Fig 2: Biological oxidants induce CAPZA1 expression by enhancing histone H3 acetylation of the CAPZA1 promoter. (A) AGS cells were incubated with hydrogen peroxide or di-tert-butyl peroxide for 2 hours, and then the CAPZA1 expression level was analyzed. (B) AGS cells were incubated with NAC for 24 hours before treatment with hydrogen peroxide or di-tert-butyl peroxide, and then CAPZA1 expression was analyzed. (C) AGS cells were incubated with NAC for 24 hours before treatment with hydrogen peroxide or di-tert-butyl peroxide. Thereafter, these cells were analyzed by a ChIP assay with an anti–histone H3 (acetyl K9) antibody or IgG obtained from rabbit serum. Real-time PCR showed the relative enrichment of the CAPZA1 promoter in the DNA fragments pulled down by an anti–histone H3 (acetyl K9) antibody. Data are presented as the means ± SD of 3 independent assays. *P < .05. P values were calculated by a 1-way analysis of variance. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 3: Representative immunostaining results for co-localization of CAPZA1 and CD44v9 in gastric mucosa of Mongolian gerbils infected with H pylori. CD44v9-positive cells were detected in gastric mucosa of 2 of 5 Mongolian gerbils infected with H pylori (no. 3 and 4). Red indicates CAPZA1 and green indicates CD44v9. The white box contains CD44v9-positive cells and is enlarged at the bottom of each panel. Scale bar: 20 μm.

Fig 4: Relationships between CAPZA1 and ESRP1 expression and nuclear translocation of ß-catenin in human gastric mucosa. (A) Immunostaining of CAPZA1 and ESRP1 in human gastric tissues (cases 6, 7, 9, and 11; CD44-positive gastric mucosa). Red indicates CAPZA1 and green indicates ESRP1. Scale bar: 20 µm. (B) Immunostaining of CAPZA1 and ß-catenin in human gastric tissues. Red indicates CAPZA1 and green indicates ß-catenin. White arrowheads indicate nuclear ß-catenin. Scale bar: 20 µm. (C) Schematic representation of the proposed mechanism by which CD44v9 expression is induced in CAPZA1-overexpressing cells infected with H pylori. (1) Overexpression of CAPZA1 enhances expression of ß-catenin. (2) Overexpression of CAPZA1 induces high expression of ESRP1. (3) Infection of CAPZA1-overexpressing cells with H pylori aberrantly enhances nuclear translocation of ß-catenin owing to accumulation of CagA, leading to induction of CD44total expression. (4) High expression of ESRP1 owing to overexpression of CAPZA1 promotes alternative splicing of CD44total to generate CD44v9.

Fig 5: Effect of eradication therapy on CAPZA1 expression. (A) Immunostaining analysis of CAPZA1 and CD44v9 in human gastric tissues. Cases 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 were a 51-year-old woman, a 62-year-old man, a 60-year-old woman, a 60-year-old man, a 54-year-old man, a 68-year-old woman, a 66-year-old man, an 80-year-old woman, a 39-year-old man, and a 74-year-old woman, respectively. Cases 6–10 were H pylori–positive. Cases 11–15 had received eradication therapy. Red indicates CAPZA1 and green indicates CD44v9. Scale bar: 20 µm. (B) The CAPZA1 staining intensity per cell in human gastric mucosa was quantified using image analysis software (Tissue Quest version 4.0; TissueGnostics). The data points indicate the average CAPZA1 staining intensity per cell in each individual image. The box-and-whisker plot presents the full range of variation, interquartile range, and median values. P values were calculated by the Student t test. (C) Representative enlarged fields containing CD44v9-positive cells in human gastric mucosa of cases 6, 7, 9, and 11. Scale bar: 20 µm.