1. Anti-fibrotic Effects of Synthetic Oligodeoxynucleotide for TGF-β1 and Smad in an Animal Model of Liver Cirrhosis 
Jung-Yeon Kim, Hyun-Jin An, Woon-Hae Kim, Mi-Gyeong Gwon, Hyemin Gu, Yoon-Yub Park, Kwan-Kyu Park 
Molecular Therapy - Nucleic Acids 
Volume 8, Pages (September 2017)
DOI: /j.omtn
Copyright © 2017 The Authors Terms and Conditions

2. Figure 1 Construction of Synthetic TGF-β1/Smad ODN
Target sites for TGF-β1 were selected via the sequential overlap simulation of secondary structures using the S-Fold program. TGF-β1/Smad ODN ligates the antisense TGF-β1 ODN and Smad decoy ODN.
Molecular Therapy - Nucleic Acids 2017 8, DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2 Confirmation of FITC-Labeled TGF-β1/Smad ODN in HSC-T6 Cells
(A) Fluorescence activity was detected in both cytoplasm and nucleus with FITC-labeled ODN deposition. Scale bar, 50 μm. (B) Transfection efficiency of FITC-labeled TGF-β1/Smad ODN through flow cytometry. Effect of Smad decoy, TGF-β1 antisense ODN, and TGF-β1/Smad ODN on Smad transcription activity and expression levels of TGF-β1 in HSC-T6 cells. (C–F) Luciferases activity in TGF-β1-treated HSC-T6 cells (C), electrophoretic mobility shift assay (EMSA) (D), real-time PCR (E), and western blot assay (F). *p < 0.05 versus normal control. †p < 0.05 versus TGF-β1-treated group.
Molecular Therapy - Nucleic Acids 2017 8, DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3 TGF-β1/Smad ODN Effectively Inhibits Activated HSCs by TGF-β1
(A) Morphology of TGF-β1-treated HSC-T6 cells with Scr ODN or TGF-β1/Smad ODN. (B) CCK-8 assay shows that TGF-β1/Smad ODN inhibits proliferation of activated HSC induced by TGF-β1. (C) Representative immunofluorescence images show the expression of α-SMA (red) and Hoechst (blue) in HSC-T6 cells. Scale bar, 50 μm. *p < 0.05 versus normal control. †p < 0.05 versus TGF-β1-treated group. (D and E) Effect of TGF-β1/Smad ODN on expressions of (D) fibrogenesis-related protein through (E) Smad signaling pathway in TGF-β1-treated HSC-T6 cells using western blot analysis.
Molecular Therapy - Nucleic Acids 2017 8, DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
TGF-β1/Smad ODN Suppresses EMT and Fibrosis-Related Protein in TGF-β1-Induced AML12 Cells
(A) Effect of TGF-β1/Smad ODN on morphologic change of AML12 cells treated with TGF-β1. (B and C) Effect of TGF-β1/Smad ODN on expressions of EMT and fibrosis-related protein (B) through Smad signaling pathway (C) using western blot analysis
Molecular Therapy - Nucleic Acids 2017 8, DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 5
Effect of TGF-β1/Smad ODN on E-cadherin and Collagen I Expression in TGF-β1-Treated AML12 Cells
Immunofluorescence double staining for E-cadherin (green) and collagen I (red) localization. Cells were counterstained with Hoechst (blue). Scale bar, 50 μm.
Molecular Therapy - Nucleic Acids 2017 8, DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 6
TGF-β1/Smad ODN Suppresses CCl4-Induced Liver Damage and Collagen Accumulation
(A and B) Histological section of murine liver stained with (A) H&E stain, (B) Masson’s trichrome staining after 8 weeks of CCl4 administration. Histological examinations are performed at 200× magnification under light microscopy. (C) Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly decreased by TGF-β1/Smad ODN treatment compared with Scr ODN treatment in CCl4-induced mice. *p < 0.05 versus normal control. †p < 0.05 versus CCl4+Scr ODN-treated group.
Molecular Therapy - Nucleic Acids 2017 8, DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

8. Figure 7
TGF-β1/Smad ODN Significantly Inhibits the Pro-inflammatory Cytokine in CCl4-Induced Mouse Model
(A and B) ELISA results demonstrate that TGF-β1/Smad ODN significantly inhibits the pro-inflammatory cytokine in CCl4-induced mice: IL-6 (A) and IFN-γ (B) expression. *p < 0.05 versus normal control. †p< 0.05 versus the CCl4+Scr ODN-treated group. (C) Western blot results show that TGF-β1/Smad ODN inhibits expressions of TNF-α, IL-1β, and IL-6.
Molecular Therapy - Nucleic Acids 2017 8, DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

9. Figure 8
TGF-β1/Smad ODN Significantly Inhibits the Fibrogenesis-Related Gene in CCl4-Induced Mouse Model
(A and B) Immunochemical staining results showed that TGF-β1/Smad ODN inhibits expression of fibronectin (A) and FSP-1 (B) in CCl4-induced mouse model. Histological examinations are performed at 200× magnification under light microscopy. (C) Western blot results show that TGF-β1/Smad ODN inhibits expressions of TGF-β1, α-SMA, and collagen I.
Molecular Therapy - Nucleic Acids 2017 8, DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

10. Figure 9
TGF-β1/Smad ODN Significantly Inhibits the Smad Signaling Pathway in CCl4-Induced Mouse Model
(A and B) Immunofluorescence staining results showed that TGF-β1/Smad ODN inhibits expression of TGF-β1 (green) (A) and phosphorylation of Smad2/3 (green) and Smad4 (B) in CCl4-induced mice. Scale bar, 50 μm. (C) Western blot results showed that TGF-β1/Smad ODN inhibits expressions of phosphorylated Smad2/3, Smad4, and Smad7.
Molecular Therapy - Nucleic Acids 2017 8, DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions