Nu7441

Cell culture, reagents and treatments. T98G and MCF7 cells were cultured in DMEM (Cellgro), 10% fetal bovine serum, and 1% penicillin/streptomycin (Cellgro). PC3 cells were cultured in Ham's F-12K medium (Gibco), 10% fetal bovine serum and 1% penicillin/streptomycin (Cellgro). All cultures were maintained in 5% CO2 at 37°C. Unless indicated otherwise, cells were treated with 25 ug/mL cisplatin (Sigma) or 15 ug/mL doxorubicin (Sigma) overnight. For DNA-PK inhibition, cells were treated with 10 nM NU7441 (STEMCELL) for 30 minutes before being treated with cisplatin. Immunofluorescence analyses. Cells were plated in duplicate on coverslips. Treated and untreated cells were fixed with 4% paraformaldehyde-PBS for 15 min and blocked with 0.3% Triton X-100-5% goat serum-PBS for 1 h. Cells were then incubated with rabbit anti-γ-H2AX (Cell Signaling 9718) at 1:400 dilution or rabbit anti-53BP1 (Cell Signaling 2675S) at 1:500 dilution in 0.3% Triton X-100-1% BSA-PBS overnight at 4°C. The following day, cells were incubated with 1:500 Alexa Fluor 488-anti-rabbit secondary antibody (Life Technologies) in 0.3% Triton X-100-1% BSA-PBS for 1 h. Coverslips were then washed and mounted as described above. Slides were examined on a Zeiss AxioImager A2 microscope.