Alisertib

For pharmacological inhibition of PLK1, BI2536 (Selleckchem), BI6727 (Volasertib, Selleckchem), and poloxin (Selleckchem) were added to cells at the indicated final concentrations in growth medium. For inhibition of Aurora A kinase, Alisertib (MLN8237, Selleckchem) was added to cells at the indicated final concentrations in growth medium. Arrest of cells in S-phase and mitosis was achieved by supplementing growth medium with 3 µM aphidicolin (Sigma) or 330 nM nocodazole (Sigma), respectively, for 16 h. Arrest of cells at the G₂/M boundary was achieved by growth medium with 9 µM RO-3306 (Selleckchem) for 20 h. Release from cell cycle arrest was performed by three washes with pre-heated (37 °C) growth medium.

Alisertib was purchased from Selleck (Houston, TX, USA). Anti-β-actin primary antibody was purchased from Sigma Aldrich (St. Louis, MO, USA). Antibody against human AURKA (#14475) was purchased from Cell Signaling Technology (Beverly, MA, USA). Immobilon Western Chemiluminescent HRP detection kit was from Millipore (Burlington, MA, USA). Cell counting kit-8 (CCK-8) was from Dojindo Laboratories (Kyushu, Japan). Caspase-Glo 3/7 assay kit was from Promega (Madison, WI, USA). Annexin V-FITC Apoptosis Detection kit was from BD Pharmingen (Franklin Lakes, NJ, USA). Lipofectamine RNAiMAX was from Invitrogen (Carlsbad, CA, USA). Cell culture medium was obtained from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Biological Industries (Kibbutz Beit Haemek, Israel). Cell extraction buffer was from Life Technologies (Grand Island, NY, USA). Alisertib was dissolved in DMSO to make a stock solution of 10 mM.

Cells were grown in DMEM medium (RPE-1) or RPMI 1640 medium (HL60, K562, KG1α, and THP1) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and maintained at 37° C with 5% CO2. For experiments where PLK4 expression was induced, media was supplemented with 2 μg/mL Doxycycline for 36 hours. Alternatively, cells were treated with 30 μM Cytochalasin B for 16 h to induce cytokinesis failure and generate tetraploid cells with double the normal centrosome number [8 (link)]. Drug treatments to subsequently inhibit AurA kinase (alisertib; MLN8054, Aurora A inhibitor 1, MK-5108 (VX-689): Selleckchem) were performed at the indicated concentrations for 16-18 hours for immunofluorescence and FACs analysis, or as otherwise indicated. Unless otherwise noted, all experiments to inhibit Aurora A kinase were performed with the highly specific Aurora A kinase inhibitor alisertib at a concentration of 100 nM.

Cells were treated with 0.5 or 1 μM TC-A2317 (Tocris, 4066), 0.5 μM alisertib (Selleckchem, S1133) or 50 nM paclitaxel (EMD Millipore, 580555) for indicated times. All drugs are dissoved in DMSO as a vehicle. The final concentration of vehicle in culture medium was 0.1%.

Tozasertib, alisertib, and nutlin-3 were purchased from Selleck Chemicals (Houston, Tx, USA), cisplatin and vincristine from Gry-Pharma GmbH (Kirchzarten, Germany), and doxorubicin from Cell-Pharm GmbH (Bad Vilbel, Germany).