Fig 3.
(A) Genomic DNA real-time PCR for the Cdh1 gene in adenocarcinomas arising in Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice to evaluate LOH. Log2 ratio of each tumor to Cre-negative gastric mucosa is depicted. LOH was defined as the average log2 ratio of three probes (for exons 6, 8, and 10) of tumor to normal DNA < −1.5 (broken red line). (Bi) Methylation-specific PCR of the gastric carcinomas shown in Fig 2A. (Bii) Bisulfite genomic sequencing analyses of the Cdh1 promoter in these gastric adenocarcinomas. Open and closed circles represent unmethylated (U) and methylated (M) CpG sites, respectively (TSS, transcription start site). A black bar indicates a CpG site targeted by (Bi). (C) E-cadherin immunostaining (top panels; Inset, E-cadherin-positive adjacent normal gastric mucosa) and real-time RT-PCR for gastric adenocarcinomas from two Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice (#5 and #11) treated with 5-aza-deoxycitidine (5-Aza). Gastric mucosa of a Cre-negative mouse (WT) and a gastric adenocarcinoma from untreated Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mouse (Mutant) were used as references. E-cadherin was not significantly up-regulated after 5-Aza treatment. (D) Western blot analysis for E-cadherin of the tissue explants of a gastric adenocarcinoma from a Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mouse after 24-h exposure to 0.1 μM and 0.3 μM of trichostatin A (TSA). Acetyl-histone H3 lysine 9 (H3K9ac) was immunoblotted as a positive control. (E) E-cadherin immunostaining of an early-onset human gastric cancer. Adjacent normal tissue of the same patient was positive (inset). (F) The CDH1 real-time RT-PCR expression level of each human gastric cancer relative to adjacent normal tissue. (G) Methylation-specific PCR analysis of human gastric cancers for CpG sites of CDH1 promoter (U, unmethylated; M, methylated CpG sites). (H) Bisulfite genomic sequencing analysis of the Cdh1 promoter in the gastric cancer with a germline missense CDH1 mutation.