ATM monoclonal antibody was bought from Santa Cruz Biotechnology (Santa Cruz, CA,

USA). BCIP/NBT alkaline phosphatase substrate kit IV was purchased from Vector laboratories (Burlingame, CA, USA). TUNEL apoptosis detection kit was bought from Roche Company (Shanghai, China). Cell lines and mice Hep-2 cell line was obtained from the laboratory of Head and Neck at Sichuan University. The cells were maintained in RPMI-1640 medium, supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, and 100 U/mL penicillin G in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Female BALB/c-nu/nu mice, aged 3-4 weeks, weighing 18-22 g, were obtained from the animal centre of West China Medical School and were maintained in the animal #Compound C cell line randurls[1|1|,|CHEM1|]# facility at West China Medical School, Sichuan University in accordance with nation’s related regulations and animal welfare requirements. Synthesis of oligodeoxynucleotides (ODNs) and selection of target sequences AS-ODNS, sense (Sen) and mismatch

(Mis) ODNs were synthesized by Shanghai Sangon Biological Engineering Technology & Services (Shanghai, China). The sequences were as follows: AS (5′-GTACTAGACTCATGGTTCACAATTT-3′); Sen (5′-AAATTGTGAACCATGAGTCTAGTAC-3′) and Mis (5′-AAAATGTAAACCATAAGTCTAGAAC-3′). All the ODNs were chemically modified to phosphorothioate ODNs by substituting the oxygen molecules of the phosphate backbone with sulfur. Transfection of ODNs in Hep-2 cells Hep-2 cells at a density of 2 × 105 cells/ml were plated in 6-cell plates for overnight incubation. Cells were maintained in next RPMI-1640 medium supplemented click here with 10% FBS at 37°C and 5% CO2. After grew to 70-80% confluent, cells were replenished with incomplete RPMI-1640 medium, then treated with ATM AS-ODNs, ATM

Sen-ODNs and Mis-ODNs. The procedures were as follows: 0.8 ug of ATM AS-ODNs, Sen-ODNs, Mis-ODNs and 2 mg/ml Lipofectamine 2000 were added to Opti-MEM I medium separately, and incubated for 5 min at room temperature. Then liposome and ODNs were mixed and incubated at room temperature for 20 min. Hep-2 cells were washed again with Opti-MEM I medium before transfection. The liposome ODNs complexes were carefully plated on the cells, and incubated at 37°C, 5% CO2. After 6 hours transfected cells were washed twice with PBS. With the medium replaced with fresh RPMI-1640 medium supplemented with 10% FBS, the cells were incubated at 37°C overnight. A second ODNs incubation was performed before cells were exposed to radiation. Real-time quantitative PCR analysis According to the manufacturer’s recommendations total RNAs were extracted from cultured Hep-2 cells using Trizol reagent. One-step RT-PCR was performed in LightCycler-RNA Amplification Kit SYBR Green I. ATM was amplified with the sense primer: (5′-GACCGTGGAGAAGTAGAATCAATGG-3′ and the anti-sense primer: 5′-GGCTCTCTCCAGGTTCGTTTGC-3′).