Data were analyzed using Summit software. Caspase 37 activity was quantified by using Caspase Glo 37 Assay from Promega. Cell viability was addressed by using CellTiter AQueousOne Solution Cell Proliferation Assay, a colorimetric method based on the reduction of a tetrazo lium compound selleck kinase inhibitor by NADPH or NADH produced by de hydrogenase enzymes in metabolically active cells. Levels of reduced Inhibitors,Modulators,Libraries glutathione were quantified by using GSH Glo Glutathione Assay following the ma nufacturers instructions. Nuclear and cytoplasmic protein fractions were Inhibitors,Modulators,Libraries obtained by using NE PER Nuclear and Cytoplasmic Extraction Kit. Experiments in hypoxia were 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 or the following chemicals ERK kinases inhibitor U0126 . PI3K inhibi tors LY294002 and wortmannin . and AKT Inhibitors,Modulators,Libraries inhibitor GSK690693. After Inhibitors,Modulators,Libraries 16 hours incubation, RNA was collected and qRT PCR was performed. Protein extracts were also col lected for western blot analysis. Quantitative real time polymerase chain reaction Total RNA was extracted using RNEasy mini kit and mRNA levels were quantified by qRT PCR using Taqman Gene Expression Assays. SYBR Green Master Mix was used with optimized primers for GA PDH at final concentration of 300 nM. b Actin was used as reference gene for the experiments in hypoxia.

All experiments were per formed at least by triplicate. Additional cell lines Additional MSC lines with activated RAS or RAS downstream effectors were generated by infection of MSC4 with the retroviral vector pBabe hygro encoding constitutive active RAS, the membrane targeted RAF 1, and Inhibitors,Modulators,Libraries myristoylated AKT, all kindly provided by Dr. Pablo Rodriguez Viciana. Immortal human mammary epithelial cells, obtained from Dr. Rodriguez Viciana, were cultured in DMEMF 12 containing 5% horse serum and supplemented with EGF, hydrocortisone, cholera toxin and insulin, all from Sigma. HMEC expressing different oncogene combinations were generated after infection with the same retroviral vectors used for the generation of MSC lines. Breast cancer cell lines MDA MB 231 and MCF 7 were cultured in DMEM containing 10% fetal bovine serum. Human umbilical vein endothelial cells were obtained from Promocell and cultured selleck products with Endothelial Cell Growth Medium according to suppliers instructions. Public transcriptome data The expression level of Nrf2 in many types of cancer was and The Cancer Genome Atlas databases of cancer expression data.