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  • br Materials and Methods br

    2020-08-28

    
    Materials and Methods 1267
    The human gastric cancer cell line AGS was purchased 1270
    from American Type Culture Collection (Rockville, MA) and 1271
    and MKN-II SalvinorinA were maintained in RPMI 1640 medium 1274
    expression in WT-A10 cells was induced by culture in the 1279
    absence of doxycycline for 24 hours.16 MKN28 cells cultured 1280
    Bacterial Strains and Culture Conditions 1284
    inson, Franklin Lakes, NJ) containing 7% sheep blood and 1289
    which were maintained using AnaeroPack MicroAero 1291
    (Mitsubishi Gas, Tokyo, Japan). KS numbered strains were 1292
    isolated from gastric biopsy specimens and stored at -80 C 1293
    12 Tsugawa et al Cellular and Molecular Gastroenterology and Hepatology Vol. -, No. -
    Drugs and Antibodies
    Figure 7. Effect of eradi-
    apy. Red indicates CAPZA1 and green in-dicates CD44v9. Scale bar: 20 mm. (B) The CAPZA1 staining intensity per cell in human gastric mucosa was quantified using image analysis software (Tissue Quest version 4.0; Tis-sueGnostics). 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) Repre-sentative enlarged fields
    Figure 8. Relationships between CAPZA1 and ESRP1 expression and nuclear translocation of b-catenin in human gastric mucosa. (A) Im-munostaining of CAPZA1 and ESRP1 in human gastric tissues (cases 6, 7, 9, and 11; CD44-positive gastric mucosa). Red in-dicates CAPZA1 and green indicates ESRP1. Scale bar: 20 mm. (B) Immuno-staining of CAPZA1 and b-catenin in human gastric tissues. Red indicates CAPZA1 and green in-dicates b-catenin. White arrowheads indicate nu-clear b-catenin. Scale bar:
    20 mm. (C) Schematic rep-resentation of the proposed mechanism by which CD44v9 expression is induced in CAPZA1-overexpressing cells infec-ted with H pylori. (1) Over-expression of CAPZA1 enhances expression of b-catenin. (2) Overexpression of CAPZA1 induces high expression of ESRP1. (3) Infection of CAPZA1-overexpressing cells with H pylori aberrantly en-hances nuclear trans-location of b-catenin owing to accumulation of CagA, leading to induction of CD44total expression. (4) High expression of ESRP1 owing to overexpression of CAPZA1 promotes alterna-tive splicing of CD44total to generate CD44v9.
    1:500; Merck Millipore) antibodies were used for immu-nohistochemistry, and an anti–b-catenin antibody (610154, 1:50; BD Biosciences) was used for fluorescence immuno-cytochemistry. Cells were grown to subconfluence and transfected with ESRP1 siRNA 1 (SI04337956, Hs_RBM35A_3; Qiagen) and ESRP1 siRNA 2 (SI04178944, Hs_RBM35A_1; Qiagen) using Lipofectamine 2000 (11668027; Thermo Fisher Scientific) according to the manufacturer’s instructions. As a nonsilencing control cells were transfected with AllStars Negative Control siRNA (SI03650318; Qiagen). 
    In Vitro Infection of H pylori 1521
    pylori G27) or G27 cagPAI-deleted isogenic mutant strain (H 1524
    pylori G27 DcagPAI) for 5 hours (multiplicity of infection, 1525
    In Vivo Infection of H pylori 1528
    14 Tsugawa et al Cellular and Molecular Gastroenterology and Hepatology Vol. -, No. -
    1533 each). Seven months later, the animals were killed and
    1534 their stomachs were excised. To confirm H pylori infection,
    1535 the number of viable colony-forming units was determined
    1536 by plating samples on Nissui Helicobacter agar (Nissui
    1537 Pharmaceutical, Tokyo, Japan). Levels of malondialdehyde
    1538 and protein carbonylation were measured using a TBARS
    1540 tein Carbonyl Enzyme-Linked Immunosorbent Assay Kit
    1544 ChIP was performed using a Simple ChIP Plus Sonication 1545Q18 Chromatin IP Kit (56383S; Cell Signaling Technology)
    1546 following the manufacturer’s instructions. Cross-linking was
    1547 performed using a 1% formaldehyde solution in phosphate-
    1548 buffered saline. Before immunoprecipitation, each input frac-
    1549 tion was saved and used as a positive control. The supernatants
    1550 were immunoprecipitated with anti–histone H3 (acetyl K9)
    1552 Sigma-Aldrich) at 4 C overnight. Then, the resulting enriched
    1553 genomic DNA samples were measured by qPCR using the
    1554 EpiTect ChIP-qPCR Primer Assay kit for CAPZA1
    1556 site). These ChIP qPCR primers were predesigned, and qPCR
    1557 assays were optimized to measure genomic DNA in the region
    1561 Human gastric adenocarcinoma tissue specimens (case