OP9/ephrin-B2 enhanced vascular network formation from E9.5 P-Sp explants.

(A) OP9 cells were stably transfected with expression plasmid DNA pCAGneo–ephrin-B2 (OP9/ephrin-B2) or expression vector pCAGneo alone (OP9/vector), and individual G418-resistant clones were selected. To check protein expression, cells were lysed in a lysis buffer, and an equal amount of each sample was subjected to SDS-PAGE and immunoblotted by anti–Lerk-2 PolyAb. (B) P-Sp explants were cocultured with OP9/ephrin-B2 or OP9/vector stromal cells. After the indicated period, the cultures were immunostained by anti–PECAM-1/CD31 MoAb. The formation of vascular networks was enhanced more on the OP9/ephrin-B2 stromal cells than on OP9/vector stromal cells; vb indicates vascular bed; vn, vascular network. The bar indicates 500 μm (day 4 and day 7) and 200 μm (day 10 and day 14). The dotted line indicates the border of vascular bed and vascular network. (C) Quantitative analysis of vascular network area by NIH Image software. The results represent the mean ± SD of triplicate cultures. ░, OP9/ephrin-B2; ■, OP9/vector; *P < .05; **P < .01.