High glucose dmem culture medium

NIH 3T3 fibroblasts (ATCC CRL-1658), B16-F1 mouse melanoma cells (ATCC CRL-6323) and derived mutants were cultivated at 37 °C and 5% CO₂ in high-glucose DMEM culture medium (Lonza, Cologne, Germany) supplemented with 1% penicillin-streptomycin (Biowest, Nuaille’, France), 10% FBS (Biowest) and 2 mM UltraGlutamine (Lonza). A total of 3 h after seeding onto 35 mm diameter wells (Sarstedt, Nümbrecht, Germany), the cells were transfected with 1 µg (B16-F1) or 3 µg (NIH 3T3) plasmid DNA using JetPRIME transfection reagent (PolyPlus) at a ratio of 1 µg of DNA to 2 µL of the transfection reagent, according to the manufacturer’s protocol. At 4 h post transfection, the transfection mixture was replaced with fresh culture medium. Cells stably transfected with pEGFP-C1-Puro-CapZβ were maintained with, additionally, 1.5 µg/mL puromycin. Used cell lines were routinely authenticated following common guidelines by local authorities.

B16-F1 mouse melanoma cells (CRL-6323) and mouse embryonic NIH 3T3 fibroblasts (CRL-1658) were purchased from ATCC. The mouse embryonic fibroblast cell line MV^D7, which expresses a temperature-sensitive version of large T antigen, was derived from Mena/VASP-deficient mice (Bear et al., 2000 (link)). For this study, MV^D7 cells were additionally immortalized with a retrovirus transducing a wild-type variant of SV40-large T (LT) antigen using standard procedures, to allow cultivation at 37°C. Expression of Evl in original and MV^D7 cells immortalized with wild-type, SV40-LT antigen was unchanged, as assessed by immunoblotting. B16-F1, NIH 3T3 and wildtype SV40-LT-immortalized MV^D7 cells were cultured at 37°C and 5% CO₂ in high-glucose DMEM culture medium (Lonza, Cologne, Germany) supplemented with 10% FBS (Biowest), 2 mM UltraGlutamine (Lonza) and 1% Penicillin-Streptomycin (Biowest). B16-F1 cells were transfected with 1 µg plasmid DNA and MV^D7 and NIH 3T3 cells with 3 µg plasmid DNA, respectively using JetPRIME transfection reagent (PolyPlus, Illkirch, France) in 35 mm diameter wells (Sarstedt, Nümbrecht, Germany) according to the manufacturer’s protocol. Used cell lines are routinely authenticated following common guide lines by local authorities. Absence of mycoplasma in cell lines was routinely checked by the VenorGeM Mycoplasma Detection Kit (Sigma, St. Louis, MO).