Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: (A) Spontaneous frequency of phospho-53BP1 foci in non-stimulated and unirradiated PBLs of five volunteers (V1-V5). Error bars signify SD (5 × 3 i.e., five individuals with triplicate blood sampling). More than 300 PBLs were analyzed for estimating the spontaneous frequency of phospho-53BP1 in each volunteer. (B) Representative PBLs with (i) no foci (ii) one focus (iii) two foci (iv) numerous foci (could be apoptotic cell).

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Sampling

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: (A) Phospho-53BP1 foci, decay kinetics, in the blood sample of five volunteers [two females (with age 22 and 24 y) and three males (with age 25 y, 31 y and 35 y)] after irradiation with 1, 2, 3, and 4 Gy. Error bars represents SEM ( n = 5 × 3, i.e., five volunteers with triplicate sampling). Number of cells analyzed/scored > 300/dose/time point/volunteer. (B) Illustration of the disappearance of phospho-53BP1 foci at 1, 2, 4, 8, 16, and 24 h of incubation after irradiation with 1, 2, 3, and 4 Gy. Dispersion of the number of phospho-53BP1 foci in PBLs irradiated with 1 Gy (cumulative data of all five volunteers) after incubation of (C) 1 h (D) 2 h (E) 4 h (F) 8 h (G) 16 h, and (H) 24 h.

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Irradiation, Sampling, Incubation

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: Fitting parameters for phospho-53BP1 foci decay for doses 1, 2, 3, and 4 Gy up to 24 h of incubation after irradiation.

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Incubation, Irradiation

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: Half-lives of phospho-53BP1 foci for doses of 1, 2, 3, and 4 Gy estimated using the formula T1/2 = 0.693*t1.

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques:

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: Dose-response curves for 60 Co-γ-rays-induced-phospho-53BP1 foci formation post-irradiation incubation of (A) 1 and 2 h (B) 4 and 8 h (C) 16 and 24 h. (D) Estimated minimum detection limits of the dose-response curves established at 1, 2, 4, 8, 16, and 24 h of incubation, following ISO recommendations (3 sigma of the background frequency) (E) Variation of the slope (53BP1 foci/cell/Gy) of the established dose-response curves, generated at 1, 2, 4, 8, 16, and 24 h of incubation, post-irradiation. (F) Microscopic images showing an increasing number of phospho-53BP1 foci in the nucleus of PBLs with increasing doses (0 to 2.0 Gy after 1 h and 3 and 4 Gy after 4 h of incubation, post irradiation). (G) Representative images of monocytes (kidney-shaped nucleus) with and without 53BP1 foci (these cells were excluded from the scoring of 53BP1 foci).

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Irradiation, Incubation, Generated

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: (A) Colocalization of phospho-53BP1 and γH2AX foci in the nucleus of human lymphocyte exposed with various doses (at 1 h incubation post irradiation). Correlations established between dose-response curves generated for phospho-53BP1 foci and γH2AX foci (data not shown) at dose points (B) 0.05 Gy (C) 0.1 Gy (D) 0.25 Gy (E) 0.5 Gy (F) 1 Gy (G) 2 Gy (H) 4 Gy with varying incubation time points (1–24 h) after irradiation. Correlations were established between dose-response curves generated for phospho-53BP1 foci and γH2AX foci, at (I) 1 h (J) 2 h (K) 4 h (L) 8 h (M) 16 h and (N) 24 h of incubation after irradiation with various doses (0–4 Gy).

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Incubation, Irradiation, Generated

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: Graphical illustration of the estimated doses of blinded samples (Z1–Z6) by immunofluorescence (phospho-53BP1 and γH2AX) and cytogenetic (dicentric and reciprocal translocation) assays.

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Immunofluorescence, Translocation Assay

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: Comparative evaluation of established response curve (phospho-53BP1) by estimating biological doses of 6 dose blinded samples by phospho-53BP1, γH2AX, dicentric, and translocation assays.

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Translocation Assay, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains

doi: 10.3390/ijms23126593

Figure Lengend Snippet: Sub-cellular distribution of transiently expressed GCaMP3.0 variants . Human embryonic kidney cells (HEK293) were either non-transfected with a GCaMP3.0 encoding construct ( A1 – E1 ) or with GCaMP3.0cyto ( A2 – E2 ), GCaMP3.0pm ( A3 – E3 ), GCaMP3.0mito ( A4 – E4 ), GCaMP3.0mom ( A5 – E5 ), or GCaMP3.0nuc ( A6 – E6 ). Expression of GCaMP3.0 variants was detected by their green fluorescence ( A2 – A6 ). For identificationof the Golgi apparatus in cells, they were transfected with Golgi-RFP ( B1 – B6 ). The endoplasmic reticulum was stained with primary rabbit anti-calnexin antibodies (dilution 1:100) and secondary donkey anti-rabbit-A594 (dilution 1:1000) antibodies ( C1 – C6 ). Labeling of mitochondria was performed with MitoTracker DeepRed FM ( D1 – D6 ). Fluorescent images were obtained by confocal imaging of the samples. Composite images are depicted in ( E1 – E6 ). The scale bar denotes 8 µm.

Article Snippet: The following primary antibodies were used: rabbit (rb)-anti-calnexin (dilution 1:100; #2674, Cell Signaling Technology, Danvers, MA 01923, USA) and (rb)-anti-MAP2 (dilution 1:500; #AB5622, Chemicon/Millipore, Darmstadt, Germany).

Techniques: Transfection, Construct, Expressing, Fluorescence, Staining, Labeling, Imaging

Journal: bioRxiv

Article Title: The chromatin associated Sin3B is a critical regulator of DNA damage repair through the engagement of the Non-Homologous End Joining

doi: 10.1101/2022.03.09.483624

Figure Lengend Snippet: A. Sin3B +/+ and Sin3B -/- T98G cells were synchronized in different phases of the cell cycle before being treated with 25 ug/mL of cisplatin for 12 followed by Annexin V staining followed by flow cytometry analysis. Annexin V positive cells were then quantified. Error bars represent SD; n = 3. Significance was determined by two-way ANOVA followed by a Tukey’s test. **** P <0.0001. B. Cells were treated with 25 ug/mL of cisplatin for 4 h. Cells were then fixed, stained for 53BP1 and imaged. Representative images of the data quantified in 2C. C. Quantification of cells with 53BP1 foci. Error bars represent SD; n = 3. Significance was determined by two-way ANOVA followed by a Sidak’s test. *** P <0.001. D. T98G cells containing the EJ5-GFP reporter were analyzed for GFP positivity, 72 hours after nuclease induction. The percentage of GFP + cells were determined for each individual condition and subsequently normalized to transfection efficiency. Error bars represent SD; n = 3. Significance was determined by Student’s t-test. ** P <0.01. E. T98G Sin3B +/+ and Sin3B -/- TLR cell lines were created and then infected with lentivirus containing SceI and the template for eGFP reconstitution. After 72 hours, percentage of GFP + cells were determined by flow cytometry for each individual condition and subsequently normalized to infection efficiency. Error bars represent SD; n = 3. Significance was determined by Student’s t-test. * P <0.05. F. Similar to E, RFP + cells were determined by flow cytometry. Error bars represent SD; n = 3. Significance was determined by Student’s t-test. **** P <0.0001. G. Sin3B +/+ and Sin3B -/- T98G cells were treated with 25 ug/mL of cisplatin for 12 h and 10 nM NU7441. Cells were then subjected to Annexin V staining followed by flow cytometry analysis. Annexin V positive cells were then quantified. Error bars represent SD; n = 3. Significance was determined by two-way ANOVA followed by a Tukey’s test. **** P <0.0001.

Article Snippet: Treated and untreated cells were fixed with 4% paraformaldehyde–PBS for 15 min and blocked with 0.3% Triton X-100–5% goat serum–PBS for 1 h. Cells were then incubated with rabbit anti-γ-H2AX (Cell Signaling 9718) at 1:400 dilution or rabbit anti-53BP1 (Cell Signaling 2675S) at 1:500 dilution in 0.3% Triton X-100–1% BSA–PBS overnight at 4°C.

Techniques: Staining, Flow Cytometry, Transfection, Infection

Journal: Nucleic Acids Research

Article Title: Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ

doi: 10.1093/nar/gkab980

Figure Lengend Snippet: Phospho-53BP1 foci formation is impaired following irradiation in cells expressing unphosphorylable ala-Ku70; inhibition of RNA Poll II impairs foci formation in cells expressing the phosphorylable ser-Ku70. ( A) Immunofluorescence labelling of γ-H2AX and p-53BP1 (S1778) foci in cells expressing ser-Ku70 or ala-Ku70. Foci were assessed in the untreated control (Control) or at 1 h post-irradiation (2Gy) without (IR) or with α-amanitin pre-treatment (2 h prior irradiation at 50 μg ml –1 ). Scale bar (white) corresponds to 10 μm. (B) Each analysis was performed on at least 100 cells, and at least 10 images of each condition were analysed. Confocal microscopy optical slice sections of 8–20 μm were recorded from the apical to the basal pole of the cells, with each acquisition containing 26 stacks. Images were prepared and stacked with ImageJ software (Bethesda, MD) by using the stacks tool. Then TIFF images were converted into 8 bits before performing γ-H2AX and p53BP1 foci counts. Cell Profiler software (Cambridge, MA) was used for the detection and scoring of foci in p53BP1 and γ-H2AX images. The representation of data as a box plot was performed using GraphPad Prism 7. For statistical analysis, to compare the number of γ-H2AX and p53BP1 foci as well as the size of p53BP1 foci, in each condition, a Mann–Whitney rank test based on at least 100 observations was performed. (C) The specific inhibition of RNA Pol II by α-amanitin delays the level of γ-H2AX in ser-Ku70-expressing cells. Cells were untreated (–) or pre-treated with α-amanitin (AM) at 50 μg ml –1 or with 5,6-dichloro-1b- d -ribofuransylbenzimidazole (DRB) at 50 μM for 2 h and then irradiated at 4 Gy. Total protein extracts were done at 2 or 4 h post-irradiation of the cell culture. After SDS-PAGE, membranes were probed with anti-γ-H2AX (ser-139) antibodies. Anti-Ku70 (clone N3H10), and anti-H2AX were probed as controls.

Article Snippet: Anti-pKu70 was generated in mouse hybridoma cells by BioGenes GmbH (Berlin, Germany); mouse anti-Ku70, clone N3H10 (ThermoFisher Scientific); rabbit anti-Ku70, ARG57851 (Arigo Biolaboratories); mouse anti-phospho-histone H2AX, clone JBW301 (Merck-Millipore), rabbit anti phospho-53BP1 (ser1778, #2675, Cell Signalling); rabbit anti-ubiquitin (ab137031, Abcam); mouse anti-ubiquitin, clone P4D1 (Cell Signalling); mouse anti-ubiquitinated proteins, clone FK2 and clone FK1 (Merck-Millipore); rat anti-RNA polymerase 2, CTD Ser5ph (Cosmo Bio Co. LTD); rabbit anti-phospho RNA Polymerase II (S5) (A304-408A-M-2, Bethyl); rabbit anti-NEDD8 (Cell Signalling); and rabbit anti-Rad6 (ab31917, Abcam).

Techniques: Irradiation, Expressing, Inhibition, Immunofluorescence, Confocal Microscopy, Software, MANN-WHITNEY, Cell Culture, SDS Page

Journal: Nucleic Acids Research

Article Title: Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ

doi: 10.1093/nar/gkab980

Figure Lengend Snippet: ( A ) pKu70 promotes genome stability after genotoxic stress. Human mammary epithelial (HME) cells expressing the ser-Ku70 or ala-Ku70 form were irradiated at 4 Gy. Following 24 h post-irradiation culture, metaphase cells were probed for chromosomal aberrations by the multi-FISH approach. Examples of 4 representative metaphase cells are shown. (Upper panel, left) Untreated (NT) ser-Ku70-expressing cells showing ins(1), ins(8–10), del(11), der(18)t(11:18), +20, der(22)t(10;22); (upper panel, right) ala-Ku70-expressing cells showing the same aberrations as in the left panel with an additional der(13)t((5;13) translocation. After irradiation (lower panels), only new translocation events (compared to untreated conditions) were considered. Other markers of genome instability are shown: dmin (double minute); del (deletion); ace (acentric fragment); C-Frag (chromosomal fragment); and mar (marker). ( B) . Schematic hypothetic pathway underlying the involvement of pKu70 in accurate cNHEJ. Ku70 interacts with ATM/DNA-PKcs kinases, resulting in its phosphorylation and interaction with RNA Pol II, which initiates bidirectional copying of complementary RNAs from the damage site with specific topological domains marked by 53BP1-RIF1. Newly synthesised DDRNAs are used in the next step by accurate DNA polymerases (encoded by POLD4 and POLD2; Figure ). Neddylation-dependent ubiquitylation precedes pKu70 dissociation from the repair complex. Unphosphorylable ala-Ku70 does not recruit RNAPII, exhibits a defect in phospho-53BP1 foci formation that allows distant end-junctions. Otherwise, its interaction with Rad6, a ubiquitin-conjugating enzyme that acts as a promotor of unfaithful DNA repair, hypothetically through Rad6/Rad18 association with translesion Y-family polymerases (i.e. Polη and Rev), may also compromise accurate repair and genome stability.

Article Snippet: Anti-pKu70 was generated in mouse hybridoma cells by BioGenes GmbH (Berlin, Germany); mouse anti-Ku70, clone N3H10 (ThermoFisher Scientific); rabbit anti-Ku70, ARG57851 (Arigo Biolaboratories); mouse anti-phospho-histone H2AX, clone JBW301 (Merck-Millipore), rabbit anti phospho-53BP1 (ser1778, #2675, Cell Signalling); rabbit anti-ubiquitin (ab137031, Abcam); mouse anti-ubiquitin, clone P4D1 (Cell Signalling); mouse anti-ubiquitinated proteins, clone FK2 and clone FK1 (Merck-Millipore); rat anti-RNA polymerase 2, CTD Ser5ph (Cosmo Bio Co. LTD); rabbit anti-phospho RNA Polymerase II (S5) (A304-408A-M-2, Bethyl); rabbit anti-NEDD8 (Cell Signalling); and rabbit anti-Rad6 (ab31917, Abcam).

Techniques: Expressing, Irradiation, Translocation Assay, Marker