1. Volume 128, Issue 7, Pages 2042-2053 (June 2005)
Interferon-γ Inhibits Hepatitis B Virus–Induced NF-κB Activation Through Nuclear Localization of NF-κB–Inducing Kinase 
Sung Gyoo Park, Hyun Mi Ryu, Seong-Oe Lim, Yong-Il Kim, Soon B. Hwang, Guhung Jung 
Gastroenterology 
Volume 128, Issue 7, Pages (June 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Analysis of NF-κB activation by each component of HBV. NF-κB activation was analyzed by reporter assay through cotransfection with the pNF-κB-luci reporter plasmid. (A) After transfecting each plasmid (pHBV1.2× for HBV replication, pHBV1.2×(-X) for HBV replication without HBx expression, pCMV-HA/HBx for HBx expression, pCMV-HA/Pol for HBV pol expression, pCMV-HA/HBs for HBs expression, and pCMV-FLAG/HBc for HBc expression) into HepG2 cells, luciferase activities were estimated. (B) Expressions of HBx in HepG2 cells transfected with pHBV1.2×, pHBV1.2×(-X), and pCMV-HA/HBx were tested by immunoblot analysis with anti-HBx rabbit polyclonal antibody. To test whether the activation of NF-κB induced by HBV replication (C) and HBx (D) is dependent on NIK, we analyzed NF-κB activity by cotransfecting pFLAG-NIK/DN into HepG2 cells transfected with pHBV1.2× or pCMV-HA/HBx. pUC119 transfection was used for the control. In the reporter assays, pNF-κB-luci and pCMV-β-Gal were cotransfected for monitoring NF-κB activity and normalizing transfection efficiency. Data represent the mean ± standard error of the mean. *P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
NIK nuclear localization occurs only in liver tissue, independent of tissue origin (nontumor versus tumor), obtained from serologically HBsAg(+) patients. Liver tissue specimens (nontumor and tumor sections) were obtained from serologically HBsAg(+) patients (n = 4) and HBsAg(−) patients (n = 4) and embedded in paraffin for immunohistochemical analysis. (A) The embedded tissues were analyzed with the anti-NIK antibody. The nuclei were stained with hematoxylin. Arrows indicate examples of stained NIK in nuclei. (B) In controls, nuclei were stained with hematoxylin, and cytoplasms were stained with eosin. This analysis shows the results from non-tumor and tumor tissues obtained from patients of each serological type.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
IFN-γ induces NIK nuclear localization in HepG cells but not in HepG2 cells. (A) HepG2 cells and (B) HepG cells were treated with IFN-γ (1000 U/mL) for 2 days. In the case of HepG cells, treatment with IFN-α (1000 U/mL) was used as the control. After IFN-γ or IFN-α treatment, localization of NIK in each cell was visualized by fluorescence microscopy with the anti-NIK rabbit polyclonal antibody as described in Materials and Methods. After treatment of (C) IFN-γ or (D) IFN-α, the levels of NIK in the cytoplasmic and nuclear regions were estimated by immunoblot analysis with anti-NIK rabbit polyclonal antibody. The following controls were used: NPM (nucleophosmin B23, nucleolar protein) for the nuclear region control, β-actin for the cytoplasmic region control, and total crude extracts of HepG2 cells transfected with pFLAG-NIK for the positive control. (E) After treatment with IFN-γ (1000 U/mL) for 2 days, expression of the HBc protein was analyzed by fluorescence microscopy with the anti-HBc rabbit polyclonal antibody in HepG cells. HepG2 cells were used for the negative control. In these fluorescence microscopy analyses, nuclei were stained with DAPI.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
The effect of IFN-γ on the localization of other kinases was tested by examining IKK-α and IKK-β localization with NIK. One day after HepG cells were transfected with (A) pFLAG-IKK-α, (B) pFLAG-IKK-β, or (C) pFLAG-NIK, cells were treated with IFN-γ (1000 U/mL). Localization of each expressed protein and expression of the HBc protein were observed by double staining with anti-FLAG mouse monoclonal (M2) and anti-HBc rabbit polyclonal antibodies. The stained cells were visualized by fluorescence microscopy. HepG2 cells were transfected with either (D) pHBV1.2×, (E) pCMV-HA/HBx, or (F) pHBV1.2×(-X) and pFLAG-NIK, treated with IFN-γ (1000 U/mL) for 2 days and (D and F) double stained with anti-FLAG mouse monoclonal (M2) and anti-HBc rabbit polyclonal antibodies or (E) double stained with anti-FLAG mouse monoclonal (M2) and anti-HBx rabbit polyclonal antibodies. The stained cells were visualized by fluorescence microscopy. For these fluorescence microscopy analyses, the nuclei were stained with DAPI. For IFN-γ treatment of cells, fresh medium containing IFN-γ (1000 U/mL) was replaced daily.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
IFN-γ inhibits NF-κB activation induced by HBV, which depends on STAT-1 signaling. (A) Transfection of pHBV1.2×, pHBV1.2×(−X), and pCMV-HA/HBx into HepG2 cells induced NF-κB activation. The effect of IFN-γ on NF-κB activity was analyzed by reporter assay after treating cells with IFN-γ (250, 1000, and 4000 U/mL). In these reporter assays, pNF-κB-luci and pCMV-β-Gal were cotransfected for monitoring NF-κB activity and normalizing transfection efficiency. (B) The effect of STAT-1 on NF-κB activation in HepG2 cells transfected with pHBV1.2× was tested by luciferase reporter assays. HepG2 cells were cotransfected with pHBV1.2× and either pSTAT-1 or pSTAT-1/DN and then treated with or without IFN-γ. HepG2 cells transfected with pUC119 were used as controls. (C) HepG cells were transfected with pSTAT-1 or pSTAT-1/DN and treated with or without IFN-γ (1000 U/mL). In these reporter assays, pNF-κB-luci and pCMV-β-Gal were cotransfected for monitoring NF-κB activity and normalizing transfection efficiency. HepG2 cells were used as controls. Data represent the mean ± standard error of the mean. *P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Within 2 days of IFN-γ treatment, IFN-γ did not significantly reduce levels of cytoplasmic HBV DNA and HBV transcripts and expression levels of the HBx protein and HBV capsids. (A) After treatment with IFN-γ for 2 days (2D) or 4 days (4D), levels of cytoplasmic HBV DNA and HBV transcripts were estimated by using “real-time” fluorescence-based PCR as in Materials and Methods. (B) Expression levels of HBx and HBV capsid in HepG cells were estimated by immunoblot analysis with the anti-HBx or anti-HBc rabbit polyclonal antibodies, respectively. In the case of HBx, β-actin were used for the internal control. Intensities were analyzed by 1D Image Analysis software (Kodak Digital Science, Rochester, NY). These experiments were done in triplicate and repeated 2 or 3 times. Data represent the mean ± standard error of the mean.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
NF-κB activations mediated by overexpression of IKK-α and IKK-β were inhibited by NIK/DN and IFN-γ in HepG cells. (A) The effect of IFN-γ treatment (1000 U/mL for 2 days) on NF-κB activity induced by transfection of pFLAG-NIK was analyzed by reporter assay. (B) The effects of NIK/DN coexpression and IFN-γ treatment (1000 U/mL for 2 days) on NF-κB activity induced by transfection of either pFLAG-IKK-α or pFLAG-IKK-β were analyzed by reporter assays. In these reporter assays, pNF-κB-luci and pCMV-β-Gal were cotransfected for monitoring NF-κB activity and normalizing transfection efficiency. Data represent the mean ± standard error of the mean. *P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 8
EMSA analysis of the effect of IFN-γ on NF-κB activation in HepG cells. (A) The NF-κB activity of HepG2 cells and HepG cells was estimated by EMSA with and without IFN-γ (250, 1000, and 4000 U/mL) treatment. (B) Shifted NF-κB/NF-κB probe complexes were confirmed by supershift analysis with anti-p50 antibodies (Ab) as in the Materials and Methods section.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

10. Figure 9
NIK nuclear localization is strongly associated with the inhibition of NF-κB activation by IFN-γ. (A) Two NIK deletion-mutation plasmids were constructed: pCMV-HA/NIK( ), which contains the NLS region, and pCMV-HA/NIK( ), which does not contain the NLS region. (B) Overexpression of each NIK protein by transfection of pFLAG-NIK, pCMV-HA/NIK( ), and pCMV-HA/NIK( ) into HepG cells activated NF-κB. The effects of IFN-γ on NF-κB activation were analyzed by reporter assays in which pNF-κB-luci and pCMV-β-Gal were cotransfected for monitoring NF-κB activity and normalizing transfection efficiency. The graphs represent the mean fold induction. For pFLAG-NIK and pCMV-HA/NIK( ) transfections, values of luciferase activity obtained with IFN-γ treatment (1000 U/mL) were significantly different from values obtained without IFN-γ treatment (P < 0.01); in contrast, for transfection of pCMV-HA/NIK( ), activity was not significantly different (P = 0.19). The localization of expressed proteins, (C) NIK( ) and (D) NIK( ), with (bottom panel) or without (top panel) IFN-γ treatment, was examined by fluorescence microscopy after each expressed NIK deletion mutation was double stained with anti-HA mouse monoclonal and anti-HBc rabbit polyclonal antibodies. The stained cells were visualized by fluorescence microscopy.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

11. Figure 10
IFN-γ inhibits HBV-induced NF-κB activation, which is mediated by NIK nuclear localization. This mechanism is specific to HBV.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions