Sulforhodamine B assay One of the melanoma cell lines in the current study was found not to metabolise the MTS reagent. Therefore, we performed an SRB assay to calculate an IC50 to E6201 for this line. The SRB assay was per formed as previously described. Briefly, after drug treatment cells were then fixed with 25 uL of cold trichloroacetic acid for 60 selleck chem Veliparib minutes. Cells were subsequently washed five times with H2O and air dried. Next, cells were stained with 50 uL of 0. 04% SRB in 1% acetic acid and incubated at room temperature for 30 minutes. Unbound SRB was removed by washing five times with 1% acetic acid and air dried. Finally, bound SRB stain was solubilized in 100 uL of 10 mM Tris buf fer before taking an optical density measurement at 570 nm using the BioTek microplate reader.

PI3K and MAPK pathway activation Cell lines in the panel were plated at a density of 500,000 cells per well on day 0 in a 6 well plate. On day 1, cells were washed twice with PBS, serum starved in DMEM containing 0. 2% FBS and protein lysates were collected 16 hours after serum starvation. 50 ug of total protein were analysed Inhibitors,Modulators,Libraries on a 3 8% SDS PAGE. Phosphory lated Akt and phosphorylated ERK1/2 proteins were probed for with phospho specific antibodies from Cell Signaling Technology. Immunoblots were then stripped and re probed for total Akt and ERK1/2. The ratio of phosphorylated Akt or ERK1/2 to total Akt or ERK1/2 respectively was calculated by densitometry using Image J software and scored as follows negative 0 15%. 15 50%, 50 100%.100% of phosphorylated protein relative to total protein levels.

On additional Western blots, PTEN and GAPDH proteins were probed for with antibodies from Cell Signaling Technology and Abcam respectively. Cell cycle Inhibitors,Modulators,Libraries analysis Cells were plated in triplicate in 100 mm2 plates. The next day, cells were treated with 200 nM E6201 or 0. 01% DMSO. After 48 hours of treatment, cells were fixed in 80% ethanol for 2 hours, washed with ice cold PBS, and then resuspended in 500 uL cell cycle staining buffer. DNA content was evaluated by flow cytometry as an indicator of Inhibitors,Modulators,Libraries cell cycle progression. Cell cycle ana lysis was performed using ModFit software. The percentage of G1 arrest was calculated as the percent increase in cells in G1 relative to the percent of cells in G1 in DMSO con trol samples as follows x 100.

Cell death analysis by Annexin V staining Annexin V FITC staining was Inhibitors,Modulators,Libraries used to measure phospha tidylserine exposure on cells undergoing apoptosis according to the manufacturers instructions. Inhibitors,Modulators,Libraries http://www.selleckchem.com/products/U0126.html 2. 5 105 cells were plated per well in a 6 well plate. Cells were treated with 200 nM E6201 or 0. 01% DMSO 24 hours after plating. After 72 hours, floating and attached cells were collected and resuspended in Annexin binding buffer. Following the addition of 500 ng/mL Annexin V FITC and 1 ug/mL propidium iodide, cells were analysed for Annexin positive cells using a CyAn ADP flow cytometer and Summit software, version 4. 3.