BLM and PICH in Centromere Disjunction Figure 2. BLM localizes to centromeres in mitotic cells. (A) Endogenous BLM localizes to centromeres in HeLa cells. Wide-field microscopy after immunofluorescence staining on chromosome spreads obtained by cytocentrifugation of BLM siRNA-transfected or control siRNA-transfected HeLa S3 cells. Staining is shown for BLM (green), CREST (red) and chromosomes (blue). Single chromosome magnifications are shown (upper panels). Proteins levels were assessed by immunoblotting, probing the same membrane with anti-BLM (ab-476) antibody and then with anti-b actin antibody, as a loading control (lower left panel). Quantification of centromeric fluorescence signals for BLM (normalized according to the CREST signal) for a total of 20 centromeres in control cells (siCtrl) and 24 centromeres in BLM-depleted cells (siBLM) from two independent experiments, demonstrate the specificity of ab476 BLM antibodies (lower right panel). Data are means and SD normalized with respect to controls. Scale bar = 5 mm. (B) Centromeres detected with CREST serum (red) from all the chromosomes of HeLa siCtrl cells and HeLa siBLM cells from Figure 2A were analyzed for BLM signals (green). Chromosomes were visualized by DAPI staining (blue) (left panels). The same analysis was performed on a total of 9 HeLa siCtrl cells and 9 HeLa siBLM cells from two independent experiments: the percentage of centromeres giving a BLM signal is shown (right panel). doi:10.1371/journal.pone.0033905.g002 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e33905