Figure 1.

JPH3 expression and methylation in colorectal and gastric cancer cell lines and primary tumors. (A) CpG methylome study identified JPH3 as a methylated target in HCT116. JPH3 gene structure, promoter and exon 1 (UCSC Genome Browser NCBI36/hg18) are shown on the top panel. e1: exon 1. Positive methylation signal peak (blue) in HCT116 was identified by MeDIP-chip. (B) Broad expression of JPH3 in a normal adult tissue panel by semi-quantitative RT-PCR (primers detecting both variant 1 and 4), with GAPDH as a control (upper). Representative immunohistochemical staining for JPH3 in normal colonic and gastric mucosa. Original magnification: 200x, 400x (lower). (C) Structure of the JPH3 promoter CpG island (CGI). CpG sites are shown as short vertical lines. MSP primer sites and BGS region analyzed are also indicated. (D) Downregulation and silencing of JPH3 in colorectal and gastric cancer cell lines as a result of its CGI methylation, with RT-PCR primers detecting both variant 1 and 4. (E) Pharmacological demethylation with 5-Aza and TSA restored JPH3 expression in methylated and silenced cell lines. Representative results are shown. (F) Representative BGS results of cell lines. Vertical lines indicate individual CpG sites. Cloned BGS-PCR products were sequenced and each colony is shown as an individual row, representing a single allele of the CGI. Filled circles represent methylated and open circles unmethylated CpG sites. U: unmethylated; M: methylated. A: 5-Aza; T: TSA; CRC, colorectal cancer; GsCa, gastric cancer.