Fig. 2

Analysis of ligand-induced Notch signaling using S³ technology. a Schematic depiction of the co-culture system used to analyze the Notch downstream response. The human MDA-MB-231 cells express robust levels of the Notch1 receptor and are co-cultured with mouse 3T3-L1 cells, which in some experiments are transfected with the Delta-like 4 (DLL4) ligand. b Analysis of 12xCSL-Luc activity for various combinations of co-culture of 3T3-L1 and MDA-MB-231 cells, where the latter are transfected with the Notch reporter 12xCSL-Luc. Note the increase in reporter activity where 3T3-L1 cells transfected with the DLL4 ligand are co-cultured with MDA-MB-231 cells, and that this increase is abrogated by the addition of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Relative luciferase units (RLU) were normalized to beta-galactosidase values before fold change analysis. *p < 0.05, **p < 0.01 (Student’s t-test). Replicates per treatment group (n = 3) are from one culture split prior to transfection and measurement. c Fold change of expression levels (RPKM) for four genes (GPR1, MTHFS, SGK3 and NME2) from MDA-MB-231 cells co-cultured with 3T3-L1 cells transfected with DLL4 or green fluorescent protein (GFP) in the presence (+DAPT) or absence (-DAPT) of DAPT, as indicated. d Principal component analysis (PCA) of the genome-wide transcriptomes in MDA-MB-231 cells in response to DLL4 ligand-stimulation and DAPT treatment, as described in the figure. e Expression levels of human DLL4 in the co-cultures of MDA-MB-231 and 3T3-L1 cells, as described. Note the high level of DLL4 expression in cells transfected with a human DLL4 plasmid (the two bars to the right, light green)