) were described previously. All cell lines were cultured in Dulbecco’s modified Eagle’s medium ; supplemented with 10% fetal calf serum , 1 mM sodium pyruvate, and 100 U of penicillin/streptomycin ; and maintained at 37°C and 5% COQuantitative reverse transcription polymerase chain reaction was performed using SYBR Green on a LightCycler 480 . Levels were normalized to the geometric mean of two housekeeping genes . Primer sequences are provided in table S1.
For time-lapse imaging, cells were seeded at optimized density in μ-Slide 8 Wells . The next day, fresh media containing SiR-DNA was added 1 hour before the start of imaging. For mitotic progression, before imaging, fresh media was added to remove SiR-DNA. Cells were imaged every 3 min for 16 hours. In case of SAC analysis, cells were treated either with Taxol or nocodazole with or without AZ3146 before imaging.
For relative amounts of protein distribution , we used chromosome spreads, as they enable a more spatially resolved analysis of subchromosomal protein distribution. For this, mean fluorescence intensities of each protein were measured on FIJI using a circle of 10-pixel diameter that was placed around the centromere and chromosome arms of the same chromosome. Approximately five isolated chromosomes were measured per spread after random selection .
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