Gene set enrichment analysis software was employed to compare genes up or down regulated in cells stably expressing WT or KR PR to cells expres sing inducible iWT or iKR PR. Using the Affymetrix expression data, four gene sets were created, genes up or down regulated 2. 0 fold by iWT with R5020, and genes up or down regulated 2. 0 selleck chemicals 17-AAG fold by iKR with R5020. Similarly, two GSEA for matted Inhibitors,Modulators,Libraries datasets were created from the Illumina expres sion data, the first dataset compares the two phenotypes, and the second com pares the two phenotypes. GSEA was performed using those Illumina datasets and queried for enrichment of the Affymetrix gene sets. GSEA was executed using the default settings, except the permutation type was set to Gene set with 1,000 permutations, and the metric for ranking genes was set to Diff of Classes because our dataset contained log scale data.

Chromatin immnunoprecipitation Chromatin immunoprecipitation assays were per formed according to the ChIP IT Express instruction manual. Cells were plated at 15 �� 106 cells per 15 cm cul ture dish in cMEM for two days, then serum starved in modified IMEM for two days. Cells were treated with R5020 or vehicle for one Inhibitors,Modulators,Libraries or four hours. For T47D cells expressing inducible PR, AP21967 was added during the starvation step. Chromatin was sheared using a Bioruptor sonicator, Inhibitors,Modulators,Libraries for 30 minutes. Immunoprecipitations were prepared with 60 ul of sheared chromatin, 2 ug antibody and immunoprecipi tated overnight. Using the purified ChIP and input DNA, relative recruitment was determined by qPCR in tripli cate.

Assays were performed on a Roche LightCycler II using SYBR green master mix. Target locus quantifica tion was normalized Inhibitors,Modulators,Libraries as a percentage of the input DNA quantification. To assay H3K4me2 levels, nucleosomes were isolated using micrococcal nuclease. In 15 cm dishes, 12 �� 106 cells were plated in cMEM, serum starved in modified IMEM and induced with AP21967 treatment for two days. One day later, cells were treated with R5020 for four hours and chromatin was harvested and immunoprecipitated as previously described. Cell proliferation and apoptosis assays Cell proliferation was measured using MTT assays. In 24 well plates, 1 �� 104 cells well were plated in cMEM for two days cells were washed and steroid starved in modified IMEM supplemen ted with 5% dextran coated charcoal treated FBS for one day before the addition of R5020.

At days 0, 2, 4, and 6, cell proliferation was determined by adding 60 ul MTT to each 0. 5 ml Inhibitors,Modulators,Libraries cell culture well for three hours, Imatinib Mesylate manufacturer medium was carefully removed and solubili zation solution PBS was added to lyse the cells. Lysate absorbance was measured using a plate reader. The 650 nm measurements were subtracted from 570 nm measure ments and sample means were normalized to day zero.