2C) and supported the augmentation of HCV PF-01367338 datasheet replication by STAT3. Furthermore, activation of STAT3 by way of exogenous cytokine treatment with LIF, a known activator of STAT3, resulted in a significant increase in STAT3 phosphorylation at Y705, as expected (Fig. 2D), while pretreatment with LIF for 24 hours prior to infection with JFH-1, resulted in a marked 2-fold increase in HCV RNA levels (Fig. 2E). These

results indicate that activated STAT3 acts to either directly assist HCV replication or potentially induce the expression of specific STAT3-dependent genes that are in turn able to create an environment that is favorable for HCV replication. Collectively, the above experiments show that activation of STAT3 results in enhanced HCV replication. To extend these observations,

selleck the converse sets of experiments were performed using both an siRNA knockdown approach and a panel of chemical inhibitors that block STAT3 activation. To validate our knockdown approach STAT3 siRNA and a control siRNA were transfected into Huh-7 cells and total STAT3 determined by immunoblot. Despite numerous attempts, we were only able to reduce STAT3 expression by ∼50% (Fig. 3Ai). To determine the effect of STAT3 siRNA knockdown on HCV replication, Huh-7.5 cells were transfected with STAT3 siRNA or a control scrambled siRNA, and infected with HCV JFH-1. The knockdown of STAT3 with siRNA significantly decreased HCV RNA levels by ∼50% (Fig. 3Aii). These results confirm previous findings in the literature, where a genome-wide siRNA screen of Huh-7 cells infected with HCV JFH-1 revealed STAT3 as a candidate host factor involved in HCV replication.[1] Next we used a number of commercial STAT3 inhibitors: (1) AG490 is a JAK-2 protein tyrosine kinase (PTK) inhibitor that indirectly inhibits Y705 phosphorylation of STAT3; (2) STA-21 is a novel selective small molecule inhibitor of STAT3, which binds to the SH-2 domain of STAT3 and specifically prevents dimerization

of STAT3 and DNA binding[18]; and (3) S31-201 is a cell-permeable inhibitor of STAT3 that targets oxyclozanide the STAT3-SH2 domain and blocks STAT3 dependent transcription.[19] Supporting Fig. 2 outlines the STAT3 signaling cascade and demonstrates the specific points where these inhibitors exert their function. The effects of STA-21-mediated inhibition of STAT3 on HCV replication were first investigated in an established HCV infection. HCV genomic replicon cells and JFH-1-infected Huh-7.5 cells treated with STA-21 (10 μM) for 24 hours demonstrated an approximate decrease in HCV RNA of 50% (Fig. 3B) and 70% (Fig. 3C), respectively. Given these findings, it appears that STAT3 activation, or STAT3-dependent gene expression, are involved in augmenting HCV replication at the RNA level.