Cells were cultured at 37 C in a humidified atmosphere of 95% air 5% CO2. Cell viability assays For cell viability assays, no cells were plated in triplicate in a 96 well microtiter plate and allowed to grow for 24 hours to an approximate confluence of 30%. MEK162 and and RAF265 were provided as a gift by Novartis Pharmaceuticals. Trametinib was purchased from Selleckchem. For drug inhibition studies RAF265, MEK162 and trametinib were used to treat mel anoma cells at various concentrations. Cell viability was evaluated at 72 hours using the CellTiter Glo Luminescent Cell Viability Assay, according to the manufacturers in structions and luminescence was mea sured using a Victor X multilabel plate reader. The IC50 values were determined by the XLfit soft ware.

Experiments Inhibitors,Modulators,Libraries were conducted Inhibitors,Modulators,Libraries three times and the results represent the average from these independent experiments. Western blotting and antibodies To assess the effect of MEK1 2 inhibition on phospho ERK1 2 or PARP cleavage, melanoma cells were treated with MEK162 or DMSO. Cells were lyzed in RIPA buffer supplemented with phosphatase and protease inhibitors and protein concentration was determined using Bradford reagent. Protein samples were boiled in Laemli buffer, resolved using 4 20% gradient Criterion XT precast gels and blotted onto nitrocellulose mem branes. To detect phospho ERK1 2 levels, membranes were probed with rabbit polyclonal phospho ERK1 2 T202 Y204 antibodies and reprobed with mouse monoclonal antibody recognizing total ERK1 2. Rabbit polyclonal antibody raised against cleaved PARP was used to detect the 89 kDa PARP cleavage product.

For loading control, membranes were stripped in Restore Western Blot Stripping Buffer and reprobed using anti B actin mouse monoclonal antibody. Representative results are shown. Clonogenic survival assays YUVON, YUROB, Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries YUKSI, YUMAC, YUDOSO and YUKIM cells were plated at 1,000 per well in six well plates to provide an optimal counting density. Cells were treated with increasing concentrations of MEK162 and cultured for one to two weeks until well defined colonies had formed, re placing culture medium every three days. Cells were fixed and stained with 0. 25% w v crystal violet in 80% methanol solution. Digital images of six well plates were captured, and colonies were counted using ProtoCOL software.

Data points are an average of four independent experiments and error bars repre sent the standard error of mean. Annexin V and propidium iodide labeling Apoptosis in MEK162 treateded melanoma cells was measured using Inhibitors,Modulators,Libraries annexin V Alexa Fluor 488 conjugate apoptosis kit according to the manufactur ers instructions. Flow cytometry was performed with a FACScalibur, and results were analyzed with FlowJo software. Experiments were conducted selleck chem Wortmannin twice independently with similar results. Re sults of one of the experiments are shown in Figure 3. Statistical methods JMP version 5.