Ma1 080

To examine HBcAg or HCV core expression in HepG2.2.15 or JFH-1 Huh7.5 cells after co-culture with PBMCs, immunofluorescence staining was performed as previously described (61 (link)). Briefly, after 48 h of co-cultivation, an average sample of 2 × 10⁵ cells were seeded into a 35 mm confocal dish. After overnight, the cells were rinsed with PBS, fixed with 4% paraformaldehyde (PFA) at 4°C for 30 min, permeabilized with 0.5% Tween-20 for 10 min, blocked with normal 5%BSA (amresco) for 1 h, and sequentially incubated with HBcAg (1:200, Catalog: ab8637, Abcam) and HCV core (1:200, Catalog: MA1-080, Thermo Fisher Scientific) antibodies overnight at 4°C and secondary antibodies at room temperature for 1 h. Nuclei were counterstained with DAPI (1 µg/mL, Catalog: D1306, Thermo Fisher Scientific) for 5 min. Images were captured using a fluorescence microscope (Olympus IX51, Tokyo, Japan).

Huh-7.5.1 cells were cultivated on 12-well plates coated with poly-L-lysine (Sigma Aldrich, St. Louis, MO, USA) for 24 h, then inoculated with viruses for 4 h. Two days after infection, the cells were fixed for immunocytochemistry. The fixed cells were treated with 1% bovine serum albumin solution and then with an anti-HCV core antibody (mouse) (MA1-080, Thermo Fisher Scientific, Waltham, MA, USA) and 2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole trihydrochloride trihydrate (Hoechst 33342, Thermo Fisher Scientific, Waltham, MA, USA). An Alexa 555-labeled anti-mouse antibody (Thermo Fisher Scientific, Waltham, MA, USA) was used to visualize the HCV core protein. Fluorescence microscopy was performed on a Zeiss Axio Scope A1 microscope.

Uninfected and HCV infected Huh-7.5 cells were seeded in two-well chamber slides (Thermo Fisher Scientific) at a density of 2 X 10⁴ cells per well. After overnight incubation, cells were transfected with 500 ng of pSTAT1-GFP or pSTAT2-GFP plasmid using FuGENE 6 (Roche Diagnostics, Indianapolis, IN) transfection reagent. At 48 hours, culture was treated with either IFN-α or IFN-λ (2.5 x IC₉₀₎ for one hour and then fixed with 4% paraformaldehyde. HCV core staining was performed using a monoclonal antibody at a dilution of 1:200 (MA1-080; Thermo Fisher Scientific, Rockford, IL) and secondary Alexa 594 labeled goat anti-mouse antibody at a dilution of 1:500 (Life Technologies, Carlsbad, CA). After the staining Hoechst 33342 nuclear dye (Calbiochem, Darmstadt, Germany) was added to the samples at 1 μg/ml, and incubated for five minutes in PBS. The translocation of GFP as well as the HCV core was monitored using a Leica TCS SP2 confocal microscope equipped with three lasers (Leica Microsystems, Exton, PA). Optical slices were collected at 512 × 512 pixel resolution.