Thymidine

For time-lapse live cell imaging, HeLa cells were transfected with GFP-BAF, and seeded (1 × 10⁴) onto a 4-well glass-bottom dish (Thermo Scientific™ Nunc™ Lab-Tek II Chambered* Coverglass, 154526). The cells were treated with 2 mM thymidine (Sigma-Aldrich, T9250) to arrest at the G1/S transition. 20 h later, the cells were washed with thymidine-free medium and cultured in complete medium for 7 h. Then, the cells were cultured again in medium containing 9 μM RO3306 (Enzo, ALX-270_463) to arrest at the G2/M transition for 2 h. The cells were released from RO3306 treatment and stained with Hoechst 33342 for the visualization of chromosomes. After 30 min, the cells were treated with 50 μM H₂O₂ or 100 nM okadaic acid. Fluorescence images were acquired every 3 min using a Nikon eclipse Ti with a 20 × 14 NA Plan Apochromat objective. Images were captured with an iXonEM + 897 electron-multiplying charge-coupled device camera and analyzed using NIS elements Ar microscope imaging software.

UTA6 and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS and antibiotics, penicillin and streptomycin. Cells were plated onto plastic dishes for large-scale cell culture and glass-bottomed dishes (LabTek) or 13 mm round coverslips for imaging. For inhibition studies, cells were treated with 10 µM MG132 (1748, TOCRIS). Double-thymidine block for synchronisation was performed on 60% confluent cell cultures by treating cells with 2 mM thymidine (ACROS organics) for 24 h, releasing cells from the thymidine treatment for 12 h, treating cells with a second round of 2 mM thymidine for 12 h and finally releasing them for thymidine treatment for 9–10 h for mitotic cell enrichment. For large-scale prometaphase cell enrichment, soon after the second round of thymidine release, 5 µM DMA was added (Enzo Life Sciences) and 14 h later rounded up cells were collected by shake-off. For anaphase cell enrichment, prometaphase cells were washed with warm DMEM and released into drug free medium for 45 min.