Fig. 5

Effects of Sunitinib and HM-3 on the levels of the active form of RhoGTPases and cytoskeleton within the target cells. a-d To test the levels of active form RhoGTPases in EAhy926 cells, Rhotekin RBD (Rho binding domain) or PAK-1 PBD (Rac1 binding domain) were used to extract active form Rac1 (a) or RhoA (b) from EAhy926 cell lysates after the cells were treated with different concentrations of Sunitinib or HM-3 as indicated. Immunoblotting was performed with specific Rac1 or RhoA antibodies. Cells that had not been treated were used as a positive control. The levels of GTP-Rac1 (c) and GTP-RhoA (d) in B16F10 cells were assayed in an analogous way. Quantification of GTP-Rac1 or GTP-RhoA signal ratios is indicated. e Effect of Sunitinib and HM-3 on the EAhy926 cytoskeleton. The images for cytoskeleton of EAhy926, B16F10 or MDA-MB-231 cells were indicated with black, red or blue words respectively. For EAhy926 images, cells were treated with 0.015 or 8 nM Sunitinib, 4.5 or 17.8 μM HM-3 at room temperature for 1 h. After fixation, nonspecific binding sites were saturated with 5% BSA. Fluorescein isothiocyanate labeled phalloidin was used to visualize actin stress fibers. For B16F10 and MDA-MB-231 images, cells were treated with 2 nM or 64 nM Sunitinib, 4.5 μM or 71 μM HM-3 at room temperature for 1 h. Cells that had not been treated were used as a control