP analysis of RHCG in esophageal cell lines with or without 5-aza-dC treatment, and paired primary ESCCs/nontumor tissues (T, tumor tissue; N, non-tumor tissue; M: methylation; U: unmethylation).