The M10 cell line was found to be mutated www.selleckchem.com/products/Bicalutamide(Casodex).html in BRAF and wild type in NRAS and PIK3CA. Drugs and reagents TMZ was kindly provided by Merck Sharp Dohme Merck Co. Inc. and was al ways prepared freshly in culture medium, because the drug readily decomposes in aqueous solution. The NFB inhibitor NEMO binding domain peptide was purchased from Alexis Corporation as a solution of 10 mM in phosphate buffered saline. The MGMT inhibitor BG was purchased from Sigma Aldrich and dissolved in ethanol. Both inhibitors were stored as stock solutions at ?80 C, and diluted in culture Inhibitors,Modulators,Libraries medium just before use. In all assays, with the exception of those performed with 293T L cells, BG was used at the final concentration of 10 uM, added to the cells 2 h before TMZ and left into the culture up to the end of TMZ treatment.

At the concen tration Inhibitors,Modulators,Libraries used, BG did not affect cell proliferation. 3 2,5 diphenylte Inhibitors,Modulators,Libraries trazolium bromide, Inhibitors,Modulators,Libraries and 4 6 Diamidino 2 pheny lindole were purchased from Sigma Aldrich. MTT was prepared at a concentration of 5 mgml in PBS, and stored at 4 C. Reagents for SDS polyacrylamide gel electrophoresis were all purchased from Bio Rad Laboratories. NFB luciferase reporter assay HCT1163 6, HCT116, M10, pUSE2 and KD12 cells were seeded into 24 well plates and allowed Inhibitors,Modulators,Libraries to adhere at 37 C for 18 h. The cells were then transiently co transfected with 3 ug or 0. 075 ug or 0. 3 ug of the NF��B responsive firefly luciferase reporter pNFB Luc and 250 ng or 6. 25 ng or 25 ng of the Renilla luciferase expression vector pRL null using Lipofectamine 2000 Reagent according to the manufacturers protocol.

Twenty four hours after trans fection, cells were exposed to 50 inhibitor U0126 uM TMZ plus BG or to BG alone as a control. After 48 and 72 h of culture, the cells were lysed and luciferase assays were per formed using the Dual LuciferaseW Reporter Assay according to the manufacturers instructions. For each sample, firefly luciferase activity was normal ized to Renilla luciferase activity and then to the total protein amount used in the assay. To evaluate the effect of NBD peptide on NFB activ ity, M10 cells were plated and co transfected with the pNF kB Luc and pRL null vectors as described above. Twenty four hours after transfection, 50 uM NBD pep tide was added to the cultures. After additional 24 h of incubation, untreated and NBD peptide treated cells were exposed to 50 uM TMZ plus BG or to BG alone. Seventy two hours after drug addition, the cells were lysed and luciferase assays were performed as described above. Enzyme Linked Immunosorbent Assay M10 cells were seeded in 100 mm plates and allowed to adhere at 37 C for 18 h. The cells were then exposed to 50 uM TMZ plus BG or BG alone for 48 or 72 h.