Journal: The Journal of Cell Biology

Article Title: Decreased Dicer expression elicits DNA damage and up-regulation of MICA and MICB

doi: 10.1083/jcb.200801169

Figure Lengend Snippet: DNA damage accumulation in Dicer knockdown cells. (A) DNA damage assayed by immunostaining for γ-H2AX and RPA70. Mean ± SD indicates the fraction of γ-H2AX– or RPA-positive cells in mock-transfected (M), control siRNA-transfected (C), Dicer siRNA1-transfected (D1), and Dicer siRNA2-transfected (D2) cells. Bar, 10 μM. (B) Immunoblot analysis revealed an increase of Chk1 phosphorylation on S345 in Dicer knockdown cells. (C, top) Representative comet assay showing formation of DNA strand breaks (formation of a “comet tail”). (bottom) Mean ± SD indicates the fraction of cells containing a comet tail. (D) Levels of DNA damage–induced transcripts determined by real-time RT-PCR. (E) Transcription of DNA damage–induced genes assayed by RNAPol-ChIP. Data in D and E represent means ± SD from three independent experiments.

Article Snippet: Cells were fixed and stained with anti–phospho-histone H2AX (S139) antibody (R&D Systems) and anti-RPA70 (Cell Signaling Technology).

Techniques: Knockdown, Immunostaining, Transfection, Control, Western Blot, Phospho-proteomics, Single Cell Gel Electrophoresis, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: Decreased Dicer expression elicits DNA damage and up-regulation of MICA and MICB

doi: 10.1083/jcb.200801169

Figure Lengend Snippet: DNA damage progresses in parallel with the kinetics of up-regulation of MICA and MICB in Dicer knockdown cells. Cells were transfected with Dicer siRNAs. (A) γ-H2AX (▪) and RPA70 (•) staining was performed at the indicated time points. (B) Levels of MICA (▪), MICB (•), and Dicer (broken lines) transcripts were measured by real time RT-PCR. Means ± SD of three independent experiments are shown.

Article Snippet: Cells were fixed and stained with anti–phospho-histone H2AX (S139) antibody (R&D Systems) and anti-RPA70 (Cell Signaling Technology).

Techniques: Knockdown, Transfection, Staining, Quantitative RT-PCR

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Combining [ 177 Lu]Lu-DOTA-TOC PRRT with PARP inhibitors to enhance treatment efficacy in small cell lung cancer

doi: 10.1007/s00259-024-06844-1

Figure Lengend Snippet: Determination of γH2AX foci formation and clonogenic survival in H446 cells. (A) Exemplary images of H446 cells after mono- or combination therapy directly after treatment or after a 23 h post-incubation time. (B) Number of γH2AX foci per cell after mono- or combination therapy directly after treatment or after a 23 h post-incubation time in medium. Displayed are mean ± SEM from 3 individual experiments. (C) Surviving fractions of H446 cells mono- or combination therapy. Displayed are mean values from 2 independent experiments

Article Snippet: 1:150 dilution), γH2AX (pSER139, Novus #NB100-2280, 1:500 dilution) with optimized protocols of the Comparative Experimental Pathology Core Facility.

Techniques: Incubation

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Combining [ 177 Lu]Lu-DOTA-TOC PRRT with PARP inhibitors to enhance treatment efficacy in small cell lung cancer

doi: 10.1007/s00259-024-06844-1

Figure Lengend Snippet: Immunohistochemistry of H69 tumors. Exemplary stainings of H69 tumors obtained from the single dose PRRT study ( n = 1 animal/group) for SSTR2, PARP1, γH2AX (DNA damage marker) and cleaved caspase 3 (apoptosis marker) are displayed. Scale bar = 100 µm. Brown color indicates positivity for the respective marker

Article Snippet: 1:150 dilution), γH2AX (pSER139, Novus #NB100-2280, 1:500 dilution) with optimized protocols of the Comparative Experimental Pathology Core Facility.

Techniques: Immunohistochemistry, Marker

Journal: bioRxiv

Article Title: Imaging-Guided Metabolic Radiosensitization of Pediatric Rhabdoid Tumors

doi: 10.1101/2024.08.09.607364

Figure Lengend Snippet: (A) Whole tissue imaging and ( B ) confocal micrographs of γ-H2AX immunofluorescence staining of all treatment groups on Day 9. (Insets) Stained A-204 tissue at high magnification. Scale bar = 50 µm for confocal images and scale bar = 1000 µm for whole tumor sections. (C) Quantification of γ-H2AX staining in whole tumor sections ( a ) and confocal micrographs ( b ) of all treatment groups. (D) H&E staining for heart, liver, kidney and muscle from various treatment groups. Scale bar=100 µm. Data represented as mean ± SD.

Article Snippet: Alexa Fluor 488 AffiniPure Donkey Anti-Rat lgG(H+L) (Lot 163136) and Rhodamine (TRITC) AffiniPure Donkey Anti-Rat lgG(H+L) (Lot 157518) were obtained from Jackson ImmunoResearch Inc. Histone H2AX antibody (NB100-384) was purchased from Novus Biologicals and secondary antibody Donkey anti-Rabbit lgG (H+L) Dylight594 (SA5-10040) from Invitrogen.

Techniques: Imaging, Immunofluorescence, Staining

Journal: Cell reports

Article Title: A P53-Independent DNA Damage Response Suppresses Oncogenic Proliferation and Genome Instability

doi: 10.1016/j.celrep.2020.01.020

Figure Lengend Snippet: (A) Mre11 suppresses oncogene-induced γH2AX foci formation in both p53-proficient and p53-deficient pMMECs. Bar graphs show quantification of the percent of nuclei containing ≥5 γH2AXfoci in the different genotypes. Representative images (right) of the nuclei containing γH2AX foci are shown. White bar indicates 5 μm. (B) Bar graphs depicting the fold change in tail DNA percent for both alkaline (left) and neutral (right) COMET assays in pMMECs with the genotypes shown post-Cre-sgRNA transduction. Representative images of alkaline and neutral COMETs in R26Cas9+sgControl, R26Cas9/Myc+sgControl, and R26Cas9/Myc+sgMre11 pMMECs are shown. Data are represented as mean ± SEM. (C) Mre11 suppresses oncogene-induced R-loop formation independently of Trp53. Scatterplot shows a quantification of the nuclear S9.6foci in pMMECs from the genetic backgrounds shown after transduction with Cre-sgControl versus Cre-sgMre11. Representative images (right) of the nuclei containing S9.6 foci are shown. White bar indicates 5 μm. (D) S9.6 (R-loop) foci after RNase H1 overexpression in R26CasaRb1fl/flTrp53fl/fl pMMECs transduced with Cre-sgControl or Cre-sgMre11. (E and F) Additionally, RNase H1 overexpression counteracts the increase in (E) γH2AX and (F) p-RPA2 foci seen in Mre11 hypomorphic R26CasaRb1fl/flTrp53fl/fl pMMECs. ***p < 0.001; ****p < 0.0001. p values are calculated using a two-tailed Mann-Whitney test. See also Figure S4.

Article Snippet: Rabbit Anti-phosphorylated Histone H2AX (g-H2AX) Polyclonal Antibody (1:500 for IF) , Trevigen , Cat# 4418-APC-100.

Techniques: Transduction, Over Expression, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: A P53-Independent DNA Damage Response Suppresses Oncogenic Proliferation and Genome Instability

doi: 10.1016/j.celrep.2020.01.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Anti-phosphorylated Histone H2AX (g-H2AX) Polyclonal Antibody (1:500 for IF) , Trevigen , Cat# 4418-APC-100.

Techniques: Virus, Recombinant, Modification, Adhesive, Single Cell Gel Electrophoresis, TA Cloning, Sequencing, Generated, Software

Journal: bioRxiv

Article Title: Polymerase Eta Recruits FANCD2 to Common Fragile Sites to Maintain Genome Stability

doi: 10.1101/2025.01.06.631600

Figure Lengend Snippet: (A) Measuring expression levels of phospho-ATR protein, phospho-Chk1 Ser317 to assess replication checkpoint activation using western blotting. Expression levels of H3 were used as a loading control and (B) DNA fiber analysis of 50uM hydroxyurea (HU) treated to assess replication fork stalling in pol eta −/− , PIP*, UBZ*, F1* and pol eta+ /+ cells. The fork rate (CldU/IdU ratio) is indicated, n=150. The p-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. Scale bar 10 μm; (C) Analysis of number of gH2AX foci (red) in the nucleus per cell in Pol eta −/− , PIP*, UBZ*, F1* and Pol eta +/+ cells when exposed to Aphidicolin (Aph). Representative images are on the top, n=250; The p-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. (D) Measurement of DNA single strand breaks by alkaline Comet assay in Pol eta −/− , PIP*, UBZ*, F1* and Pol eta +/+ cells treated with Aphidicolin (APH). The p-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. Lengths were measured using the Adobe Photoshop Tools. (E) Time course experiment to measure recovery of cells after release into drug free media over the course of 48hours. Cells were collected at six time points (0, 4, 8, 12, 24, 28 hours) and expression levels of phospho-Histone H2AX Ser139 were measured by Immunofluorescence staining. The p-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. Scale bar 10 μm.

Article Snippet: Primary Antibodies used-Primary antibodies used were anti-FANCD2 (1:1000, NB100-182, Novus), anti-cGAS (1:1000 D1D3G, Rabbit mAb), anti-Phospho-Histone H2A.X (Ser139), anti-XPV DNA Pol eta (Novus NBP1-87165, 1:2000), anti-BRCA1 (Sigma 07-434, 1:1000), anti-BRCA2 (Merck Millipore OP95, 1:1000) and anti-RAD51 (CosmobioUSA BAM-70-002-EX, 1:1000).

Techniques: Expressing, Activation Assay, Western Blot, Control, Alkaline Single Cell Gel Electrophoresis, Immunofluorescence, Staining

Journal: Biomedical research (Tokyo, Japan)

Article Title: Glycochenodeoxycholic acid promotes proliferation of intestinal epithelia via reduction of cyclic AMP and increase in H2AX phosphorylation after exposure to γ-rays.

doi: 10.2220/biomedres.33.159

Figure Lengend Snippet: Fig. 5 Implication of DNA strand breaks in the increase of glycochenodeoxycholic acid (GCDCA)-induced IEC-6 cell proliferation in response to γ-radiation. (A) Phosphorylation of histone H2AX induced by GCDCA (200 μM) in IEC-6 cells after γ-irradiation. (B) Quantitative analysis of p-H2AX on Western blots. (C) GCDCA (200 μM) suppressed G1 ar- rest in IEC-6 cells after exposure to γ-rays. Values shown are means ± SEMs (n = 5). Values with different superscript letters are significantly different in each treatment (P < 0.05). An asterisk indicates a significant difference from control (Ctl).

Article Snippet: Mouse anti-histone H2AX was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Irradiation, Western Blot, Control

Journal: Genes

Article Title: In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls-Part 2: Adaptive Response.

doi: 10.3390/genes13122283

Figure Lengend Snippet: Figure 6. The influence of the DNA samples on the γH2AX levels in HSFs and PBL. (A). a1—The most typical examples of the γH2AX assay with FCA in HSFs. The data from the device are given for sz-HSF3. a2—Proportion of R1(γH2AX) fraction cells. a3—Analysis of relative changes in R1(γH2AX) levels in the presence of DNA samples compared to control. a4—Changes in the R1(γH2AX) levels in sample of HSFs (n = 10). a5—Index γH2AX (R2): the values of the medians of γH2AX (R2), normalized to the control signal value. Average values for three measurements and standard deviation are given. a6—Analysis of relative changes in γH2AX (R2) levels in the presence of DNA samples compared to control. a7—Dependence of R1(8-oxodG) on R1(γH2AX). a8—sz- HSF4 staining with two types of antibodies with different labels: γH2AX (PB450) and 8-oxodG (PE). (B). b1—The most typical examples of the γH2AX assay with FCA in lymphocytes. b2,b3—Changes in the R1(γH2AX) index and γH2AX (R2) index in sample of lymphocytes (n = 4). b4—PBL staining with two types of antibodies with different labels: γH2AX (PB450) and 8-oxodG (PE).

Article Snippet: Inc. ’, Boston, MA, USA) or NOX4-FITC (Q9NPH5, ‘Cusabio Technology’, Houston, TX, USA), BRCA1-FITC (NB100-598F, ‘Novus Biologicals, LLC’, Centennial, CO, USA), γH2AX-pb450 (nb100-384AF405, ‘Novus Biologicals, LLC’, Centennial, CO, USA), SOD-PE (sc-11407 and rabbit IgG-PE sc-3753, ‘Santa Cruz Biotechnology, Inc. ’, Santa Cruz, CA, USA), or HIF1A-PE (NB100-479, ‘Novus Biologicals, LLC’, Centennial, CO, USA and rabbit IgG-PE sc-3753, ‘Santa Cruz Biotechnology, Inc.’, Santa Cruz, CA, USA).

Techniques: Control, Standard Deviation, Staining