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. 2018 Aug 13;7(8):59. doi: 10.1038/s41389-018-0069-z

Fig. 3. Notch3 upregulates GATA-3 expression by binding to the core element of the GATA-3 promoter.

Fig. 3

a–c Schematic representation of the three CSL-binding element-containing primers (Regions 1 and 2 containing respective single CSL-binding elements, region 3 containing both CSL-binding elements) and negative control primers (region 1 does not contain a CSL-binding element) used for ChIP assays. Bands were seen in regions 1, 2, and 3 of the PCR product but not in the negative control region 4 (b); bands were dramatically decreased in regions 1, 2, and 3 of the PCR product when cells were treated with siNotch3 (c). d Probes 1 and 2 represent two biotin probes containing the core element of the CSL-binding sites of the GATA-3 promoter. Supershift bands (lane 3) were seen in the presence of an anti-Notch3 antibody, but not in the presence of an anti-IgG antibody (lane 4). Competition assays were performed using a 100-fold (lane 5) excess of unlabeled oligonucleotide containing the core element of the CSL-binding element of the GATA-3 promoter. A 100-fold excess of unlabeled mutation oligonucleotide containing the mutated core element of the CSL-binding element (lane 6) was used for mutation competition assays. Nuclear extracts from MCF-7 cells were not added to lane 1. e Notch3 was silenced in MCF-7 cells by siRNA and then co-transfected with the GATA-3 promoter or a mutated GATA-3 promoter (deleting both CLS-binding elements) construct containing Firefly luciferase, and internal control plasmid, pRL-SV40, containing Renilla luciferase. Firefly luciferase/Renilla luciferase values were used to indicate promoter activity. Each sample was tested in triplicate. *P < 0.05. f N3ICD was overexpressed in MDA-MB-231 cells and co-transfected with the GATA-3 promoter or a mutated GATA-3 promoter (deleting both CSL-binding elements) construct containing Firefly luciferase and internal control plasmid, pRL-SV40, containing Renilla luciferase. Firefly luciferase/Renilla luciferase values were used to indicate promoter activity. Each sample was tested in triplicate. *P < 0.05