Journal: Nucleic Acids Research
Article Title: Naphthalene diimide-naphthalimide dyads promote telomere damage by selectively targeting multimeric G-quadruplexes
doi: 10.1093/nar/gkaf301
Figure Lengend Snippet: Biological activity evaluation of RHPS4 and Pyridostain. ( A , B ) Immunofluorescence evaluation of G4 structures in human cervical HeLa (A) and osteosarcoma U2OS (B) cancer cell lines. The cells were treated with the telomere-directed pentacyclic acridine 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and the non-telomere-directed N,N′-bis (quinolinyl)pyridine-2,6-dicarboxamide (Pyridostatin, PDS) at the final concentration of 0.5 μM for 24 h and then processed for immunofluorescence with the specific antibody for G4 structures (anti-BG4). Left panel s : representative images acquired by confocal microscopy (magnification 63×). G4 structures are labeled in red and the nuclei are stained with DAPI (blue). 4× enlargements of selected regions (white squares) are shown. Scale bars indicate 20 μm. Right panels: Quantification of the number of G4 structures per cell. At least 30 cells for condition were counted. The histograms represent the mean values ± S.D. of three independent experiments. * P < .05, ** P < .01, and *** P < .001. ( C , D ) Cell viability assays performed in human foreskin-derived fibroblast (BJ), expressing the human telomerase reverse transcriptase hTERT and SV40 early region (BJ-EHLT) or only hTERT (BJ-hTERT) and treated with PDS and RHPS4 at the final concentrations of 0.1, 0.5, and 1 μM for 48 (C) and 96 (D) h. The results were expressed as the percentage of cell viability over the untreated cells. (E–G) BJ-EHLT cells were treated with the indicated compound for 24 h at a final concentration of 0.5 μM and, successively, processed for telomeric FISH combined with immunofluorescence experiments using the antibody against γH2AX, a marker of the DNA damage. ( E ) Representative images acquired by confocal microscopy (magnification 63×). Scale bars indicate 10 μm. ( F ) Percentage of γH2AX positive cells. ( G ) Quantitative analysis of the number of TIF per cell and of the percentage of TIF positive cells. Cells with at least four γ-H2AX/Telo colocalizations were scored as TIF positive. The histograms represent the mean values ± S.D. of three independent experiments. * P < .05, ** P < .01, *** P < .001, and **** P < .0001.
Article Snippet: BJ-hTERT cells were obtained infecting primary BJ cells with a retrovirus carrying hTERT (Addgene plasmid #1773); BJ-EHLT derived from the transformation of BJ fibroblasts with hTERT and SV40 early region [ ].
Techniques: Activity Assay, Immunofluorescence, Concentration Assay, Confocal Microscopy, Labeling, Staining, Derivative Assay, Expressing, Reverse Transcription, Marker
Addgene inc is a verified supplier 
![Biological activity evaluation of RHPS4 and Pyridostain. ( A , B ) Immunofluorescence evaluation of G4 structures in human cervical HeLa (A) and osteosarcoma U2OS (B) cancer cell lines. The cells were treated with the telomere-directed pentacyclic acridine 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and the non-telomere-directed N,N′-bis (quinolinyl)pyridine-2,6-dicarboxamide (Pyridostatin, PDS) at the final concentration of 0.5 μM for 24 h and then processed for immunofluorescence with the specific antibody for G4 structures (anti-BG4). Left panel s : representative images acquired by confocal microscopy (magnification 63×). G4 structures are labeled in red and the nuclei are stained with DAPI (blue). 4× enlargements of selected regions (white squares) are shown. Scale bars indicate 20 μm. Right panels: Quantification of the number of G4 structures per cell. At least 30 cells for condition were counted. The histograms represent the mean values ± S.D. of three independent experiments. * P < .05, ** P < .01, and *** P < .001. ( C , D ) Cell viability assays performed in human foreskin-derived fibroblast (BJ), expressing the human telomerase reverse transcriptase hTERT and SV40 early region (BJ-EHLT) or only hTERT (BJ-hTERT) and treated with PDS and RHPS4 at the final concentrations of 0.1, 0.5, and 1 μM for 48 (C) and 96 (D) h. The results were expressed as the percentage of cell viability over the untreated cells. (E–G) BJ-EHLT cells were treated with the indicated compound for 24 h at a final concentration of 0.5 μM and, successively, processed for telomeric FISH combined with immunofluorescence experiments using the antibody against γH2AX, a marker of the DNA damage. ( E ) Representative images acquired by confocal microscopy (magnification 63×). Scale bars indicate 10 μm. ( F ) Percentage of γH2AX positive cells. ( G ) Quantitative analysis of the number of TIF per cell and of the percentage of TIF positive cells. Cells with at least four γ-H2AX/Telo colocalizations were scored as TIF positive. The histograms represent the mean values ± S.D. of three independent experiments. * P < .05, ** P < .01, *** P < .001, and **** P < .0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0870/pmc12000870/pmc12000870__gkaf301fig7.jpg)