RAPL contributes to the angiogenic effect of Rap1a. Endothelial cells transfected with siRNAs targeted against RAPL or scrambled control. (A) Twenty-four hours later, expression of RAPL mRNA was assessed by RT-PCR; GAPDH serves as loading control. (B) Statistical summary of spheroid assays with RAPL siRNA or scrambled transfected endothelial cells. Spheroids were stimulated with or without 50 ng/mL bFGF (n = 3). Cumulative length of all sprouts originating from each spheroid was quantified after 24 hours. Statistical summary represents the mean plus or minus SEM (*P < .05 vs scrambled siRNA, #P < .05 vs scrambled siRNA + bFGF). (C) Forty-eight hours after transfection, migration assays on fibronectin were performed (n = 5). Data are presented as mean of migrated cell % of control plus or minus SEM (*P < .05 vs scrambled siRNA, #P < .05 vs scrambled siRNA + VEGF). (D) RT-PCR analysis of RAPL and Rap1aV12 expression by RT-PCR. GAPDH serves as loading control. (E) Statistical summary and representative micrographs of spheroid assay performed to analyze the effect of simultaneous RAPL silencing and Rap1aV12 overexpression on sprouting capacity of HUVEC (n = 5). Data are given as mean plus or minus SEM (*P < .05 vs scrambled siRNA + Rap1aV12; **P < .05 vs scrambled siRNA + pcDNA3.1; #P < .05 RAPL siRNA + pcDNA3.1). 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).