2009;137:472C84
2009;137:472C84. chaperone DAXX prevents CENP-A mislocalization and rescues the reduced interkinetochore distance and CIN phenotype in CENP-A overexpressing cells. In summary, our results establish that CENP-A overexpression and mislocalization result in a CIN phenotype in human cells. This study provides insights into how overexpression of CENP-A may contribute to CIN in cancers and underscore the importance of understanding the pathways that prevent CENP-A mislocalization for genome stability. and scaled manually to 8-bit using linear LUT and the same range of scaling for all the images. Quantitative immunofluorescence analysis To Pirarubicin Hydrochloride calculate fluorescence intensities, boxes of 8 8 pixels were Pirarubicin Hydrochloride drawn on centromeric region as ascertained by bright foci of CENP-A and/or CREST and on non-centromeric region as ascertained by the signal outside the centromeric region on a chromosome (chromosome spreads) or chromosomes aligned around the metaphase plate. For background, four boxes of 8 8 pixels were drawn at four random areas within the cytoplasm in the same cell. The maximum intensity values from all drawn areas were obtained using data inspector tool in to draw a straight collection between the brightest pixels of CENP-A or Nuf2 on two sister chromatids. Only congressed pairs of kinetochores in MG132 arrested metaphase cells were included for analysis. Orientation between two centromeric/kinetochore markers and focal plane were used as a basis Pirarubicin Hydrochloride for considering two kinetochores as a pair. For example, to consider two sister kinetochores as a pair in a cell immunostained with Nuf2 (outer kinetochore marker) and CENP-A (inner kinetochore marker), sister kinetochores should reside in the same focal plane and should orient Nuf2 towards spindle pole and CENP-A towards equatorial plate. The length of each collection was then calibrated based on a models/pixel and assigned in m. Interkinetochore distance was measured for at least 10 kinetochore pairs in a single cell and 8-15 cells from two impartial experiments. Average values from more than 100 kinetochore pairs were calculated and used as the imply to calculate the SEM across areas measured. Statistical analysis and R-lab, respectively. SUPPLEMENTARY MATERIALS FIGURES AND Furniture Click here to view.(1.2M, pdf) Acknowledgments We are grateful to Don Cleveland, Aaron Straight, Iain Cheeseman, Mary Dasso and Alexie Arnautouv for the nice gift of antibodies and guidance, Kathy McKinnon of the National Cancer Institute, Vaccine branch, FACS soreThomas Reid and Danny Wangsa for technical guidance, Anna Roschke and users of our laboratory for discussions and comments around the manuscript. Abbreviations CINChromosomal instabilityRPE1Retinal Pigmental EpithelialSACSpindle Assembly CheckpointCCANConstitutive Centromere Associated NetworkMAD1Mitotic Arrest DeficientKMNKnl1 Mis12 Ndc80GFPGreen Fluorescent ProteinNEBDNuclear Envelope BreakdownPFAParaformaldehydeANAAnti-Nuclear AntibodyPBSTPhosphate Buffered Saline TweenDAPI4,6-diamidino-2-phenylindole Contributed by Author contributions Experiments were Pirarubicin Hydrochloride designed and conceived by R.L.S. and M.A.B. All experiments were performed and analyzed by R.L.S. with help from M.I.S. for Figures ?Figures1A1A and ?and1B1B and G.S.A. for Figures ?Figures4B,4B, ?,4C4C and ?and5A.5A. Cell lines were generated by K.M.S., T.K. provided technical guidance for microscopic analysis and D.R.F. provided cell lines and intellectual contributions Gsk3b for the work. Manuscript was written by R.L.S. and M.A.B. All the authors read and agreed on the publication of this manuscript. CONFLICTS OF INTEREST No potential discord of interest FUNDING R.L.S., G.S.A., M.I.S. and M.A.B. were supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health. K.M.S. was supported by Department of Defense Visionary Postdoctoral Fellowship (W81XWH-13-1-0106). D.R.F. was supported by NIH R01GM111907 Recommendations 1. Weaver BA, Cleveland DW. Does Anauploidy cause cancer? Curr Opin Cell Biol. 2006;18:658C67. 0.1016/j.ceb.2006.10.002. [PubMed] [Google Scholar] 2. Holland AJ, Cleveland DW. Losing balance: the origin and impact of aneuploidy in cancer. EMBO Rep. 2012;13:501C14. doi:?10.1038/embor.2012.55. [PMC free article] [PubMed] [CrossRef] [Google Scholar] 3. Lengauer C, Kinzler KW, Vogelstein B. Genetic Instability in colorectal cancers. Nature..