2)), mouse anti-cyclin B1 (1500 for IF, sc-245 from Santa Cruz), mouse anti-Rad21 (11000 for IB, 53A303 from Upstate), mouse anti-Topo II alpha (11000 for IB and IF, 3D4 from Assay Style), mouse anti-Topo II alpha (1250 for IP, KiS1 from Millipore), anti–actin (110000 for IB; Sigma), anti-GAPDH (110000 for IB; Millipore), anti-PICH (1200 for IF and 1750 for IB, Perform1P from Abnova) and anti-CENP-A (1200 for IF, Stomach13939 from Abcam)

2)), mouse anti-cyclin B1 (1500 for IF, sc-245 from Santa Cruz), mouse anti-Rad21 (11000 for IB, 53A303 from Upstate), mouse anti-Topo II alpha (11000 for IB and IF, 3D4 from Assay Style), mouse anti-Topo II alpha (1250 for IP, KiS1 from Millipore), anti–actin (110000 for IB; Sigma), anti-GAPDH (110000 for IB; Millipore), anti-PICH (1200 for IF and 1750 for IB, Perform1P from Abnova) and anti-CENP-A (1200 for IF, Stomach13939 from Abcam). immunoblotting, probing the same membrane with anti-BLM (ab-476) and anti-PICH antibodies L-NIL and with anti- actin antibody, being a launching control (higher left -panel).(TIF) pone.0033905.s004.tif (770K) GUID:?FCF1E981-4F56-4480-8292-6B36C0E488F4 Body S4: BLM-downregulated and PICH-downregulated HeLa cells screen non disjunction of centromeres. HeLa cells had been transfected for 72 hours with Rad21 Rabbit Polyclonal to MASTL siRNAs and transfected with control siRNAs, BLM siRNAs or PICH siRNAs. BLM, Rad21 and PICH proteins amounts had been evaluated by immunoblotting, probing the same membrane with anti-BLM (ab-476), anti-Rad21 and anti-PICH antibodies and with anti- actin antibody, as a launching control (still left -panel). Chromosome spreads had been performed and sorted based on their phenotype: X-shapes, imperfect disjunction or comprehensive disjunction. We examined 500 spreads from two indie experiments for every cell series. The frequency of every phenotype, in each one of the three cell lines, is certainly proven in the histogram (correct panel). Bars signify SD.(TIF) pone.0033905.s005.tif (721K) GUID:?4DB1EBD6-5C2A-4B79-A586-C68D8EF0C803 Figure S5: siRNA-mediated Rad21 downregulation was similarly effective in every conditions. HeLa or GFP-BLM cells were transfected for 72 hours using the indicated siRNAs. Rad21 mRNA amounts were dependant on invert transcription quantitative PCR in every conditions. Histograms signify the amplification of Rad21 mRNA in one test in triplicate for GFP-BLM and BS cells and so are the mean of the amplification of Rad21 mRNA in triplicate in two independent experiments for HeLa cells. Bars represent s.e.m.(TIF) pone.0033905.s006.tif (648K) GUID:?100CA461-923B-4298-87ED-06490C984053 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. L-NIL The nucleus was visualized by DAPI staining (blue). Scale bar?=?5 m. (B) Total number of UFBs detected and scored in PICH-downregulated cells. UFBs positive for PICH only or for BLM L-NIL only or for both PICH and BLM were scored in three independent experiments including a total of 129 cells transfected with control siRNAs and 129 cells transfected with PICH siRNAs.(TIF) pone.0033905.s007.tif (1.5M) GUID:?710E7409-73B5-4567-BD6B-D68B7A534E1D Abstract Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their L-NIL integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment L-NIL of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase II, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase II in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase II inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase II, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges. Introduction The centromere is a highly differentiated chromosomal structure consisting, in human cells, of -satellite DNA repeats [1]. It plays an essential role in cell division, particularly in kinetochore assembly, and in ensuring the segregation of equal number of chromosomes to daughter cells during mitosis [2]. The maintenance of centromere stability is thus.