NRP2 knockdown inhibited both in vitro and in vivo tumor growth. (A & B), NRP2 expression was knocked down by NRP2 shRNA in 143B cells. Knockdown efficiency was determined by real-time PCR (A) and Western blotting (B). (C), By MTT assay, NRP2 knockdown suppressed anchorage-dependent growth of osteosarcoma 143B cells. (D) Soft agar assay. NRP2 knockdown did not reduce the number of colony formed by 143B cells. 143B cells transfected with ShNRP2 formed smaller colony than control vector transfected cells as shown in the representative images of soft agar (insert). (E) & (F) NRP2 knockdown inhibited the in vivo tumor growth in xenograft nude mice model. 1 × 106143B cells were inoculated in NCR nu-nu nude mice. Tumor size was measured every 3 days and a tumor growth curve was created (E). Points, mean tumor volume; Bars, SEM. (F), Representative picture of tumor harvested at day 21. (G), the knockdown of NRP2 expression in tumor samples was confirmed by immunofluorescence staining using NRP2 antibody. Significant differences are indicated by: (*) p < 0.05, (**) p < 0.01. Column: mean value; Error bars: SEM.