activity was inhibited in cultured cells by treatment with 4,5,6,7-tetrabromo-2-azabenzamidazole, initially a gift from Prof. E. Pinna, latterly from Sigma.43 TBB was diluted directly into culture medium to a working concentration of 100 M from a 10 mM stock in DMSO. Caspase assays. Cells were seeded at 1 x 105 per well in 24-well plates, and cultured for 16 h before induction of apoptosis. Cells were treated with 250 ng/ml recombinant TRAIL for the times indicated, then lysed in 150 200 l of MPER buffer containing 1 mM EDTA, 1 g/ l pepstatin A, 1 mM AEBSF for 45 minutes and centrifuged at 13,000 rpm to remove debris. Caspase activity assays were performed in a 96-well microtiter plate: 5 g of caspase-3 substrate was incubated with 20 l of cell lysate and 200 l of reaction buffer at 37C for 1 h. Fluorescent emission was measured using a Spectrofluorometer with excitation and emission wavelengths set at 390 and 450 nm respectively. 546 Cell Cycle Volume 10 Issue 3 GST pull downs. All genes encoding GST tagged recombinant proteins were cloned in pGEX4T1, and protein expression induced in BL21 E. coli with 0.5 mM IPTG at 1820C, using standard methods, see reference 42. Recombinant proteins were bound to glutathione-sepharose 4B beads in lysis buffer, 0.05% triton X-100, 100 M PMSF and 1 g/ml CLAP) by incubation for 1 h at 4C with rotation. For in vitro translation proteins were transcribed and translated using the T7 or T3 T NT coupled reticulocyte lysate system with approximately 0.04 mCi of 35 S methionine as tracer. After binding to the beads, GST-lysates were washed with PBS then incubated with Dynamic mechanisms in the eukaryotic cell exist to ensure high fidelity segregation of genetic material during cell division. Chromosome segregation is delayed by cell cycle MedChemExpress ONX-0914 checkpoints until DNA replication is complete and sister chromatids are fully aligned at the metaphase plate. Mitotic checkpoint failure, in the absence of cell death, results in aneuploid progeny Key words: Bub1, mitotic checkpoint, Aurora B, chromosome segregation, aneuploidy Submitted: 09/15/11 Revised: 09/19/11 Accepted: 09/20/11 http://dx.doi.org/10.4161/cc.10.21.18156 Correspondence to: Jan M. van Deursen; Email: [email protected] when chromosome segregation is imbalanced. Aneuploidy refers to an abnormal karyotype that is not a multiple of the haploid complement. Although aneuploidy is a common feature of tumors and correlates with poor clinical prognosis,1-4 how aneuploidy contributes to the oncogenic process is currently a matter of debate. Inaccurate chromosome segregation during mitosis is one potential source of aneuploidy PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19822626 in tumors.5 The rate at which whole chromosomes are gained or lost is referred to as whole chromosomal instability. The mitotic checkpoint delays anaphase onset until all kinetochores are properly attached to the spindle, thereby protecting against chromosome missegregation.6 Recent mouse models have shown that aberrant chromosome segregation not only can correspond to tumorigenesis in certain contexts, but may in fact act as an initiator.7-13 Moreover, kinetochores that 
are aberrantly attached to microtubules are detected and resolved by the error correction machinery, of which the principal component is the mitotic kinase Aurora B.14-18 While reduced Aurora B activity is known to cause both misaligned and lagging chromosomes during mitosis,19-25 less is known about the impact of increased Aurora B activity on segregation fidelity. More
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