5 μM of 1 (○), 2 (▲) and 3 (×) or vehicle (□). The number of viable cells was determined daily. Three independent experiments were evaluated. Error bars indicate SD. b: Histograms show the percentage of growth inhibition of BJ-EHLT and BJ-hTERT cells treated with 0.5 μM of each compound versus untreated samples at the indicated times. Consequently, the ability of the Ku-0059436 ic50 new-generated G-quadruplex ligands 2 and 3 to cause telomere uncapping has been investigated. To this aim, a two-steps analysis was performed to establish, in a first case, if the compounds are able to induce DNA damage and, secondly, if the DNA

damage is localized at the telomeres. Immunofluorescence analysis performed to evaluate the phosphorylation of H2AX, a hallmark of DNA double-strand break, showed that all the compounds activated a DNA damage response pathway (Figure  6a). However, the quantitative analysis revealed that the compound 2 induced a percentage of cells positive for γH2AX quite similar to compound 1, while, consistently with the above reported data on cell proliferation (Figure  5), 3 is less potent this website than the lead compound (*P < 0.05), in activating the damage response pathway.

Selleck MAPK Inhibitor Library Figure 6 Activation of DNA damage response. Human transformed BJ-EHLT fibroblasts were treated with 0.5 μM of 1, 2 and 3 for 24 hrs, then fixed and processed for IF analysis with anti-γH2AX antibody, and counterstained with DAPI to mark nuclei. a: Representative images of IF at 63× magnification. b: Histograms shows the percentage of γH2AX-positive cells scored by immunofluorescence C1GALT1 analysis

(*P < 0.05). These results encouraged us to undertake further studies aimed to investigate the telomere specific effects of the ligands, analyzing whether γH2AX was phosphorylated in response to dysfunctional telomeres. Deconvolution microscopy showed that, similarly to 1, some of the damaged foci induced by 2 and 3 co-localized with TRF1, an effective marker for interphase telomeres, forming so-called Telomere dysfunction Induced Foci (TIFs) [34] (Figure  7a). Quantitative analysis revealed that treatment with 2 increased the percentage of cells with more than four γH2AX/TRF1 co-localizations (indicated as TIF-positive cells), at comparable levels with respect to 1, while 3 had a significant but less pronounced effect. Consistently with these results, while 1 and 2 induced a superimposable number of TIFs per nucleus (ca. eight) the mean of telomere foci induced by 3 was reduced to six (Figure  7b, c). Figure 7 Induction of telomere damage and aberrations. Cells untreated or treated with 1, 2 and 3 for 24 hrs were fixed and processed for IF analysis against TRF1 and γH2AX. a: Representative images of IF were acquired with a Leica deconvolution microscope (magnification 100×). Enlarged views (2.5×) of treated merged images are reported. Histograms represent the Percentage of TIFs-positive cells. b: and average number of TIFs per nucleus.