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Fig. 3 | Breast Cancer Research

Fig. 3

From: Decoding breast cancer tissue–stroma interactions using species-specific sequencing

Fig. 3

Analysis of two different modes of Notch ligand presentation. a Schematic depiction of activation of Notch by immobilized ligand (Fc-DLL4) or with Fc as control. b Analysis of 12xCSL-Luc activity in MDA-MB-231 cells cultured on immobilized Fc-DLL4 or Fc alone as control, and in the presence or absence of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), as indicated. Note the increase in reporter activity when cells are cultured on Delta-like 4 (DLL4) and that this activity is abrogated by the addition of 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) of four genes (P2RY11, MOB4, FAM183A and PRSS22) in the MDA-MB-231 cells in response to DLL4 ligand-stimulation and DAPT treatment, as described in the figure. 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 indicated. e Comparison of Notch response signatures derived by DLL4 presented from co-cultured cells (left) or immobilized DLL4 (right). In the upper left circle are the 164 genes that are >2-fold upregulated in MDA-MB-231 cells by DLL4 on 3T3-L1 cells and the lower left circle denotes the 164 genes that are downregulated by DAPT. The overlap between these two categories (63 genes) are genes that are both upregulated by DLL4 and downregulated by DAPT, i.e. the Notch signature. For the immobilized ligand there are 76 genes that are upregulated by immobilized DLL4, and 84 genes are downregulated by DAPT. The overlap is 29 genes, which are both upregulated by immobilized DLL4 and downregulated by DAPT. Comparison of the “co-culture” and “immobilized” signatures identifies only one gene in common (the middle section of the figure). *p < 0.05 (Fisher’s exact test). f Fold change of expression levels (RPKM) from four well-established Notch target genes (NRARP, HES4, HES1 and SNAI1) in the MDA-MB-231 cells in response to DLL4 ligand-stimulation by co-culture (upper row) or DLL4 immobilized ligand (immob. lig., lower row), respectively, and in the presence or absence of DAPT as indicated. GFP green fluorescent protein

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