BLM and PICH in Centromere Disjunction Figure 4. BLM-deficient cells and PICH-downregulated display non disjunction of centromeres and impaired recruitment of active Topo IIa to centromeres. (A) GFP-BLM and BS cells were transfected for 72 hours with Rad21 siRNAs and transfected either with control siRNAs or with PICH siRNAs. BLM, PICH and Rad21 proteins 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 (lower left panel). Chromosome spreads were performed and sorted on the basis of their phenotype: X-shapes, incomplete disjunction or complete disjunction. The scale bars indicate 5 mm (upper left panel). This classification is based on the intensity profiles of centromeres (upper right panel). We analyzed 500 spreads from three independent experiments for each cell line. The frequency of each phenotype, in each of the three cell lines, is shown in the histogram (lower right panel). Bars represent SD. (B) GFP-BLM, BS and GFP-I841T cells were transfected for 72 hours with Rad21 siRNAs and the same experiments as in (A) (right panels) were carried out. We checked the levels of BLM and Rad21 proteins by western blotting (left panels). doi:10.1371/journal.pone.0033905.g004 effect of the depletion of BLM and PICH on CEN-8 signal volume, suggesting that these proteins are involved in the same regulatory pathway. These results also confirmed the involvment of BLM and PICH in centromeric DNA structure before anaphase onset. BLM and PICH are Required for Complete Centromere Disjunction As BLM localizes to centromeres (Figures 1 and 2), we investigated whether, in addition to its potential role in resolving UFBs during anaphase [5,15], BLM might be involved in PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e33905