Cell lines The melanoma cell lines used in this study and their mutational status are listed in Table 1. This panel was chosen from a larger cohort Belinostat HDAC of well characterized melan oma cell lines to enrich for common and rare mutation genotypes, such as joint BRAF and RAS wildtype status and wildtype PTEN status, in order to increase the likeli Inhibitors,Modulators,Libraries hood of detecting significant associations. Cells were grown in DMEM plus 10% foetal calf serum. Melanoma cell lines prefixed with MM. as well as BL, NK14, WSB, A375 and SKMEL13, were kindly pro vided by Dr Nick Hayward of the Queensland Institute of Medical Research, Brisbane, Australia. Those cell lines prefixed with UACC were originally obtained from the Arizona Cancer Center Tissue Culture Shared Resource, University of Arizona, Tucson, USA and were kindly provided by Dr Jeffrey Trent along with the WM35, M91 054 and M92 001 cell lines.
We would also like to thank Inhibitors,Modulators,Libraries the Australasian Biospecimen Network and Chris Schmidt for the D17 and D35 cell lines. Mutational analysis Mutational analysis was generally performed as previ ously reported using Sanger sequencing. Sequencing pri mers for each gene were as previously reported. BRAF, NRAS, KRAS, PTEN, CDKN2A and TP53. Those primers used to sequence HRAS and CDK4 in this study are available on request. E6201 IC50 calculation Each cell line was plated in triplicate in 200 uL DMEM containing 10% FBS at a density of 3,000 cells per well in 96 well plates. Six hours after cells were seeded, E6201 was added in half log dilutions in triplicate. An equivalent concentration of DMSO was added to untreated wells as a vehicle control.
In vitro cell proliferation assays were performed Inhibitors,Modulators,Libraries using an MTS assay or SRB assay four days after the addition of E6201. IC50 values were calculated using Inhibitors,Modulators,Libraries nonlinear regression curve fit with Prism 4 software. The MTS assay was used for all cell lines except MM329, as this cell line failed to effectively metabolize the MTS reagent. the SRB assay was used in place of the MTS assay in this case. We confirmed in several other melanoma cell lines that both proliferation assays pro duced comparable IC50 results. MTS assay For the MTS assay, media was removed and 120 ul of media containing 20 ul of MTS and PMS was added to each well and incubated for 3 hours at 37 C. Absorbance at 490 nm was measured using a BioTek Synergy HT Multiple Detection micro plate reader.
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 Inhibitors,Modulators,Libraries 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 minutes. Cells were subsequently washed five times with H2O and air dried. Next, cells were stained with kinase inhibitor 17-DMAG 50 uL of 0.