Figure 2
Nicastrin (NCST) monoclonal antibodies (mAbs) and gamma secretase inhibitor (GSI) Pfizer can reverse epithelial to mesenchymal transition (EMT) process. (A) Binding of anti-NCST mAbs to endogenous cells surface Nicastrin of tamoxifen-resistant (TAM-R) and MCF7 cells. Non-permeabilised cells were incubated with 50Ā Ī¼g/ml of mAbs1/2, followed by incubation with the secondary anti-rat FITC antibody. Rat immunoglobulin G (IgG) was used as control. Binding was assessed by FACS. (B) TAM-R cells were pre-incubated for 30Ā minutes with 50Ā Ī¼g/ml of mAb1/2, or 10Ā Ī¼M GSIPF (PF03084014). For invasion assay, cells were seeded in the upper compartment on top of matrigel-coated membrane and allowed to invade for 72Ā hrs. For motility assay, matrigel was excluded. Pre-treated cells were seeded on 6-well plates for 54Ā hrs, then harvested and counted. A total of 50,000 were transferred to the upper compartment for 18Ā hrs. The results are representative of three biological and two technical replicates. (C, D) qRT-PCR showing mAbs and GSIPF treatment modulate EMT and Notch-responsive genes. Cells were treated as in B and analysed 72Ā hrs later. microRNA (mRNA) levels are represented as fold induction normalised to GAPDH and compared to control. Results represent three biological as well as three technical replicates of each. (E) Representative western blot showing downregulation of Nicastrin and Notch proteins following mAbs and GSIPF treatments. IQGAP1 was chosen as EMT representative protein. Quantitation represents the average from three biological experiments (bars represent standard deviation (SD)).