Inhibition of Gas1 expression by siRNA prevented the antiapoptotic effect of VEGF. (A) Cells were incubated with siRNA as indicated in “Materials and methods” and then treated with VEGF (80 ng/mL for 24 hours) or control medium. Expression of Gas1 was evaluated by Western blot. Gas1 siRNA inhibited the expression of the protein by 70% in resting conditions (OD values: 2267 vs 7528 for control and siRNA treated cells, respectively) and by 50% in VEGF-treated cells (OD values: 6288 vs 12 430 for control and siRNA treated cells, respectively). (B) Cells were incubated with Gas1 siRNA and VEGF as described above. Cell apoptosis was quantified by measuring DNA fragmentation as oligonucleosomes. Data are means ± SD of a quadruplicate of a typical experiment. Two independent experiments gave comparable results. (C) Allantois explants were cultured, transfected with siRNA, and treated with VEGF (80 ng/mL for 24 hours) or control medium as described in “Materials and methods” (original magnification, × 100). VEGF is required for the formation of a well-organized vascular network as evidentiated by labeling with an anti–PECAM-1 mAb. In contrast, in absence of the growth factor, endothelial cells are unable to form vessels and many of them undergo apoptosis evaluated as DNA fragmentation (TUNEL) (C-D). Gas1 siRNA reduced the protective effect of VEGF and double-labeling PECAM-1/TUNEL demonstrates apoptotic cells in newly forming vascular structures at the periphery of the network (arrowheads and the insert). The number of apoptotic cells in allantoises (D) has been quantified as described in “Materials and methods.” Data are means ± SD of triplicate of a typical experiment. Two independent experiments gave comparable results.