Fig. 3: ERβ regulates PML promoter activity.

a Luciferase activity of PML promoter in cell lines co-transfected with WT-ERβ, pGL3-PML-prom(human), and pRL-TK (Renilla luciferase construct), and treated with either DMSO or DPN. The figure represents relative fold change in luciferase readings, normalized against Renilla reporter activity. b Schematic representation of wild type ERβ gene mentioning all its domains and its mutant construct pcDNA ERβ (Δ144-225), with its DNA binding domain (DBD) deleted. ERβ DBD can occupy the ERE sites (consensus sequence TCAAGGTCA) on ERβ target promoters. c Luciferase activity of PML promoter in MDA MB 468 cells transfected with either WT-ERβ or the mutant construct pcDNA ERβ (Δ144-225), along with pGL3-WT-PML-prom(human) and pRL-TK. d Luciferase activity of PML promoter co-transfected with either scrambled siRNA or siRNA against ERβ, pGL3-PML-prom(human) and pRL-TK. e Luciferase activity of PML promoter co-transfected with pGL3-PML-prom(human) and pRL-TK and treated with DMSO, DPN or ICI. f Schematic representation of human (−1447 to +250) and mouse (−800 to TSS) PML promoters containing ERE sites. Chromatin immunoprecipitation (ChIP) assay performed using the indicated antibodies in g human MDA MB 468 and MDA MB 453 and h mouse 4T1 cells. RNA Polymerase II and IgG served as positive and negative controls respectively and PS2 promoter served as the control for ERβ binding. GAPDH and Actin promoters served as positive controls for Pol II in human and mouse ChIP assays respectively. ChIP assay performed on i MDA MB 468 and MDA MB 453 cells and j 4T1 cells transfected with either scrambled siRNA or with siRNA against ERβ. ‘Neg Con’ stands for non-genomic intragenic regions serving as negative control for the same. k Schematic representation of the human WT pGL3 PML promoter and the three ΔERE deletion constructs. l Luciferase activity measured in MDA MB 468 cells co-transfected with either WT ERβ along with pGL3-WT-PML-prom(human) or its deletion constructs and pRL-TK. Data are normalized to Renilla luciferase activity and represented as fold activity with respect to control cells. Error bars represent mean (±) s.d. calculated from three independent experiments. P < 0.0001 is represented as **** for highly significant and NS denotes ‘not significant’