Fig. 5

ERα-36 regulates PR transcriptional activity. a HeLa cells were transiently transfected with MMTV-LUC reporter plasmid and expression vectors encoding PR (10 ng) and ERα-36 (from 50 to 200 ng) using Lipofectamine 2000. Transfected cells were grown in a hormone-free medium for 48 h in the presence or absence of 10 nM R5020, and extracts of the harvested cells were tested for luciferase activity using the Promega luciferase assay kit. The results were normalized as indicated and presented as the mean ± SEM of at least three independent experiments. The p value was determined using Student’s t test. *p < 0.05, ***p < 0.001. b–d Clones of T47D were treated, or not (Eth), 6 h with 10 nM of R5020. Total RNA was prepared and cDNAs analyzed by RT-qPCR with specific primers for SGK1, STAT5A, FKBP5, PDK4, DUSP1, and RGS2. The values were normalized against 28S mRNA and represent the mean ± SEM of three experiments. The p value was determined comparing each ERα-36 KO clones to the corresponding condition in the WT using Student’s t test. **p < 0.01, ***p < 0.001. e–g T47D clones, grown in a charcoal-stripped serum for 48 h and then treated with 10 nM R5020 for 1 h, were subjected to ChIP assay using an anti-PR antibody. The precipitated DNA fragments were used for qPCR analysis using specific primers for the indicated promoters. The results are expressed relative to the signal obtained from input chromatin. The mean ± SEM of at least three experiments is shown. The p value determined by comparing each ERα-36 KO clone to the corresponding condition in WT cells using Student’s t test was not significant