Fig 2: Interleukin (IL)-8 mRNA and protein expression in AGS cells infected with wild-type (ATCC 60190) and cytotoxin-associated gene pathogenicity island (cagPAI)-depleted (CagA− and CagE−) Helicobacter pylori. (A) IL-8 mRNA expression was decreased by infection with cagPAI knockdown strains compared to wild-type H. pylori in AGS cells at 5 hours after infection. (B) The IL-8 mRNA levels were significantly higher in the wild-type H. pylori-infected cells than in the cagPAI-depleted H. pylori-infected cells. (C) The knockdown mutants displayed a reduced capacity to mediate IL-8 release compared with the wild-type strain. (D) IκB-α degradation and IκB-α phosphorylation were reduced in the AGS cells infected with cagPAI-depleted H. pylori compared with those infected with the wild-type strain. (E) Nuclear factor κB (NF-κB) binding in AGS cells infected with the cagPAI knockdown strain was reduced compared with binding in cells infected with the wild-type strain. The error bars indicate the standard error of the mean of triplicate samples, which were representative of three independent experiments. *p<0.05.

Fig 3: Analysis of promoter methylation, mRNA and protein expression of claudin-11 in gastric cell lines.This figure illustrates the claudin-11 promoter methylation, mRNA expression levels and protein expression in gastric cells lines. A) Quantitative methylation-specific PCR (qMSP) for CLDN11. Genomic DNAs isolated from immortalized human normal gastric epithelial cells (HFE145) and GC cell lines AGS, SIIA, MKN28, KATOIII, and SNU-1 obtained from ATCC were analyzed by qMSP. This Figure illustrates that the promoter region of CLDN11 gene is hypermethylated in all GC cell lines relative to HFE145 cells. B) CLDN11 mRNA expression in gastric cell lines. Total RNAs from different gastric cell lines were subjected to quantitative real-time RT-PCR analysis. As can be seen in this figure, HFE145 cells expressed very high levels of CLDN11 mRNA, while all five cancer cell lines tested had very low or undetectable CLDN11 mRNA expression. C) Western blot analysis of claudin-11 expression in gastric cell lines. Total cell lysates obtained from various gastric cell lines were probed with the anti-claudin-11 antibody. This figure illustrates that while the immortalized normal gastric epithelial cell line, HFE145, expressed abundant claudin-11 protein, it could not be detected in various GC cell lines. Anti-β-actin antibody was used as a loading control.