Annexin V assay Cells were treated with DMSO or VX 680 for 48 h prior to collecting and resuspending in binding buffer. Annexin V FITC and propidium iodide were added to each sample accord ing to the manufacturers protocol. 4, 6 diamidino 2 phe nylindole was used to visualize nuclei. 20 25l of cell suspension was transfered onto glass microscope slides respectively, and viewed immediately sellectchem using a fluorescence microscope. Western blot assay Western blot assay was performed as described previously. Antibodies used were mouse anti GAPDH, rabbit anti Bcl 2, rabbit anti cleaved caspase 3, mouse anti cleaved PARP, rabbit anti phosphorylated Akt, mouse anti phospho GSK3, mouse anti IB,rabbit anti GSK3 , goat anti Akt1, rabbit anti Bcl xL and rabbit anti Aur A.
Generation of Inhibitors,Modulators,Libraries stable transfection cell lines Myr Akt1 and pUSE plasmids were generously provided by Xiao feng Zhu. Transfections were conducted according to manufactur ers recommendations. Tca8113 cell clones stably transfected with plasmid were selected in 400g ml G418. Transient transfection and cotransfection Inhibitors,Modulators,Libraries Transient transfection of Aur A and its vector control pCS2 or cotransfection of Aur A or pCS2 with siRNA against Akt1 or its control were conducted according to manufacturers recommendations. Lysates were prepared 48 h after transfection. Cells were treated with API or wortmannin for 24 h prior to collecting for Western blot. RNA mediated interference siRNA for downregulating Aur A or Akt1 expression was done by the transfection of RNA oligonucleotides with lipofectamine 2000.
The sequence for siRNA against Aur A was and siRNA against Akt1 was. Lysates were prepared 36 h after transfection. Transwell migration assay Transwell assay was performed as described previously. Briefly, cells were incubated in serum or serum free media containing Inhibitors,Modulators,Libraries desired drugs for 16 h. The migrated cells in five fields were counted, and the average of each chamber was determined. Statistics Statistical analysis was performed using SPSS version 13. 0. The 2 test and Students t test was used to make a statistical comparison between groups. P 0. 05 was considered statistically significant. We performed each study at least three times under iden tical conditions. Abbreviations The abbreviations used are PI3K phosphatidylinositol 3 kinase. Aur A Aurora A. TSCC tongue squamous cell car cinoma. IGF 1 insulin like growth factor 1.
API 2 Akt protein kinase B signaling inhibitor 2. GSK 3 glycogen synthase kinase 3. IKK IB kinase. IBIkappaB. NFBnuclear factor B. Competing interests The authors declare that they have no competing interests. Authors contributions JY carried out protein studies, apoptosis analysis, tran Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries swell assays, immunofluorescence staining, statistical analysis and co wrote the manuscript. MY drafted the manuscript, contributed to the study design and partici pated in the GW572016 performed the protein studies, apoptosis analysis, transwell assays, immunofluorescence staining, statistical analysis.