Nucleolin mediates the antiendothelial activities of endostatin. (A-B) In the presence or absence of ES (10 μg/mL), HMEC proliferation assay (n = 6) and migration assay (n = 5) were performed with saline (control), nonimmune IgG (40 μg/mL), or anti-NL antibody (40 μg/mL), respectively. The cell number in proliferation assay was evaluated by MTT assay. (C) HMECs were transfected with blank plasmid, negative control (NC, vector encoding scrambled shRNA), and shRNA-NL plasmid, respectively. The NL expression of transfected HMECs was detected by IB. Actin blot serves as loading control. Densitometry analysis of IBs showed the relative expression level (NL/actin). (D,E) In the presence or absence of ES (10 μg/mL), cell proliferation assay (n = 6) and migration assay (n = 5) were performed with HMECs transfected with blank plasmid, NC, and shRNA-NL plasmid, respectively. After 48 hours, the cell number in proliferation assay was evaluated by MTT assay. (F-I) HMECs were transfected with siRNA-NL, siRNA-integrin α₅, siRNA-integrin α_v, and siRNA-MMP2, respectively. A scrambled siRNA serves as negative control (NC). The expression of NL, integrin α₅, siRNA-integrin α_v, and siRNA-MMP2 were detected by IB with indicated antibodies, respectively. Actin blot serves as loading control. Densitometry analysis of IBs showed the relative expression level of target proteins. (J) In the presence or absence of ES (10 μg/mL), cell proliferation assay was performed with HMECs transfected with NC, siRNA-NL, siRNA-integrin α₅, siRNA-integrin α_v, and siRNA-MMP2, respectively. After 48 hours, the cell number was evaluated by MTT assay (n = 6). Error bars are SD.