Role of HDAC5 for sprout formation and secretion of angiogenic factors. (A) Sprout formation from endothelial cell spheroid cultures incubated with conditioned medium from HUVECs transfected with scrambled siRNA or HDAC5 siRNA for 42 hours (n = 9). (B) Boyden chamber migration of nontransfected HUVECs toward a conditioned medium. The medium was derived from HUVECs transfected with scrambled siRNA or HDAC5 siRNA for 48 hours. The number of migrated cells per field after 5 hours is given. Addition of exogenous VEGF (50 ng/mL, 5 hours) served as control. *P < .05 versus conditioned medium of scrambled siRNA-transfected HUVECs (n = 4). (C) Enzyme-linked immunosorbent assay measurement of FGF2 release into medium incubated for 2 days with HUVECs transfected with siRNA against HDAC5 (n = 4). (D) Capillary-like sprout formation from HUVEC spheroid cultures stimulated with exogenous FGF2 (30 ng/mL) after transfection of scrambled siRNA or HDAC5 siRNA for 24 hours (n = 4). (E) Capillary-like sprout formation from HUVEC spheroid cultures in the presence of a neutralizing FGF2 antibody (4 μg/mL) or IgG control after transfection of scrambled siRNA or HDAC5 siRNA for 24 hours (n = 5-7). (F) Sprout formation from spheroid cultures of scrambled versus HDAC5 siRNA-transfected HUVECs cotransfected with either of 2 independent Slit2 siRNA oligonucleotides (n = 2). (G) Capillary-like sprout formation from HUVEC spheroid cultures in the presence of R5, an IgG_2b monoclonal antibody to the first immunoglobulin domain of Robo1, which neutralizes activation by Slit2, or 12CA5 IgG control antibody with no antagonistic effect at the Robo1 receptor after transfection of scrambled siRNA or HDAC5 siRNA for 24 hours. *P < .05 versus HDAC5 siRNA with 12CA5 IgG control antibody (n = 4-10). Representative images are shown on the right. Pictures were taken using an Axiovert 100M microscope, an AxioCam camera, a Plan-NEOFLUAR 10×/0.30∞/0.17 objective, and the AxioVision Rel. 4.6.3 Sp1 software (Carl Zeiss).