We previously reported that telomestatin inhibits the binding of POT1 to the telomeric G-overhang in a coupled transcription and translation assay [42]. The use of purified recombinant POT1 protein under bandshift conditions indicated that telomestatin also inhibited the binding of POT1 to longer oligonucleotides (59-G3(T2AG3)7-39, IC50 of 500 nM [47]) and to Wt26 (IC50 of 100 nM) (Figure 2C). Interestingly, these values are in the same range that of telomestatin for inhibition of Topo III binding to telomere oligonucleotides (60– 300 nM). Since telomestatin was shown to uncap POT1 from telomeres in cell culture [42,48,49], we speculated that telomestatin may also alter recruitment of Topo III to telomeres. Treatment with telomestatin reduces levels of Topo III, TRF2, and BLM and disorganizes APBs in MRC5-V1 ALT cells Topo III co-localizes with telomeric proteins at APBs in ALT cells [39]. To examine the effects of telomestatin treatment on the recruitment of Topo III to these nuclear bodies in cultured cells, we stably transfected a YFP-tagged Topo III construct into MRC5-V1 ALT cells [39]. Using a D6 antibody directed against Topo III, it was shown that the tagged protein localizes at the same sites as does the endogenous protein ([39,50], see also Figure 4A). Therefore, it can be used to investigate the cellular effect of telomestatin on Topo III localization. The YFP-Topo III fluorescence signal, which mostly corresponded to large nuclear foci, was decreased after telomestatin treatment (Figure 4A). The immunofluorescence signal of the D6 antibody was also decreased in telomestatin-treated cells, indicating that telomestatin alters Topo III protein levels (Figure 4A). Further analysis by western blot showed that Topo III protein levels were decreased by telomestatin treatment in a dose- and time-dependent manner (Figure 4B). After 72 hours of treatment with 2 mM telomestatin, Telomestatin Uncaps Telomeres we observed a nearly complete loss of Topo III protein (relative to actin). Since Topo III, BLM, and TRF2 form a complex in ALT cells [39], we next investigated if telomestatin treatment had an effect on the BLM and TRF2 protein levels. As shown in Figure 4B&C, the Topo III decrease was associated with a significant decrease in BLM and TRF2 protein levels, but there was not significant induction of apoptosis as measured by caspase 3 cleavage (result not shown). This suggests that, like siRNA inhibition of Topo III translation [39], telomestatin triggers Topo III/TRF2/BLM complex degradation in ALT cells. To further analyze the effect of telomestatin on organization of APBs in MRC5-V1/YFP-Topo III cells, we treated cells with 2 mM telomestatin for 48 h, because these conditions measurably reduce Topo III, TRF2 and BLM protein levels but cells remain viable. This allowed us to perform nuclear localization studies by microscopy. Microscopic examination of treated cells showed important changes in the nuclear organization of YFP-Topo III at APBs. Telomestatin reduced the co-localization of YFP-Topo III with PMLs (Figure 5A) and with TRF2 (Figure 5B). Double staining with anti-TRF2 and with anti-PML antibodies allowed us to visualize these APBs components in MRC5-V1/YFP-Topo III cells. After telomestatin treatment, YFP-Topo III, TRF2, and PML were observed in separate foci (Figure 6). Similar loss of co- localization was observed between YFP-Topo III and two other factors of the shelterin complex, TIN2 and TRF1 (Figure 7 A and B). After telomestatin treatment, only 20% of YFP-Topo III co- localized with PML and TRF2 and only about 45% of YFP-Topo III co-localized with TRF1 or TIN2 (Figure 8A). Interestingly, telomestatin did not qualitatively alter the co-localization between YFP-Topo III and BLM (Figure 8A and B); however, BLM protein levels decreased after telomestatin treatment as shown by western-blot (Figure 4B), suggesting that telomestatin does not Figure 6. Co-localization of Topo III with PML and TRF2 is inhibited by telomestatin treatment. Representative images are of untreated MRC5-V1/YFP-Topo III cells and cells treated with 2 mM telomestatin for 48 h; YFP-tagged Topo III is shown in green, TRF2 is shown in red (detected by immunofluorescence), and PML is shown in blue (detected by immunofluorescence). doi:10.1371/journal.pone.0006919.g006 PLoS ONE | www.plosone.org 7 September 2009 | Volume 4 | Issue 9 | e6919