Fig 4.

Blocking of vascular endothelial growth factor (VEGF) and chemokine (C-X-C motif) receptor 4 (CXCR4) signals synergistically suppresses cell proliferation in synovial sarcoma (SS). (a) Immun-oblot analysis of CXCR4 under 2-D or spheroid culture conditions (left), and quantification of CXCR4 mRNA by quantitative RT-PCR under 2-D or spheroid culture conditions from day 1 to day 7 (right panel) (n = 3; *P < 0.05). A.U., arbitrary unit (the same l applies hereafter); M.W., molecular weight. (b) Immunofluorescence staining of Yamato-SS spheroids with a CXCR4 antibody (green). Nuclei are counterstained with Hoechst33342 (blue). Scale bar = 100 μm. (c) Quantification of VEGFA, VEGFR2, CXCL12, and CXCR4 mRNA by quantitative RT-PCR in Yamato-SS spheroids treated with pazopanib (Pazo, 250 nM) for 24 h (n = 3; *P < 0.05). (d) Rescue experiments with exogenously added recombinant human VEGF-A (10 ng/mL) by soft agar assay after treatment with CXCR4-specific inhibitor AMD3100. Colonies >200 μm are shown. (n = 3; *P < 0.05, **P < 0.005). (e,f) Soft agar assay of Yamato-SS cells treated with bevacizumab (Bev, 0.2 μg/mL) (e) or Pazo (50 nM) (f) in combination with AMD3100 (0.5 μM). Colonies >200 μm were counted (n = 3; *P < 0.05, **P < 0.005).