Figure 5.

Stable Nicastrin (NCST) overexpression activates Notch pathways and epithelial to mesenchymal transition (EMT) in MCF7 cells. (A) Representative western blot of NCST, Notch receptors and IQGAP1 levels in control (CT) and NCST overexpressing MCF7 cells. ActinB was used as loading control. (B) qRT-PCR showing Nicastrin overexpression effect on Notch1, Notch4, Notch-responsive genes, and EMT genes (C). microRNA (mRNA) levels were quantified relative to GAPDH. The mean of three different experiments is shown. (D) Representative images showing E-cadherin expression in MCF7 CT and MCF and NCST cells at cell-cell contact. (E) CT and MCF7 NCST cells were stained with Crystal Violet and 10X images were taken with bright-field microscope when cells were 50% confluent (bars represent 400 μM). Boyden chamber-based assay was used to determine the migratory capacity of MCF7 NCST compared to MCF7 CT cells. Cells were allowed to migrate for 18 hrs before the insert was fixed, cut, and mounted in Mowiol infused with DAPI. 4X images were taken with a fluorescent microscope. (Bars represent 1,000 μM). The results are representative of three biological and two technical replicates.