Telomestatin Uncaps Telomeres alter the interaction between these two proteins. These results indicate that the G-quadruplex ligand telomestatin decreases the amount of Topo III/BLM/TRF2 complex present in cells and disrupts the formation of APBs. Co-immunoprecipitation experiments were also performed to study whether the interaction of TRF2 or BLM with Topo III is altered by telomestatin treatment. In the complex precipitated by the Topo III D6 antibody, we detected TRF2 and BLM by immunoblotting (Figure S1 and [39]). After 48 hours of treatment with 2 mM telomestatin, we observed a nearly complete loss of TRF2 protein associated with Topo III. In contrast, BLM associated with Topo III is not affected by telomestatin treatment. These results confirm the immunofluorescence observations. Telomestatin induces a massive DNA damage response at telomeres in ALT cells To further determine whether telomestatin-induced ABP disrup- tion affected telomere integrity, we tested MRC5-V1/YFP-Topo III cells for DNA damage that co-localized with telomeres. Telomere dysfunction-induced foci (TIFs) can be detected due to the presence of c-H2AX, a phosphorylated variant of histone 2A that associates with DNA double-strand breaks [51]. Cells treated with 2 mM telomestatin Figure 7. Telomestatin induces a de-localization of Topo III from TIN2 and TRF1. Representative images are of untreated MRC5-V1/YFP- Topo III cells or cells treated for 48 h with 2 mM telomestatin. A, Topo III was detected by fluorescence of YFP-tagged Topo III (green) and TIN2 was detected by immunofluorescence (red). Dapi staining of DNA is shown in blue. B, Topo III was detected by fluorescence of YFP-tagged Topo III (green) and TRF1 was detected by immunofluorescence (red). Dapi staining of DNA is shown in blue. The amount of co-localization between Topo III and TIN2 or Topo III and TRF1 observed in untreated cells was decreased by telomestatin treatment. doi:10.1371/journal.pone.0006919.g007 PLoS ONE | www.plosone.org 8 September 2009 | Volume 4 | Issue 9 | e6919