The promoter activities of RANTES and PRDII in transfected cells mock infected or infected with HCV for the indicated periods were determined using the luciferase reporter assay, as previously described.12, 14 Detailed methods selleck screening library have been presented elsewhere.7, 12, 15 The following polyclonal (pAb) and monoclonal (mAb) antibodies were used: anti-IκBα (C-21) and anti-p65 (C-20) pAbs and anti-Lamin A/C (636) mAb (Santa Cruz Biotechonology, Santa Cruz, CA); anti-HCV NS5A (9E10) mAb (a gift

from Charles Rice, Center for the Study of Hepatitis C, The Rockefeller University); anti-actin mAb (Sigma-Aldrich, St. Louis, MO); anti-ISG15 (a gift from Dr. Arthur Haas, Louisiana State University), and anti-ISG56 pAbs12; and peroxidase-conjugated secondary antirabbit and antimouse pAbs (Southern Biotech, Birmingham, AL). The protein bands were visualized by enhanced chemiluminescence (Millipore, Billerica, MA), followed by exposure to X-ray films. 7.5-TLR3 cells (∼5 × 106) were mock infected or infected with HCV (multiplicity of infection [MOI] = 0.5) for 48 hours, then were processed for chromatin immunoprecipitation (ChIP) assay, as described previously.14 The antibodies used for ChIP were

from Santa Cruz (anti-p65) and Active Motif (control immunoglobulin G). The ChIP-enriched samples were subjected to qPCR quantification of various chemokine and Trefoil family factor 1 (TFF1) (negative control) promoter sequences using BMN 673 nmr specific primers presented online in Supporting Table 3. To investigate whether TLR3 plays a role in hepatoceullar proinflammatory response to HCV

infection, we determined the production of cytokines/chemokines in HCV-infected 7.5-TLR3 cells, which were derived from Huh7.5 cells by stably reconstituting the expression of human TLR3. As controls, we studied Huh7.5 cells expressing the control vector (Vect) and mutant TLR3s defective for signaling as a result of incapability of dsRNA binding (H539E and N541A). All these cells are RIG-I defective, and the effects observed were the result of activation of the TLR3 pathway in the absence of RIG-I. We have previously utilized these cell lines to demonstrate that TLR3 senses HCV infection and triggers a weak ISG response, thereby moderately reducing HCV propagation DOK2 when cells were infected at low MOIs.12 We infected cells with JFH1 virus at an MOI of 0.2 for 72 hours, then harvested the culture supernatants to measure cytokine/chemokine production by a Bio-Plex Multiplex Cytokine Assay. At this MOI, HCV did not replicate differentially among these Huh7.5 derivatives,12 and all the cells were infected at 72 hours, as determined by immunostaining of NS5A (data not shown). Compared to mock-infected cells, six chemokines/cytokines were strongly induced by HCV infection in 7.5-TLR3 cells, including RANTES (37-fold), MIP-1β (25-fold), MIP-1α (17-fold), IL-6 (13-fold), IP-10 (10-fold), and tumor necrosis factor alpha (TNF-α) (10-fold) (Fig. 1).