Fetal bovine serum (fbs)

The human prostate adenocarcinoma cell lines, LNCaP, PC3 and DU145 were purchased from the American Type Culture Collections (ATCC, Manassas, VA) and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS, Clontech) and antibiotics cocktail (100 U/mL penicillin and 100 μg/mL Streptomycin; HyClone/GE Healthcare Life Science). For doxycycline induction, LNCaP cells were cultured in the medium supplemented with tet system-approved fetal bovine serum (tetracycline-free FBS, Clontech) and simultaneously transduced with lentiviral vectors, FUW-tetO-EGFP or FUW-tetO-TSPX variants and the transactivator, FUW-M2rtTA. The transduced cells were cultured under non-induced conditions, in the absence of doxycycline (Dox). To induce the expression of TSPX and/or EGFP in the transduced cells, cells were cultured in the presence of 1 μg/mL Dox (Sigma-Aldrich). For cell proliferation analyses, cells were seeded at 2×10³ cells/well in 96 well plates and cultured in the presence or absence of 1 μg/mL Dox. The cell viability was monitored at the indicated time points by using the CellTiter 96 Aqueous One Cell Proliferation Assay kit (Promega), a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-fophenyl)-2H-tetrazolium (MTS) based assay system, according to the manufacturer’s instructions.

Cell-free concentrated KSHV (rKSHV.219) and EBV were isolated from latently rKSHV.219-infected iSLK.219 cells or latently EBV-infected Akata-BX1 cells, respectively. Virus production was stimulated by replacing iSLK.219 maintenance media with DMEM containing 10% FBS (Clontech), 1% Pen-Strep, 3 μg/mL doxycycline (Thermo Fisher), and 1 mM sodium butyrate (Sigma-Aldrich) for KSHV, and Akata-BX1 maintenance media with RPMI containing 10% FBS (Clontech), 1% Pen-Strep, 25 µg/mL goat anti-human IgG (Jackson Labs) for EBV. The supernatant was harvested 72 h or 120 h after induction, centrifuged to pellet cell debris, and filtered through a sterile 0.45 μm filter (Millipore Sigma). In total, 30 mL of the filtered supernatant was layered over a 5 mL cushion of 20% sucrose (Sigma-Aldrich), and the virus was pelleted by ultracentrifugation in a SW32Ti rotor (Beckman Coulter) at 114,000 × g for 2.5 h at 4 °C. After decanting the supernatant, virus pellets were resuspended in 3 mL PBS (Thermo Fisher).

The tetracycline-inducible cell line HEK T-Rex^TM-293 (ThermoFisher) was cultured under the following conditions: cells were maintained in DMEM/F-12 50/50 medium containing 10% fetal bovine serum (tetracycline-free, Takara, Mountain View, CA), penicillin (100 units/ml), streptomycin (100 g/ml), and blastcidine (2 μg/ml) in a humidified atmosphere containing 5% CO₂. Cells were passaged twice each week, maintaining subconfluent cultures. Stably transfected cells were cultured as above with the addition of hygromycin (50 μg/ml) and Zeocin (20 μg/ml).