Cell culture, transfection, protein extraction and immunoblotting Human embryonic kidney cells HEK293, stably expressing the tetracycline repressor protein (T-REx HEK293 Cell Line, Invitrogen, USA), and human hepatoma cells Huh7.5 [65] were used throughout this study. Cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Biological Industries, Israel) in a humidified 5% CO2 incubator at 37uC. The calcium-phosphate transfection method was applied for introducing 2 mg of the plasmid ‘‘pmCherry-NS3 activated MazF’’ or the plasmid ‘‘pmCherry-uncleavable MazF’’ into T-REx HEK293 cells inducibly expressing EGFP-Full NS3-4A [10] seeded 1.56106 cells per 60 mm plate 24 h before transfection. Stable transfectants, inducibly expressing EGFP-Full NS3-4A and consti- tutively expressing mCherry-NS3 activated MazF (denoted ‘‘Tet- NS3/activated MazF cells’’) or mCherry-uncleavable MazF (denot- ed ‘‘Tet-NS3/uncleavable MazF cells’’) were selected in a medium containing 1 mg/ml of G418 (A.G. Scientific, USA). Cell clones that express high level of the cleavable construct or the uncleavable control were identified by fluorescence microscopy and isolated. For protein extraction, 48 h post-transfection the cells were washed with PBS, scraped and lysed in a buffer containing 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 10 mM Tris(HCl) pH 7.5, and protease inhibitors cocktail (Sigma, Israel). Following 30 minutes of incubation on ice, lysates were cleared by centrifugation at 20,000 g for 10 minutes, at 4uC. For immunoblotting, protein samples were electrophoresed on 12% SDS/polyacrylamide gel, transferred to nitrocellulose and detected using mouse monoclonal anti-mCherry antibody (Clontech, USA), mouse monoclonal anti-GFP antibody (Santa-Cruz, USA) or mouse monoclonal anti-actin antibody (Abcam, USA), followed by horseradish peroxidase (HRP)-conju- gated goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories, USA) and enhanced chemiluminescence (ECL) detection using SuperSignal West Pico Chemiluminescent Substrate (Thermo SCIENTIFIC/Pierce, USA). HCV infection Virus assays were carried out with an inter-genotypic chimeric hepatitis C virus (HCV) produced by replacing the core-NS2 segment of the JFH-1 virus genome with the comparable segment of the genotype 1a H77 virus. This chimeric virus, HJ3-5 (Kindly provided by Prof. Stanley Lemon, University of Texas at Galveston), contains two compensatory mutations that promote its growth in cell culture as described previously [40]. HCV RNAs were transcribed in vitro and electroporated into cells essentially as described previously [66,67]. In brief, 10 mg of in vitro-synthesized HCV RNA was mixed with 56106 Huh7.5 cells in a 2-mm cuvette and pulsed twice at 1.4 kV and 25 mF. Cells were seeded into 12- well plates or 25-cm2 flasks, and passaged at 3-to 4-day intervals posttransfection by trypsinization and reseeding with a 1:3 to 1:4 split into fresh culture vessels. When infectivity reached ,50%, as was monitored by immunofluorescent staining with anti HCV core protein mouse monoclonal antibody (Affinity BioReagents, USA) and Cy2-conjugated goat anti-mouse IgG (Jackson ImmunoRe- search Laboratories, USA) (see ‘‘visualizing HCV infected cells (infectivity assay)’’ below), the mixed culture (of uninfected and HCV infected cells in 1:1 ratio) was taken for cytotoxicity assays. Fluorescence/Immunofluorescence Microscopy Confocal fluorescence microscopy analysis of T-REx HEK293 cells inducibly expressing EGFP-full NS3-4A and Zymoxin-Mediated Removal of HCV Infected Cells constitutively expressing mCherry-NS3 activated or uncleavable MazF. 16105 Tet-NS3/activated MazF or Tet- NS3/uncleavable MazF cells were seeded on poly-L-lysine coated cover-slips in a 24 well-plate. 12 h later, the cells were supplemented with 1 mg/ml of tetracycline, or left untreated. 24 h later, the cells were fixed with 4% formaldehyde in PBS. Uninduced cells were permeabilized with Triton X-100 (0.1% in PBS) for 5 minutes, blocked with 90% fetal calf serum/10% PBS at room temperature for 25 minutes, incubated with 1:200 diluted rabbit-polyclonal anti-calnexin antibody (Sigma, USA) as primary antibody for 1 h and followed by 1:200 diluted Cy2- conjugated anti-rabbit IgG (Jackson ImmunoResearch Laborato- ries, USA) secondary antibody for 30 minutes. Nuclei of induced and uninduced cells were then stained by Hoechst 33258 for 1 h at room temperature. Slides were washed with PBS, mounted in Mowiol 4-88 solution (Calbiochem, USA) (immunostained unin- duced cells) or ImmuGlo Mounting Medium (IMMCO Diagnos- tics, USA) (induced Tet-NS3/uncleavable MazF cells) and examined using a Zeiss LSM 510 META laser scanning confocal microscope. Visualizing T-REx HEK293 cells inducibly expressing EGFP-full NS3-4A (supplemented with different tetracycline concentrations) and constitutively expressing mCherry-NS3 activated MazF or mCherry-uncleavable MazF. 16105 Tet- NS3/activated MazF or Tet-NS3/uncleavable MazF cells were seeded on poly-L-lysine coated cover-slips in a 24 well-plate. 12 h later, cells were supplemented with 10 ng/ml or 1000 ng/ml of tetracycline, or left untreated. 36 h later, cells were fixed with 4% formaldehyde in PBS. Following nuclear staining by Hoechst 33258 for 1 h at room temperature, Slides were washed with PBS, mounted in ImmuGlo Mounting Medium and examined using a fluorescence microscope. Visualizing HEK293 cells infected with recombinant adenovirus encoding for mCherry-NS3 activated MazF or mCherry-uncleavable MazF. 36105 HEK293 cells were seeded per well in 6 wells plate. When the culture reached 90% confluence, the growth medium was replaced by fresh medium containing 10 fold dilutions of recombinant adenoviruses encoding for mCherry-NS3 activated MazF or mCherry-uncleavable MazF, starting from 2.56106 PFU per well. After 36 h, cells were fixed with 4% formaldehyde in PBS and examined using a fluorescence microscope. Visualizing HCV infected cells (infectivity assay). Huh7.5 cells infected with HCV HJ3-5 chimeric virus were seeded into 8- well chamber slides (Nalge Nunc, USA). After 24 h, cells were fixed and permeabilized with 1:1 acetone/methanol mixture and stained with 1:300 diluted mouse monoclonal antibody C7-50 (Affinity BioReagents, USA) specific for the HCV core protein followed by staining with 1:100 diluted Cy2-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, USA). Nuclei were then stained with DAPI (Sigma, Israel) and slides were washed with PBS, mounted (SouthernBiotech, USA) and examined using a fluorescence microscope. Visualizing zymoxin-treated mixed culture of uninfected and HCV infected cells. See ‘‘Cytotoxicity assay of recombinant adenoviruses encoding for mCherry-NS3 activated MazF or mCherry-uncleavable MazF on a mixed culture of uninfected and HCV-infected Huh7.5 cells’’ below. Colony formation assay 7.56105 HEK293 T-REx cells were seeded per well in 6 wells plate. 24 h later, cells were transfected with 2 mg of the plasmids ‘‘pmCherry-NS3 activated MazF’’, ‘‘pmCherry’’ or ‘‘pEGFP- MazF’’ encoding for mCherry-NS3 activated MazF, mCherry PLoS ONE | www.plosone.org 14 February 2012 | Volume 7 | Issue 2 | e32320