P2P-R inhibits estrogen-dependent transcription. (A) Functional assays documenting that the transient transfection of FLAG-tagged P2P-R into MCF-7, Ishikawa, HeLa and 293T cells co-represses estrogen-induced transcription. MCF-7 and Ishikawa cells, that contain endogenous ERα, were transfected with 300 ng of luciferase reporter construct (pGl2-ERE2X-TK) plus renilla luciferase plasmid (15 ng/well), with or without co-transfection with 1 μg of FLAG-tagged P2P-R(25-1560). HeLa and 293T cells, that lack endogenous ERα, were also co-transfected with 50 ng pSG5-ERα. Cells were then incubated for 18 h in phenol-red free DMEM with 5% CS-FBS in the presence of DMSO or 100 pM 17-beta estradiol (E2). Data are expressed as Relative Luciferase Activity (RLA) of E2-dependent transcription (see Methods). Experiments were performed in triplicate, bars denote standard error. (B) Characteristic domains of the P2P-R protein (1-1560 aa) and definition of the five P2P-R constructs used in transcription assays. A, ring type zinc finger domain, B, proline-rich domain, C, SR-like domain, D, Rb1 binding domain, E, p53 binding domain, and F, lysine-rich domain. (C) Functional assays measuring the effects of expression of specific P2P-R domains on E2-dependent transcription in MCF-7 cells. Cells were transfected with 300 ng of pGl2-ERE2X-TK plus renilla luciferase plasmid (15 ng/well) and 1 μg of 3 × FLAG-myc-CMV™-29 expressing the respective P2P-R segments and then incubated for 18 h in phenol-red free DMEM with 5% CS-FBS in the presence of DMSO or 100 pM E2. Data are presented as % Relative Luciferase Activity (RLA) of E2-dependent transcription. The value of RLA in the presence of the expression vector only (3 × FLAG-myc CMV™ -29) was set to be 100 (control) so that the data can be presented as % RLA. Standard error determinations were calculated based on three replicate experiments.