TNF induces jagged-1 expression. (A) 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 3 similar experiments. (B) Cells were cultured as for panel A, and gene expression was analyzed by qRT-PCR. Copy number was normalized to GAPDH. Means and SD for triplicate samples are shown. One of 2 similar experiments. (C) ECs were cultured in the presence or absence of TNF (10 ng/mL) for 4 hours and then harvested for analysis of jagged-1 expression by FACS. An isotype-matched, nonbinding antibody was used as a control. (D) 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. Data were normalized to GAPDH. One of 3 similar experiments. (E) ECs were allowed to sprout into fibrin gels for 6 days at which time the gels were prepared for laser capture microdissection (LCM). Five hundred tip cells, and considerably more trunk cells, were captured and RNA was prepared for qRT-PCR. Expression of PDGFB and jagged-1 in tip cells versus trunk cells was determined by qRT-PCR. Data were normalized to GAPDH. Means and SD shown are for triplicate samples. For both genes, P < .01, tip versus trunk, by Student t test. (F) Sprouts in fibrin gels were stained in situ for jagged-1 expression. Phase-contrast images of the tips of sprouts are shown in the top panels. The corresponding immunofluorescent images are shown in the bottom panels. Control Ab staining on the left; jagged-1 staining on the right. Confocal fluorescence images were captured on a Carl Zeiss MicroImaging LSM 510 Meta microscopic system (10×/0.45 and 40×/1.20 Apochromat objectives).