Hesperadin

HT1080 6TG cells were a kind gift from Eric Stanbridge (University of California, Irvine) and Saos-2 were provided by Roger Reddel (CMRI). IMR90 cells were purchased from ATCC and IMR90 E6E7 derived in the Karlseder laboratory. Phoenix cells were purchased from ATCC. Identity of all cell lines were verified by Cell Bank Australia using short tandem repeat profiling. HT1080 6TG, HeLa, Saos-2 and derivatives were cultured at 37 °C, 10% CO₂, and atmospheric oxygen, in DMEM (Life Technologies) supplemented with 1% non-essential amino acids (Life Technologies), 1% Glutamax (Life Technologies), and 10% bovine growth serum (Hyclone). IMR90 and derivatives were cultured at 37 °C, 10% CO₂, and 3% O₂, in DMEM, supplemented with 1% non-essential amino acids, 1% Glutamax, 10% fetal bovine serum (Life Technologies). The following compounds were used in cell treatments: dimethyl sulfoxide (DMSO, Sigma-Aldrich), colcemid (Life Technologies), Taxol (Sigma), reversine (Selleck Chemicals), KU-55933 (Calbiochem), Hesperadin (Selleck chemicals), Aphidicolin (Sigma-Aldrich) and Hydroxyurea (Sigma-Aldrich). All cell lines were tested for mycoplasma contamination (MycoAlert, LT07-118, Lonza) and were found to be negative.

HeLa and MCF7 cells were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM, WelGENE Inc.) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. All cell lines tested negative for mycoplasma contamination by PCR. siRNAs were transfected into HeLa cells using DharmaFect 1 (Dharmacon, Inc.). DNA transfection was performed using Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer’s instructions. For inhibition experiments, the cells were incubated with the Haspin kinase inhibitor CHR-6494 (10 nM) (Calbiochem), the Aurora B inhibitor hesperadin (2 μM) (Selleckchem), the Cdk1 inhibitor RO3306 (1 μM) (Enzo Life Sciences), the proteasome inhibitor MG132 (0.5 μM) (Sigma-Aldrich), and the PRMT6 inhibitor MS023 (20 μM) (Sigma-Aldrich).

E14 and ZHBTc4 ESCs were cultured as described (Jang et al., 2012 (link)) in 0.1% gelatin (Sigma-Aldrich, St. Louis, Missouri ) coated plates. ZHBTc4 ESCs were kindly provided by Hitochi Niwa (RIKEN, Japan). The mouse ESC medium was composed of DMEM (Hyclone, Logan, Utah) and 15% (v/v) fetal bovine serum (FBS; Gibco, Grand Island, New York), supplemented with 2 mM L-glutamine, 55 μM β-mercaptoethanol, 1% (v/v) nonessential amino acids, 100 U/ml penicillin, 100 μg ml^-1 streptomycin (all from Gibco), and 1000 U/ml ESGRO (Millipore, Germany). Aurora kinase inhibitors AT9283, Hesperadin and MLN8237 were purchased from Selleckchem (Houston, Texas) and okadaic acid from Sigma.