Antiserum from 12P-KLH-vaccinated mice inhibited proliferation, matrigel tube formation and migration of HUVECs. (A) MTT assay of vascular endothelial cells treated with the indicated groups: anti-KLH, anti-control-KLH, anti-12P-KLH. 12P was used to block the function of anti-12P-KLH and Avastin was used as the positive control. All assays were carried out in triplicate and the error bars indicate standard deviation. Statistical analysis was performed using the Student’s t-test (*p < 0.05). (B) Inhibition of tube formation of HUVECs treated with antibodies was detected on Matrigel. HUVECs (1 × 105/ml) were seeded on top of the matrigel with complete RPMI 1640 medium containing antibodies and cultured for 8 h. The representative photographs were taken using a light microscope (200× view field). (C) HUVECs were seeded in the upper chamber of a Millicell insert with RPMI 1640 containing antibodies and incubated with 10% FCS RPMI 1640 medium containing 5 ng/ml VEGF in the lower chamber for 12 h. The cells were fixed and stained with 4,6-diamidino-2-phenylindole (DAPI). The migrated cells were visualized under a microscope. A representative image of DAPI staining is shown. (D) Data of C was analyzed by Student’s t-test. *p < 0.05. All of the assays were carried out in triplicate and the error bars indicate standard deviation.