Tunel assay

Staining and wash steps were performed on an orbital shaker. Cells were washed twice in PBS and then fixed in 4% PFA/PBS for 10 min. After another 2 washes with PBS, they were treated with 0.1% Triton-X/PBS for 10 min, then blocked overnight at 4 °C in blocking buffer (1% BSA/PBS). Primary antibodies were incubated at 1:200 in blocking buffer for 2 h, then washed thrice with PBS for 10 min each. Secondary antibodies (Alex Fluor, Thermo Fisher Scientific) were incubated at 1:500 in blocking buffer for 1 h in the dark. Cells were counterstained with DAPI/PBS for 10 min, then washed twice in PBS for 10 min. Primary antibodies (Santa-Cruz and Abcam) used were as follows: mouse anti-HBs (sc-23944); mouse anti-HBc (sc-23946); rabbit anti-HBx (ab39716); rabbit anti-NTCP (sc-98484); goat anti-HNF4α (sc-6556X). TUNEL assay (R&D Systems, Minneapolis, Minesotta, USA) for cytoplasmic rcDNA was performed with manufacturer’s protocol. Images were acquired using the EVOS M5000 Cell Imaging System (Invitrogen). The cccDNA fluorescence intensity for each cell was quantified by ImageJ v1.53s.

Cell viability was determined by water soluble tetrazolium (WST)-1 assay (Sigma-Aldrich). 5000 Cells were seeded in 96-well plates, allowed to attach and treated as described above. WST-1 reagent diluted 1:10 in growth medium was added to each well. After incubation at 37 °C in a 5% CO₂ incubator for 3 h, absorbance at 450 nm was measured using a microplate reader (Tecan, Crailsheim, Germany). As a non-metabolic readout for cell viability we stained AML12 cells with the fluorescence nucleus dye DAPI (Invitrogen; ProLong™ Gold Antifade Mountant with DAPI) and counted cells with and without signs of cytotoxicity (viable cells in percent).²² Cell proliferation was measured using a colorimetric cell proliferation ELISA (Roche), according to the manufacturer’s protocol. This assay measures 5-bromo-2′-deoxyuridine (BrdU) incorporated in replicating cells. The reaction product was quantified by measuring the absorbance at 450 nm using a microplate reader. Double strand breaks as indicator of cell apoptosis were measured via TUNEL assay (R&D Systems, Minneapolis, USA) in AML12 cells treated with 5-FU. The reaction product was quantified by measuring the absorbance at 450 nm.

A total of 5.0 × 10⁴ (24 h) and 2.0 × 10⁴ (48 h) A637 cells, as well as 4.0 × 10⁴ (24 h) and 2.0 × 10⁴ (48 h) RD-ES cells, were seeded into 100 µL of cell suspension in a 96-well plate. CPP treatment was performed indirectly by treating 200 µL of full medium with CPP for 5 s in a 24-well plate. Then, 100 µL of the treated medium was added to the seeded cells. Cells treated with 200 µL of the full medium treated with argon for 5 s served as controls. After the CPP treatment, the cells were treated with DOX and VIN at their respective IC₂₀ concentrations, and control with argon and the cytostatic drugs was also performed. Controls with untreated cells (1 negative and 1 positive; nuclease treated) were included on each plate. A corresponding second plate was treated in parallel to normalize the absorption values to cell numbers. The TUNEL assay (R&D Systems, Minneapolis, MN, USA) was performed 24 h or 48 h after treatment according to the manufacturer’s protocol using the TECAN multimode plate reader. The relative TUNEL signals of cells treated with CPP, cytostatic or combination therapy were normalized to the mean relative TUNEL signals of cells treated with argon (control).