Azd 1152

A total of 1–2 × 10⁶ HEK293T cells were seeded on a 60-mm dish. After attaching to the dish (10–24 h), cells were transfected with 6 μg DNA per plate, mixed with 36 μg linear PEI (L.PEI). Cells were harvested at 24 to 48 h post transfection with 300 μl extraction buffer (20 mM Tris-HCl pH 8.0, 225 mM NaCl, 0.5 mM EDTA, 1% NP-40, 5% glycerol,1 mM DTT, and protease inhibitor cocktail [Sigma-Aldrich]). Cell extracts were sonicated and centrifuged at 4 °C for at least 15 min (>16,000g). Anti-NMIIB antibodies were incubated with protein A/G beads (Santa Cruz Biotechnology) prewashed with 300 μl extraction buffer on a rotator at 4 °C for 1.5 to 2 h. The beads–antibodies mix was washed three times with extraction buffer. The cell extracts were added to the bead–antibody mix and were incubated for 1.5 to 2 h on rotator at 4 °C. Then, the mix was washed three times in extraction buffer and analyzed by Western blotting using antibodies for NMIIB, GFP, or Borealin.
For the coimmunoprecipitation in the presence of AZD, 1–2 × 10⁶ HEK293T cells were seeded on a 60-mm dish. After attaching to the dish (10–24 h), cells were transfected with 6 μg DNA per plate, mixed with 36 μg L.PEI. Before harvesting, 100 nM AZD-1152 (Sigma-Aldrich) was added for 1 h. Cells were harvested with extraction buffer containing AZD. Cells were sonicated, and the coimmunoprecipitation assay was carried out as above.

Immortalized Ku70^−/− MEFs were obtained from S. Matsuyama (Case Western, Cleveland). Hela and IMR-90 cells were obtained from ATCC. All cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO₂. Ku70^−/− MEFs stably re-expressing Ku70 WT and mutants were generated by infecting Ku70^−/− MEFs with recombinant MSCV retrovirus followed by puromycin selection as previously described26 (link). To assess proliferation rates, the Ku expressing MEFs were seeded in triplicate in 6-well dishes. At each time point cells were trypsinized, counted using a hemocytometer, and the mean number of cells was determined. Percent growth was obtained by dividing the number of cells at each time point by the number of cells at day 1. For irradiation experiments, cells were plated the night before at 50–70% confluency. Irradiations were performed with a Faxitron RX-650 at a dose rate of 1.42 Gy/min (10 Gy treatment) or 3.8 Gy/min (40 Gy treatment). For ATM inhibitor treatments, MEFs were incubated with 10 μM of KU-55933 (Selleck Chemicals, Houston, TX) for 1 hour prior to 4 Gy of irradiation. For Aurora B inhibitor treatments, MEFs were incubated with 20 nM of AZD-1152 and 50 nM ZM447439 (Sigma-Aldrich) for 48 hours.

Thymidine, nocodazole, phalloidin-rhodamin, propidium iodide and AZD-1152 were from Sigma–Aldrich. RNase A was supplied by Euromedex.