Cells were grown at 37��C in http://www.selleckchem.com/products/Bicalutamide(Casodex).html a 5% CO2 atmosphere within a humidified incubator. JFH1 system Cell supernatant containing infectious JFH1 HCV 2a virus was harvested from day 7�C20 and frozen at ?80��C as previously described [27]. Culture media from Huh 7.5.1 (80% confluent) in 10 cm dish was removed and replaced by 3 ml of thawed JFH1 viral stock. After 3 h medium was replaced by DMEM. Titration of viral infection was done as previously described [28]. In brief, Huh7.5.1 cells were seeded in 96-well plates at 1��104 cells/well. Supernatant was serially diluted 10-fold in complete growth medium and used to infect the seeded cells (6 to 8 wells per dilution). Following 2 or 3 days of incubation the cells were immunostained for HCV core protein.

Wells that expressed at least one core expressing cell were counted as positive, The TCID50 was calculated using the TCID50 calculator http://www.med.yale.edu/micropath/pdf/infectivity%20calculator.xls as previously described [29]. Multiplicity of infection (MIO) was calculated using a conversion formula: http://www.bioon.com./book/biology/e-protocol/cell/miopfu.htm. Quantification of HCV RNA by RT-PCR was done as previously described [27]. In brief: Total RNA was used to quantify HCV. JFH copy number was calculated according to a standard curve run in each RT-PCR experiment. The standard curve was calculated using a series of 10-fold dilutions of previously titrated JFH-1 plasmid. Titration of JFH-1 was done with Abbott RealTime HCV assay (Abbott, Illinois, USA), with previously published primers (Table 1).

Viral quantization was done repeatedly throughout the experiments to ensure equal MIO. In order to avoid nutrient stress, cells were split when reaching approximately 80% confluence and growth media was regularly exchanged. Equal numbers of cells were used for each experiment. Table 1 Primers used for real-time PCR. Mice C57BL/6J HCV-Tg mice expressing the entire HCV 1b ORF under alpha-1 antitrypsin promoter were kindly provided by Prof. N. La Monica (IRBM, ��P. Angeletti��, Pomezia, Italy) [30]. C57BL/6J wt mice (Harlan, Jerusalem, Israel) were used as controls. Animal care and experiments were approved and conducted in accordance with the Hebrew University-Hadassah Animal Authority guidelines (Ethical approval number: MD-1012389-5). Male mice, 10�C12 weeks old, 24�C26 gr were kept in the SPF unit in the animal facility at the Hebrew University Medical School.

Mice were treated i.p with tunicamycin (Fermentek, Jerusalem, Israel) Dacomitinib injection of 1 mg/kg (volume of 20 ��l/1gr mouse) every other day (total 2�C3), and were sacrificed 3 days following the last injection. Control mice received 5% dextrose injection in the same volume. Mice were anesthetized with Ketamine 5 mg/kg (Kepro, Holland) + Xylazine 5%, (Kepro), terminally bled from the heart and sacrificed by cervical dislocation.