Vx 680

HeLa S3 and HEK293 cells were cultured at 37 °C in 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) (Cellgro, Herndon, VA) supplemented with 10% Fetal Bovine Serum (FBS) (Mediatech, Herndon, VA), and 1% Penicillin-Streptomycin (Mediatech). HeLa-Flag-YY1 cells are stably transfected with pCS2(+)-Flag-YY1, the generation of this cell line has been previously described²⁸ . Cells were arrested at pro-metaphase of mitosis by adding nocodazole (100 ng/ml) or Taxol (100 nM) (Sigma) for 16–18 hours; cells were then collected by shake-off. For thymidine-nocodazole synchronisation, cells were blocked in thymidine (Sigma) at 2.5 mM for 18 hours, released for 3 hours, and then nocodazole was added for 12 hours. Cells were then collected, washed, and re-plated into fresh media to progress through mitosis. For the inhibition of Aurora A activity, VX-680 (LC Laboratories, Woburn, MZ) was added, for 15 minutes, to cells blocked in mitosis by nocodazole. For Aurora A knockdown, cells were transfected with control scrambled siRNA or Aurora A siRNA (50 nM) using DharmaFECT reagent (Dharmacon, Chicago, IL). After 36 hours of knockdown, the cells were blocked in mitosis with nocodazole for 18 hours. For transfection of mammalian expression plasmids, the Polyjet transfection reagent (SignaGen, Rockville, MD) was used according to the manufacturer’s instructions.

An MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay (a water-soluble form of the MTT assay) was performed as described in our previous study (12 (link)). OSU-03012 was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). VX-680 was purchased from LC Laboratories (Woburn, MA, USA). The stock solutions of the inhibitors were prepared at 10 mM in DMSO, and stored at −20°C.