Figure 4

TSN inhibits osteosarcoma cells invasion and reserves EMT phenotype. (a) 143B and MG63.2 cells were seeded into the upper chamber of the transwell inserts. After 12 h incubation, images were obtained, Scale bar, 40 μm. Cell invasion was quantified manually. The bars indicate the mean±s.d. Statistically significant differences (Student’s t-test), *P<0.05; **P<0.01; ***P<0.001. (b) 143B cells were seeded on FITC-conjugated gelatin (green) for 12 h. Phalloidin (red) was used to indicate F-actin and nuclei was stained with DAPI (blue). Gelatin degradation areas were the black areas underneath the cells, Scale bar, 10 μm. The bars indicate the mean±s.d. Statistically significant differences (Student’s t-test), ***P<0.001. (c) 3D culture assay. 143B cells were plated onto a layer of Matrigel. DMEM plus 10% Matrigel as well as indicated concentrations of TSN was then added. The on-top mixture was changed every other day. Four days later, cells were photographed, Scale bar, 40 μm. (d) After treatment with TSN for 72 h, a western blot assay was performed. (e) After treatment with TSN for 72 h, the level of N-cadherin (green) and E-cadherin (green) were detected by IF staining. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.