Fro zen tissue samples were derived from patients diagnosed with either ductal carcinoma, infiltrating ductal carci noma, lobular carcinoma, or metastatic carcinoma. Spe cimens were analyzed find more info by the University of Minnesota clinical pathology department and scored for ER and PR expression using standard clinical histological methods. Tumor samples were harvested individually for protein or mRNA using standard methods buffer, tri reagent and total PR, phospho Ser294 PR and ERK1 2 protein expression levels were measured by western blotting. All specimens were obtained from patients with informed consent and approval from University of Minnesota Institutional Review Board. Cell culture, expression vectors and western blotting T47Dco parental cell lines were characterized previously.

T47D cells stably expressing PR were created by molecular cloning of cDNAs encoding either WT, K388R, S294A, or K388R S294A PR Inhibitors,Modulators,Libraries into a pIRES neo3 expression vector, followed by transfection of vectors into T47D Y cells using FuGENE HD. Single cell clones were expanded under high G418 selection and maintained in low G418 selection. These cells were maintained in complete minimal essential medium supplemented with 5% fetal bovine serum, 1% non essential amino acids, 1% penicillin streptomycin, 6 ng ml insu lin. T47D cells expressing inducible PR were described pre viously. Inducible PR expression was achieved by adding AP21967 to cell culture medium for a mini mum treatment time of two days. MCF 7 cell lines expressing PR were created by transfection of pIRES neo3 vectors containing cDNA inserts encoding either WT or KR PR into cells using FuGENE HD.

Single cell Inhibitors,Modulators,Libraries clones were expanded under high G418 selection and maintained in low G418 selection. MCF 7 cells were maintained in MEM supplemented with 5% FBS, 1% penicillin streptomycin. BT 474 cells were main tained in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS, 1% penicillin streptomycin. SDS PAGE was performed using 8% gels and western Inhibitors,Modulators,Libraries blotting analysis was performed as pre viously described. For antibody information, see Additional file 1. Gene expression profiling T47D cells stably expressing pIRES Inhibitors,Modulators,Libraries neo3 empty vector, WT or KR PR were serum starved in modified improved MEM for one day, treated with R5020 or vehicle control for six hours before RNA extraction using a RNeasy kit.

Six Inhibitors,Modulators,Libraries hours of progestin treatment allowed for substantial PR depen dent gene expression as compared to prior studies. DNase I treated RNA samples from duplicate experiments were prepared for expression analysis Y-27632 using the Illumina HT 12v4 bead chip platform according to the manufactures protocols. Data were analyzed within R software using the Bioconductor package called lumi where raw intensities were log2 transformed and quantile normalized. Differentially expressed genes were analyzed using the limma package, where empirical Bayes was used to better estimate the variance of the genes.