Thymidine

HeLa cells were grown in Dulbecco’s Modified Eagle’s Medium (Lonza), supplemented with 10% fetal bovine serum (Sigma), 1% penicillin/streptomycin (Sigma) and 1%w/v L-glutamine at 37 °C. For interphase arrest, cells were first treated with 2 mM thymidine (Santa Cruz, 296542 A), released for 8 hours and then treated again with 2 mM thymidine for 17 hours. For mitosis synchronization, cells were treated with 10 µM STC (Sigma, 164739-5G164739) for 12 hours. To induce monopolar cytokinesis, cells were subsequently treated with 30 µM Purvalanol A (Tocris Bioscience, 1580) for 15 minutes and then washed immediately and extensively with PBS.
To induce synchronization in bipolar cytokinesis, following double thymidine blocking, the cells were released for 7 hours, then incubated with 0 ng/ml, 10 ng/ml and 25 ng/ml of Nocodazole (Calbiochem, 487928) for 4 or 5 hours. Next, cells were extensively washed with PBS. Mitotic cells were collected by gentle shaking and incubated for 50 or 60 minutes in the new plate.

Cells were synchronised at prometaphase by treatment with 5 µM S-trityl-L-cysteine (STLC; 164739, Sigma) for 20 h. For biochemistry, cells were synchronized using 2 mM thymidine (#296542A, Santa Cruz Biotechnology) for 16 h and released into fresh complete medium for 8 h, followed by either 16 h of 2 mM thymidine (interphase block) or 20 h of 5 µM STLC (prometaphase block) incubation.

HeLa S3 cells (ATCC CCL-2.2) were maintained in DMEM high glucose supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Lonza) at 37°C in a humidified atmosphere containing 5% CO₂. The arrest of cell cycle progression during interphase was achieved by double thymidine block. Briefly, 2 mM thymidine (296542; Santa Cruz Biotechnology) was added to the cell culture medium, and the cells were incubated for 16 h at 37°C. Then, the cells were incubated in complete medium without thymidine for 8 h. After the second thymidine block of the cells for 16 h, most of the cells were arrested in the G₁ phase of interphase. For mitotic arrest, the cells were incubated in complete medium without thymidine for 8 h after the second thymidine block. Then, 10 ng/ml of nocodazole (487928; Calbiochem) was added to the cell culture medium and the cells were incubated for 5 h. At the end of the incubation with nocodazole, the cells were arrested in prometaphase. For cytokinesis arrest, the cells were incubated in complete medium for 1 h after nocodazole treatment. To induce monopolar cytokinesis, after the second thymidine block, the cells were incubated in complete medium containing 10 μM S-trityl-L-cysteine (STC) (164739; Sigma-Aldrich) for 16 h. Then, 100 μm purvalanol-A (1580; Tocris Bioscience) was added to the cell culture medium and the cells were incubated for 15 min.