Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect
J. Clin. Invest. Erica Salvati, et al. 117:3236 doi:10.1172/JCI32461 [Go to this article.]

Figure 4
RHPS4 specifically and rapidly delocalizes POT1 from telomeres. Transformed BJ-EHLT fibroblasts were treated with RHPS4 and double stained with the indicated antibodies. (A) Representative confocal images showing merged TRF1 (green) and TRF2 or POT1 (red) staining in untreated and treated cells. (B) Percentages of cells with more than 4 colocalizations per nucleus of TRF1 and TRF2 (black bars) and of TRF1 and POT1 (gray bars). Error bars indicate SD. (C) Western blot analysis of TRF2 and POT1 in untreated (–) and RHPS4-treated cells (+) lysed with various concentrations of KCl (150, 300, and 450 mM). (D) Representative confocal images showing merged γ-H2AX (red) and TRF2 (green) or γ-H2AX (green) and POT1 (red) staining in untreated and RHPS4-treated cells. Original magnification, ×63; ×126 (enlarged panels). (E) Percentage of TIF-positive cells using TRF2 (black bars) and POT1 (gray bars) as telomeric proteins. On average, more than 80 cells were screened per point in 4 independent experiments. Error bars indicate SD. (F) Percentages of cells with more than 4 TRF1/TRF2 colocalizations. Error bars indicate SD. (G) BJ-EHLT fibroblasts were treated with RHPS4 for the indicated times or transfected with an expression vector carrying TRF2^ΔBΔC cDNA and processed for telomeric overhang assay under native conditions (left). Subsequently, the DNA was denatured in the gel and rehybridized with the same probe (right). Where indicated, the DNA was incubated with Exonuclease I (ExoI). Signals were measured using ImageQuant software by integration of the entire lane.