Fig. 2. EphA2-deficiency impairs ephrin-A1-induced vascular assembly in vitro. (A) Lung microvascular endothelial cells (MPMEC) isolated from wild-type (+/+), heterozygous (+/–), or EphA2-deficient (–/–) mice were plated on a thin layer of growth-factor-reduced Matrigel in the presence or absence of ephrin-A1 to examine and quantify vascular assembly. After 9 hours, the endothelial cells were photographed. Scale bar, 20 µm. (B) The numbers of intersections between endothelial cell branches were counted. Four fields per culture were scored for each condition and data are means±s.d. of three independent experiments. Significant differences in assembly for EphA2^-/– cells stimulated with ephrin-A1 (^*) compared to other experimental conditions are indicated for P<0.01 using ANOVA analysis. (C) EphA2-deficient (–/–) MPMEC were transduced with recombinant adenoviruses encoding wild-type EphA2 (Ad-EphA2) or control ß-galactosidase (Ad-ßgal). After 48 hours, the cells were plated on a thin layer of growth factor-reduced Matrigel in the presence or absence of ephrin-A1 for vascular assembly assay and photographed after 9 hours. Scale bar, 20 µm. For MPMEC (–/–) Ad-EphA2, the left hand panels show bright-field images, and the right hand panels show fluorescence images of identical fields displaying co-expression of GFP from the adenovirus plasmid. (D) The numbers of intersections between endothelial cell branches were counted. Four fields per culture were scored for each condition and data are presented as means±s.d. of three independent experiments. Significant differences in assembly are indicated where P<0.01 (using Student's t-test) for EphA2^-/– Ad-EphA2 (^**) versus EphA2^-/– Ad-ßgal and for EphA2^-/– Ad-EphA2 + ephrin-A1 (^***) versus EphA2^-/– Adßgal + ephrin-A1. (E) Immunoblot analysis of ßgal or EphA2 expression in lysates from MPMEC transduced with recombinant adenoviruses.