Hylation status at each CpG site in CpG island of hepcidin promoter is shown. Methylated sites are indicated by filled dark circles and unmethylated sites by empty white ones. N: nontumor tissues; T: tumor tissues. (D) Huh7.5-JFH1 cells were treated with or without Trichostatin A (TSA), and hepcidin expression was analyzed by qRT-PCR. The data are presented as mean 6 SD from three independent experiments. doi:10.1371/journal.pone.0046631.gFigure 3. Hepcidin peptide inhibits HCV replication. (A) The synthesized hepcidin peptide with cyclized-disulfide bond between cysteine was shown. (B) and (C) Huh7.5 cells were incubated with JFH1 (B) or HCV-JC1 virus for 16 hours (C), then treated with or hepcidin peptide or control peptide for 3 days, and HCV mRNA levels were analyzed by qRT-PCR. GAPDH was used as the internal control in the PCR reaction. (D) and (E) FLneo cells 
were treated with or without hepcidin for 3 days and 1326631 5 days, and HCV mRNA expression was analyzed by RT-PCR (D) or qRT-PCR (E). The data are presented as mean 6 SD from three independent experiments. doi:10.1371/journal.pone.0046631.gHepcidin Exhibits Antiviral Activity against HCVfrom immunostaining assay show that over-expression of hepcidin resulted in a significant decrease in JC1 protein level, whereas down-regulation of hepcidin led to an increase in JC1 protein expression (Fig. 4D). We also knocked down hepcidin transiently in Huh7.5 cells and then explored the effect of hepcidin downmodulation on HCV replication. Hepcidin shRNA could effectively knockdown hepcidin in Huh7.5 cells which showed a reduction of about 70 at the mRNA level at 72 hours posttransfection. Knockdown of hepcidin induced an increase of HCV replication in Huh7.5 cells (Fig. 4E).Hepcidin Antiviral Activity is Associated with STAT3 ActivationAlthough it is not completely understood how intracellular antiviral activity is established, JAK-STAT signaling has been known to play a key role in IFN-induced antiviral activity. We thus examined whether hepcidin induces antiviral activity through JAK-STAT signaling. Firstly, we tested whether hepcidin treatment can affect STAT1 and STAT3 phosphorylation. Huh7.5 cells were treated with 20 mM of hepcidin peptide for 30 min, 1 h and 4 h, followed by protein extraction. Then we analyzed STAT1 and STAT3 protein phosphorylation status by Western blot analysis using a monoclonal antibody 3PO manufacturer specific for phosphorylated STAT or an antibody specific for total STAT. As shown inFigure 4. Modification of hepcidin expression in Huh7.5 cells affects HCV virus replication. (A) Western blot analysis of hepcidin expression in Huh7.5-vector, Huh7.5-Hepc and Huh7.5-antiHepc stable cell lines. (B,C) qRT-PCR analysis of HCV mRNA expression in JFH1 infected Huh7.5-vector, Huh7.5-hepc, Huh7.5-antihepc cells (B); and HCV-JC1 infected Huh7.5-vector, Huh7.5-hepc, and Huh7.5-antihepc cells (C). The data are presented as mean 6 SD from three independent experiments. (D) Immunostaining for HCV (JC1) NS5A in Huh7.5-vector, Huh7.5-hepc and Huh7.5antihepc cells on day 7 post infection. The original magnification is 2006. (E) Knockdown of hepcidin mRNA expression in Huh7.5 by shRNA transfection and endogenous hepcidin expression was analysed by qRT-PCR. The effect of hepcidin knockdown on HCV-JFH1 replication was CP21 assayed on day 3, day 5 and day 7 post-infection. doi:10.1371/journal.pone.0046631.gHepcidin Exhibits Antiviral Activity against HCVfigure 5A, phosphorylated STAT3 was up-regulated by.Hylation status at each CpG site in CpG island of hepcidin promoter is shown. Methylated sites are indicated by filled dark circles and unmethylated sites by empty white ones. N: nontumor tissues; T: tumor tissues. (D) Huh7.5-JFH1 cells were treated with or without Trichostatin A (TSA), and hepcidin expression was analyzed by qRT-PCR. The data are presented as mean 6 SD from three independent experiments. doi:10.1371/journal.pone.0046631.gFigure 3. Hepcidin peptide inhibits HCV replication. (A) The synthesized hepcidin peptide with cyclized-disulfide bond between cysteine was shown. (B) and (C) Huh7.5 cells were incubated with JFH1 (B) or HCV-JC1 virus for 16 hours (C), then treated with or hepcidin peptide or control peptide for 3 days, and HCV mRNA levels were analyzed by qRT-PCR. GAPDH was used as the internal control in the PCR reaction. (D) and (E) FLneo cells were treated with or without hepcidin for 3 days and 1326631 5 days, and HCV mRNA expression was analyzed by RT-PCR (D) or qRT-PCR (E). The data are presented as mean 6 SD from three independent experiments. doi:10.1371/journal.pone.0046631.gHepcidin Exhibits Antiviral Activity against HCVfrom immunostaining assay show that over-expression of hepcidin resulted in a significant decrease in JC1 protein level, whereas down-regulation of hepcidin led to an increase in JC1 protein expression (Fig. 4D). We also knocked down hepcidin transiently in Huh7.5 cells and then explored the effect of hepcidin downmodulation on HCV replication. Hepcidin shRNA could effectively knockdown hepcidin in Huh7.5 cells which showed a reduction of about 70 at the mRNA level at 72 hours posttransfection. Knockdown of hepcidin induced an increase of HCV replication in Huh7.5 cells (Fig. 4E).Hepcidin Antiviral Activity is Associated with STAT3 ActivationAlthough it is not completely understood how intracellular antiviral activity is established, JAK-STAT signaling has been known to play a key role in IFN-induced antiviral activity. We thus examined whether hepcidin induces antiviral activity through JAK-STAT signaling. Firstly, we tested whether hepcidin treatment can affect STAT1 and STAT3 phosphorylation. Huh7.5 cells were treated with 20 mM 
of hepcidin peptide for 30 min, 1 h and 4 h, followed by protein extraction. Then we analyzed STAT1 and STAT3 protein phosphorylation status by Western blot analysis using a monoclonal antibody specific for phosphorylated STAT or an antibody specific for total STAT. As shown inFigure 4. Modification of hepcidin expression in Huh7.5 cells affects HCV virus replication. (A) Western blot analysis of hepcidin expression in Huh7.5-vector, Huh7.5-Hepc and Huh7.5-antiHepc stable cell lines. (B,C) qRT-PCR analysis of HCV mRNA expression in JFH1 infected Huh7.5-vector, Huh7.5-hepc, Huh7.5-antihepc cells (B); and HCV-JC1 infected Huh7.5-vector, Huh7.5-hepc, and Huh7.5-antihepc cells (C). The data are presented as mean 6 SD from three independent experiments. (D) Immunostaining for HCV (JC1) NS5A in Huh7.5-vector, Huh7.5-hepc and Huh7.5antihepc cells on day 7 post infection. The original magnification is 2006. (E) Knockdown of hepcidin mRNA expression in Huh7.5 by shRNA transfection and endogenous hepcidin expression was analysed by qRT-PCR. The effect of hepcidin knockdown on HCV-JFH1 replication was assayed on day 3, day 5 and day 7 post-infection. doi:10.1371/journal.pone.0046631.gHepcidin Exhibits Antiviral Activity against HCVfigure 5A, phosphorylated STAT3 was up-regulated by.
