Figure 2. GSEA analysis of Jag1flox;Nfatc1Cre and Dll4flox;Nfatc1Cre expression profiles, compared against their respective WT controls. An angiocrine factor gene set consisting of 52 genes was obtained from a previous publication (see Author response table 1 and Nolan et al. Dev Cell. 2013 Jul 29; 26(2)). Of the 52 angiocrine factors, 38 were detected as expressed in the Dll4 contrast (14,302 genes), and 34 in the Jag1 contrast (13,982 genes), as represented by the Venn diagrams at the upper left corner (panel A). GSEA results indicated that angiocrine factors were enriched among the genes that are more expressed in both Jag1 and Dll4 mutant backgrounds, as indicated by the respective enrichment profiles (panels B and C). In these profiles, red to blue gradient stripes represent the complete collections of expressed genes, sorted by decreasing fold change value in mutant versus WT contrasts: red colours indicate higher expression in mutant background (positive fold change), and blue colours indicate higher expression in WT background (negative fold change). Angiocrine factors, represented by black segments perpendiculary oriented along the gradient stripes, tend to concentrate in positive regions, as indicated by the green enrichment curves. Normalized enrichment score (NES) was therefore positive for both contrasts (1.54 and 0.93, for the Jag1 and Dll4 contrasts, respectively), although enrichment was statistically significant only for the Jag1 analysis (FDR = 0.04). Black rectangles indicate the groups of genes that contribute mostly to calculated NES values, which casually consisted in 11 genes for both analyses. The two leading edge subsets shared 7 genes (VEGFA, PDGFB, ANGPT2, EDN1, FGF9, CCL9 and BMP6), as represented by the Venn diagrams at the lower left corner (panel F). The central scatterplot (panel D) compares the fold change rank of the set of 34 angiocrine factors that were detected in both contrasts, and indicates (with a colour code) which of them were detected as differentially expressed (with p-value < 0.05) in each of the contrasts. Six leading edge subset angiocrine factors were detected as differentially expressed in at least one contrast, and four of them (VEGFA, BMP6, EDN1 and PDGFB) were detected in both. Heatmaps represent relative expression values for 38 angiocrine factors in Dll4 KO samples and their respective WT controls (panel E), and 34 angiocrine factors in Jag1 KO samples and the respective WT controls (panel G). Red and blue colours represent expression values above and below average, respectively. The four genes with asterisks in panel E are those that were detected as expressed only in the DLL4 contrast.