Phosphorylation of PR Ser294 drives SUMO-deficient PR gene expression and promoter selectivity in MCF-7 and T47D cells. (A) Relative expression level (copy number) of PR target genes in breast cancer patient cohorts. (B) Relative gene expression levels of selected PR target genes in MCF-7 cells stably expressing either empty vector (PR-null), WT or SUMO-deficient K388R PRs. Cells were co-treated with progestin R5020 and/or antiprogestin RU486 for six hours and mRNA levels were measured using RT-qPCR (see Materials and methods). (C) Relative gene expression levels of the same PR target genes (as in parts A-B) were measured using RT-qPCR in five vector-matched T47D cell lines stably expressing PRs: empty vector (null), wild-type (WT) PR, K388R mutant (KR) PR, S294A mutant (SA) PR, and K388R and S294A double mutant (KRSA) PR. Cells were treated with R5020 for six hours. (D) T47D cells expressing WT PR were treated cells with epidermal growth factor (EGF) for two days and treated with R5020 for 3, 24, or 48 hours. Relative MAP1A and RGS2 mRNA levels were measured using RT-qPCR. (E) Parental T47Dco cells were pretreated with EGF for 20 minutes prior to 24 hours of R5020 treatment. Relative RGS2 mRNA levels were measured by RT-qPCR. Data are represented as mean of n = 3 +/- SD and significance calculated using Student's t-test. n, number; PR, progesterone receptor; SD, standard deviation; SUMO, small ubiquitin-like modifier; WT, wild type.