Cell culture experiments

Mammalian cell lines were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and antibiotics in a humidified incubator at 37°C with 5% CO₂. Lentiviruses were produced in human embryonic kidney 293T cells, by calcium phosphate cotransfection (CalPhos mammalian transfection kit, Clontech Laboratories) of packaging vectors (pCMV-VSV-G envelope and pCMV-dR8.2 dvpr) together with either pTRIPZ-V2THS_12364 (doxycycline inducible human Aurora A shRNA, Dharmacon) or a pHAGE2-based lentiviral construct (82) expressing an shRNA-resistant 2xFLAG-tagged Aurora A (wild type or mutant) driven by a native Aurora A promoter fragment consisting of −367 to +356 base pairs around the transcriptional start site (53). Virus-containing media were filtered through 0.45-μm filters and used to transduce HeLa cells. HeLa cells stably transduced with inducible shAurora A were selected with puromycin (2 μg/ml). Doubly transduced HeLa cells were produced by subsequent transduction of these cells with pHAGE2-expressing shRNA-resistant 2xFLAG-tagged Aurora A–internal ribosomal entry site–green fluorescent protein (GFP), followed by selection with blasticidin (25 μg/ml) and sorting for GFP⁺ cells. Knockdown of endogenous Aurora A in transduced HeLa cell lines was induced by treatment with doxycycline (2 μg/ml) for 48 hours. The cells were then arrested by addition of nocodazole (10 μM), harvested 15 hours later, and stored as frozen cell pellets. Lysates were produced by sonication of the cell pellets in phosphate-buffered saline supplemented with 1% SDS, 1 mM NaF, 1 mM Na₃VO₄, 10 mM β-glycerol phosphate, and 10 mM EDTA. Total and phospho–Aurora A in the lysates were detected by Western blotting, using rabbit monoclonal α–Aurora A antibody (1G4, Cell Signaling Technology) and α–phospho–Aurora A (pT288) antibody (D13A11, Cell Signaling Technology), respectively, and IRDye fluorescent secondary antibodies.