Melanoma Cell Line Cultivation and PI3K Inhibition

The Wistar Melanoma (WM) cell lines were kindly provided by Dr. Meenhard Herlyn (Wistar Institute, Philadelphia, PA) and have been described elsewhere 19 . FEMX-1 cell line was established from a patient treated for melanoma at the Norwegian Radiumhospital, Oslo University Hospital 20 (link). Normal human melanocytes were isolated and cultured as previously described 21 (link). All cells were cultured in RPMI 1640 medium (Bio Whittaker, Verviers, Belgium) supplemented with 5% fetal bovine serum (FBS, PAA Laboratories, Linz, Austria) and 2 mmol/L l-glutamine (GibcoBRL, Paisley, UK) and maintained at 37°C in a humidified 5% CO₂ atmosphere. The cells were routinely checked for mycoplasma infections and authenticated using the Powerplex 16 kit (Promega, Madison, WI). For inhibition of the PI3K/AKT signaling the cells were treated with 25 μmol/L PI3K inhibitor LY294002 (Cell Signaling Technology Inc, Beverly, MA).