#ab13830); HCV NS5A (9E10, kindly provided by Dr. Charles Rice); HSP90α/β (H-114) (rabbit polyclonal, Santa Cruz Biotechnology, sc-7947); HSP90 (rabbit polyclonal, Cell Signaling, Cat. #4874) Confocal imaging was performed with

a Leica TCS SP2 confocal microscope (Leica Microsystems, Wetzlar, Germany), and Leica confocal software was used for the acquisition of Selleck JAK inhibitor images. Immunofluorescence images were acquired using an Olympus BX51 fluorescence microscope and the Nixon NIS-Element BR 3.10 software for image analysis. GW182-body sizes and numbers were quantified using ImageJ 1.46 software (National Institutes of Health) for 35 cells in each experimental condition selected randomly from different fields and slides from four Z-VAD-FMK independent

repeat experiments. Cytotoxicity of DMAG, HSP90 siRNA, and GW182 siRNA was performed using an LDH-Cytotoxicity Assay Kit (Abcam, Cat. #65393) according to the manufacturer’s specifications. Data are presented as the mean ± SEM and were analyzed using a two-tailed Student t test; P < 0.05 was considered statistically significant. Analysis of variance was used when comparing variation with more than two experimental means. GWB-associated proteins have recently been shown to be essential host factors for HCV replication.16, 20-22 This finding prompted us to surmise that ethanol might modulate HCV replication by affecting the expression of GWB proteins. We found that

among the GWB components, GW182 mRNA and protein were selectively up-regulated by ethanol exposure (Fig. 1A,B) compared with no changes in the expression of EIF2C2 (Ago2), DDX3X, DDX6, and PALT1 mRNA (Supporting Fig.1A-E). Ethanol treatment up-regulated HCV RNA levels as well as HCV protein expression in genotype 2 J6/JFH 1 virus infection (Fig. 1C,D). Fluorescence microscopy analysis revealed that endogenous GW182 was localized to intensely stained, punctate perinuclear cytoplasmic structures consistent with GWBs, and within 24 hours of ethanol exposure the number of GWBs (GW182 antibody as marker) 上海皓元医药股份有限公司 was significantly increased compared with untreated cells (Fig. 1E, Supporting Fig. 2A-D). To determine the effect of GW182 on HCV RNA accumulation, we used fluorescein isothiocyanate–conjugated control or GW182 siRNAs to ensure transfection efficiency (data not shown), and knock-down was confirmed by decreased GW182 mRNA (Fig. 2A). Compared with the siRNA control, GW182-specific siRNA-transfected J6/JFH1-infected Huh7.5 cells showed significantly reduced expression of HCV RNA (Fig. 2B) and protein (Fig. 2C). Next, we sought to evaluate the mechanisms by which GW182 could facilitate HCV replication and asked whether GW182 influenced miR-122 abundance. We found a significant reduction in miR-122 levels after transfection of J6/JFH1-infected Huh7.