Culturing and Transfecting HeLa and RPE1 Cells

HeLa-Kyoto (K) cells were grown in a humidified incubator at 37°C and 5% CO₂ in DMEM (Gibco) containing 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin supplemented with 0.1 µg/ml puromycin (Invitrogen) for maintenance of the eGFP-CENP-A cell line (Jaqaman et al., 2010 (link)). The HeLa H2B-GFP cell line was maintained in nonselective medium. The hTERT-RPE1 eGFP-CENP-A cell line was maintained in DMEM/F-12 medium containing 10% FCS, 2.3 g/l sodium bicarbonate, 100 U/ml penicillin, and 100 µg/ml streptomycin. siRNA oligonucleotides (53 nM) were transfected using oligofectamine (Invitrogen) according to the manufacturer’s guidelines and analyzed at 48 h. The following sequences were used: control, 5′-GGACCUGGAGGUCUGCUGU-3′; Ska1, 5′-CCGCUUAACCUAUAAUCAA-3′; and MCAK, 5′-GAUCCAACGCAGUAAUGGU-3′. For drug treatments, cells were treated with 2 µM ZM447439 (Tocris Bioscience) for 30 min or 100 nM taxol (Sigma-Aldrich) for 1 h before live-cell imaging.