U2OS, HeLa, and human embryonic kidney 293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 IU/ml), and streptomycin (100 μg/ml) at 37°C in a 5% CO2 atmosphere. HeLa Kyoto H2B-mCherry/EGFP-Lamin A cells were maintained in DMEM containing 10% FBS, penicillin (100 IU/ml), streptomycin (100 μg/ml), G418 (500 μg/ml), and puromycin (0.5 μg/ml). Transient transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were synchronized in M phase either by incubation for 16 hours with nocodazole (500 ng/ml) or by incubation with 10 μM RO-3306 (Enzo Life Sciences) for 16 hours, followed by transfer into fresh medium with 20 μM MG132 (EMD Millipore) for 2 hours. M-phase arrested cells were collected by mitotic shake-off after 16 hours of treatment. SAC inactivation was achieved through incubation with the MPS1 inhibitor, AZ3146 (Sigma), for 4 hours.

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