53bp1 goat if  (R&D Systems)

Journal: Cancers

Article Title: Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition

doi: 10.3390/cancers13225699

Figure Lengend Snippet: Pesticide exposure for 21 days induces DNA damage in normal BM-MSCs. ( A ) DNA damage with double strand breaks, quantified by ℗-S139-γH2AX immunoreactivity by flow cytometry, increases in BM-MSCs after pesticide exposure for 21 days (left panel, n = 6), as shown in a representative experiment (middle panel). Comet assays reveal increased nucleus DNA migration after exposure of BM-MSCs to pesticides for 21 days, as shown in a representative experiment (right panel). ( B ) ℗-S139 γH2AX, 53BP1, MDC1 triple immunostaining in BM-MSCs indicates a higher number of double positive 53BP1/℗-S139 γH2AX signals in the nucleus of exposed cells (FOCI) and a loss of MDC1 staining. Data are expressed as percent of the control ± SEM; *, p < 0.05; **, p < 0.01; Friedman followed by post hoc Dunn’s multiple comparisons test. Scalebar: 50 µm ( A ) and 10 µm ( B ).

Article Snippet: After 21 days, cells were fixed with 4% paraformaldehyde, permeabilized (PBS/Triton X-100 0.1%, 10 min, room temperature), blocked with gelatin from cold water fish skin 5%, Triton X-100 0.1% (Sigma-Aldrich) in PBS, and were incubated overnight at 4 °C with anti-phospho-S139 γH2AX monoclonal mouse antibody (Abcam) at 1/1000, anti-MDC1 polyclonal rabbit antibody (Novus Biologicals, Centennial, CO, USA) at 1/500 and anti-53BP1 polyclonal goat antibody (R&D systems) at 1/1000, followed by a secondary anti-mouse antibody labeled with TRITC (Jackson Immunoresearch, Ely, UK), Alexa Fluor ® 647 conjugated anti-goat (Jackson Immunoresearch) and FITC-conjugated anti-rabbit (Jackson Immunoresearch) at 1/2000 each at room temperature for 2 h. Fluorescence images were acquired with a Leica TCS SP8 (Leica, Wetzlar, Germany) confocal microscope and analyzed using ImageJ software.

Techniques: Flow Cytometry, Migration, Pesticides, Triple Immunostaining, Staining

Journal: Cancer Nanotechnology

Article Title: Development of Fe 3 O 4 core–TiO 2 shell nanocomposites and nanoconjugates as a foundation for neuroblastoma radiosensitization

doi: 10.1186/s12645-021-00081-z

Figure Lengend Snippet: Radiosensitizing effects of bare nanocomposites and DOPAC-coated nanoconjugates. a , c SK-N-AS and b , d SK-N-DZ cells were irradiated in the presence of bare (Bare-NCs) or DOPAC-coated (DOPAC-NCs) nanoconstructs of different concentrations. Curves were generated by adjusting cell viabilities to 100% for non-irradiated cells in each nanoconstruct treated group. A statistically significant radiosensitizing effect was observed at 250 nM bare nanocomposites in both cell lines, particularly at 10 Gy. Datapoints presented are average of 5 biological replicates and are representative of at least two independent MTS experiments. Error bars indicate mean ± SD. e Annexin V/propidium iodide flow cytometry assay of SK-N-AS cells 24 h after irradiation (0 or 10 Gy) preceded by treatment with 250 nM DOPAC-nanocomposites. H 2 O 2 was the positive control. Three independent experiments were done, with 3 biological replicates per experiment. Con = cells not exposed to nanoconjugates; NC = DOPAC nanoconjugate treatment; f percentage of SK-N-AS cells with > 20 foci per nucleus, for untreated or treated with 250 nM bare nanocomposites or 250 nM DOPAC-nanoconjugates for one hour and irradiated as indicated. 53BP1 foci were stained by immunocytochemistry while the nuclei were counterstained with propidium iodide (PI). At least 100 cells were counted for each treatment group per replicate. N = total number of biological replicates from 4 independent experiments of 1–2 replicates each. There was a significant increase in the percentage of cells with > 20 foci after 2 Gy treatment. g Representative images of cells shown in f . Error bars indicate mean ± SD. * < 0.05 significance level, ** < 0.01 significance level, *** < 0.001 significance level when treatment sample is compared to untreated and/or unirradiated control

Article Snippet: Slides were incubated for 1 h with primary antibody against 53BP1 (ab21083—Abcam, Cambridge, UK) used at 1:200 dilution, washed and incubated for 45 min with fluorescent secondary antibody (Alexa Fluor 488—Goat Anti-Rabbit, ab150077, Abcam, Cambridge UK).

Techniques: Irradiation, Generated, Flow Cytometry, Positive Control, Staining, Immunocytochemistry

Journal: eLife

Article Title: Phosphorylation-mediated interactions with TOPBP1 couple 53BP1 and 9-1-1 to control the G1 DNA damage checkpoint

doi: 10.7554/eLife.44353

Figure Lengend Snippet:

Article Snippet: Antibody , Goat polyclonal anti-53BP1 , Bethyl Laboratories, Inc , Cat#A303-906A RRID: AB_2620256 , (1:100) dilution IF.

Techniques: Stable Transfection, Expressing, Transfection, Construct, Recombinant, Plasmid Preparation, Sequencing, Cloning, Mutagenesis, Antibody Labeling, Imaging, Western Blot, Produced, Protease Inhibitor, In Situ, Software, Microscopy, Sonication