Fig 2.

Effects of HCV proteins in the regulation of STAT3, MMP-2, and Bcl-2 expression. (A) Huh7 cells were cotransfected with the reporter pGL3-APRE-Luc containing the luciferase gene under the control of the STAT3 promoter and pCMV-Flag2A, pCMV-core, pCMV-E1, pCMV-E2, pCMV-P7, pCMV-NS2, pCMV-NS3, pCMV-NS4A, pCMV-NS4B, pCMV-NS5A, or pCMV-NS5B expressing the corresponding HCV proteins. Luciferase activity was measured as described in Materials and Methods. Results are expressed as the mean ± SD of independent experiments performed in triplicate and normalized using a β-galactosidase assay. *, P < 0.05 versus pCMV-Flag2A; Rel. Lucif. Act., relative luciferase activity. (B) Huh7 cells were transfected with pCMV-NS4B or pCMV-Flag2A. At 48 h posttransfection, total RNA was isolated and used as the template for RT-PCR using primers specific to Bcl-2, MMP-2, STAT3, SOCS3, and β-actin. (C) Huh7 cells were transfected with pCMV-NS4B at different concentrations, as indicated. Proteins were detected by Western blot analysis using antibodies to p-STAT3, STAT3, MMP-2, Bcl-2, SOCS3, and β-actin. (D) Huh7 cells were transfected with pCMV-NS4B or pCMV-Flag2A at the indicated concentrations. MMP-2 activity was measured by gelatin zymography at 48 h posttransfection. (E and F) Huh7 cells were transfected with pCMV-NS4B or pCMV-Flag2A. The STAT3 protein levels in whole-cell lysates, the cytoplasm, and the nucleus were determined by Western blot analysis using antibodies to STAT3. (G) Effect of NS4B on the translocation of STAT3 from the cytosol to the nucleus. Huh7 cells were transfected with pCMV-NS4B or control vector for 48 h. After fixation, the cells were immunostained with antibody for STAT3. The nuclei were stained by DAPI.