Figure 2: CD146 is essential for Wnt5a-induced endothelial cell migration and JNK activity.

(a) Knockdown of CD146 blocks Wnt5a-induced HUVEC migration; 5000 HUVECs depleted of CD146 were seeded in 8 mm transwell plates and incubated with Wnt5a or control. Knockdown efficiency and the expression level of siRNA-resistant form of CD146 were determined by western blotting, and migrated cells were stained and counted. Data are presented as mean values±s.d. of three experiments. Significant difference was determined by Student’s t-test (*P<0.05; **P<0.01). (b) Knockdown of CD146 inhibits Wnt5a-induced JNK activity. HUVECs were transfected with CD146 siRNA or control siRNA for 48 h. Transfected cells were treated with Wnt5a or control CM for a further 1 h, and the phosphorylation of JNK was assessed by western blotting. β-actin was used as a loading control. (c) CD146-specific antibodies AA1 and AA2 block Wnt5a-induced HUVECs migration. 6,000 cells were pre-incubated with 50 μg ml⁻¹ mIgG, AA1 or AA2 for 1 h followed by addition of Wnt5a CM. Cells were cultured for 10 h, and migrated cells were stained and counted. Data are presented as mean values±s.d. of three experiments. Significant difference was determined by Student’s t-test (**P<0.01; NS, no significant difference). (d) CD146-specific antibodies AA1 and AA2 block Wnt5a-induced JNK activity. HUVECs were pretreated with antibodies against the ECD of CD146 for 4 h, Wnt5a-induced JNK activity was determined by western blotting using P-JNK antibodies. β-actin was used as a loading control. (e) CD146 activates JNK. HEK293T cells were collected with ectopic expressed CD146 at 36 h and cell lysate was subjected to western blotting. JNK activity was demonstrated with antibody against p-JNK. Dvl2 and empty vector-transfected cells were used as positive and negative controls, respectively. β-actin was used as a loading control. (f) JNK inhibitor blocks CD146-promoted cell migration. CD146-HEK293T cells or mock cells seeded in 8 mm transwell plates were incubated with 10 mm JNK inhibitor SP600125or dimethyl sulphoxide. Migrated cells were stained and counted (scale bar, 150 μm). Data are presented as mean values±s.d. of three experiments. Significant difference was determined by Student’s t-test (*P<0.05; **P<0.01).