Tet on advanced gene expression system

All cell lines used were of human origin and were routinely cultivated in DMEM medium supplemented by 10% FBS and 10 µg/µL ciprofloxacin (Sigma, Piscataway, NJ, USA) in a humified atmosphere with 5% CO₂ at 37 °C. Transfections were performed using polyethylenimine (Polysciences, Inc., Warrington, PA, USA) or GenMute transfection reagent (SignaGen Laboratories, Frederick, MD, USA) in the case of DNA and siRNA, respectively, according to manufacturer’s protocol. Stable cell lines were prepared by lentiviral transduction using the second-generation packaging system (pLVX constructs, Tet-On Advanced Gene expression system, Clontech, Mountain View, CA, USA) and enriched by cell sorting, based on EGFP expression. In the case of IFN-treated cells used for morphology studies, RNA and protein lysates, cells were pre-treated with 10 ng/mL recombinant human IFNs (Peprotech, Cranbury, NJ, USA) for 48 hours and IFNs were also present during subsequent 3D cultures (overall exposure time 96 h). Ruxolitinib (Sigma) was used at a concentration of 10 µM, dasatinib (LC Laboratories, Woburn, MA, USA) at 1 µM and doxycycline (Sigma, Piscataway, NJ, USA) at 250 ng/mL. The siRNA sequences targeting IRF9 mRNA were: siIRF9_1: 5′-GCAGAGACUUGGUCAGGUC-3′ and siIRF9_2 5′-CACAGAAUCUUAUCACAGU-3′.