1. Volume 126, Issue 2, Pages 529-540 (February 2004)
Hepatitis C virus core and nonstructural proteins induce fibrogenic effects in hepatic stellate cells 
Ramó Bataller, Yong-han Paik, Jeffrey N. Lindquist, John J. Lemasters, David A. Brenner 
Gastroenterology 
Volume 126, Issue 2, Pages (February 2004)
DOI: /j.gastro

2. Figure 1
Expression of mRNA encoding the putative HCV receptors CD81, LDL receptor, and complement 1q (C1q) receptor. Quiescent human HSCs (2 days in culture after isolation from a normal liver), human HSCs activated in culture (7 and 14 days in culture), and human HSCs activated in vivo (2 days in culture after isolation from a HCV-induced cirrhotic liver) were studied. No albumin mRNA was amplified in any of the samples (not shown). β-Actin was amplified to demonstrate equal mRNA loading.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
(A ) Effect of recombinant core and NS3 proteins on [Ca2+]i in human HSCs. Cells were loaded with the Ca2+-sensitive dye Fluo-4 (10 μmol/L) and then stimulated with buffer, core protein (10 ng/mL), or NS3 protein (10 ng/mL). Changes in [Ca2+]i were assessed with a 510 Zeiss confocal microscopy. Changes in cell fluorescence were assessed every 10 seconds. A change in cell color from blue to green-red reflects an increase in [Ca2+]i. Preincubation of cells with a blocking antibody against complement 1q (C1q) receptors, but not an isotype-matched antibody (not shown), attenuated core protein-induced increase in [Ca2+]i. (B) Quantification of [Ca2+]i changes in fluo-4-loaded HSCs using a fluorometer at 485–535 nm.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Effect of recombinant core and NS3 proteins on ROS formation in human HSCs. (A ) Cells were loaded with the redox sensitive dye 2′,7′-dichlorofluorescein diacetate (DCFDA, 10 μmol/L) and then stimulated with buffer, core (10 ng/mL), and NS3 protein (10 ng/mL). (B) Quantification of changes in DCFDA-loaded HSCs using a fluorometer at 485–535 nm. Both core and NS3 proteins induced a rapid increase in cell fluorescence. Core protein-induced ROS formation was attenuated by diphenylene iodonium (DPI, 1 μmol/L), a NADPH oxidase inhibitor, and by the antioxidant N-acetylcysteine (NAC, 10 μmol/L). Similar regulation was observed with NS3 protein (not shown). This graph is representative of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Core and NS3 recombinant proteins stimulate intracellular signaling pathways in human HSCs. (A ) Effect of core and NS3 protein on NF-κB-dependent gene expression, as assessed by luciferase reporter gene assay. ∗P < 0.05 vs. untreated. (B) Effects of core (10 ng/mL) and NS3 protein (10 ng/mL) on phosphorylation of ERK-2, c-Jun, p38 MAPK, and AKT. Cells were stimulated with buffer or HCV proteins for 15 minutes. Twenty-five micrograms of proteins were subjected to Western blot analysis (see Materials and Methods section). α-Tubulin was measured to demonstrate equal protein loading. Figure is representative of 3 independent experiments. (C ) Effect of core and NS3 proteins on AP-1 DNA binding, as assessed by electrophoretic mobility shift assay (see Materials and Methods section). Figures are representative of 3 independent experiments. Relative expression is shown beneath each line.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Effect of adenoviral expression of HCV core and NS3-NS5 proteins on DNA synthesis in human HSCs. (A ) Cells were serum starved for 24 hours and then infected with either control adenovirus or adenovirus encoding core and NS3-NS5 proteins for 24 hours. DNA synthesis was estimated as 3H-thymidine incorporation. Results are the mean ± SE of 4 independent experiments done in triplicate. ∗P < 0.05 vs. cells incubated with control adenovirus. (B) Effect of different inhibitors on adenovirus core-induced increase in DNA synthesis. Cells were incubated for 12 hours with DMSO, the ERK inhibitor PD98059 (5 μmol/L), the PI3K inhibitor LY (2 μmol/L), the antioxidant N-acetylcysteine (NAC, 10 μmol/L), the Ca2+ chelator BAPTA-AM (10 μmol/L), the Ras antagonist SCH66336 (1 μmol/L), the p38 MAPK inhibitor SB (1 μmol/L), and the JNK inhibitor SP (20 μmol/L). Results are expressed as mean ± SE of 3 independent experiments done in triplicate. ∗P < 0.05 vs. cells incubated with DMSO.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
(A ) Effect of infection with recombinant adenovirus encoding HCV core and NS3-NS5 proteins on chemokine secretion in human HSCs. Cells were serum starved for 24 hours and then infected with either control adenovirus or adenovirus carrying core and NS3-NS5 proteins for 24 hours. Supernatants were collected, and a sandwich ELISA was performed for human interleukin-8 (IL-8), MCP-1, and RANTES. Results are expressed as fold secretion with respect to cells infected with control adenovirus. Results are mean ± SE of 4 independent experiments done in duplicate. ∗P < 0.05 vs. cells incubated with control adenovirus. (B) Effect of different treatments on adenovirus NS3-NS5-induced increase in chemokine secretion. Cells were infected for 24 hours with a NF-κB super repressor adenovirus or a control adenovirus or incubated for 12 hours with DMSO, the ERK inhibitor PD98059 (5 μmol/L), the antioxidant N-acetylcysteine (NAC, 10 μmol/L), the Ca2+ chelator BAPTA-AM (10 μmol/L), the p38 MAPK inhibitor SB (1 μmol/L), and the JNK inhibitor SP (20 μmol/L). Results are expressed as mean ± SE of 3 independent experiments done in duplicate. ∗P < 0.05 vs. cells incubated with a control adenovirus. ∗∗P < 0.05 vs. cells incubated with DMSO. (C ) Effect of adenovirus-driven NS3-NS5 protein on cell adhesion molecule expression in human HSCs. Cells were infected with either control adenovirus and NS3-NS5 adenovirus for 24 hours, and VCAM-1 expression was assessed by flow cytometry (see Materials and Methods section). Figure is representative of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Effect of infection with recombinant adenoviruses encoding HCV core and NS3-NS5 proteins on phenotypic activation in rat primary HSCs. Cells were infected with adenoviruses at day 2 after isolation. (A ) Phase contrast photography of HSCs at day 4 after isolation. Cells infected with adenoviruses encoding core protein showed increased cell spreading and features of phenotypic activation to myofibroblastic cells (arrows). (B) Quantification of smooth muscle α-actin, a marker of cell activation, by Western blotting in rat HSCs. Cells were infected with adenoviruses at day 2 and whole cell extracts obtained at day 4. Ten micrograms of proteins were blotted with an antismooth muscle α-actin antibody. Figures are representative of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
Fibrogenic effects of infection with recombinant adenoviruses encoding HCV core and NS3-NS5 proteins on primary rat HSCs. Cells were infected with adenovirus at day 2 after isolation and mRNA and cell supernatants collected at day 4. (A ) Measurement of procollagen α1(I) mRNA levels by RNase protection assay (see Materials and Methods section). Results are normalized to GADPH expression. Figure is representative of 3 independent experiments. ∗P < 0.05 vs. cells infected with control adenovirus. (B) Measurement of bioactive TGFβ1 in cell supernatants. Results are the mean ± SE from 3 independent experiments. ∗P < 0.01 vs. cells infected with control adenovirus.
Gastroenterology  , DOI: ( /j.gastro )