depleted media Transformation assays Soft agarose colony formati

depleted media. Transformation assays Soft agarose colony formation by anchorage independent growth and tumor enografts were previously described. The animal e periments were conducted in accord ance with institutional guidelines under the approved pro tocols. For the in vivo tumor growth e periments, Kaplan Meier survival plots were generated, selleck bio and from the survival data a log rank test was used to demonstrate Inhibitors,Modulators,Libraries significant differences between groups. Antibodies and reagents The following antibodies were used for immunoblot Inhibitors,Modulators,Libraries ting rabbit polyclonal C 20, mouse monoclonal clone 1 F3, and rabbit monoclonal EP1808Y for Nrf2.

Actin was from Calbiochem MerckMillipore, NQO1 was from Novus Biologicals, G6PD was from Bethyl, HIF 1 was from BD Biosciences, Cleaved PARP, total AKT, phosphorylated AKT, total ERK1 2, phosphorylated ERK1 2, Cul3, Keap1, HSP90 and Lamin A C antibodies were all from Cell Signaling Technology, GAPDH was from Advanced Immunochemical Inc, Inhibitors,Modulators,Libraries Secondary antibodies were from DAKO. N acetyl L cysteine, ascorbic acid, tert butylhydroqui none, camptothecin, etoposide and staurosporine were all obtained from Sigma. Cell treatments Apoptosis was induced by treatment with 5 uM camp tothecin for 24 hours, 1 uM etoposide for 48 hours, and 1 uM staurosporine for 3 hours. The percentage of apoptotic cells was measured by flow cytometry after double staining with Anne in V and Propidium Iodide using the FITC Anne in V Apoptosis Detec tion Kit following the manufacturers instructions. Data were analyzed using Summit software. Caspase 3 7 activity was quantified by using Caspase Glo 3 7 Assay from Promega.

Cell viability was addressed by using CellTiter AQueousOne Solution Inhibitors,Modulators,Libraries Cell Proliferation Assay, a colorimetric method based on the reduction of a tetrazo lium compound by NADPH or NADH produced by de hydrogenase enzymes in metabolically active cells. Levels of reduced glutathione were quantified by using GSH Glo Glutathione Assay following the ma nufacturers instructions. Nuclear and cytoplasmic protein fractions were obtained by using NE PER Nuclear and Cytoplasmic E traction Kit. E periments in hypo ia were GSK-3 performed as previously described. In the inhibition studies for the RAS downstream sig naling pathways, breast cancer cell lines MDA MB 231 and MCF 7 were seeded onto 6 well plates and 24 hours later washed with PBS and subjected to free serum standard media.

24 hours later the cells were incubated with free serum standard media containing DMSO EPZ-5676 molecular weight or the following chemicals ERK kinases inhibitor U0126, PI3K inhibi tors LY294002 and wortmannin, and AKT inhibitor GSK690693. After 16 hours incubation, RNA was collected and qRT PCR was performed. Protein e tracts were also col lected for western blot analysis. Quantitative real time polymerase chain reaction Total RNA was e tracted using RNEasy mini kit and mRNA levels were quantified by qRT PCR using Taqman Gene E pression Assays. SYBR Green Master Mi was used with optimized forward primers for GA PDH

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