The experiment was repeated three times, and a mean value was presented. Colony formation, matrigel invasion and flow cytometric analysis Colony Formation 500 cells were grown in a 60 mm dish with culture medium. The cells were treated with X ray irradiation at doses of 1 Gy or 2 Gy, respectively, after 12 hours of incubation. The cells Crenolanib purchase were then continuously cultured until visible colonies were formed. Only those containing 50 cells were counted. The rate of colony formation was indicated by the ratio of the number of clones over the number of seeded cells. The experiment was repeated three times, and a mean value was presented. Matrigel cell invasion assay Briefly, in each upper chamber, 5��105 cells were grown in serum free culture medium. The lower chambers were filled with RPMI 1640 medium containing 10% fetal calf serum.

After being incubated for 24 hours, the cells that migrated through Inhibitors,Modulators,Libraries the pores were fixed with methanol for 30 minutes and stained with hematoxylin. For each filter, the number of cells was counted microscopically in 5 random fields under a 200��magnification. The experiment was repeated three times, and a mean value was presented. Flow cytometric analysis for cell apoptosis Cells Inhibitors,Modulators,Libraries were collected at 72 hours after X ray treatment and then immediately stained with the Annexin V FITC/PI double staining kit before being analyzed by the FACSCalibur Flow Cytometer with Cell Quest 3. 0 software to determine the level of cell apoptosis. The experiment was repeated three times, and a mean value was presented.

Xenograft to nude mice Four week old male BALB/c nude mice were obtained from the animal facility. All the mice were handled in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes Inhibitors,Modulators,Libraries of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the China Medical University. All efforts were made to minimize suffering of the experimental animals. The mice were randomly divided into 4 groups. Each mouse was inoculated subcutaneously in the right axilla with 5��106 human lung cancer cells suspended in 0. 2 ml sterile PBS. The large dimension and short dimen sion of the subcutaneous nodules were measured with a vernier caliper every 3 days, Inhibitors,Modulators,Libraries and the tumor volume was calculated by the formula, V W2 L ��/6, before being plotted into the growth curve for each group.

Four weeks after inoculation, the mice were sacrificed, and the tumor nodules from each mouse were completely excised and measured. The rate of tumor growth inhibition was calculated according to the formula Inhibitors,Modulators,Libraries /mean tumor weight of control group��100%. Statistical analysis SPSS version 13. 0 for Windows was utilized to analyze the data. The Mann Whitney U test and Students t test were Brefeldin A used to examine the statistical difference of experi mental data between the groups.