FIG. 3.
Blimp1 represses ERα gene expression in breast cancer cells. (A) Nuclear extracts, prepared from ZR-75 cells transfected with Blimp1 expression vector, were used in competition EMSAs with the Blimp1 element from the c-MYC gene as a probe and a 50-fold molar excess of oligonucleotides containing the c-MYC or putative ERα promoter Blimp1 sites 1, 2, or 3 (P1 to P3). (B) Nuclear extracts, prepared from ZR-75 cells transfected with EV (−) or Blimp1 expression vector (+), were used in EMSAs with the putative ERα gene P1, P2, and P3 Blimp1 sites as a probe. (C) Nuclear extracts, prepared from ZR-75 cells transfected with Blimp1 expression vector, were used in supershift EMSAs in the absence (−) or presence (+) of 200 ng of Blimp1 antibody and the putative ERα gene Blimp1 P2 site as a probe. (D) ERα promoter A and B regions with confirmed Blimp1 site indicated with the element sequence given below, where the core is underlined. (E) ChIP analyses were performed on MCF-7 cells transiently transfected with Blimp1 (top panels) or Hs578T cells (bottom panels) using anti-Blimp1 antibody or the corresponding normal IgG as indicated. The DNA, extracted from the immunoprecipitates or the input, was amplified by PCR using pairs of primers specific to the confirmed Blimp1 site of ERα promoter (Blimp1 site) (left panels) or an upstream region as negative control (right panels). (F) ZR-75 and MCF-7 cells were transiently transfected with 5.0 μg of Vxy-puro TBlimp vector expressing the 35-kDa dnBlimp protein or EV DNA, and WCEs were subjected to immunoblotting for ERα, dnBlimp, and β-actin. (G) ZR-75 and MCF-7 cells were transiently transfected with 5.0 μg of Blimp1 expression vector or EV DNA, and WCEs were subjected to immunoblotting for ERα, Blimp1, and β-actin. Densitometry indicated the ERα levels in the ZR-75 and MCF-7 cells were reduced to 28.0 ± 3.4 and 73.9 ± 5.0, respectively, upon ectopic Blimp1 expression compared to EV DNA, set at 100. (H) ZR-75 cells were transiently transfected with 5.0 μg of vector expressing either Blimp1 or dnBlimp protein (left or right panels, respectively) or the corresponding EV DNA, and RNA was subjected to RT-PCR for ERα and GAPDH. (I) MCF-7 cells carrying an inducible C4bsrR(TO-RelB) RelB construct, plated in the absence or presence of 1 μg of doxycycline/ml, were transiently transfected with 6.0 μg of dnBlimp expression vector. After 48 h, WCEs were analyzed for levels of ERα, RelB, dnBlimp, and β-actin.