Erk1 2 u0126

Human melanoma cell lines: primary WM115 (VGP) and metastatic: WM266–4 (derived from metastatic site – right thigh skin). These cells lines feature the specific V600D mutation in the B-RAF gene, as well as express PTEN loss of function including hemizygous deletion and wild type for N-ras, c-KIT and CDK4. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics: penicillin and streptomycin. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. Cells were treated with inhibitors of: 1/AKT – MK-2206 (Selleck) at 2 μM concentration, 2/ MEK – AS-703026 (Selleck) at 10 μM concentration, 3/ PI3K – LY294002 (Cell Signalling TM) at 20 μM concentration, 4/ ERK1/2 – U0126 (Cell Signalling TM) at 10 μM concentration, and 5/ mTOR – everolimus (Selleck) at: 20 nM, 2 μM, 5 μM and 10 μM concentrations. The incubation time of melanoma cells with inhibitors were 24 and 48 h. Cells were obtained from the ESTDAB Melanoma Cell Bank (Tubingen, Germany).

Human melanoma cell lines: WM793 [vertical-growth phase (VGP)]—Lu1205 (metastatic; biopsy taken from the lung; selection in mice; a culture from the primary site (sternum area) from the same donor as WM793). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics: penicillin and streptomycin. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. Cells were treated with inhibitors: (1) PI3K-LY294002 (Cell Signaling TM)—20 μM concentration, (2) ERK1/2-U0126 (Cell Signaling TM)—10 μM concentration, (3) mTOR—rapamycin (Selleck)—5 nM concentration, (4) mTOR—everolimus (Selleck)—5 nM concentration, (5) B-RAF-GDC-0879 (Selleck)—2 μM concentration, (6) GSK-3β-CHIR-99021(Selleck)—2 μM concentration. Cells were obtained from the ESTDAB Melanoma Cell Bank (Tubingen, Germany).