If AZD1152 or other AURKB inhibitors might be shown to boost the therapeutic index for androgen resistant prostate cancer, this would have a substantial clinical effect. All cells were incubated at 37 C in 95-pound air/5% CO2. AZD1152 was obtained from AstraZeneca. Western Immunoblotting Imatinib Glivec Cells were treated with different concentrations of AZD1152. These were gathered at various times and then washed with ice-cold PBS twice prior to the addition of lysis buffer including phosphatase inhibitor cocktail and protease inhibitor cocktail I. Protein concentration was quantified by the Bio Rad process. Similar quantities of protein were loaded in to each well and divided by 12. 510-525 or 15,000-25,000 SDS PAGE gel, followed by transfer onto PVDF membranes. Membranes were blocked with 5% nonfat dry milk in PBST for 1 h at room temperature. The blots were then incubated with anti Aurora B, anti phosphohistone H3, and anti Actin for 1 h at 4 C. Goat anti rabbit IgG secondary was incubated for 45 min at room temperature. Western blots were developed using the Lymph node chemiluminescence detection system in line with the manufacturers protocol and autoradiography. Cell Cycle Analysis Cells were seeded in 10 cm2 dishes 24 h before AZD1152 treatment and then treated with different doses of AZD1152 for 48 h. The cells were then obtained by trypsinization, fixed with 70-75 ethanol, and stored over night at fi20 H. Propidium iodide was then added, and the cells were incubated supplier Lonafarnib at room temperature for 5 min. The number of cells in each stage of the cell cycle was determined and calculated as a percentage of the total cell population. Clonogenic Assay Cells were treated with AZD1152 or DMSO. 8 Gy/min utilizing a 137Cs irradiator. After irradiation, the method was modified and cells were incubated at 37 C for 8 days. Cells were stained for 30 min with one of the methylene blue in water and then set for 30 min with 70-75 methanol. After staining, colonies were counted by eye using a cutoff of 50 viable cells. The surviving fraction was determined as / plating performance, where PE was understood to be /. After 48 h of incubation, cells were irradiated with either 0 or 5 Gy. Both 30 min or 6 h after irradiation, the cover slips were washed with cold PBS, and cells were fixed with 401(k) formaldehyde for 10 min at room temperature. Cells were then washed twice in PBS and added to cover slips in ice cold wells. Then 2 ml of ice-cold Triton X 100 solution was added. After 15 min, the cover slips were washed 3 times in PBS.