Melanoma Cell Line Cultivation and Characterization

The present study used eight melanoma cell lines (listed in Table SI). Their mutational status (BRAF and NRAS mutations) is shown in Table SII. The origin of cells has been described previously (31 (link),32 (link)). Cells were cultivated in RPMI medium (MilliporeSigma) supplemented with 10% FCS (Thermo Fisher Scientific, Inc.), glutamine and antibiotics (MilliporeSigma) at 37°C and 5% CO₂ in 100% humidity. All cell lines were authenticated and tested for mycoplasma using a mycoplasma detection kit (MP0035; MilliporeSigma). Cells were passaged every 72–96 h using a trypsin-EDTA solution. When plated, cells (5×10⁵) were seeded from a stable culture into 12-well plates and incubated for 24 h at 37°C prior to inhibitor treatment or transfection, unless specified otherwise. Normal human melanocytes were purchased from Cascade Biologics Inc. and cultivated according to the manufacturer´s instructions. The generation of inducible melanoma cell lines in which melanoma-associated transcription factor (MITF) protein can be downregulated by the addition of doxycycline (Tet-on system) has been previously described (32 (link)).