Cell synchronization

We used several methods for cell synchronization. To synchronize cells at the G1/S transition, we applied thymidine-hydroxyurea blocks. HeLa cells were pre-synchronized in S phase by incubation with 2.0 mM thymidine (Sigma-Aldrich) for 20 h, released in drug-free medium for 8 h, then cells were resynchronized at the G1/S transition by incubation with 1.5 mM hydroxyurea (Sigma-Aldrich) for 13 h. When needed, plasmid DNA was transfected at 2 h after release from thymidine. The G1/S cells were then washed, incubated in fresh medium for the appropriate time, and used for experiments.

For mitotic time course analysis, cells were arrested at prometaphase using hydroxyurea-monastrol or hydroxyurea-nocodazole blocks. The cells were pre-synchronized at the G1/S transition by incubation with 1.5 mM hydroxyurea for 20 h, released for 3 h and then synchronized at the early phase of mitosis by treatment with medium containing 100 nM monastrol (Sigma-Aldrich), an inhibitor of the mitotic kinesin Eg5 (Kapoor et al., 2000) or 100 ng/ml nocodazole for 6 h. Cells at prometaphase were collected by the shake-off method (Jackman and O'Connor, 2001), aliquoted, and cultured in fresh medium for the indicated period. The cells were then immediately lysed in sample buffer.