VE-465 induces cell death in the presence of paclitaxel. Cells were treated for 96 hours with differing doses of VE-465 in the presence of 15 ng/mL paclitaxel. (A) PTX10 cells (B) 1A9 cells. Analysis was performed as described in Figure 3. The sub G0/G1 population represents apoptotic cells. Each time point represents data from at least 3 independent experiments. Paclitaxel and VE-465 did not synergize to cause apoptosis in PTX10 (C) or 1A9 (D) cells. Percent of apoptotic cells are plotted for cells treated for 96 hrs with VE-465 alone or VE-465 and 15 ng/mL paclitaxel. Triangles – cells treated with increasing concentrations of VE-465. Squares – cells treated with increasing concentrations of VE-465 in the presence of 15 ng/mL paclitaxel. (E) Caspase 3/7 assays of PTX10 cells treated with 10–100 nM of VE-465 alone or in combination with 15 ng/mL paclitaxel. Confirming flow cytometry data, combination treatment with paclitaxel and VE-465 did not synergistically increase apoptosis in the PTX10 cell line. (F) Caspase 3/7 assays of 1A9 cells treated with 1–3 nM of VE-465 alone, 15 ng/mL paclitaxel alone, or in combination with 15 ng/mL paclitaxel. A dose of 3 nM VE-465 alone induced 2-fold more apoptosis than 15 ng/mL paclitaxel, whereas combined 3 nM VE-465 and 15 ng/mL paclitaxel synergistically induced 4.5-fold more apoptosis than 15 ng/mL paclitaxel alone. (* = p-value less than 0.0025 by students T-test.) (G) Immunoblot of 1A9 cells treated with increasing concentrations of VE-465 for 96 hours. The kinase activity of Aurora-A and Aurora-B is suppressed in a dose-dependent manner consistent with the known Ki values of VE-465. Phosphorylation of the Aurora-A target p53 (S315) is inhibited at doses of 1 nM and higher whereas auto-phosphorylation of Aurora-B (T232) is only inhibited at doses exceeding 25 nM.