DXC 930 P camera. Centromeric disjunction classification was evaluated by retrieving intensity profiles with the ‘‘plot profile function’’ of ImageJ applied to distance-calibrated images, along a line placed over the primary constriction region. For chromosome immunolabeling, chromosome spreads were obtained by cytocen- trifugation and processed as previously described [27]. Topo II ChIP Analysis Cells (106) were treated with 0.5 mM etoposide for 15 min. They were then lysed in 1.5 ml of buffer containing 1% Sarkosyl, 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, and protease inhibitor mixture (Complete Mini, Roche) by 10 passages through a 21G needle. CsCl was added from a concentrated stock solution (7 M), to give a final concentration of 0.5 M. DNA was fragmented by sonicating lysates with a Bioruptor sonicator (Diagenode), with 20- second pulses (20 seconds on/20 seconds off), for 15 minutes at the high-power setting. Under these conditions, DNA fragments of 0.5 to 3 kb were generated, as shown by agarose gel electrophoresis. Before immunoprecipitation (IP), we added 3 volumes of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 100 mM NaCl, 0.1% Triton X-100, and protease inhibitor mixture to 1 volume of lysate. The resulting mixture was then centrifuged for 15 min at 16 000 6 g at 4uC. Preclearing was then achieved by adding 60 ml of Protein G plus agarose beads (Santa Cruz) and rotating the tubes for 2 h at 4uC. The unbound fraction recovered by centrifugation was used for IP reactions. It was incubated overnight, at 4uC, with gentle shaking, with 5 mg of specific anti- Topo II antibody (KiS1) or control antibody (mouse IgG). Protein G plus agarose beads (Santa Cruz) were added and the mixture was incubated for 1 h. The beads were collected by centrifugation and washed three times with 150 mM NaCl buffer (10 mM Tris- HCl pH 7.5, 10 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, protease inhibitor). Three rounds of elution were then carried out with 500 mM NaCl buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 500 mM NaCl, 0.1% Triton X-100, protease inhibitor), with the beads collected by centrifugation between elutions. The DNA fragments bound to the beads were treated with 50 mg/ml RNase A, at 55uC, for 30 min, then with 200 mg/ml proteinase K at 55uC overnight. They were then purified with the MiniElute PCR Purification kit (QIAGEN), according to the manufacturers’ instructions. DNA fragments were quantified with the Quant-IT Pico Green dsDNA kit (InvitrogenTM, Molecular ProbesH), according to the manufacturer’s protocol. Alpha-satellite DNA (20 pg) from chromosome X or chromosome 17 (DXZ1 and D17Z1, respectively) [31] or from pericentromeric DNA satellite 3 [32] was amplified by semiquantitative PCR. PCR products were analyzed by agarose gel electrophoresis and quantified with Image J (NIH) software. The signal obtained from the GFP-BLM cells was taken as 1. Electron Microscopy BS and GFP-BLM cells grown at low density on coverslips were fixed by incubation with 2% paraformaldehyde, 2.5% glutaralde- hyde in 0.1 M cacodylate buffer for 24 h and postfixed by incubation with 1% (wt/vol) OsO4 supplemented with 1.5% (wt/ vol) ferrocyanide. They were then dehydrated in ethanol and embedded in Epon. Ultrathin sections were prepared with a Reichert UltracutS ultramicrotome (Leica) and viewed with a TEM CM120 Philips electron microscope after counterstaining with uranyl acetate and lead citrate. Statistical Methods Significance was assessed with Student’s t-test. For all tests, p,0.05 was considered statistically significant. BLM and PICH in Centromere Disjunction Supporting Information Table S1 (XLS) Sequences of the siRNAs used in this study. Figure S1 GFP-BLM localizes to the PML body. Coloca- lization of GFP-BLM (green) with PML (red). Nuclei were visualized by DAPI staining (blue). Scale bar = 5 mm. (TIF) Figure S2 Kinetochore structure is not profoundly affected in BLM-deficient cells. CENP-A immunostaining of BS and GFP-BLM metaphase cells revealed a similar localization in the two types of cells. Chromosomes are visualized by DAPI staining (red). Scale bar = 5 mm. Similar results were obtained with 5 representative kinetochore/centromere proteins (AURORA B, CENP-I, HEC, BUB1 and BUBR1). (TIF) Figure S3 Structural defects at the centromeres in BLM- and PICH-deficient HeLa cells. Comparison of the volume of the centromeric FISH signal detected on chromosomes 8 from HeLaV-siCtrl (defined as 1), HeLa V-siPICH, HeLash-siBLM and HeLash-siBLM-siPICH cells (right panel). We analyzed between 18 and 27 metaphase cells from two independent experiments for each cell line. BLM and PICH protein levels were assessed by immunoblotting, probing the same membrane with anti-BLM (ab- 476) and anti-PICH antibodies and with anti-b actin antibody, as a loading control (upper left panel). (TIF) Figure S4 BLM-downregulated and PICH-downregulat- ed HeLa cells display non disjunction of centromeres. HeLa cells were transfected for 72 hours with Rad21 siRNAs and transfected with control siRNAs, BLM siRNAs or PICH siRNAs. BLM, PICH and Rad21 protein levels were assessed by immunoblotting, probing the same membrane with anti-BLM (ab-476), anti-PICH and anti-Rad21 antibodies and with anti-b actin antibody, as a loading control (left panel). Chromosome spreads were performed and sorted on the basis of their phenotype: X-shapes, incomplete disjunction or complete disjunc- tion. We analyzed 500 spreads from two independent experiments for each cell line. The frequency of each phenotype, in each of the three cell lines, is shown in the histogram (right panel). Bars represent SD. (TIF) Figure S5 siRNA-mediated Rad21 downregulation was similarly effective in all conditions. GFP-BLM or HeLa cells were transfected for 72 hours with the indicated siRNAs. Rad21 mRNA levels were determined by reverse transcription quantitative PCR in all conditions. Histograms represent the amplification of Rad21 mRNA from one experiment in triplicate for GFP-BLM and BS cells and are the mean of the amplification of Rad21 mRNA in triplicate in two independent experiments for HeLa cells. Bars represent s.e.m. (TIF) Figure S6 Centromeric UFBs are not detectable in PICH-deficient cells. (A) (Upper panels) GFP-BLM cells were transfected for 72 hours with PICH or control siRNAs. UFBs were detected by immunostaining of BLM and PICH and quantified (164 UFBs from 129 siCtrl cells and 21 UFBs from 129 siPICH cells were scored in three independent experiments, respectively). Bars represent SDs. (Lower panel) Representative example of a UFB in anaphase cells revealed by BLM staining. The nucleus was visualized by DAPI staining (blue). Scale bar = 5 mm. (B) Total number of UFBs detected and scored in PICH-downregulated PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e33905