T47D cells expressing inducible PR were plated in 10 cm dishes in cMEM and induced with AP21967. Cells were washed, induced, and serum starved for four days. Cells were then treated with R5020 for six hours before adding doxorubicin selleck chemicals to dishes for 24 hours. Protein was harvested using standard RIPA lysis buffer, subjected to SDS PAGE and western blotting using cleaved PARP and PR antibodies. Beta actin western blotting was per formed Inhibitors,Modulators,Libraries for sample loading controls. Cell viability after treatment with cytotoxic doxorubicin was determined by measuring the concentration of ATP, which is directly proportional to viable cell number, using Cell Titer Glo bioluminescence assays. T47D cells expres sing WT or KR PR were plated in 24 well dishes containing cMEM.

Cells were washed and ster oid starved in modified IMEM supplemented Inhibitors,Modulators,Libraries with 5% DCC FBS for one day. Cells were treated with R5020 for six hours before doxorubicin was added to the wells. After four days, cell viability was determined by adding Cell Titer Glo substrate and luminescence was measured using a plate reader. Sample means were nor malized to day zero. Oncomine data analysis The relative expression of individual PR target genes in human breast tumor samples was determined by searching the Oncomine database. Individual PR target genes were queried in The Cancer Genome Atlas Breast 2 dataset. Oncomine output data was sorted to isolate cancer versus normal associations and reported as the copy number unit expression values for blood, normal breast and breast carcinoma sam ples using box and whiskers plots.

For each analysis, specific breast carcinomas specified for each gene are, Invasive Lobular Breast Carcinoma, Invasive Ductal and Lobular Carcinoma, Intraductal Cribriform Breast Adenocarcinoma, and Mucinous Breast Carci noma. Multiple breast cancer concepts, as described in the Oncomine database, were associated with the ligand dependent KR WT gene signature. According to Oncomine, Inhibitors,Modulators,Libraries concepts are derived from gene expression microarrays or gene copy number datasets derived from tumor cohorts or cancer cell line experiments. Specifically, concepts are a list of genes from various published datasets that are defined by some criteria. The LD gene signature was created by normalizing the gene expression values in the R5020 treatment group to the R5020 treatment group, then Inhibitors,Modulators,Libraries comparing those normalized fold change values between the KR and WT PR expressing cell lines.

This analysis identified 151 LD genes upregulated 1. 5 fold in cells expressing SUMO deficient PR versus WT PR expressing cells. The ligand independent gene signature was created Inhibitors,Modulators,Libraries by normalizing the gene expression values in R5020 treatment group in WT or KR expres sing cells to the R5020 treatment group in the PR null Tofacitinib baldness expressing cells, then comparing those normalized fold change values between the KR and WT expressing cell lines.