Figure 4

NOTCH signaling inhibition decreases the migratory behavior of MDA-MB-231 cells. (A) Western blot showing Notch one intracellular domain (N1ICD) expression in MDA-MB-231 cells after 48 h of treatment with DAPT or RO4929097 (RO). Equal amounts of protein loading were validated with an anti-SMC3 antibody. (B) Notch activity measured with the 10 × CBF1 reporter. The value for DAPT and RO4929097 treated cells is relative to the activity in cells treated with DMSO. Data are mean ± SEM of quadruplicates in three independent experiments (*P <0.05, **P <0.005 determined by Student's t-test). (C) Representative images of migration towards IGF-1 50 ng/ml of MDA-MB-231 cells with DAPT or RO4929097 or the vehicle DMSO. (D) Migration index obtained in both conditions. Data are mean ± SEM of duplicates in three independent experiments (***P <0.001 determined by Student's t-test). (E) Migration index quantification of MDA-MB-231 cells transfected with an empty vector (pCDNA3) or a dominant negative version of CBF1 (DN-CBF1) towards IGF-1 (50 ng/ml). Data are mean ± SEM of duplicates in three independent experiments (*P <0.05 determined by Student's t-test). CBF1, CSL, Suppressor of Hairless; DAPT, N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester; DMSO, dimethyl sulfoxide; MCF-7, Michigan Cancer Foundation-7 breast cancer cell line; MDA-MB-231, Breast cancer cell line derived from metastatic site (pleural effusion); N1ICD, Notch one intracellular domain