Figure 3

Knockdown of NRP2 inhibited the tumor invasion, migration and lung metastasis of osteosarcoma. (A), Migration assay. The BD chamber system without Matrigel coating was used to evaluate the migration of shNRP2 and control vector transfected osteosarcoma 143B cells. The migration of 143B cells was significantly inhibited by NRP2 knockdown. (B), Matrigel invasion assay was performed in BD chamber system coated with Matrigel, using shNRP2 and control vector transfected osteosarcoma 143B cells. There were less NRP2 depleted 143B cells invaded through the matrigel coated porous membrane. (C), Knockdown of NRP2 in 143B cells reduced the lung metastasis of osteosarcoma in an orthotopic lung metastasis mouse model. Mouse lungs were fixed in Bouin’s solution, and the number of lung surface metastatic nodules was counted and graphed. Each group contained 10 mice and the experiment was repeated 3 times (D), Representative photographs of lungs with metastatic nodules of osteosarcoma. (E), NRP2 knockdown significantly reduced both 143B and Saos-2 cells adherence to the endothelial monolayer. The mean cell number was calculated from 10 fields (×100). (F), Representative images of GFP transfected tumor cells adhering to the endothelial monolayer. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. Column: mean value; Error bars: SEM.