FIG. 5.

Reduced in vitro tube-forming capacity of Smad4-deficient ECs. (A) Morphology and purity of isolated embryonic ECs. The upper panels show the typical cobblestone-like shape of sorted Tie2-positive cells in culture. The middle panels show >90% EC purity as verified by DiL-Ac-LDL uptake. The lower panels show the endothelial property of PmT-transformed ECs by VE-cadherin (CD144) immunostaining. (B) RT-PCR and Western blot analysis of Smad4 expression in E9.5 YS, embryos proper, sorted Tie2-positive cells, and PmT-transformed embryonic ECs is shown from left to right in the upper part of the panel. In the lower part of the panel, “+T” indicates stimulation by 10 ng of TGF-β1/ml for 20 h. Smad4 expression is reduced in the mutant embryos and absent in the mutant ECs. (C) Tube-like structures of isolated ECs on Matrigel observed after 8 h of incubation. The mutant ECs exhibit less tube formation than that of controls. (D) Quantification shows a significant 35% reduction of tube number (left) and a remarkable 44% decrease of total tube length (right) in the mutant ECs. The data are means ± the standard error of the mean. Scale bar: 27 μm (top images of panel A), 14.5 μm (middle and lower images of panel A), or 150 μm (C).