Journal: Biomolecules

Article Title: Synergistic Effects of UVB and Ionizing Radiation on Human Non-Malignant Cells: Implications for Ozone Depletion and Secondary Cosmic Radiation Exposure.

doi: 10.3390/biom15040536

Figure Lengend Snippet: Figure 3. γH2AX staining in human non-malignant cells following co-exposure to protons and UVB. Results are presented as mean effect (value) ± SD for two independent experiments. The red line designates the mean value in control samples. (a) γH2AX foci per nucleus in Hs27 cels at 1 h, 4 h, and 24 h after exposure to 0.5 Gy protons or UVB (50 J/m2 or 100 J/m2) as single stressors, or to combined

Article Snippet: Samples were double stained with an antibody against histone H2AX (p S139) (80312T, Cell Signaling Technology, Danvers, MA, USA) and an antibody against 53PB1 (88439, Cell Signaling Technology, Danvers, MA, USA) in 0.5% BSA and 0.2% gelatin in PBS.

Techniques: Staining, Control

Journal: Biomolecules

Article Title: Synergistic Effects of UVB and Ionizing Radiation on Human Non-Malignant Cells: Implications for Ozone Depletion and Secondary Cosmic Radiation Exposure.

doi: 10.3390/biom15040536

Figure Lengend Snippet: Figure 4. γH2AX staining in human non-malignant cells following co-exposure to gamma rays and UVB. Results are presented as mean effect (value) ± SD for two independent experiments. The red line designates the mean value in control samples. Scale bar is indicative of the nucleus size. (a) γH2AX foci per nucleus in Hs27 cells at 1 h, 4 h, and 24 h after exposure to 0.5 Gy gamma rays or 100 J/m2 UVB as single stressors, or to combined challenges (protons first, and UVB 20 min thereafter) (stdevcontrol = 0.45 foci/nucleus); (b) pan-nuclear γH2AX staining in Hs27 cells at 1 h, 4 h and 24 h after exposure to 0.5 Gy gamma rays or UVB 100 J/m2 as single stressors, or to combined challenges (gamma rays first, and UVB 20 min thereafter) (stdevcontrol = 3.47%); (c) γH2AX foci per nucleus in HaCaT cells at 1 h, 6 h, and 24 h after exposure to 0.5 Gy gamma rays or 50 J/m2

Article Snippet: Samples were double stained with an antibody against histone H2AX (p S139) (80312T, Cell Signaling Technology, Danvers, MA, USA) and an antibody against 53PB1 (88439, Cell Signaling Technology, Danvers, MA, USA) in 0.5% BSA and 0.2% gelatin in PBS.

Techniques: Staining, Control

Journal: Biomolecules

Article Title: Synergistic Effects of UVB and Ionizing Radiation on Human Non-Malignant Cells: Implications for Ozone Depletion and Secondary Cosmic Radiation Exposure.

doi: 10.3390/biom15040536

Figure Lengend Snippet: Figure 5. Complex DNA damage detection. The red line designates the mean value in control samples. (a) Representative electron micrographs of γH2AX immunogold localization in the nuclei of Hs27 fibroblasts co-exposed to 100 J/m2 and 0.5 Gy of gamma rays processed 24 h post-irradiation. Cell arrows indicate single immunogold particles (15 nm gold) and boxes indicate immunogold particles in very close proximity (clusters); (b) representative electron micrographs of γH2AX immunogold localization in the nuclei of non-irradiated Hs27 fibroblasts processed 24 h post-irradiation. Cell arrows indicate single immunogold particles (15nm gold) and boxes indicate immunogold particles in very close proximity (clusters); (c) number of DSBs induced by co-exposure to 100 J/m2 and 0.5 Gy of gamma rays 24 h post-irradiation. γH2AX marker was used for the detection of DSBs and analysis was performed by TEM analysis and Image J. Results indicate mean number of γH2AX particles per nuclear area µm2. N: nucleus, NM: nuclear membrane. Scale bar: 500 nm; original magnification: 20,000×; (d) colocalization percentages γH2AX and 53BP1 staining in normal Hs27 fibroblasts at 1 h, 4 h, and 24 h after exposure to 0.5 Gy gamma rays or 100 J/m2 UVB as single stressors, or to combined challenges. Results are presented as mean effect (value) ± SD for two independent experiments. (stdevcontrol = 2.01%); (e) fluorescence images of Hs27 cells (i) unirradiated, (ii) exposed to 100 J/m2

Article Snippet: Samples were double stained with an antibody against histone H2AX (p S139) (80312T, Cell Signaling Technology, Danvers, MA, USA) and an antibody against 53PB1 (88439, Cell Signaling Technology, Danvers, MA, USA) in 0.5% BSA and 0.2% gelatin in PBS.

Techniques: Control, Irradiation, Marker, Membrane, Staining, Fluorescence

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 NDI-NIs and DEG-NDI-NIs. ( A ) 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 the indicated compounds for 48 h at the final concentrations of 0.1, 0.5, and 1 μM. The results were expressed as the percentage of cell viability over the untreated cells. (B–D) 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. ( B ) Percentage of γH2AX positive cells. ( C ) Quantitative analysis of the number of telomere induced foci (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. ( D ) Representative images of the experiment in panels (B) and (C), acquired by confocal microscopy (magnification 63×). Scale bars indicate 10 μm. The histograms represent the mean values ± S.D. of three independent experiments. * P < .05, ** P < .01, *** P < .001, and **** P < .0001.

Article Snippet: Briefly, cells were grown on glass coverslips, treated for 24 h and processed for IF using the mouse mAb anti-phospho-histone H2AX (γH2AX; Millipore, Burlington, Massachusetts, USA, 05–636) as primary antibody and the anti-mouse IgG (H + L), F (ab’)2 Fragment (Alexa Fluor 488 Conjugate, Cell Signaling Technology, Danvers, Massachusetts, USA, #4408) as secondary antibody.

Techniques: Activity Assay, Derivative Assay, Expressing, Reverse Transcription, Concentration Assay, Immunofluorescence, Marker, Confocal Microscopy

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: Briefly, cells were grown on glass coverslips, treated for 24 h and processed for IF using the mouse mAb anti-phospho-histone H2AX (γH2AX; Millipore, Burlington, Massachusetts, USA, 05–636) as primary antibody and the anti-mouse IgG (H + L), F (ab’)2 Fragment (Alexa Fluor 488 Conjugate, Cell Signaling Technology, Danvers, Massachusetts, USA, #4408) as secondary antibody.

Techniques: Activity Assay, Immunofluorescence, Concentration Assay, Confocal Microscopy, Labeling, Staining, Derivative Assay, Expressing, Reverse Transcription, Marker

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: Antitumoral efficacy of NDI-NIs in advanced 3D models. Advanced 3D spheroids were established from human colorectal cancer cells (HCT116) by growing the cells within drops of a methylcellulose scaffold matrix (day 0). After 2 days of culture (day 2), the spheres were treated with the indicated compound at final concentrations of 1, 2, and 5 μM. ( A ) Time course analysis of tumor spheroids growth, starting from 24 h after treatment (day 3), monitored by Incucyte ® S3 Live-Cell Analysis System (Essen BioScience, Ann Arbor, MI), 10× magnification. The results were expressed as the percentage of the spheroids area upon treatment relative to their own area before the compound administration. The histogram represents the mean values ± S.E.M. of at least four spheres. ( B ) Representative images of tumor spheroids growth described in panel (A). ( C ) Histological and immunohistochemical analysis of the spheroids generated from HCT116 cell lines and grown as in panel (A). Upper panels , representative images of immunostained sections. Lower panels, quantification of Ki-67, cleaved-caspase and γH2AX levels, expressed as percentage of positive cells. Thirty fields for condition were analyzed. The histograms represent the mean values ± S.D. of three independent experiments. * P < .05, ** P < .01, *** P < .001, and **** P < .0001.

Article Snippet: Briefly, cells were grown on glass coverslips, treated for 24 h and processed for IF using the mouse mAb anti-phospho-histone H2AX (γH2AX; Millipore, Burlington, Massachusetts, USA, 05–636) as primary antibody and the anti-mouse IgG (H + L), F (ab’)2 Fragment (Alexa Fluor 488 Conjugate, Cell Signaling Technology, Danvers, Massachusetts, USA, #4408) as secondary antibody.

Techniques: Cell Analysis, Immunohistochemical staining, Generated