Figure 7

Rac1 was essential for bFGF-induced motility of ESFT cells. (A) Rac1 activation in RD-ES cells was assessed using glutathione S-transferase (GST)-p21-binding domain (PBD) beads to isolate GTP-bound Rac1. RD-ES cells were stimulated by bFGF (10 ng ml⁻¹) with or without pre-treatment with SU5402 (20 μ M), LY294002 (50 μ M), or rapamycin (50 ng ml⁻¹) for 2 h. bFGF increased the activation of Rac1, whereas SU5402 and LY294002 inhibited Rac1 activation. In contrast, rapamycin did not affect bFGF-induced Rac1 activation. Rac1 activity was indicated by the amount of GTP-bound Rac1 normalised to the amount of total Rac1 in whole-cell lysate. The mean value of Rac1 activity relative to serum-starved control cells was indicated as relative Rac activity. (B and C) The effects of a Rac1 inhibitor and rapamycin on the bFGF-induced motility of RD-ES cells were investigated in a wound-healing assay (panel B) and chemotaxis assay (panel C). The bFGF-induced motility of RD-ES cells was inhibited by the Rac1 inhibitor (20 μ M) but not by rapamycin (20 ng ml⁻¹). Data are depicted as mean±s.d. of at least three independent experiments. ^***P<0.001, #, not significant. (D) Effects of a specific Rho inhibitor, C3, on bFGF-induced chemotaxis. bFGF-induced chemotaxis of RD-ES cells was not reduced by C3 (7.5 μg ml⁻¹). In contrast, chemotaxis of a human osteosarcoma cell line, MG-63, in response to serum was inhibited by C3. In chemotaxis assay, 2 × 10⁵ cells were plated onto the upper chamber. Data are depicted as mean±s.d. of at least three independent experiments. ^***P<0.001 vs control. #, not significant.