Braf v600e

The human melanoma cell line A375 (MITF wild type, BRAF V600E, NRAS wild type) was obtained from American Type Culture Collection (Manassas, VA) and was grown in Dulbecco’s modified Eagle’smedium (DMEM, Euroclone, Milano, Italy) containing 2 mM glutamine, 100 UI/ml penicillin, 100 μg/ml streptomycin and 10% FBS (Euroclone, Milano, Italy). A375–M6 melanoma cells (M6) were isolated from lung metastasis of SCID bg/bg mice i.v. injected with A375 cells and grown in the same conditions of A375. A375 and M6 were independently validated by STR profiling by the DNA diagnostic centre BMR Genomics (Padova, Italy). Cells were amplified, stocked, thawed and were kept in culture for a maximum of 4 months. ECFCs were isolated from > 50 ml human umbilical cord blood (UCB) of healthy newborns as described previously [24 (link)], were selected as CD45⁻, CD34⁺, CD31⁺, CD105⁺, ULEX⁺, vWF⁺, KDR⁺ cells [24 (link)] and were grown in EGM-2 culture medium (Lonza), supplemented with 10% FBS. Human Microvascular Endothelial Cells (HMVECs) were purchased from Lonza and were grown in the same conditions of ECFCs.

The human melanoma cell lines A-375 and SK-MEL-28, carrying BRAF^v600e mutation, were obtained from the American Type Culture Collection (ATCC, Manassas VA). The cultures were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4 mM l-glutamine; 4500 mg/L glucose; 1 mM sodium pyruvate and 1500 mg/L sodium bicarbonate, in the presence of 10% FBS; and 1% penicillin–streptomycin. To ensure the quality and integrity of human cell lines, cells from the initial thawed vials were used for up to a maximum of 10 passages in all the experiments, as recommended by the supplier. Cells were tested for the presence of mycoplasma (EZ-PCR Mycoplasma Test Kit; Biological Industries, Beth Haemek, Israel) with negative results.