1. Large Hepatitis Delta Antigen Modulates Transforming Growth Factor-β Signaling Cascades: Implication of Hepatitis Delta Virus–Induced Liver Fibrosis 
Soo–Ho Choi, Sook–Hyang Jeong, Soon B. Hwang 
Gastroenterology 
Volume 132, Issue 1, Pages (January 2007)
DOI: /j.gastro
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2. Figure 1
(A) Schematic diagram of functional domains in small hepatitis delta antigen (SHDAg) and large hepatitis delta antigen (LHDAg). CCS, coil-coiled sequence; NLS, nuclear localization signal; ARM, arginine-rich motif; HLH, helix-loop-helix; RBD, RNA-binding domain; IS, isoprenylation site. (B, C) LHDAg amplifies transforming growth factor-β (TGF-β)–induced transcriptional activity. Huh7 cells were transfected with either SBE4-luc or 3TP-lux reporter plasmid together with the indicated plasmids. At 24 hours after transfection, cells were either left untreated or treated with human TGF-β (5 ng/mL) for 24 hours, and then luciferase activities were measured. The data shown represent triplicate experiments. (D) LHDAg enhances TGF-β–mediated transcriptional activation in a dose-dependent manner. Huh7 cells were cotransfected with the indicated plasmids, together with selected amounts (2 and 5 μg) of either SHDAg or LHDAg. At 36 hours after transfection, cells were harvested, and SBE-luc reporter activity was determined. (E) LHDAg activates TGF-β–induced Smad-DNA complex formation. Huh7 cells were transfected with empty vector, SHDAg, and LHDAg expression plasmid, respectively. At 24 hours after transfection, cells were stimulated with human TGF-β (5 ng/mL) for 24 hours, and nuclear extracts were assessed for the formation of Smad and DNA complex by using TGF response sequence as a probe.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Both small hepatitis delta antigen (SHDAg) and large hepatitis delta antigen (LHDAg) interact with Smad3 in vitro and in vivo. (A) In vitro glutathione S-transferase (GST) pull-down assay. Huh7 cells were transfected with either SHDAg-Myc or LHDAg-Myc expression plasmid. At 36 hours after transfection, cell lysates were incubated with either GST or GST–Smad fusion proteins purified from Escherichia coli. Bound proteins were precipitated by glutathione beads and detected by Western blot (WB) analysis using anti-Myc monoclonal antibody (top panel). Both SHDAg and LHDAg expressions in Huh7 cells were verified by anti-Myc antibody (middle panel). GST-fusion proteins used in binding assay were verified by staining with Coomassie brilliant blue (CBB; bottom panel). (B) In vivo coimmunoprecipitation assay. Cos7 cells were transfected with the indicated combinations of expression plasmids paired with recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). At 12 hours after transfection, cell lysates were immunoprecipitated with anti-Flag monoclonal antibody, and bound proteins were detected by Western blot analysis by using anti-Myc monoclonal antibody (top panel). Both HDAg and Smad protein expressions were verified by using the same cell lysates by Western blotting with either anti-Myc monoclonal antibody for HDAgs (middle panel) or anti-Flag monoclonal antibody for Smad proteins (bottom panel). (C) Reciprocally, cell lysates used in B were immunoprecipitated with anti-Myc antibody, and bound proteins were Western blotted with anti-Flag antibody. IP, immunoprecipitation.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
(A) Hepatitis delta antigen (HDAg) interacts with Smad3 through amino acids 68–107 of HDAg. Schematic diagram shows both wild-type and mutant forms of Myc-tagged HDAg. aa, amino acids (B) Huh7 cells were transfected with HDAg-Myc expression plasmids. At 36 hours after transfection, cell lysates were incubated with glutathione S-transferase (GST)–Smad3 fusion protein purified from Escherichia coli. Bound proteins were precipitated by glutathione beads and detected by Western blot (WB) analysis by using anti-Myc monoclonal antibody (top panel). HDAg expressions in Huh7 cells were verified by anti-Myc antibody (middle panel). GST-Smad3 protein used in binding analysis was verified by staining with Coomassie brilliant blue (CBB; bottom panel). (C) Schematic diagram shows both wild-type and mutant forms of Smad3. (D) Smad3 interacts with HDAg through MH2 domain of Smad3. Huh7 cells were transfected with either wild-type or mutant forms of Flag-tagged Smad3 expression plasmids. At 36 hours after transfection, total cell lysates were incubated with either GST–small hepatitis delta antigen (SHDAg) or GST–large hepatitis delta antigen (LHDAg) fusion protein purified from E. coli. Bound proteins were precipitated by glutathione beads and detected by Western blot analysis by using anti-Flag monoclonal antibody (top, middle, and right panels). Protein expressions of both wild-type and mutants of Smad3 in Huh7 cells were verified by anti-Flag antibody using total cell lysates (top, left panel). Both GST-SHDAg and GST-LHDAg used in the binding assay were verified by staining with CBB (bottom 2 panels).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
(A) c-Jun–induced activator protein-1 (AP-1) activity is synergistically elevated by large hepatitis delta antigen (LHDAg). Huh7 cells were transfected with empty vector, Flag-c-Jun (2 μg), small hepatitis delta antigen (SHDAg; 2 μg), and LHDAg (2 μg) expression plasmid in the absence or presence of c-Jun. At 24 hours after transfection, cells were harvested, and AP-1-luc reporter activity was determined (top panel). Protein expressions of exogenous c-Jun and HDAgs were verified in the bottom two panels. (B) LHDAg activates c-Jun–induced AP-1 activity. Huh7 cells were transfected with the indicated expression plasmids. At 24 hours after transfection, nuclear extracts were purified and assessed for the formation of AP-1 and DNA complex. (C) HDAgs directly interact with c-Jun. In vitro GST pull-down assay. Huh7 cells were transfected individually with plasmid expressing Flag-JNK1, Flag-c-Jun, and c-Fos-HA. At 36 hours after transfection, total cell lysates were incubated with either GST (lane 1) or GST-HDAg (lanes 2 and 3) fusion proteins purified from Escherichia coli. Bound proteins were precipitated by glutathione beads and detected by Western blot analysis using either anti-Flag monoclonal antibody (top and middle panels) or anti-HA polyclonal antibody (bottom panel). Input proteins are shown in lane 4.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Large hepatitis delta antigen (LHDAg) abrogates the inhibitory effect of c-Jun on transforming growth factor-β (TGF-β)–mediated signaling cascade. (A) Huh7 cells were cotransfected with c-Jun plasmid together with selected amounts (0.5, 1, and 2 μg) of either small hepatitis delta antigen (SHDAg) or large hepatitis delta antigen (LHDAg). At 24 hours after transfection, cells were either left untreated or treated with human TGF-β (5 ng/mL) for 24 hours, and then luciferase activities were determined (upper panel). Protein expressions of c-Jun and HDAgs in Huh7 cells were verified by corresponding antibodies (lower two panels). (B) HDAgs inhibit Smad3–c-Jun complex formation. Huh7 cells were transfected with the indicated combinations of expression plasmids. Total cell lysates were immunoprecipitated with anti-Myc antibody, and then bound proteins were Western blotted (WB) with anti-Flag antibody (top panel). The protein expressions of Smad3, c-Jun, and HDAgs were verified by Western blotting with anti-Myc, anti-Flag, and anti-HDAg antibodies, respectively (bottom three panels). (C) Alternatively, in vitro–translated Flag-c-jun (224–331 aa) and HDAgs were incubated with either GST or GST-Smad3 protein. Glutathione S-transferase–bound proteins were detected by autoradiography (top panel). Protein expressions of in vitro–translated c-jun and HDAgs were shown in bottom panel.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Isoprenylation of large hepatitis delta antigen (LHDAg) is essential for the enhancement of transforming growth factor-β (TGF-β)–mediated signal transduction. (A) Huh7 cells were cotransfected with either constitutively active TGF-β receptor 1 (TβR-I; T204D) or c-Jun and various HDAg expression plasmids. At 36 hours after transfection, cells were harvested, and both SBE-luc (top left panel) and AP-1 reporter (top right panel) activities were determined. The protein expressions of transfected plasmids in the same cell lysates were verified as shown in bottom panels. (B) Huh7 cells were transfected with empty vector, LHDAg, and L-C211S expression plasmid, respectively. At 24 hours after transfection, cells were stimulated with human TGF-β (5 ng/mL) for 24 hours, and nuclear extracts were assessed for the formation of activated Smad–DNA complexes by using 32p-labeled TGF-β–response sequence as a probe. (C) Huh7 cells were transfected with the indicated expression plasmids. At 24 hours after transfection, nuclear extracts were prepared and assessed for the formation of AP-1 and DNA complex.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
Large hepatitis delta antigen (LHDAg) enhances the expression level of endogenous plasminogen activator inhibitor (PAI)-1 protein. Huh7 cells were transfected individually with empty vector, small hepatitis delta antigen (SHDAg), and LHDAg expression plasmid (2 μg each). At 24 hours after transfection, cells were either left untreated or treated with human transforming growth factor-β (TGF-β; 5 ng/mL) for 24 hours, and then total cell lysates were Western blotted with anti–PAI-1 antibody (BD Biosciences). The protein expressions of HDAgs and β-actin in the same cell lysates were verified by Western blot analysis (lower 2 panels).
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Coexpression of large hepatitis delta antigen (LHDAg) and hepatitis B virus X (HBx) synergistically activates transforming growth factor-β (TGF-β) and AP-1 signal-transduction pathways. (A) Huh7 cells were either transfected individually with vector, HBx, or LHDAg or were cotransfected with HBx and LHDAg (1 and 2 μg) expression plasmids. At 24 hours after transfection, cells were either left untreated or treated with human TGF-β (5 ng/mL) for 24 hours, and then SBE-luc activity was measured. (B) Huh7 cells were cotransfected with the indicated combinations of expression plasmids. At 24 hours after transfection, cells were harvested, and AP-1 activity was determined.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions