Transcriptional regulation of PIP by AR and CREB1. (A) Luciferase reporter assay. The transcriptional activation of PIP promoter by PRLR, AR, CREB1, PRLR + AR, and PRLR + CREB1 expression constructs was assessed using Dual-Luciferase assays in MCF-7 cells and relative response ratios are reported. Co-transfection with the PIP reporter vector and an empty pcDNA vector was used as a control (CTL). *P <0.01, is compared to the control group. (B) Induction of PIP expression following DHT treatment. PIP expression was assessed using qPCR following DHT treatment at 30 minute, 1 hour, 3 hour, 12 hour, 24 hour, and 48 hour time-points in MDA-MB-453 and HCC-1954 cell lines. Fold changes are measured relative to the respective control at each time point. *P <0.03, is compared to the control group (dashed line). Error Bars: ± 2SEM. (C) Putative transcription factor binding sites for CREB1 in 1.5 kb promoter region of PIP. P1 (primer set 1) and P2 (primer set 2) are regions of amplification for ChIP assays. (D) ChIP assay with CREB1 antibody. The results of qPCR amplification for ChIP assays are demonstrated with two sets of primers for PIP promoter. Percentage recovery of input chromatin is shown for each primer set. *, P <0.01 is for CREB1 Ab. versus control Ab. Error Bars: ± 2SEM. (E) Western blot analysis to show CREB1 and PIP protein levels following CREB1-knockdown using siRNA in MDA-MB-453 cell line. Fold changes (RR) in band densities were measured relative to non-targeting siRNA control (CTL). Ab, antibody; AR, androgen receptor; ChIP, chromatin immunoprecipitation; DHT, dihydrotestosterone; qPCR, quantitative PCR; RR, relative risk; SEM, standard error of the mean.