Bismarck Amoah-Apraku, Mao-Zhong Fang, Nicolas J. Guzman, M.D

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A non-nucleotide–bridged DNA decoy inhibits renal epithelial nitric oxide synthase expression Bismarck Amoah-Apraku, Mao-Zhong Fang, Nicolas J. Guzman, M.D Kidney International Volume 57, Issue 1, Pages 83-91 (January 2000) DOI: 10.1046/j.1523-1755.2000.00809.x Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 1 Electrophoretic mobility gel shift assay showing competitive displacement of radiolabeled interferon-γ regulatory factor-1 (IRF-1) oligonucleotide (ODN) probe from nuclear IRF-1 protein binding by triethyleneglycol-bridged IRF-1 decoy ODN. Probe lane represents electrophoresis of radiolabeled IRF-1 ODN in the absence of nuclear extract. The control lane represents binding reaction of radiolabeled IRF-1 ODN probe to IRF-1 in nuclear extracts from untreated cells. Treatment with IFN increases nuclear IRF-1. Incubation of nuclear extracts from IFN-treated cells with bridged IRF-1 decoy ODN (dc) displaces the IRF-1 ODN probe from its binding to IRF-1, whereas bridged scrambled ODN (sc) has no effect. Kidney International 2000 57, 83-91DOI: (10.1046/j.1523-1755.2000.00809.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 2 Electrophoretic mobility gel supershift assay using an anti–IRF-1 monoclonal antibody (1/250) in nuclear extracts of murine convoluted tubule (MCT) cells treated with IFN (100 U/mL for 2 hours). An addition of the antibody causes a shifted band (second lane from the left) that is competitively displaced by the IRF-1 decoy ODN (dc) but not by the scrambled ODN (sc). Kidney International 2000 57, 83-91DOI: (10.1046/j.1523-1755.2000.00809.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 3 Effect of increasing concentrations of bridged IRF-1 decoy ODN (A) and scrambled ODN (B) on nitrite production by MCT cells (*P < 0.05 vs. L/I, N = 12; L/I, LPS plus IFN treatment). Kidney International 2000 57, 83-91DOI: (10.1046/j.1523-1755.2000.00809.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effects of bridged IRF-1 decoy (dc) and scrambled (sc) ODNs on (A) iNOS protein and (B) steady-state iNOS mRNA levels in MCT cells treated with a combination of LPS (1 μg/mL) and IFN (100 U/mL) for 12 and 6 hours, respectively. Kidney International 2000 57, 83-91DOI: (10.1046/j.1523-1755.2000.00809.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 5 Effect of bridged IRF-1 decoy (dc) and scrambled (sc) ODNs on transcriptional activity of the iNOS promoter in MCT cells following induction with LPS and IFN (N = 4; L/I, LPS plus IFN treatment). Kidney International 2000 57, 83-91DOI: (10.1046/j.1523-1755.2000.00809.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 6 Effect of bridged IRF-1 decoy (dc) and scrambled (sc) ODNs on (A) IRF-1 protein and (B) steady-state IRF-1 mRNA levels in MCT cells treated with a combination of LPS (1 μg/mL) and IFN (100 U/mL). IRF-1 protein was measured after 6 and 10 hours of LPS and IFN treatment, and iNOS mRNA was measured after 2 hours of similar treatment. Kidney International 2000 57, 83-91DOI: (10.1046/j.1523-1755.2000.00809.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 7 Effect of bridged IRF-1 decoy (dc) ODN on LPS stimulation (1 μg/mL for 2 hours) of MCP-1 mRNA expression. Kidney International 2000 57, 83-91DOI: (10.1046/j.1523-1755.2000.00809.x) Copyright © 2000 International Society of Nephrology Terms and Conditions