Fig. 1

An assay to identify Notch-regulated genes in basal-like breast cancer cell lines. A A schematic representation of the Notch signalling pathway. Binding of the Notch receptor to a ligand presented on the neighbouring cell leads to S2 proteolytic processing (S2), which is followed by S3 processing in the membrane (S3) by the γ-secretase complex. S3 processing results in the liberation of the Notch intracellular domain (NICD) and its translocation into the nucleus. In the nucleus, NICD together with CSL and MAML forms a ternary transcriptional complex that regulates expression of Notch downstream genes. B Schematic depiction of the assay to identify Notch-regulated genes to establish a Notch transcriptomic signature. Six basal-like breast cancer cell lines (as indicated) were seeded in three replicates. Notch activation (“Notch on”) was achieved by cultivating the cell lines on immobilized ligands, and inhibition of Notch signalling (“Notch off”) was achieved by adding the y-secretase inhibitor (GSI) DAPT, which blocks S3-cleavage. Cultivation of the cells after addition of DMSO was carried out as control (“ground state”). Bulk transcriptomes were captured after 8 and 72 h of culturing under the different conditions. C Expression of known Notch target genes (HES1, NRARP) was evaluated after 8 or 72 h in the BT-20 and MDA-MB-468 cell lines, as indicated (*p < 0.05, **p < 0.01 and ***p < 0.001). D Principal component analysis of the transcriptomic data from the six cell lines under “Notch on”, “Notch off” or “ground state” conditions at 8 or 72 h, as indicated. A total of 12,459 genes were used for the PCA. E Correlation of the principal components to different experimental parameters in the transcriptomic dataset from the six basal-like cell lines. The Horn’s and Elbow represent metrices to identify the optimal number of principal components [10, 11]