Zm447439

The function of the two kinases Mps1 and AurkC was investigated using two inhibitory drugs, namely Mps1i or compound 5 (kindly supplied by Prof. Geert Kops Hubrecht Institute, Utrecht, the Netherlands) (Kwiatkowski et al., 2010 (link), p. 359–368) and AURKi or ZM447439 (CAS 331771, Sigma-Aldrich Chemical Co., St. Louis, Missouri, United States). In order to evaluate the function of Mps1 and AurkC on spindle assembly, mature oocytes displaying a polar body were first incubated for 10 min at 38°C with 5% CO₂ in IVM medium containing 20 μM Nocodazole (M1404, Sigma-Aldrich Chemical Co., St. Louis, Missouri, United States) to depolymerize the MII-spindle. Following Nocodazole washout, the oocytes were left to re-form their spindle for 120 min in IVM medium with different concentrations of either MPS1i (0, 200, or 500 nM) or AURKi (0, 5, or 10 μM). The oocytes in the control group were not treated with Nocodazole or inhibitors.

The drugs used for screening in this study are listed in Supplementary Table 1.
Reversine, SP600125, ZM 447439, BI 2536, STLC, Dimethylenastron, SB203508, RO 3306, Cdk1 Inhibitor III, Roscovitine, NSC 95397, IPA3, Y-27632, ITX-3, Nocodazole, Paclitaxel, Blebbistatin, Cytochalasin B, MG 132 and Velcade were purchased from Sigma–Aldrich (St. Louis, MO, USA). AZ 3146 and Mps-BAY2a were obtained from Tocris (Bristol, United Kingdom). MLN8237 (Alisertib), AZD1152-HQPA (Baraserib) and SB743921 were purchased from Selleckchem. GSK923295 is from MedChem express.
Monoclonal antibodies against α-tubulin, CDK1, AURKA, PLK1, KIF11 (Sigma-Aldrich); CCNB1, CCNE1, TP53, CDKN1A, JNK1, JNK2 (Cell signaling); EB1, COFILIN (Santa Cruz), TTK, BUBR1 (Abcam). Polyclonal antibodies against PRC1 (Santa Cruz), Tpx2 [69 (link)]; Kif23 [70 (link)] were used.

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.

U2OS (ATCC, Manassas, VA, USA), U2OS-CASP2^−/−, U2OS-CASP2^−/−/CASP3^−/−, A549, A549-CASP2^−/− and stable cell lines were maintained in Dulbecco’s Modified Eagles Medium (DMEM, Sigma-Aldrich) supplemented with 10% foetal bovine serum (JRH Biosciences, Lenexa, KS, USA), 0.2 mM l-glutamine (Sigma-Aldrich), 15 mM HEPES (Sigma-Aldrich) and 100 µM penicillin/streptomycin (Sigma-Aldrich) in a humidified incubator at 37 °C with 10% CO₂. A549 and A549-CASP2^−/− cell lines were provided by Prof. Villunger (Medical University of Innsbruck, Austria). Where indicated, cells were treated with 100 nM BI2536 (Axon Medchem, Netherlands), 2 µM ZM447439 (Sigma-Aldrich), 400 nM AZD1152-HQPA (Sigma-Aldrich), or 50 µM blebbistatin (Selleck Chemicals, Houston, TX, USA) for 24 or 48 h. MEFs were maintained in DMEM (Sigma-Aldrich) supplemented with 0.2 mM l-glutamine (Sigma-Aldrich), 15 mM HEPES (Sigma-Aldrich), 10% foetal bovine serum (JRH Biosciences) and 50 µM β-mercaptoethanol (Sigma-Aldrich), non-essential amino acid mix (Sigma-Aldrich), 100 µM penicillin/ streptomycin (Sigma-Aldrich). Cells were cultured in a humidified incubator at 37 °C with 10% CO₂. Transfection of plasmid DNA was performed using Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. All cell lines were tested for mycoplasma contamination.