Fig. 4

Endothelial Dll1 instructs macrophage maturation in vitro. a Quantitative RT-PCR analysis of p3 primary human aortic EC (HAEC, A) or coronary microvascular (MV) EC. n = 3 independent samples in duplicates/group, error bars represent s.e.m., **p < 0.01, Student’s unpaired t-test. b Representative flow cytometry from d3 culture of human CD14⁺ monocytes in the presence of 10 ng/ml M-CSF or HAEC (AEC). n = 4 independent experiments. c, d HAEC were transfected with anti-DLL1 siRNA (Si-DLL1) or scambled control (Scr) before co-culture with CD14⁺ monocytes. c. Quantitative RT-PCR analysis of CD11b⁺ isolated from co-culture normalized to expression levels of input monocytes, n = 3 independent experiments, error bars represent s.e.m. **p < 0.01, *p < 0.05, Student’s paired t-test. d BrdU incorporation (6 h) in CD11b⁺ cells from co-culture, n = 3 independent experiments performed with duplicate measurements, error bars represent s.e.m. *p < 0.05, Student’s unpaired t-test. e Protein concentration of CSF1 in co-culture supernatants (A: arterial EC, MV: microvascular EC) or macrophage (MF) supernatants by ELISA, nd: not detectable. n = 3 independent experiments performed in triplicates, error bars represent s.e.m. ***p < 0.001 one-way ANOVA with Bonferroni post-test. f Number of CD11b⁺ macrophages recovered after co-culture of CD14⁺ monocytes with HAEC treated with isotype (α-Con) and CSF1 neutralizing (α-CSF1) antibody. n = 2 independent experiments performed in triplicates, error bars represent s.e.m. p < 0.0001, Student’s unpaired t-test g, h Effect of HAEC (A) or MVEC (MV) on CD14⁺ monocytes. g Representative flow cytometry and profile of gated population after d3 of co-culture. n = 3 independent experiments. h Transcriptional profile of CD11b⁺ cells isolated after 3d of culture with EC (A vs MV) or CSF1, normalized to expression levels of input monocytes (d0). n = 3 independent experiments, error bars represent s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05, one way ANOVA with Bonferroni multiple comparison test