Raf265

The TT cell line (MTC, human) was obtained from the European Collection of Cell Cultures (Sigma-Aldrich, Milano, Italy) and cultured in RPMI 1640 (Gibco - Life Technologies, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS; Gibco), L-glutamine (2 mM) and penicillin-streptomycin (100 IU/ml–100 μg/ml respectively). Adherent monolayer cells were maintained in T-75 culture flasks and incubated at 37°C with 5% CO₂ until they achieved 85% confluence. Cells were detached using 0.25% trypsin-EDTA (Sigma-Aldrich) and plated in T-75 flasks at a density of 2 × 10⁶ cells.
RAF265 was kindly provided by Novartis International (Basel, Switzerland), SB590885 and ZSTK474 were purchased from Selleck Chemicals (Houston, TX, USA). The powders were dissolved in a 10 mM stock solution in dimethyl sulfoxide (DMSO), following the manufacturer’s instructions.

SkMel28 cells (American Type Culture Collection; ATCC; Rockville, MD), A375, DM13, DM331, DM6, HT144, SKMel24, VMM12, VMM15, VMM17, VMM18, and VMM5A (kind gift from Dr. Craig Slingluff, University of Virginia) were propagated in RPMI Medium 1640 (Invitrogen, Grand Island, NY) supplemented with 5% or 10% fetal bovine serum (FBS; Gemini Bio-Products, West Sacramento, CA). All cultures were maintained in a humidified chamber at 37°C with 5% CO₂. OncoMap analysis was conducted at the Broad Institute to identify the mutational status of over 30 known oncogenes and tumor suppressor genes. The cell lines were authenticated by comparing the tumor mutation profile determined by OncoMap with published reports.
Antibodies were obtained from the following sources: anti-phosphoERK and anti-total ERK (Sigma-Aldrich), and anti-tubulin (Calbiochem, Gibbstown, NJ).
Sources of combination inhibitors are shown in Supplementary Table S1. PLX4720 was a gift from Plexxikon, RAF265 a gift from Novartis and PD325901 a gift from Pfizer.

Raf265 was provided by Novartis Pharmaceuticals Corporation. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless specified below.