To this end, we investigated the role of activated STAT3 in the context of the full HCV life cycle, including entry, replication, and egress. We present evidence that STAT3 may enhance HCV replication by way of control of MT dynamics and we hypothesize indirectly through STAT3-dependent gene expression. These studies emphasize the need for further investigations into the role of STAT3 in the life cycle AZD1208 supplier of HCV and suggest that targeting STAT3 therapeutically may limit disease progression in those with CHC. Moreover, the ability of HCV to constitutively activate STAT3

and the oncogenic nature of STAT3 suggest that HCV activation of STAT3 could be responsible in part for the increased incidence of HCC in individuals chronically infected with HCV. pRc/CMV-STAT3-C-FLAG (Jacqueline F Bromberg, Rockefeller University, NY) and pXJ40-STMN1-Myc (Dominic Chi Hiung Ng, University of Melbourne, Australia), were generous gifts. pSTAT3-Luc was purchased from Panomics (Santa Clara, CA) and transfection of all plasmids was performed using Fugene6 (Roche, Indianapolis, IN). The human hepatoma cell lines Huh-7, Huh-7.5 (Charles Rice, Rockefeller University, Daporinad research buy NY), NNeoC-5B, and NNeo3-5B[7] were maintained as described.[8] Huh-7.5 cells stably expressing STAT3-C were generated

using pRc/CMV-STAT3-C and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 800 μg/mL G418 (Geneticin) (Gibco, Life Technologies). The relative luciferase activity of STAT3 promoter elements were measured

using the Luciferase Assay System (Promega, Madison, WI). Cells were seeded at a density of 7 × 104 cells/well and cotransfected with pSTAT3-luc and pRL-TK the following day and 24 hours later cells were infected with HCV JFH-1 (multiplicity of infection [MOI] = 0.01). At 48 hours postinfection cell lysates were harvested as per the manufacturer’s instructions and luciferase output was measured using a Glow Max Luminometer (Promega). Infectious JFH-1[9-11] and Jc1-Myc[12] were prepared as described. Infectivity titers were ascertained as described,[11] with minor differences. Huh-7.5 cells were seeded into 96-well trays at 2 × 104 cells/well and cultured Loperamide overnight before infection for 3 hours with viral supernatant. Cell monolayers were then washed with phosphate-buffered saline (PBS) and returned to culture for 3 days before fixation and indirect immunofluorescent labeling of HCV antigens and determination of viral titers, expressed as focus-forming units (ffu/mL). All experiments involving real-time PCR were performed using RNA extracted from cells cultured in 12-well plates. For this, Huh-7, Huh-7.5, or STAT3-C stable cells were seeded at 8 × 104/well, 24 hours prior to transfection/infection.