1. Volume 122, Issue 4, Pages 1020-1034 (April 2002)
Interferon-α activates multiple STAT signals and down-regulates c-Met in primary human hepatocytes 
Svetlana Radaeva, Barbara Jaruga, Feng Hong, Won–Ho Kim, Saijun Fan, Hongbo Cai, Stephen Strom, Youhua Liu, Osama El–Assal, Bin Gao 
Gastroenterology 
Volume 122, Issue 4, Pages (April 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Primary mouse and rat hepatocytes respond very well to IFN-γ but not to IFN-α. Primary rat hepatocytes, mouse hepatocytes, and mouse spleen cells were isolated and cultured for 24 hours, followed by a 30-minute stimulation with rat or mouse IFN-α (250 U/mL), IFN-β (250 U/mL), or IFN-γ (10 ng/mL). Whole-cell extracts were prepared and subjected to Western blot analysis by using anti–phospho-STAT1 or anti-STAT1 antibodies. Both p91 STAT1 and p84 STAT1 were detected.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

3. Fig. 2
IFN-α activates STAT1, STAT2, STAT3, and STAT5 in primary human hepatocytes, HCC HepG2 and Hep3B cells. (A) Primary human hepatocytes were transfected with an IFN-responsive reporter plasmid DNA (pISG54-luciferase), followed by stimulation with various concentrations of IFN-α for 24 hours. Luciferase activities were measured and expressed as fold change to control. (B) Primary human hepatocytes were treated with IFN-α (250 U/mL), IFN-β (250 U/mL), and IFN-γ (10 ng/mL) for 30 minutes. Whole-cell extracts were prepared and subjected to Western blot analysis by using anti–phospho-STAT1 or anti–phospho-STAT3 antibodies. (C) Primary human hepatocytes, HCC Hep3B and HepG2 cells were treated with IFN-α (500 U/mL) for various time-points as indicated. Whole-cell extracts were prepared and subjected to Western blot analysis by using anti–phospho-STAT1, anti–phospho-STAT3, and anti–phospho-STAT5. For the STAT2 activation, whole-cell extracts were immunoprecipitated with an anti–phospho-tyrosine antibody and blotted with anti-STAT2 antibody. (D) Primary human hepatocytes were treated with different hIFN-α for 30 minutes. Whole-cell extracts were prepared and subjected to Western blotting by using anti–phospho-STAT1, anti–phospho-STAT3, or anti–phospho-STAT5 antibodies.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Adult primary mouse hepatocytes express predominantly inhibitory soluble IFNAR2a, whereas primary human hepatocytes express predominantly functional full-length IFNAR2c. (A) Mouse liver RNA was isolated and subjected to RT-PCR by using mIFNAR primers as described in the Materials and Methods section. (B) HepG2, Hep3B, and primary human hepatocytes were treated with IFN-α (500 U/mL) for 24 hours. Total RNA was then isolated and subjected to RT-PCR by using hIFNR2 primers as described in the Materials and Methods section. (C) Expression of IFNAR1 and IFNAR2 in primary human hepatocytes by fluorescence-activated cell sorter analysis. Primary human hepatocytes were stained with directly conjugated IFNAR1 or IFNAR2 monoclonal antibody or control immunoglobulin G. Histograms for IFNAR1 or IFNAR2 (solid line) are shown overlaid on the control (dotted line).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

5. Fig. 4
IFN-α up-regulates antiviral genes and inhibits VSV activity in primary human hepatocytes and hepatoma cells. (A) Primary human hepatocytes (Hep.), HepG2, Hep3B cells were treated with IFN-α (500 U/mL) for 24 hours. Total RNA was prepared and subjected to RT-PCR by using various primers as described in the Materials and Methods section, or protein extracts were prepared and subjected to Western blot analysis (*) by using antibodies as indicated. (B) Primary human hepatocytes (Hepat.), HepG2, and Hep3B cells were treated with various concentrations of IFN-α overnight, followed by infection with VSV. After 2 days, cell viability was examined by using the MTT assay kit (Sigma) as described in the Materials and Methods section.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Effects of IFN-α on cell cycle and cell proliferation of primary human hepatocytes and hepatoma cells. (A) Primary human hepatocytes and HepG2 cells were incubated with 500 U/mL of IFN-α for 12 and 24 hours, cells were then harvested and used for cell cycle analysis as described in the Materials and Methods section. A representative DNA histogram was shown (upper panel). Statistical analyses of S-phase and sub–G1-phase were obtained from 3 independent experiments and are shown in the lower panel. Values shown are means ± SEM from 3 independent experiments. *P < 0.05, **P < 0.01, in comparison with corresponding IFN-untreated controls. (B) Primary human hepatocytes from 2 different cases and HepG2 cells were incubated with 500 U/mL of IFN-α for 24 hours; DNA was then extracted and used for DNA fragmentation analysis. (C) Primary human hepatocytes, HepG2 cells, and Hep3B cells were incubated with 500 U/mL of IFN-α for 24, 48, and 72 hours, followed by measuring thymidine uptake as described in the Materials and Methods section. Values shown are means ± SEM from a representative of 3 independent experiments. *P < 0.05, **P < 0.01, in comparison with corresponding IFN-untreated groups.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
IFN-α down-regulates c-Met, followed by suppression of HGF–c-Met–induced signals and cell proliferation. (A) Primary human hepatocytes (Hep.), HepG2, Hep3B cells were treated with IFN-α (500 U/mL) for 24 hours. Cell extracts were prepared and subjected to Western blotting analysis by using anti–c-Met or anti-ERK1/2 antibodies. Western blotting from 1 of 3 independent experiments is shown in the lower panel. Statistical analyses from 3 independent experiments are shown in the top panel. *P < 0.05, in comparison with the IFN-α–untreated groups. (B and C) HepG2 and Hep3B cells were treated with IFN-α (500 U/mL) for 24 hours, followed by a 10-minute stimulation with various concentrations of HGF as indicated (B), or by a 10-minute stimulation with epidermal growth factor (5 ng/mL) or insulin (5 μg/mL) (C). Cell extracts were prepared and subjected to Western blot analysis by using anti–phospho-ERK1/2 or anti-ERK1/2 antibodies. Western blotting from 1 of 3 independent experiments is shown in the lower panel of B. Statistical analyses of ERK1/2 from 3 independent experiments are shown in the upper panel of B. *P < 0.05, **P < 0.01 in comparison with the IFN-α–untreated groups. (D) Primary human hepatocytes were treated with IFN-α (500 U/mL) for 24 hours, followed by a 24-hour stimulation with HGF (5 or 2.5 ng/mL). Then thymidine uptake was measured as described in the Materials and Methods section. Values shown are means ± SEM from a representative of 3 independent experiments. &P < 0.05, in comparison with control group; *P < 0.05; **P < 0.01, in comparison with corresponding HGF-treated group.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
IFN-α down-regulates c-Met promoter activity and Sp1 binding. (A) Hep3B cells were transfected with various c-Met constructs as described in the Materials and Methods section, followed by a 48-hour stimulation with IFN-α (500 U/mL). CAT activities were measured. Values shown are means ± SEM from a representative of 3 independent experiments. *P < 0.05, **P < 0.01, in comparison with the corresponding untreated group. (B) Hep3B cells were treated with IFN-α (500 U/mL) for various time-points, cell extracts were then prepared and subjected to a gel shift assay by using 32P-labeled Sp1 probe or by using 32P-labeled NF-κB probe, or subjected to Western blot by using an anti-SP1 antibody.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions