Behavior of a patient-derived human fibroblast line (PD-NC4) containing a mutated chromosome 4 in which the normal centromere is epigenetically silenced and a neo-centromere has formed 25 Mbp down the q arm at a site that lacks centromere repeats. Shown is a z-series of a metaphse spread stained with anti-centromere antibody (ACA, green) which labels both the epigenetically silenced centromere and the neocentromere of chromosome 4 which appears in the lower left quadrant in the z-slice displayed. Also stained for DNA with DAPI (blue). See Fig 1A in Bassett et al. 2010. Epigenetic centromere specification directs aurora B accumulation but is insufficient to efficiently correct mitotic errors. J Cell Biol 190:177-185.
Cells were metaphase-arrested with nocodazole, swollen in 75 mM KCl, centrifuged onto glass slides, and fixed with formaldehyde before processing for indirect immunofluorescence. Digital z-series images were captured with an ORCA AG CCD mounted on DMI6000B Leica microscope using a 100X 1.4 NA objective lens and subsequently processed by deconvolution.
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