Fig 2.

Knockdown of the SS18–SSX fusion gene suppresses cell proliferation and induces endothelial differentiation in synovial sarcoma (SS) cells. (a) Confirmation of SS18–SSX fusion gene knockdown by siRNA-A and siRNA-B with immunoblot analysis. Closed or open arrow heads represents SS18–SSX or SS18, respectively. M.W., molecular weight. (b) Amount of vascular endothelial growth factor-A (VEGF-A) secreted by Yamato-SS cells that had been treated with SS18–SSX siRNA under spheroid culture conditions, as determined by ELISA (n = 3). (c) VEGFA and VEGFR2 mRNA levels in Yamato-SS cells treated with SS18–SSX siRNA as determined by quantitative RT-PCR (n = 3). A.U., arbitrary unit (the same l applies hereafter). (d) Cell proliferation of Yamato-SS cells cultured in a tube formation assay treated with bevacizumab (Bev; 5 μg/mL) (left) or pazopanib (Pazo; 250 nM) (right) (n = 3; *P < 0.005, **P < 0.05). (e,f) Immunofluorescence analysis of CD31 (e), and von Willebrand factor (vWF) (f). Nuclei were counterstained with Hoechst33342 (blue). White arrowheads identify cells expressing CD31 or vWF, and phase contrast images of tube formation. Scale bar = 100 μm. (g) Quantitation of tubular formation assay in Yamato-SS cells treated with SS18–SSX siRNA, in the presence or absence of treatment with Bev (5 μg/mL, left) and Pazo (250 nM, right) (n = 2; *P < 0.005).