ERα and ERβ Mediate the E2-Induced Increase in NRF-1 mRNA Expression in MCF-7 and H1793 Cells in a Genomic Manner
A, B, C, E, and H, NRF-1 expression as determined by QRT-PCR as described in MATERIALS AND METHODS; A, MCF-7 and H1793 cells were treated with EtOH or 10 nm E2 for the time indicated and as described in MATERIALS AND METHODS; B, MCF-7 and H1793 cells were pretreated with 100 nm ICI 182,780 for 6 h before addition of 10 nm E2 for 4 h; C, MCF-7 cells were pretreated with 10 μg/ml ActD or CHX or with 50 μm PD98059 or 50 nm wortmannin for 1 h before incubation with 10 nm E2 for 4 h; D, MCF-7 cells were transfected with control siRNA or siRNA targeting ERα (siERα) or ERβ (siERβ) for a total of 48 h, as described in MATERIALS AND METHODS, and then treated with EtOH or E2 for 4 h. NRF-1mRNA expression was determined by Q-RT-PCR. Representative Western blots (40 μg WCE per lane) for ERα, ERβ, and α-tubulin in the EtOH-treated MCF-7 cells are shown. Similar results were observed in E2-treated cells. E, MCF-7 or H1793 cells were treated with EtOH, 10 nm E2, 10 nm DPN, 10 nm PPT, or 100 nm R,R-THC, alone or in combination as indicated, for 4 h. F, A representative Western blot (40 μg WCE per lane) is shown for NRF-1 expression in MCF-7 cells treated with 10 nm E2 for the indicated times. The bar graph summarizes NRF-1 protein normalized to α-tubulin from the same blot from three separate experiments. G, MCF-7 cells were either transfected with control siRNA, siERα, or siERβ for 48 h and then treated with EtOH or E2 for 48 h or not transfected and treated with EtOH or E2 for 48 h. H, Quantitation of the NRF-1 protein relative to β-actin in the same blot relative to 48-h EtOH values. As indicated, the striped bars are NRF-1 normalized to siRNA control EtOH NRF-1/β-actin values. Values with error bars are the average of three to six separate experiments ± sem. *, P < 0.05 compared with EtOH; ##, significantly different from the E2 alone value.