Flt-1^−/− ES cell cultures have increased numbers of endothelial cells.

(A) RNase protection assay using an antisense PECAM RNA probe on day 8 WT, flt-1^+/−, andflt-1^−/− attached cultures. Protected fragments were separated on a polyacrylamide-urea gel and quantified by using a PhosphorImager. Protected PECAM signal was normalized to a β-actin signal, and the normalized PECAM band densities forflt-1^+/− and flt-1^−/−samples were compared with WT (^+/+) samples. Sample 1 and sample 2 are RNAs from separate differentiations. Each bar is the average of 3 experiments performed on a particular sample. (B) Quantitative image analysis of the ICAM-2⁺ area on day 8 WT (^+/+), flt-1^+/−, andflt-1^−/− attached cultures. Each bar represents the average area stained with ICAM-2 antibody from 3 wells. This experiment was repeated (data not shown), and similar quantitative trends were obtained. (C) Fluorescent cell sorting of ICAM-2–labeled day 8 WT (^+/+), flt-1^+/−, andflt-1^−/− ES cell cultures. The plots in dotted lines are controls without primary antibody.