BLM and PICH in Centromere Disjunction Figure 5. PICH and BLM are required for the recruitment of Topo IIa to centromeres (A) ChIP-PCR analysis of Topo IIa recruitment to pericentromeric region satellite 3 (Sat3) or to the centromeric DNA of chromosomes X (DXZ1) or 17 (D17Z1) was performed in GFP- BLM and BS cells (lower panels). The histogram shows relative enrichment in DXZ1 (DXZ1/Sat3) or D17Z1 (D17Z1/Sat3) sequences for active Topo IIa in GFP-BLM (defined as 1) and in BS cells (upper panels). The results presented are from three independent experiments. Error bars indicate the SD. (B) We performed the same experiments as in (A), with GFP-BLM cells transfected for 72 hours with control siRNAs or PICH siRNAs. (C) The amount of Topo II was evaluated in each cell line by western blotting. (D) We performed the same experiments as in (A), with GFP-BLM, GFP-I841T and BS cells. doi:10.1371/journal.pone.0033905.g005 for IB and IF, 3D4 from Assay Design), mouse anti-Topo II alpha (1:250 for IP, KiS1 from Millipore), anti-b-actin (1:10000 for IB; Sigma), anti-GAPDH (1:10000 for IB; Millipore), anti-PICH (1:200 for IF and 1:750 for IB, DO1P from Abnova) and anti- CENP-A (1:200 for IF, AB13939 from Abcam). Secondary antibodies conjugated to Alexa Fluor 488, 555 and 633 (Molecular Probes, 1:200-600) were used for immunofluorescence and secondary antibodies conjugated to horseradish peroxidase (Santa Cruz, 1:5000-10000) were used for immunoblot detection. Reverse Transcription and Real-time Quantitative PCR Total RNA was extracted with the RNeasy Mini kit (Qiagen) including a DNAse digestion step. cDNAs were synthesized with 250 ng of random hexamers (Invitrogen), 2 mg of RNA and Superscript II reverse transcriptase (Invitrogen). qPCR experi- PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e33905