BLM and PICH in Centromere Disjunction Figure 6. Model of PICH/BLM-dependent decatenation of centromeric DNA. We propose that the combined action of BLM and PICH promotes the organization of centromeric chromatin, thereby rendering some centromeric catenates accessible to Topo IIa. In the absence of BLM or PICH, a defect in the recruitment of active Topo IIa results in the persistence of some catenations leading to centromeric non disjunction and the formation of additional UFBs. doi:10.1371/journal.pone.0033905.g006 ments were performed according to the MIQE Guidelines [24]. Amplification mixtures contained the cDNA template (1/100 dilution), SYBR Green Supermix 16 (BioRad) and 300 nM forward and reverse primers. Amplification was performed with the CFX96 detection system (BioRad). The primer sequences for Rad21 have been described on the website www.rtprimerdb.org (RTPrimerDB ID:8036). The relative quantities of the Rad21 cDNAs were normalized against two reference genes (RPL32, SDHA) chosen on the basis of their low M-value [25,26]. Immunofluorescence Microscopy Immunofluorescence staining was performed as previously described [27] with the inclusion of a prepermeabilization incubation with 0.5% Triton X-100 before fixation for the detection of chromatid-bound protein. Nuclear DNA was detected by mounting slides in ProlongH Gold antifade reagent supple- mented with DAPI (Invitrogen). Cell images were acquired with a 3-D deconvolution imaging system consisting of a Leica DM RXA microscope equipped with a piezoelectric translator (PIFOC; PI) placed at the base of a 63x PlanApo N.A. 1.4 objective, and a CoolSNAP HQ interline CCD camera (Photometrics). Stacks of conventional fluorescence images were collected automatically at a Z-distance of 0.2 mm (Metamorph software; Molecular Devices). Images are presented as maximum intensity projections generated with ImageJ software, from stacks deconvolved with an extension of Metamorph software [28]. FISH Analysis FISH was performed with the CEP-8 probe (CEN-8) from Abbott Vysis according to the manufacturer’s instructions with fixation of cells in paraformaldehyde. Images were acquired on a Leica SP5 confocal system, equipped with an argon laser, with 405 nm and 561 nm laser diodes, using a 636/1.4 objective. All recordings were made with the appropriate sampling frequency (5126512 images, line average of 8 and zooming set to 8, pixel size: 60.2 nm). Image processing and quantification were carried out with the freely available Image J software [29]. FISH signal volume was determined with the 3D object counter plugin to Image J software [30]. A binary 3D image was generated, and a tagged map of all objects was obtained, based on pixel connexity (one tag per independent FISH signal, 26-neighbor connexity). The number of voxels (i.e. 3D pixels) carrying each tag was retrieved, then multiplied by the unit volume of the voxel (64.5 nm 6 64.5 nm 6 200 nm), to obtain the volume of each object. More information is available from the documentation supplied for the plugin: (http://imagejdocu.tudor.lu/lib/exe/fetch. php?media = plugin:analysis:3d_object_counter:3d-oc.pdf). Chromosome Spreading and Immunolabeling Cells were left untreated or were transfected as indicated. After 48 h of transfection with an siRNA specific for Rad21, cells were transferred to slides in 6-well plates. After 24h at 37uC, colchicine was added to a final concentration of 0.1 mg/ml and the cells were incubated for 1 hour. The cells were then incubated in hypotonic solution (1:5 (vol/vol) FCS-distilled water) and fixed by incubation with a 3:1 (vol/vol) mixture of methanol and acetic acid. Cells were then stained by incubation with 2% Giemsa solution (VWR) for 16 minutes, rinsed in distilled water, dried, and mounted. Chromosomes were observed with a Leica DMRB microscope at 1006 magnification. Metaphases were captured with a SONY PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e33905