Gsk923295

HeLa cells were arrested in mitosis using either 100 ng/ml nocodazole (487928; Merck) or 10 µM STLC (164739-5G; Merck) for 2.5 h (immunofluorescence) or 18 h (immunoblotting).
For biochemical analysis, mitotic cells were harvested by shake-off, washed twice in PBS and once in Opti-MEM, both of which had been pre-equilibrated to 37°C, 5% CO₂. Cells were counted and resuspended in pre-equilibrated Opti-MEM to give 7.5 × 10⁶ cells/ml. Subsequently, cells were either treated with 0.5 µM Aurora A inhibitor MLN8237 (A4110-APE-10 mM; ApexBio), 10 µM Aurora B inhibitor ZM 447439 (A4113-APE-10 mM; ApexBio), 10 µM Aurora B inhibitor AZD1152 (A4112-APE-10 mM; ApexBio), 25–100 nM PP1/PP2A inhibitor calyculin A (1,336/100 U; TOCRIS), and 40 µM proteasome inhibitor MG-132 (474790-5MG; Merck) along with 1–10 µM MPS1 inhibitor (AZ3146; TOCRIS) or DMSO (D8418; Merck) for up to 25 min.
For immunofluorescence microscopy, nocodazole- or STLC-arrested cells were treated with 20 µM MG132 and the inhibitors described above for 30 min prior to fixation. For the CENP-E inhibitor experiment, asynchronous cultures were treated with MG132 for 3 h, or 300 nM CENP-E inhibitor (GSK923295; TOCRIS) for 3 h and MG-132 with or without Aurora A inhibitor for 30 min before fixation.

DLD-1, HeLa, RKO and HCT-116 cell lines were cultured in DMEM plus 10% fetal calf serum (LifeTechnologies), 2 mM glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin (Lonza) at 37°C in a humidified 5% CO₂ atmosphere. DLD-1 Histone-H2B-mCherry were as described previously [62 (link)]. Small molecule inhibitors dissolved in DMSO were as follows: GSK923295, Cenp-E inhibitor (in house); AZ3146, Mps1 inhibitor (Tocris); Monastrol, Eg5 inhibitor (Sigma).