1. Volume 139, Issue 1, Pages 226-238.e6 (July 2010)
Inhibitor of κB Kinase Beta Regulates Gastric Carcinogenesis via Interleukin-1α Expression 
Kei Sakamoto, Yohko Hikiba, Hayato Nakagawa, Yoku Hayakawa, Ayako Yanai, Masao Akanuma, Keiji Ogura, Yoshihiro Hirata, Klaus H. Kaestner, Masao Omata, Shin Maeda 
Gastroenterology 
Volume 139, Issue 1, Pages e6 (July 2010)
DOI: /j.gastro
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2. Figure 1
Generation of IKKβ gastric epithelium knockout mice. (A) Gastric mucosa, forestomach, and spleen of IkkβF/F and IkkβΔST mice were lysed, gel-separated, and immunoblotted with antibodies against the indicated proteins. (B) IkkβF/F and IkkβΔST mice were administered 10 mg/kg of lipopolysaccharide; after 1 hour the stomachs were extracted and stained with anti-p65 antibody. Left panel, IkkβF/F (magnification, 200×, 800×); right panel, IkkβΔST (magnification, 200×, 800×). (C) p65 nuclear-positive cells were counted, and the positive rate of IkkβΔST mice relative to IkkβF/F mice is shown (*P < .05). (D) NF-κB activation was determined by electrophoretic mobility shift assay. TF2D was used as a control for nuclear extract recovery. lps, lipopolysaccharide.
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3. Figure 2
Loss of IKKβ in the gastric epithelium inhibits gastric tumor development. (A) Schematic overview of gastric carcinogenesis model. Black areas indicate MNU (240 ppm) administration periods, and white areas indicate MNU nonadministration periods. (B) Representative stomach tumors. Left panels, IkkβF/F tumors; right panels, IkkβΔST tumors. Upper panels show macroscopic views of the tumor-bearing stomachs. Arrows indicate the tumors. Lower panels show H&E staining of tumor parts (magnification, 40×, 100×). (C) Number of tumors in stomachs. Stomachs were observed macroscopically, and tumors greater than 0.5 mm in diameter were counted. Values represent the mean ± standard error for IkkβF/F (n = 14) and IkkβΔST (n = 10) mice (*P < .05). (D) Stomach tumor sizes. The stomachs were observed macroscopically, and tumors greater than 0.5 mm in diameter were measured. Values represent the mean ± standard error for IkkβF/F (n = 35) and IkkβΔST (n = 8) mice. (E) Tumor incidence rates in IKKβF/F (85.7%; 12 of 14) and IKKβΔST (60.0%; 6 of 10) mice. (F) IKKβF/F and IKKβΔST tumors were immunoblotted with antibodies against IKKβ and β-tubulin.
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4. Figure 3
IKKβ knockdown potentiates MNU-mediated apoptosis. (A) Four hours after the oral administration of MNU (1000 ppm), stomachs were stained immunohistochemically with anti–phospho-p65 antibody (magnification, 100×, 800×). NF-κB activation level was measured by NF-κB ELISA (right panel). (B) MNU (1000 ppm) was administered orally and apoptotic cells were identified using the terminal deoxynucleotidyl transferase–mediated deoxyuridine nick-end labeling assay (upper panels; magnification, 100×). Lower panels show counterstaining with 4′,6-diamidino-2-phenylindole (magnification, 100×). (C) Total messenger RNA was isolated from gastric antrum 6 hours after a single oral dose of MNU (1000 ppm) to IkkβF/F and IkkβΔST mice. The expression levels of each gene were measured using real-time polymerase chain reaction. (D) IKKβ siRNA or control siRNA was transfected into SH101 cells, followed by treatment with 250 μmol/L MNU after 48 hours. The cells were collected at the indicated times, and NF-κB/p65 activity was quantified using ELISA. The y-axis shows p65 immunoreactivity, compared with that of recombinant p65. (E) Cells transfected with IKKβ siRNA (ikkβsi) or control siRNA (control) were treated with MNU for 48 hours, after which the number of living cells was counted. The survival rates of treated vs untreated cells are shown (*P < .05). (F) Levels of DNA released into the culture medium 48 hours after MNU stimulation were measured (*P < .05).
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5. Figure 4
Loss of IKKβ suppresses MNU-induced IL-1α expression. (A) MNU (1000 ppm) was administered orally to IkkβF/F and IkkβΔST mice, and stomachs were collected and lysed at the indicated times. Cytokine levels were measured using ELISA. (B) MNU (1000 ppm) was administered orally to IkkβF/F and IkkβΔST mice. Stomachs were stained immunohistochemically with anti–IL-1α antibody (magnification, 200×). Lower panels show counterstaining with 4′,6-diamidino-2-phenylindole (dapi) (magnification, 200×). (C) SH101 cells transfected with IKKβ siRNA (ikkβsi) or control siRNA (control) were treated with or without 250 μmol/L MNU. IL-1α levels in the culture medium were measured 12 hours after MNU stimulation (*P < .05).
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6. Figure 5
IL-1α promotes cell proliferation and inhibits apoptosis. (A) SH101 cells transfected with IL-1α siRNA (il-1αsi) or control siRNA (control) were treated with 250 μmol/L MNU for 48 hours, after which the number of living cells was counted. The survival rates of treated vs untreated cells are shown (*P < .05). (B) Cells were treated as described earlier. The degree of apoptotic induction was determined based on the amount of released DNA (*P < .05). (C) Cells were stimulated with or without 250 μmol/L MNU and/or 50 ng/mL of IL-1r antagonist (il-1ra) for 48 hours, and the degree of apoptotic induction was determined as described earlier (*P < .05). (D) AGS cells were stimulated with or without 250 μmol/L MNU and/or 50 ng/mL IL-1α for 48 hours, and the degree of apoptotic induction was determined as described earlier (*P < .05). (E) AGS cells were treated with or without IL-1α, and the cell number was determined at the indicated times (*P < .05).
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7. Figure 6
IL-1R knockout inhibits MNU-induced gastric tumor development. (A) MNU (1000 ppm) was administered orally to WT mice and IL-1 receptor knockout mice (IL-1R−/−). After 48 hours, the stomachs were removed and apoptotic levels were determined by the terminal deoxynucleotidyl transferase–mediated deoxyuridine nick-end labeling assay (upper panel; magnification, 100×). Lower panels show counterstaining with 4′,6-diamidino-2-phenylindole (magnification, 100×). (B) The number of tumors in WT and IL-1R−/− stomachs is shown. The stomachs were observed macroscopically, and tumors greater than 0.5 mm in diameter were counted. Values represent the mean ± standard error in WT (n = 14) and IL-1R−/− (n = 6) mice (*P < .05). (C) WT and IL-1R−/− stomach tumor sizes are shown. The stomachs were observed macroscopically, and tumors greater than 0.5 mm in diameter were measured. Values represent the means ± standard error in WT (n = 31) and IL-1R−/− (n = 7) mice. (D) The tumor incidence rate in each group is shown. (E) Proposed model of gastric carcinogenesis in MNU-treated mice.
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8. Supplementary Figure 1
Typical histology of stomach from the IkkβF/F and IkkβΔST mice (magnification, 100×).
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9. Supplementary Figure 2
Typical nontumor area at 40 weeks after initial MNU administration (magnification, 100×).
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10. Supplementary Figure 3
(A) Two hours after stimulation by MNU stomachs were examined by H&E staining (magnification, 100×, 400×). (B) The number of the terminal deoxynucleotidyl transferase–mediated deoxyuridine nick-end labeling (tunel)-positive cells per field is shown. (C) Total messenger RNA was isolated from corpus 6 hours after a single oral dose of MNU (1000 ppm) to IkkβF/F and IkkβΔST mice. The expression levels of each gene were measured using real-time polymerase chain reaction (*P < .05).
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11. Supplementary Figure 4
(A) MNU (1000 ppm) was administered orally to IkkβF/F and IkkβΔST, and the stomachs were stained with anti-PCNA antibody. (B) The percentage of the PCNA-positive cells in the stomach was calculated (mean ± standard deviation).
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12. Supplementary Figure 5
Cells transfected with IKKβ or control siRNA were treated with MNU for 6 hours, after which the expression levels of the indicated genes were measured using real-time polymerase chain reaction (*P < .05).
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13. Supplementary Figure 6
MNU (1000 ppm) was administrated to the mice, and the stomachs were lysed at the indicated times. Protein expression levels of cytokines were analyzed by the cytokine array. The spots in the red circles are IL-1α.
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14. Supplementary Figure 7
The number of the terminal deoxynucleotidyl transferase–mediated deoxyuridine nick-end labeling (tunel)-positive cells per field is shown. The TUNEL-positive cells were counted microscopically (magnification, 200×) (mean ± standard deviation).
Gastroenterology  , e6DOI: ( /j.gastro )
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15. Supplementary Figure 8
MNU (1000 ppm) was administered orally to mice, and stomachs were lysed at the indicated times. The lysates were gel-separated and immunoblotted with the indicated antibodies.
Gastroenterology  , e6DOI: ( /j.gastro )
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16. Supplementary Figure 9
(A) Six weeks after infection of H felis or Sydney strain 1 stomachs were examined by H&E staining (magnification, 400×). The arrows indicate infiltrated inflammatory cells. (B) The number of mononuclear cells and polymorphonuclear cells in the stomachs after 6 weeks of infection. Each cell was counted microscopically (magnification, 400×) at 5 fields/stomach (mean ± standard deviation). (C) Immunohistochemistry of phospho-IκBα in stomachs after 6 weeks of infection. Arrows indicate positive cells for phospho-IκBα (magnification, 400×).
Gastroenterology  , e6DOI: ( /j.gastro )
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