Dulbecco s modified mem

The 4T1 and TS/A spontaneous murine cells are TNBC and ER+ phenotypic lines, respectively, originated in BALB/C mice [37 (link),38 (link),39 (link),40 (link)]. The 4T1 (ATCC) cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 units of penicillin, and 100 μg/mL of streptomycin. TS/A cells (Sigma-Aldrich Inc., St. Louis, MO, USA) were grown in Dulbecco’s modified MEM (GIBCO, Life Technologies, Monza, MB, Italy) supplemented with 2 mM of glutamine, 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% heat-inactivated fetal calf serum (GIBCO, Life Technologies, Monza, MB, Italy) at 37 °C in humidified 5% of CO₂ atmosphere. Culture medium was changed every 2–3 days, and cells were passaged with 0.25% trypsin/EDTA. Cells were routinely tested for Mycoplasma using a MycoAlert mycoplasma detection kit (BioWhittaker-Lonza, Euroclone S.p.a., Milan, Italy).

BSR-T7/5 cells which stably express T7 RNA polymerase [25 (link)] were maintained in Glasgow’s minimal essential medium (MEM) (Lonza) containing 10% fetal bovine serum (FBS), 10% tryptose phosphate broth, MEM Amino Acids Solution and geneticin (1 mg/ml). Vero E6 cells were maintained in Dulbecco’s modified MEM (Gibco) containing 5% FBS. MRC-5 cells were maintained in Eagle's MEM (EMEM) (Gibco) containing 10% FBS, MEM Non-Essential Amino Acids Solution (Gibco), and 1% sodium pyruvate (Sigma). HeLa cells were maintained in EMEM containing 10% FBS. MP-12 is a highly attenuated RVFV strain obtained after 12 serial passages of an RVFV ZH548 strain in the presence of 5-fluorouracil [26 (link)]. A recombinant MP-12 strain and other MP-12-derived mutants were rescued from cDNAs as described previously [24 (link)], except that BSR-T7/5 cells were used in place of BHK/T7-9 cells. Titers of the rescued viruses were determined by using a plaque assay [24 (link)]. For the virus carrying R16H/M250K mutations in NSs, passage 0 (P0) virus obtained from plasmid-transfected BSR-T7/5 cells were serially diluted and inoculated into VeroE6 cells. The highest titer of P1 virus was selected and used for the study.