Analyzing Mitotic Chromosome Segregation

Chromosome segregation analysis was performed on MEFs stably expressing H2B-RFP, as previously described (Malureanu et al., 2009 (link)). In mitotic timing experiments, the time interval between nuclear envelope breakdown (NEBD) and anaphase onset was measured in H2B-mRFP positive cells by monitoring unchallenged mitoses. Briefly, cells undergoing NEBD were marked and monitored at two minute intervals until anaphase onset. For SAC sensitivity experiments, cells were treated with nocodazole (Sigma-Aldrich) at a final concentration of either 20 or 10 ng/ml and then monitored from NEBD to anaphase onset. nocodazole challenge experiments were performed as previously described (Malureanu et al., 2009 (link)). Briefly, nocodazole was added to a final concentration of 100 ng/ml. Cells undergoing NEBD were marked and monitored at 10 min intervals to determine when they decondensed their chromosomes. The duration of arrest in mitosis, which is defined as the interval between NEBD (onset of mitosis) and chromatin decondensation (exit from mitosis without cytokinesis), was then calculated and plotted. For checkpoint silencing and sensitivity experiments, 500 nM or 2 µM AZ3146 (Sigma-Aldrich) was added either concurrently or in sequence with nocodazole. All experiments were performed on at least three independently generated MEF lines unless stated.