Fig 1: Tpl2-selective kinase inhibitor treatment impairs VEGF-A-stimulated endothelial cell tubulogenesis and aortic sprouting. (A) Endothelial cells co-cultured with primary human fibroblasts were stimulated with VEGF-A (25 ng/ml)±Tpl2-selective kinase inhibitor (2 µM). Endothelial cell tubules were fixed and stained for the Tpl2-selective kinase endothelial cell-specific marker and cell adhesion molecule (PECAM-1) for visualization using fluorescence microscopy. (B) Quantification of endothelial tubule length from fluorescence microscopy datasets. Error bars indicate ±s.e.m. (n=3). Statistical test=two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001. (C) Endothelial sprouting from mouse aortic slices stimulated with VEGF-A (25 ng/ml)±2 µM Tpl2-selective kinase inhibitor followed by fixation, staining with FITC-conjugated endothelial-specific lectin (see the Materials and Methods) and visualization using fluorescence microscopy. (D) Quantification of endothelial sprout number from mouse aortas upon treatment with VEGF-A±Tpl2, selective kinase inhibitor. Error bars indicate ±s.e.m. (n=4). Statistical test=two-way ANOVA; ***P<0.001, ****P<0.0001. Scale bars: 1000 µm.