A Dual AP-1 and SMAD Decoy ODN Suppresses Tissue Fibrosis and Scarring in Mice  Hong-Feng Yuan, Hong Huang, Xiang-Yun Li, Wei Guo, Wei Xing, Zhi-Ya Sun, Hua-Ping Liang, Jian Yu, Dong-Feng Chen, Zheng-Guo Wang, Jin Hao, Xiang Xu  Journal of Investigative Dermatology  Volume 133, Issue 4, Pages 1080-1087 (April 2013) DOI: 10.1038/jid.2012.443 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The binding of dual-functional decoy double-stranded oligodeoxynucleotides (dsODNs) with transcription activator protein 1 (AP-1) and SMAD (small mothers against decapentaplegic). (a) Interaction of the indicated dsODNs with c-jun or SMAD3 analyzed using surface plasmon resonance. Recombinant c-jun and SMAD3 (1.25μg) were injected at the experimental times 1 and 2, respectively. (b) The DNA-binding activity of AP-1 (left) or SMAD (right) with or without antifibrosis ODN4 (AFODN4) was analyzed by supershift electrophoretic mobility shift assay (EMSA) using nuclear extracts from NIH3T3 cells treated with 5ngml−1 of transforming growth factor-β (TGF-β1). Journal of Investigative Dermatology 2013 133, 1080-1087DOI: (10.1038/jid.2012.443) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Dual-functional decoy double-stranded oligodeoxynucleotides (dsODNs) inhibited transcription activator protein 1 (AP-1) and SMAD (small mothers against decapentaplegic) transcriptional activities in L929 fibroblasts. (a) Effect of the indicated decoy dsODN on the transcriptional activities of AP-1 induced by transforming growth factor-β (TGF-β). (b) Effect of the indicated decoy dsODN on the transcriptional activities of SMAD induced by TGF-β. (c) Dosage effect of antifibrosis ODN4 (AFODN4) on the transcriptional activities of AP-1 and SMAD. (d) Effect of AFODN4 on the transcriptional activity of NF-κB in response to tumor necrosis factor-α (TNF-α). (e) Effect of AFODN4 on the transcriptional activity of STAT1/3 in response to IFN-γ. RLU, relative luminescence units. *P<0.05, **P<0.01 versus scramble dsODN without TGF-β stimulation; #P<0.05, ##P<0.01 versus scramble dsODN with TGF-β stimulation. Journal of Investigative Dermatology 2013 133, 1080-1087DOI: (10.1038/jid.2012.443) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of antifibrosis oligodeoxynucleotide 4 (AFODN4) on cell proliferation and apoptosis in transforming growth factor-β (TGF-β)-stimulated L929 fibroblasts. (a) Cell proliferation measured by cell counting after treatment with the indicated decoy double-stranded ODN (dsODN) and TGF-β. (b) Cell proliferation was measured using [3H]-thymidine proliferation assay after treatment with the indicated decoy dsODN and TGF-β. (c) Effect of AFODN4 on apoptosis was analyzed by flow cytometry. Experiments were performed at least three times and representative data are shown. *P<0.05, **P<0.01 versus scramble dsODN group. Journal of Investigative Dermatology 2013 133, 1080-1087DOI: (10.1038/jid.2012.443) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of antifibrosis oligodeoxynucleotide 4 (AFODN4) on the expression of transforming growth factor-β (TGF-β) target genes in L929 fibroblasts. (a) mRNA expression of indicated TGF-β target genes was measured by quantitative real-time reverse-transcriptase–PCR (qRT–PCR). (b) Protein expression of the indicated TGF-β targets was analyzed by immuno-blotting (IB) analysis. (c) Relative IOD of protein expression of the indicated TGF-β targets. Decoy double-stranded ODNs (dsODNs) were used at 100nM. Results are expressed as mean±SEM (n=3). #P<0.05, ##P<0.01 versus scramble dsODN without TGF-β stimulation; *P<0.05, **P<0.01 versus scramble dsODN with TGF-β stimulation. AP-1, transcription activator protein 1; Col1α2, collagen type I α2; CTGF, connective-tissue growth factor; IOD, integrated option density; PAI-1, plasminogen activator inhibitor-1; SMAD, small mothers against decapentaplegic. Journal of Investigative Dermatology 2013 133, 1080-1087DOI: (10.1038/jid.2012.443) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of antifibrosis oligodeoxynucleotide 4 (AFODN4) on tissue fibrosis in wounded mice. (a) Granulation tissues were stained for fibrosis using Masson’s trichrome stain. (b) Immunohistochemistry (IHC) of for α-smooth muscle actin (α-SMA) in the granulation tissues. The positive staining (yellow) demonstrates α-SMA-positive myofibroblasts. Scale bar=100μm; original magnification × 40. (c) Interstitial collagen index assessed by the point-counting method in Masson’s trichrome–stained sections. (d) Hydroxyproline (HP) contents of the wound bed. (e) The number of myofibroblasts (α-SMA-positive cell) per field in the wounds. (f) The expression of indicated fibrotic mediators and proinflammatory cytokines in granulation tissues was detected using immuno-blotting (IB) analysis. (g) DNA binding of transcription activator protein 1 (AP-1) and SMAD (small mothers against decapentaplegic) in granulation tissues was detected using electrophoretic mobility shift assay (EMSA). *P<0.05, **P<0.01 versus scramble double-stranded oligodeoxynucleotide (dsODN) group. Journal of Investigative Dermatology 2013 133, 1080-1087DOI: (10.1038/jid.2012.443) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions