Materials and methods The authors declared that the current research has been approved by The Ethics Committee of Nanjing University of Traditional Chinese Medicine. Reagents DMEM and fetal bovine serum were purchased from Thermo Fisher Scientific at CHINA. 3 2,5 diphenyl tetrazoliumbro mide was obtained from Sigma Aldrich. Anti Aurora B antibody and anti Histone H3 antibody were obtained from Abcam. Anti Survivin antibody was purchased from Cell Signaling. Anti Histone H3 and GAPDH antibody were obtained from Santa Cruz Biotechnology. Cell culture The human colorectal adenocarcinoma cell lines, SW48 and SW620, were obtained from the American Type Culture Collection. The cells were maintained in DMEM supplemented with 10% heat inactivated FBS at 37 C, 5% CO2, and 95% humidity.

Plasmids and transfection The full length cDNA sequence of survivin was amp lified from total RNA of SW620 cells by using Reverse Transcription PCR. The fragment was inserted into pBABE Puro vector. The control vector plasmid or the plasmid encoding inhibitor WIKI4 survivin was transfected into Phoenix Retroviral Expression System. Virus was produced and ap plied onto target cells according to the standard protocol. The cells were subjected to drug selection for 3 days to enrich for the desired cells. Silencing of Aurora A and B in cells 1. 5 × 105 cells were seeded in 60 mm plates and incu bated for 24 h before transfection. The negative control siRNA or Aurora A or B siRNA was diluted in Opti MEM I Reduced Serum Medium and mixed with Lipofectamine 2000 according to the manufacturers instructions.

The mix of DNA and Lipofectamine was added to cells. After inhibitor L-Mimosine 72 hours post transfection, expression levels of Aurora genes were determined by Real time PCR and cells were used for different assays. Ionization radiation Cells were plated in dishes, and then irradiated with X ray by using an X ray irradiator for indicated dosages. Determination of surviving fraction 2 × 105 cells were plated in a 60 mm dish. 24 hours later, the cells were exposed to different dosages of ionization radiation. After a 6 hour recovery, one percent of the cells were re plated in a new dish. After 10 days the number of colonies formed were counted. Combination effect of radiation and CCT137690 Cells were first treated with CCT137690 at different con centrations for 48 hours before they were exposed to dif ferent dosages of ionization radiation.

Cell cycle assay Cells were collected by trypsinization and washed with PBS, centifuged and then resuspended in 0. 4 ml of PBS and fixed by adding 1ml cold ethanol slowly. Cells were kept at 4 C overnight. For analysis, cell suspensions were centrifuged at 1500 rpm for 5 mins, washed with PBS and re suspended in 500 ul staining solution at 37 C for 30 mins in the dark. Cells were analyzed by flow cytometry.