Cell Synchronization and Inhibitor Treatments

HCT116 p21+/+, HCT116 p21−/−, HeLa, MDA-MB-231, MCF7 and U2OS cells were cultured as instructed. Stable HeLa 776-6, expressing shRNA targeting cyclin B1, were generated as described [18 (link), 19 (link)]. To synchronize cells in prometaphase, cells were treated with 50 ng/ml nocodazole (Sigma-Aldrich, Taufkirchen). Thymidine (Sigma-Aldrich) synchronization and release was performed as described [46 (link)]. The Plk1 inhibitor BI2536 (25 nM) was obtained from Selleck Chemicals LLC (Houston, USA), the specific Cdk1 inhibitor RO-3306 (9 μM) and the MAP cascade inhibitor PD98059 (10 μM) from Merck Millipore (Darmstadt). λ-Phosphatase (λ-PPase) was purchased from NEB (Frankfurt), MG132 (Z-Leu-Leu-al; 10 μM), cycloheximide (25 μg/ml) and DMSO from Sigma-Aldrich, the calpain inhibitor PD150606 (200 μM) from Santa Cruz (Heidelberg), and the pan-caspase inhibitor Z-VAD-FMK (Z-VAD; 20 μM) from Enzo Life Science GmbH (Lörrach). siRNA (10 to 20 nM) was transiently transfected with Oligofectamine™ (Life Technology). siRNAs targeting p21 (sense: ACACCUCCUCAUGUACAUAUU and antisense: AAUAUGUACAUGAGGAGGUGU), cyclin B1 (sense: GAAAUGUACCCUCCAGAAATT and antisense: GCUGACCCUGAAGUUCAUCUU) and Cdk2 (sense: ACACUCACCUUCUAGUCUUUU and antisense: AAGACUAGAAGGUGAGUGUUU) were manufactured by Sigma-Aldrich. Control siRNA was obtained from Qiagen (Hilden). For transient transfections with pBI-p21 and its constructs, electroporation was used (250 V, 250 μF, 500 Ω). The generation of the stable cell line HCT116 with H2B-tdTomato and the performance of time-lapse imaging are described [6 (link)]. FLAG constructs were transfected with FuGENE^® HD in a ratio 1:3 (Promega, Mannheim).