Figure 10

IFN-α induced Jak-Stat signaling in both S-Huh-7 and R-Huh-7 cells after HCV 2a infection. (A) Both S-Huh-7 and R-Huh-7 cells were infected with JFH1-GFP chimera virus for 96 hours. The phosphorylation of Stat1 protein in the infected cultures after 30 minutes of IFN-α treatment (1000 IU/ml) was determined by western blot analysis. Equal amounts of proteins were used in the blot to assay for total Stat1 and beta-actin levels. (B) IFN-α induced pStat2 protein in S-Huh-7 and R-Huh-7 cells with or without HCV 2a infection. The experiment was carried out as described in panel A. Equal amounts of proteins were used in the blot to assay for total Stat2 and beta-actin levels. (C) Demonstrates IFN-α induced nuclear localization of pStat1 in the S3-GFP and R4-GFP stable replicon cells at 24 and 72 hours by immunofluorescence microscopy using anti-mouse pStat1 (1:1000 dilution) and Alexaflour labeled anti-mouse secondary antibody (1:2000 dilution). The pStat1 protein was detected in the nucleus of S3-GFP replicon cells only at 24 and 72 hours after IFN-α treatment. The pStat1 translocation is associated with negative GFP expression after 72 hours of IFN-α treatment only in S3-GFP cells. (D) IFN-α induced ISRE-luciferase activity in S-Huh-7 and R-Huh-7 cells after HCV 2a infection. Both S-Huh-7 and R-Huh-7 cells were were infected with full-length HCV. After 72 hours, infected cells were transfected with μgm of pISRE-luciferase plasmid. After 24 hours, ISRE-luciferase activity in the cell lysates were measured in the presence and absence of IFN-α(1 IU/ml) treatment for 24 hours. The values were calculated as fold increase with respect to untreated cells.