For staining with anti-neurotactin, embryos were heat-fixed as described previously

ls were scored based on the chromosome morphology. Site-directed mutagenesis INCENP point mutants were generated by site-directed mutagenesis using the plasmid pTrAP-GgINCENP class I and transferred into the knockout cells by electroporation.Quantitative analysis Growth curves were generated by seeding the various cell lines at 2 105 cells/ml at 39C and counting the cell number every 12 h until 48 h. To avoid the effects of overgrowth, cells were diluted to 4 105 cells/ml whenever the number exceeded 106 cells/ml. The cell number at each time point was multiplied by the RS-1 web appropriate dilution factor to get a true count. To determine the multinucleation or mitotic index, a total of 500 cells were scored at each time point for each individual experiment. The multinucleation index was calculated by dividing the number of multinucleated interphase cells by the total number of interphase cells. The mitotic index was calculated by dividing the number of mitotic cells by the number of interphase and mitotic cells. To assess the distribution of mitotic phases, cells were subject to immunofluorescence using appropriate antibodies. A total of at least 200 mitotic cells were scored at each time point. The frequency of multipolar spindles was determined in an independent experiment by dividing the number of mitotic cells with more than two spindle poles by the total number of 200 mitotic cells. Each quantitation was performed independently at least three times. Quantitative immunoblotting Membranes were first incubated with primary antibodies recognizing aurora B, -tubulin, H3S10ph, and subsequently with IRDye-labeled secondary antibodies. The fluorescence intensities were subsequently determined using a charge-coupled device scanner PubMed ID: according to the manufacturer’s instructions. Quantitative real-time RT-PCR Total RNA was extracted from DT40 cells using TRizol following the manufacturer’s instructions.Spindle checkpoint assay Exponentially growing cells were treated with 10 or 100 nM taxol/0.5 g/ml nocodazole/2 M ZM447439 for 9 h. Cells were plated on polylysinecoated slides, fixed with 4% PFA, and stained by DAPI. A total of 200 cells were scored from each sample to determine mitotic index. Experiments were performed at least three times. Online supplemental material Fig. S1 shows that INCENP class I is sufficient to keep DT40 cells alive. Fig. S2 shows distribution of Survivin and Borealin in cells expressing only mutant INCENP. Fig. S3 shows distribution of aurora B and CENP-A and levels of histone H3 phosphorylation in INCENPOFF cells expressing various INCENP mutants.This process, on which all cells rely to remain viable, is usually executed with great accuracy. Its perturbation results in aberrations in chromosome numbers, which are a cause of disease and cor relate with cellular transformation. Fidelity of cell division is the result of feedback controls. The first control mechanism halts the process of cell division and imposes a mitotic arrest when chromosomemicrotubule attachment is perturbed in different ways.This was confirmed in experiments in which cells were treated with concentrations of nocodazole that cause partial or complete microtubule depolymeri zation, respectively. The addition of reversine caused a dosedependent reduction in the timing of mitotic arrest, and the override was complete at 1.0 M rever sine at either concentration of nocodazole. At lower concentrations of reversine, the effects on the duration of the checkpoint w