Figure 3. EZH2 knockdown inhibits cell growth of osteosarcoma in vitro and in vivo.

(A) The expression of EZH2 and TSSC3 in SaOS2 and U2OS cell lines transfected with lenti-shEZH2 was evaluated by qRT-PCR. (B) Decreased protein level of EZH2 and increased TSSC3 was revealed by western blotting after transfection with lenti-shEZH2, consist with that of qRT-PCR (left). Blots are representative of three experiments and were reprobed for β-actin to verify equal loading (right). Band intensities were measured by densitometry and normalised to β-actin expression. (C) Downregulation of EZH2 caused a significant growth inhibition of osteosarcoma cells as revealed by CCK-8 assay. Lenti-siEZH2 versus control group, P < 0.05. Values shown are the mean absorbance ± SD for five wells from one experiment, and are representations of three independent experiments. (D) Inhibition of colony-formation by EZH2 knockdown in osteoblast cells. Values shown are mean ± SD from three independent experiments. ***P < 0.001 bars, SD. (E) DNA content analysis revealed that EZH2 knockdown increased the percentage of cells in G1 phase and decreased the percentage of cells in G2 phase in both SaOS2 (upper) and U2OS (lower) cell lines. The figure shows relative cell percentage in each cell cycle phase. Values shown are the mean ± SD from three independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05; bars, SD. (F) Xenograft studies using lenti-shEZH2 transfected cells shows a reduced ability to form tumors in SCID mice. (G) Knockdown of EZH2 inhibited tumor growth of osteosarcoma in vivo (P < 0.05). (H) Hematoxylin-eosin and immunohistochemical staining with antibodies to EZH2, TSSC3 and Ki67 were performed on paraffin-embedded tumor sections of xenografts from siEZH2 cells and control cells. Scale bars: 100 μm. (I) Quantitation of Ki67 staining in xenografts from siEZH2 cells and control cells. **P < 0.01. Values are means ± SD from three independent experiments. Bars, SD.