Silencing of Rap1 inhibits in vitro angiogenesis. Endothelial cells transfected with siRNAs targeted against Rap1a, Rap1b, or scrambled controls. Two different sequences were used as indicated by I and II. (A) Expression of Rap1a and Rap1b mRNA was assessed by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as loading control. (B) Statistical analysis and representative images of spheroidal sprouting assay performed with endothelial cells transfected as indicated with Rap1a-, Rap1b-, Rap1a-, and Rap1b-siRNA (sequence I) or scrambled siRNA in the absence or presence of 50 ng/mL bFGF (n = 13). The mean cumulative length of sprouts per spheroid was assessed after 24 hours (*P < .05 vs scrambled siRNA, #P < .05 vs scrambled siRNA + bFGF). Bar represents 100 μm. The wells were viewed with an Axiovert 100M inverted microscope using as objective a Plan-NEOFLUAR (at 10×/0.30). Images were acquired using a digital camera Axiocam MR and were processed using AxioVision Rel 4.5 digital imaging software (all from Carl Zeiss).