As synthesized from 100 ng total RNA with Quantifast RT-PCR kit (Qiagen

As synthesized from 100 ng total RNA with Quantifast RT-PCR kit (Qiagen). For realtime PCR, we used the following SYBR Green QuantiTect Primers purchased from Qiagen: RevErba (QT00000413), RoRa (QT00072380), ARNTL (QT00068250), ARNTL2 (QT00011844), CLOCK (QT00054481), PER1 (QT00069265), PER2 (QT00011207), PER3 (QT00097713), CRY1 (QT00025067) and CRY2 (QT00094920). For HCV quantification the following primers were used: Forward 104 (59-AGA GCC ATA GTG GTC TGC GG-39) and Reverse 197R (59-CTT TCG CGA CCC AAC ACT AC-39) described in [33]. Reactions were set up in 96-well plates using a 7700 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and all samples were assayed in triplicate. Expression levels of target gene were normalized using the housekeeping control gene TATA binding protein (TBP, QT00000721).were lysed and processed for immunoblotting analysis with specific antibodies as previously described [19]. Quantitative measurements of bands were performed using the NIH-Image analysis program Scion IMAGE (Scion Corp., Frederick, MD, USA).AntibodiesRabbit and mouse polyclonal antibody directed against ARNTL2, ClOCK, PER1, PER2, CRY1, CRY2 and b-actin were from Santa Cruz Biotechnology, Santa Cruz, CA, USA. ARNTL and Rev-Erba antibodies were purchased from Millipore S.p.a., Milan, Italy. The monoclonal anti-core (C7?0) antibody was obtained from Vinci-Biochem (Florence, Italy).Statistical AnalysisResults are expressed as means 6 SE of at least three different experiments. Comparisons were made using Student’s t-test as appropriate. Differences were considered as significant at P,0.05.Results Altered Clock Genes Expression in OR6 Replicating the Full Length HCV RNAAs viruses are highly dependent on cellular machinery for replication, it was proposed that the viral replication may beWestern BlottingHuh-7 control cells and cells transfected with either genotype HCV core protein 1b, HCV core 3a or with the empty vectorHCV Alters Hepatic Clock Gene ExpressionFigure 4. qRT-PCR analysis of clock gene mRNAs expression levels in Huh-7 cells POR8 overexpressing the HCV core proteins genotype 1b and 3a. Huh-7 cells were transiently transfected with HCV core proteins 1b and 3a as previously described [17], or with GFP. 48 hours after transfection mRNA levels of Rev-Erba, Rora, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2 genes were assessed by qRT-PCR. Values were normalized against TBP as housekeeping control gene. Light gray: GFP transfected cells; 15755315 dark gray: HCV core protein genotype 3a transfected cells; black: HCV core protein genotype 1b transfected cells. Results are expressed as means 6 SE of three independent experiments. * = p,0.05 in HCV core proteins transfected versus GFP transfected control cells. doi:10.1371/journal.pone.0060527.gsynchronized to the molecular clockwork and that in turn the circadian clock may 548-04-9 biological activity influence viral replication [7]. With this premise in mind, we sought to analyze by qRT-PCR the time-qualified expression of a panel of clock genes (Rev-Erba, Rora, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2) in OR6 cells, which express full length HCV replicon of genotype 1b [16]. The latter was able to perturb clock gene expression as shown in Figure 1A and B. A significant downregulation of CRY2 mRNA levels was observed over all the six time points, while PER2 mRNA was significantly decreased at 1 h, 10 h, 16 h, 22 h and 28 h after serum shock. Rev-Erba, Rora, ARNTL mRNA levels did not show statistic.As synthesized from 100 ng total RNA with Quantifast RT-PCR kit (Qiagen). For realtime PCR, we used the following SYBR Green QuantiTect Primers purchased from Qiagen: RevErba (QT00000413), RoRa (QT00072380), ARNTL (QT00068250), ARNTL2 (QT00011844), CLOCK (QT00054481), PER1 (QT00069265), PER2 (QT00011207), PER3 (QT00097713), CRY1 (QT00025067) and CRY2 (QT00094920). For HCV quantification the following primers were used: Forward 104 (59-AGA GCC ATA GTG GTC TGC GG-39) and Reverse 197R (59-CTT TCG CGA CCC AAC ACT AC-39) described in [33]. Reactions were set up in 96-well plates using a 7700 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and all samples were assayed in triplicate. Expression levels of target gene were normalized using the housekeeping control gene TATA binding protein (TBP, QT00000721).were lysed and processed for immunoblotting analysis with specific antibodies as previously described [19]. Quantitative measurements of bands were performed using the NIH-Image analysis program Scion IMAGE (Scion Corp., Frederick, MD, USA).AntibodiesRabbit and mouse polyclonal antibody directed against ARNTL2, ClOCK, PER1, PER2, CRY1, CRY2 and b-actin were from Santa Cruz Biotechnology, Santa Cruz, CA, USA. ARNTL and Rev-Erba antibodies were purchased from Millipore S.p.a., Milan, Italy. The monoclonal anti-core (C7?0) antibody was obtained from Vinci-Biochem (Florence, Italy).Statistical AnalysisResults are expressed as means 6 SE of at least three different experiments. Comparisons were made using Student’s t-test as appropriate. Differences were considered as significant at P,0.05.Results Altered Clock Genes Expression in OR6 Replicating the Full Length HCV RNAAs viruses are highly dependent on cellular machinery for replication, it was proposed that the viral replication may beWestern BlottingHuh-7 control cells and cells transfected with either genotype HCV core protein 1b, HCV core 3a or with the empty vectorHCV Alters Hepatic Clock Gene ExpressionFigure 4. qRT-PCR analysis of clock gene mRNAs expression levels in Huh-7 cells overexpressing the HCV core proteins genotype 1b and 3a. Huh-7 cells were transiently transfected with HCV core proteins 1b and 3a as previously described [17], or with GFP. 48 hours after transfection mRNA levels of Rev-Erba, Rora, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2 genes were assessed by qRT-PCR. Values were normalized against TBP as housekeeping control gene. Light gray: GFP transfected cells; 15755315 dark gray: HCV core protein genotype 3a transfected cells; black: HCV core protein genotype 1b transfected cells. Results are expressed as means 6 SE of three independent experiments. * = p,0.05 in HCV core proteins transfected versus GFP transfected control cells. doi:10.1371/journal.pone.0060527.gsynchronized to the molecular clockwork and that in turn the circadian clock may influence viral replication [7]. With this premise in mind, we sought to analyze by qRT-PCR the time-qualified expression of a panel of clock genes (Rev-Erba, Rora, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2) in OR6 cells, which express full length HCV replicon of genotype 1b [16]. The latter was able to perturb clock gene expression as shown in Figure 1A and B. A significant downregulation of CRY2 mRNA levels was observed over all the six time points, while PER2 mRNA was significantly decreased at 1 h, 10 h, 16 h, 22 h and 28 h after serum shock. Rev-Erba, Rora, ARNTL mRNA levels did not show statistic.