The murine fibroblast cell line NIH3T3 was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum, penicillin, streptomycin and different combinations of antibiotics to select for stable vector integration (G418 [1 mg/ml], puromycin [2.5 μg/ml], hygromycin [300 U/ml]). NIH3T3 IFN-β-tGFP cells, described elsewhere (6 (link)), were transfected with either ptet.NS1-H or ptet-NS3/4A-H and the rtTA-encoding plasmid prtTA2.PGK.puro and the KRAB-transrepressor encoding plasmid pKrab.PGK.puro by lipofection using Metafectene (Biontex Laboratories) according to the manufacturer's instructions. Transfected cells were seeded at low density onto 10-cm dishes and clonal colonies were isolated. Cell clones were characterised for doxycycline-induced transgene expression and IFN-β-tGFP reporter expression. Representative clones exhibiting good inducibility upon addition of doxycycline and physiological IFN-β expression levels in the absence of doxycycline were used for further experiments. Cell lines were termed IFN-β-tGFP tet.NS1-H and IFN-β-tGFP tet.NS3/4A-H, respectively.
Fluorescent labelling of HaloTag-linked proteins was done by adding 300 nM HaloTag-ligand coupled TMR (Promega) for 30 min (for flow cytometric analysis) or supplementing medium with 20 nM HaloTag-ligand coupled TMR continuously for live-cell imaging.
Fluorescent labelling of HaloTag-linked proteins was done by adding 300 nM HaloTag-ligand coupled TMR (Promega) for 30 min (for flow cytometric analysis) or supplementing medium with 20 nM HaloTag-ligand coupled TMR continuously for live-cell imaging.