Monstrating that KLF5 is induced by H. pylori in a cagE-independent

Monstrating that KLF5 is induced by H. pylori in a cagE-independent manner. We next examined KLF5 expression in epithelial cells isolated from uninfected and infected murine gastric tissue by flow cytometry analysis. Consistent with the KLF5 immunohistochemistry (Figure 4), flow cytometry demonstrated a significant increaseFigure 4. H. pylori upregulates KLF5 expression in vivo. (A ) KLF5 expression in murine antral gastric tissue was assessed by KLF5 immunostaining in uninfected (A), H. pylori PMSS1-infected mice (B), and H. pylori PMSS1 cagE2-infected mice (C) at 11967625 4006 magnification. (D and E) A single pathologist, blinded to Salmon calcitonin treatment groups, assessed and scored KLF5 immunostaining. KLF5 immunohistochemistry (IHC) score was determined by assessing the percentage of KLF5+ epithelial cells multiplied by the intensity of epithelial KLF5 staining (1?) in both the cytoplasm and nucleus of murine gastric epithelial cells (D and E). Each data point represents an individual animal and mean values are shown. Circles designate uninfected mice, squares represent H. pylori PMSS1-infected mice, and triangles represent H. pylori PMSS1 cagE2-infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups. doi:10.1371/journal.pone.0054344.gKLF5 and H. Pylori-Mediated Gastric CarcinogenesisKLF5 and H. Pylori-Mediated Gastric CarcinogenesisFigure 5. H. pylori induces expansion of a KLF5+ cell population in vivo. (A ) KLF5 expression in murine gastric epithelial cells was assessed by flow cytometry analysis in uninfected and H. pylori-infected mice at acute time points (24, 48, 72 hours, and 1 week) and chronic time points (4 and 8 weeks) post-challenge. Percentage of KLF5+ cells at 4 weeks (A) and 8 weeks (C) and levels of KLF5 protein at 4 weeks (B) and 8 weeks (D), as determined by mean fluorescence units (MFU), were determined by flow cytometry. Data from 4 and 8 week time points were analyzed at separate times. H. pylori colonization density in mice infected for 24, 48, and 72 hours, and 1 week was assessed by quantitative culture (E). Percentage of KLF5+ cells (F) and levels of KLF5 protein (G) at 24, 48, or 72 hours, or 1 week were determined by flow cytometry. Each data point represents gastric epithelial cells analyzed from a single animal and mean values are shown. Circles designate uninfected mice, and squares represent H. pylori-infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups. doi:10.1371/journal.pone.0054344.gin the percentage of KLF5+ cells in H. pylori-infected mice at both 4 and 8 weeks (Figure 5A and 5C). Levels of KLF5 protein, as determined by mean fluorescent units (MFU), were also significantly increased in H. pylori-infected mice compared to uninfected controls (Figure 5B and 5D). Consistent with the immunohistochemistry results, infection with wild-type strain PMSS1 or the PMSS1 cagE2 isogenic mutant induced similar increases in the percentage of KLF5+ cells and levels of KLF5 protein (Figure 5A and 5B), confirming that induction of KLF5 occurs in a cagEindependent manner.To determine if KLF5 upregulation was mediated by the host inflammatory response or by the effects of H. pylori per se, we assessed the expression of KLF5 during acute H. pylori infection in vivo. C57BL/6 mice were challenged with MedChemExpress Pentagastrin Brucella broth as a negative uninfected (UI) control or H. pylori strain PMSS1 for 24, 48, or 72 hours, or 1 week. Colonization effici.Monstrating that KLF5 is induced by H. pylori in a cagE-independent manner. We next examined KLF5 expression in epithelial cells isolated from uninfected and infected murine gastric tissue by flow cytometry analysis. Consistent with the KLF5 immunohistochemistry (Figure 4), flow cytometry demonstrated a significant increaseFigure 4. H. pylori upregulates KLF5 expression in vivo. (A ) KLF5 expression in murine antral gastric tissue was assessed by KLF5 immunostaining in uninfected (A), H. pylori PMSS1-infected mice (B), and H. pylori PMSS1 cagE2-infected mice (C) at 11967625 4006 magnification. (D and E) A single pathologist, blinded to treatment groups, assessed and scored KLF5 immunostaining. KLF5 immunohistochemistry (IHC) score was determined by assessing the percentage of KLF5+ epithelial cells multiplied by the intensity of epithelial KLF5 staining (1?) in both the cytoplasm and nucleus of murine gastric epithelial cells (D and E). Each data point represents an individual animal and mean values are shown. Circles designate uninfected mice, squares represent H. pylori PMSS1-infected mice, and triangles represent H. pylori PMSS1 cagE2-infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups. doi:10.1371/journal.pone.0054344.gKLF5 and H. Pylori-Mediated Gastric CarcinogenesisKLF5 and H. Pylori-Mediated Gastric CarcinogenesisFigure 5. H. pylori induces expansion of a KLF5+ cell population in vivo. (A ) KLF5 expression in murine gastric epithelial cells was assessed by flow cytometry analysis in uninfected and H. pylori-infected mice at acute time points (24, 48, 72 hours, and 1 week) and chronic time points (4 and 8 weeks) post-challenge. Percentage of KLF5+ cells at 4 weeks (A) and 8 weeks (C) and levels of KLF5 protein at 4 weeks (B) and 8 weeks (D), as determined by mean fluorescence units (MFU), were determined by flow cytometry. Data from 4 and 8 week time points were analyzed at separate times. H. pylori colonization density in mice infected for 24, 48, and 72 hours, and 1 week was assessed by quantitative culture (E). Percentage of KLF5+ cells (F) and levels of KLF5 protein (G) at 24, 48, or 72 hours, or 1 week were determined by flow cytometry. Each data point represents gastric epithelial cells analyzed from a single animal and mean values are shown. Circles designate uninfected mice, and squares represent H. pylori-infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups. doi:10.1371/journal.pone.0054344.gin the percentage of KLF5+ cells in H. pylori-infected mice at both 4 and 8 weeks (Figure 5A and 5C). Levels of KLF5 protein, as determined by mean fluorescent units (MFU), were also significantly increased in H. pylori-infected mice compared to uninfected controls (Figure 5B and 5D). Consistent with the immunohistochemistry results, infection with wild-type strain PMSS1 or the PMSS1 cagE2 isogenic mutant induced similar increases in the percentage of KLF5+ cells and levels of KLF5 protein (Figure 5A and 5B), confirming that induction of KLF5 occurs in a cagEindependent manner.To determine if KLF5 upregulation was mediated by the host inflammatory response or by the effects of H. pylori per se, we assessed the expression of KLF5 during acute H. pylori infection in vivo. C57BL/6 mice were challenged with Brucella broth as a negative uninfected (UI) control or H. pylori strain PMSS1 for 24, 48, or 72 hours, or 1 week. Colonization effici.