TNF induces an EC tip cell phenotype. EC-coated beads were established in fibrin gels and treated with (A) PBS or (B-D) TNF (10 ng/mL) for 6 days. Cells were then cultured for a further 4 days in the absence of TNF. (A) Sprouting and lumen formation in control-treated cultures. Lumens are indicated by arrows. (B) Individual migrating tip cells induced by TNF, showing no lumen formation. Some of the tip cells have been numbered to highlight that they are not part of an organized sprout but are migrating with only minimal, and likely transitory, contacts. (C) Higher-power view of 5 tip cells showing polarity and numerous filopodia, but no lumens. (D) High-power view of tip cell stained with phalloidin to show actin fibers, and DAPI to highlight nucleus. Images in panels A-D were captured on an Olympus IX70 inverted phase-contrast/fluorescence microscope using a 10×/0.30 or a 40×/0.60 fluorescence objective and an Optronics digital camera. High-resolution images were magnified in Photoshop. (E) ECs were cultured in the presence or absence of TNF (10 ng/mL) for 2 days and then harvested for analysis of gene expression by RT-PCR. One of 2 similar experiments. (F) Cells were cultured as for panel E, and gene expression was analyzed by qRT-PCR. Copy number was normalized to GAPDH. Means and SD for triplicate samples are shown. One of 3 similar experiments. (G) ECs were cultured in the presence of TNF (10 ng/mL) for the indicated times and then harvested for analysis of gene expression by qRT-PCR. Shown are means and SD for triplicate samples. One of 3 similar experiments.