Superoxide enhances interleukin 1β–mediated transcription of the hepatocyte-inducible nitric oxide synthase gene  Paul C. Kuo, Keith Abe, Rebecca A. Schroeder  Gastroenterology  Volume 118, Issue 3, Pages 608-618 (March 2000) DOI: 10.1016/S0016-5085(00)70268-X Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Nucleotide sequence of the rat hepatocyte iNOS promoter (1845 bp; GenBank X95629). ARE and NF-κB regulatory elements are labeled. Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Structure of successive 5'-deletion constructs of the rat hepatocyte iNOS promoter. Numbers assigned to the deletion plasmids correspond to the 5'-ends of the 5'-flanking sequence of the promoter from the transcription start site. Locations of the 2 NF-κB sites are labeled for all deletion constructs. Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Nuclear run-on assay of iNOS gene transcription in control, IL-1β–, and BZT-treated hepatocytes, as described in Materials and Methods. β-Actin and λ-phage cDNA served as positive and negative controls, respectively. Blot is representative of 4 experiments. IL-1β, 100 U/mL; BZT, 10 or 100 μmol/L. Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Nuclear run-on assay of iNOS gene transcription in control, IL-1β–, and BZT-treated hepatocytes in the presence and absence of SOD, as described in Materials and Methods. β-Actin and λ-phage cDNA served as positive and negative controls, respectively. Blot is representative of 4 experiments. IL-1β, 100 U/mL; BZT, 10 or 100 μmol/L; SOD, 100 U/mL. Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) iNOS promoter activity in rat hepatocytes stimulated with IL-1β and BZT. Total CAT expression was normalized to β-galactosidase activity and total protein. Data are expressed as mean ± SD of 4 experiments (ANOVA, P = 0.001 for all concentrations of IL-1β; P < 0.05 IL-1β 10 U/mL vs. IL-1β 100 U/mL for all concentrations of BZT; P < 0.05 IL-1β 100 U/mL vs. IL-1β 1000 U/mL for all concentrations of BZT; P < 0.02 IL-1β 1000 U/mL vs. IL-1β 10 U/mL for all concentrations of BZT). (B) iNOS promoter activity in rat hepatocytes stimulated with IL-1β, BZT, and SOD (100 U/mL). Total CAT expression was normalized to β-galactosidase activity and total protein. Data are expressed as mean ± SD of 4 experiments (P < 0.05 IL-1β 10 U/mL + SOD vs. IL-1β 100 U/mL + SOD for all concentrations of BZT; P < 0.05 IL-1β 100 U/mL + SOD vs. IL-1β 1000 U/mL + SOD for all concentrations of BZT; P < 0.02 IL-1β 1000 U/mL + SOD vs. IL-1β 10 U/mL + SOD for all concentrations of BZT). Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) iNOS promoter activity in rat hepatocytes stimulated with IL-1β and BZT. Total CAT expression was normalized to β-galactosidase activity and total protein. Data are expressed as mean ± SD of 4 experiments (ANOVA, P = 0.001 for all concentrations of IL-1β; P < 0.05 IL-1β 10 U/mL vs. IL-1β 100 U/mL for all concentrations of BZT; P < 0.05 IL-1β 100 U/mL vs. IL-1β 1000 U/mL for all concentrations of BZT; P < 0.02 IL-1β 1000 U/mL vs. IL-1β 10 U/mL for all concentrations of BZT). (B) iNOS promoter activity in rat hepatocytes stimulated with IL-1β, BZT, and SOD (100 U/mL). Total CAT expression was normalized to β-galactosidase activity and total protein. Data are expressed as mean ± SD of 4 experiments (P < 0.05 IL-1β 10 U/mL + SOD vs. IL-1β 100 U/mL + SOD for all concentrations of BZT; P < 0.05 IL-1β 100 U/mL + SOD vs. IL-1β 1000 U/mL + SOD for all concentrations of BZT; P < 0.02 IL-1β 1000 U/mL + SOD vs. IL-1β 10 U/mL + SOD for all concentrations of BZT). Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Normalized CAT expression in rat hepatocytes transfected with iNOS promoter constructs and stimulated with IL-1β and BZT. Data are expressed as mean ± SD of 4 experiments. #P < 0.01 vs. control; *P < 0.01 vs. IL-1β. (B) Normalized CAT expression in rat hepatocytes transfected with iNOS promoter constructs and stimulated with IL-1β and BZT. Data are expressed as mean ± SD of 4 experiments. #P < 0.01 vs. control; *P < 0.01 vs. IL-1β. Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Normalized CAT expression in rat hepatocytes transfected with iNOS promoter constructs and stimulated with IL-1β and BZT. Data are expressed as mean ± SD of 4 experiments. #P < 0.01 vs. control; *P < 0.01 vs. IL-1β. (B) Normalized CAT expression in rat hepatocytes transfected with iNOS promoter constructs and stimulated with IL-1β and BZT. Data are expressed as mean ± SD of 4 experiments. #P < 0.01 vs. control; *P < 0.01 vs. IL-1β. Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Schematic representation of oligonucleotides selected from the region nt 1359 to nt 1305 for use in gel shift assays. Each oligonucleotide has an overlap of 3–6 nucleotides. Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Gel shift analysis using ARE2, Redox 1, and Redox 2 in IL-1β (1000 U/mL)- and BZT (100 μmol/L)-stimulated rat hepatocytes. Representative of 3 analyses. Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 Gel shift analysis using ARE2 and mARE2 in IL-1β (1000 U/mL)- and BZT (100 μmol/L)-stimulated rat hepatocytes. Representative of 3 analyses. Gastroenterology 2000 118, 608-618DOI: (10.1016/S0016-5085(00)70268-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions