Figure 3.

Notch3 regulates ERα expression by binding to CSL binding elements in the ERα promoter. (A) MCF-7 cells, silenced by control siRNA or siNotch3, were co-transfected with an ERα promoter-driven firefly luciferase (ERα-Luc) and an internal control plasmid pRL-SV40 expressing Renilla luciferase. Promoter activity was expressed as the ratio of firefly luciferase/Renilla luciferase. (B) MDA-MB-231 cells were transiently co-transfected with ERα-Luc and N3ICD at incremental doses as indicated, and Renilla luciferase was transfected as an internal control. (C) Schematic representation of wild-type ERα-Luc and mutant constructs ΔCSL (deletion of one CSL binding element) or Δ2CSL (deletion of both CSL binding elements) used in reporter assays. (D) Luciferase reporter assay with ERα-Luc or mutant ERα-Luc were performed in the absence or presence of N3ICD in MDA-MB-231 cells. Promoter activity was expressed as the ratio of firefly luciferase/Renilla luciferase. (E and F) ChIP assays. Schematic representation of the 2 CSL binding element-containing regions (Region 2 and Region 3) and negative control region (Region 1) used in the ChIP assay. PCR products were detected in the presence of anti-Notch3 antibody. (G) Sequences of probes 1 and 2 containing the GGGAA core element for EMSA. (H) EMSA using probes 1 and 2. Competition assays were performed by adding either 100-fold (lane 3) excess of unlabeled oligonucleotides containing the core CSL binding element, or 100-fold excess of unlabeled mutant oligonucleotides in the core CSL binding element (lane 4). Each reporter assay was performed in triplicate.