Journal: Genes & Development

Article Title: ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair

doi: 10.1101/gad.292516.116

Figure Lengend Snippet: ZMYM3 antagonizes the BRCA1-A complex to promote HR. ( A ) ZMYM3 promotes HR and opposes RAP80 and ABRA1 inhibition of HR. DR-GFP reporter assays were performed after depletion or codepletion of the indicated proteins by siRNAs. Data represent mean ± SD. n = 3. ( B ) Confirmation of knockdown efficiency by Western blotting from experiments performed in A . ( C ) 53BP1 depletion rescues HR defects in ZMYM3-depleted cells. Experiments were performed as in A . ( D ) ZMYM3 knockout cells are sensitive to IR and PARP inhibitors compared with parental U2OS cells. Cells were challenged with IR or PARP inhibitor as indicated and were analyzed by colony formation assays. Data represent mean ± SD. n = 3. ( E ) Western blotting analysis of knockdown efficiency in cells transfected with BRCA1 siRNA. ( F ) Epistasis analysis of ZMYM3 and BRCA1. Wild-type and ZMYM3 knockout cells either alone or in combination with siBRCA1 were challenged with IR and PARP inhibitor followed by colony formation assays. ( G ) Chromosome aberration analyses in ZMYM3 knockout and BRCA1 knockdown cells. Experiments were performed as in E. ( H ) Complementation assay of ZMYM3 knockout cells. ZMYM3 knockout cells with empty vector or wild-type ZMYM3 were analyzed as in D . (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 versus same treatment with control cells, Student's t -test.

Article Snippet: Primary antibodies used in this study were Flag M2 (Sigma, F1804), Myc (Santa Cruz Biotechnology, SC-40), H2AX (Millipore, 07-627), γH2AX (Millipore, 05-636), H2AZ (Cell Signaling, 2718S), macroH2A (Abcam, AB37264), H2A.Bbd (Millipore, 06-1319), ZMYM3 (Abcam, AB106626), ATM (Santa Cruz Biotechnology, SC-135663), pATM S1981 (Abcam, AB81292), β-tubulin (Abcam, AB6046), RAD51 (Abcam, AB88572), RAP80 (Bethyl Laboratories, A300-763A), ABRA1 (Abcam, AB139191), BRCA1 (Santa Cruz Biotechnology, SC-6954), HRP-linked anti-GST (Sigma, A7340), MBP (Abcam, AB9084), phospho-H3 S10 (Cell Signaling, 3377), 53BP1 (Novus Biologicals, NB100-304), RAD18 (Cell Signaling, 9040), RPA2 (Abcam, AB2175), pRPA2 S33 (Bethyl Laboratories, A300-246), pRPA2 S4/S8 (Bethyl Laboratories, A300-245), Chk1 (Santa Cruz Biotechnology, SC-8408), and pChk1 (Cell Signaling, 2348).

Techniques: Inhibition, Knockdown, Western Blot, Knock-Out, Transfection, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: Modulating the unfolded protein response: Impacts of radiation on the response of prostate cancer cells to ONC201

doi: 10.1101/710400

Figure Lengend Snippet: a) Cell Cycle analysis of PC3 primed to radiation (Xrad) with ONC201 for 24 hours shows an expansion of the cell population in S and G2/M phases. b) Schematic representation of the treatment in which PC3 cells have been primed with ONC201 before being radiated with single doses of Xrad. c) Number of 52BP1 + foci per cell at 24hours post irradiation. Samples were harvested as in scheme (b). d) Repair Kinetcs from the DNA damage induced by radiation. Samples were harvested as in scheme (b). e) FOCI determination through 53BP1 (red) staining at 1 and 24hours from radiation (IF). Samples were harvested as in scheme (b). One way ANOVA test has been run. The data shown in panel (c) have been run through ANOVA test on Ranks and further analysed with Dunnett’s Method. *P<0.05, **P<0.03, ***P <0.001 (±SD).

Article Snippet: Anti-53BP1 rabbit monoclonal primary antibody (Novus Biological, Centennial, USA), diluted 1:5000 in blocking buffer was added to the cells.

Techniques: Cell Cycle Assay, Irradiation, Staining

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-53BP1 , Novus Biologicals , Cat# NB100-904.

Techniques: Transduction, Recombinant, Protease Inhibitor, SYBR Green Assay, Mutagenesis, Purification, Gel Extraction, Imaging, Sequencing, Negative Control, Real-time Polymerase Chain Reaction, Software, Expressing

Journal: Cell death and differentiation

Article Title: 53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

doi: 10.1038/cdd.2009.159

Figure Lengend Snippet: Figure 1 Aggregation and phosphorylations of 53BP1 in karyogamic syncytia elicited by HIV-1 Env. (a–h) Syncytia, arising from the coculture of HeLa Env and HeLa CD4 cells for 48 h, were subjected to immunofluorescence stainings. Representative karyogamic syncytia stained with anti-53BP1, 53BP1S25P, 53BPS1778P, PML, ATMS1981P, g-H2AX antibodies and Hoechst 33342 are shown. Colocalization between 53BP1(a, d), 53BP1S25P (b, e, g) and 53BP1S1778P (c, f, h) with g-H2AX (a, b, c), PML (d, e, f) or ATMS1981P (g, h) are observed. (i) Kinetics of karyogamy (KG), of PML or of 53BP1 aggregation, of phosphorylations on serine 25 (53BP1S25P) or on serine 1778 of 53BP1 (53BP1S1778P), on serine 1981 of ATM (ATMS1981P) of H2AX, on serine 137 of H2AX (g-H2AX) and on nuclear apoptosis. (j) Quantification of PML or 53BP1 aggregation or phosphorylation of 53BP1 (53BP1S25P and 53BP1S1778P), ATM (ATMS1981P) and g-H2AX on single cells (SC), on prekaryogamic (pre-KG) and karyogamic (KG) syncytia and during apoptosis. Karyogamy and nuclear apoptosis are evaluated using Hoechst 33342 staining (X±S.D., n ¼ 3)

Article Snippet: Rabbit antibody against phospho-53BP1 (Novus Biologicals) and biotinylated goat anti-rabbit IgG were incubated with tissue sections.

Techniques: Staining, Phospho-proteomics

Journal: Cell death and differentiation

Article Title: 53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

doi: 10.1038/cdd.2009.159

Figure Lengend Snippet: Figure 3 ATM controls 53BP1 phosphorylations but not its aggregation. (a, b) 53BP1 aggregation occurs before the activation of PML, TopBP1 and ATM. HeLa CD4 and HeLa Env cells were transfected 48 h before fusion with siRNAs specific for PML, TopBP1 and ATM and then were cocultured for 36 h. Syncytia were then subjected to immunofluorescence microscopy for the detection of karyogamy (KG), apoptosis (nuclear chromatin condensation), g-H2AX foci or the aggregation of PML and 53BP1. Representative karyogamic syncytia are shown after staining with antibodies against PML and 53BP1 (a). Results are shown as means±S.D. of three independent experiments (b). (c) ATM-dependent 53BP1 phosphorylation. After 48 h transfection with specific siRNA against ATM, HeLa CD4 and HeLa Env cells were cocultured for 36 h and stained with specific antibodies against 53BP1S25P and 53BP1S1778P. The effect of ATM knockdown on 53BP1 phosphorylation was evaluated using immunofluorescence microscopy in four independent experiments (X±S.D.). (d, e) Partial inhibition of 53BP1S25P on syncytia obtained after HIV-1 infection of PHA/IL-2 lymphoblasts obtained from A-T patients. PHA/IL-2 lymphoblasts from controls or A-T patients (n ¼ 3) were infected by HIV-1, and the frequency of syncytia with 53BP1S25P þ

Article Snippet: Rabbit antibody against phospho-53BP1 (Novus Biologicals) and biotinylated goat anti-rabbit IgG were incubated with tissue sections.

Techniques: Activation Assay, Transfection, Microscopy, Staining, Phospho-proteomics, Knockdown, Inhibition, Infection

Journal: Cell death and differentiation

Article Title: 53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

doi: 10.1038/cdd.2009.159

Figure Lengend Snippet: Figure 4 53BP1 depletion induces syncytial cell death through an increase in mitoses. (a–c) HeLa CD4 and HeLa Env cells were separately transfected with 53BP1-specific siRNAs, and 48 h later the cells were cocultured for further 36 h, lysed and subjected to the immunodetection of 53BP1 and GAPDH (a). Representative karyogamic syncytia transfected during 36 h with specific siRNA against 53BP1 and stained with antibodies specific for 53BP1, 53BP1S1778P, PML, g-H2AX, cytochrome c (Cyto. c) or activated caspase-3 are shown (b). Note that 53BP1 depletion triggers cytochrome c release and chromatin condensation on Env-elicited syncytia. Effect of 53BP1 depletion on the pro-apoptotic signaling pathway induced by HIV-1 envelope and on mitosis (c). The frequency of syncytia showing nuclear aggregation of PML or 53BP1, phosphorylation events 53BP1S25P, 53BP1S1778P, ATMS1981P, g-H2AX, cytochrome c release or nuclear apoptosis (chromatin condensation) was assessed using immunofluorescence microscopy. Karyogamy (KG) and mitosis were determined using Hoechst 33342 staining (X±S.D., n ¼ 5)

Article Snippet: Rabbit antibody against phospho-53BP1 (Novus Biologicals) and biotinylated goat anti-rabbit IgG were incubated with tissue sections.

Techniques: Transfection, Immunodetection, Staining, Phospho-proteomics, Microscopy

Journal: Cell death and differentiation

Article Title: 53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

doi: 10.1038/cdd.2009.159

Figure Lengend Snippet: Figure 5 53BP1 knockdown induces mitotic catastrophe on HIV-1 Env-elicited syncytia. (a) Transient phosphorylation of 53BP1 on serine 1778 (53BP1S1778P) during normal mitosis. Detection of 53BP1S1778P during normal anaphase, normal telophase and normal cytokinesis using immunofluorescence microscopy. (b) Depletion of 53BP1 triggers abnormal mitosis and mitotic catastrophe of HIV-1 Env-elicited syncytia. HeLa CD4 and HeLa Env cells were separately transfected for 48 h with 53BP1- specific siRNAs. Syncytia arising from the coculture of these cells for 36 h were fixed and stained with specific antibody against a-tubulin and activated caspase-3 for immunofluorescence microscopy. Representative prekaryogamic and karyogamic syncytia, prophase, aberrant metaphase, telophase and mitotic catastrophe are shown. (c) Knockdown of 53BP1 leads to multipolar metaphases with multiple centrosomes. Cells were fixed and stained with antibody against g-tubulin for immunofluorescence microscopy. Representative aberrant metaphase revealing numerous centrosomes is shown. (d) Quantification of different mitotic stages and mitotic catastrophe induced by 53BP1 silencing was determined (X±S.D., n ¼ 3). (e) Inhibition of syncytial mitotic catastrophe by N9-isopropyl-olomoucine. HeLa CD4 and HeLa Env cells were separately transfected for 48 h with 53BP1-specific siRNAs, and syncytia arising from the coculture of cells for 36 h were then incubated with 10 mM of N9-isopropyl-olomoucine (N9-Olo). Cells were fixed and stained with specific antibodies against a-tubulin and activated caspase-3 for immunofluorescence microscopy. The effects of N9- isopropyl-olomoucine on mitotic catastrophe induced by 53BP1 knockdown were determined (X±S.D., n ¼ 3)

Article Snippet: Rabbit antibody against phospho-53BP1 (Novus Biologicals) and biotinylated goat anti-rabbit IgG were incubated with tissue sections.

Techniques: Knockdown, Phospho-proteomics, Microscopy, Transfection, Staining, Inhibition, Incubation

Journal: Cell death and differentiation

Article Title: 53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

doi: 10.1038/cdd.2009.159

Figure Lengend Snippet: Figure 6 53BP1 phosphorylation in vivo, in HIV-1-infected patients. (a) Phosphorylation of 53BP1 on serine 25 in syncytia detectable in frontal cortex from patients with HIV-1-associated encephalitis (HAE). 53BP1S25P phosphorylations were visualized using immunohistochemistry on histological sections from control patients or from untreated HIV-1 carriers with HAE but without opportunistic infection. The insert shows labeled giant multinuclear cells. The percentage of syncytia (X±S.E.M.) staining positively for 53BP1S25P amounted to 58±9, as determined for six different HIV-1 carriers. (b) 53BP1S25P phosphorylation in lymph nodes from controls and untreated HIV-1 carriers. The insert reveals bonafide labeled syncytia. (c) 53BP1S25P in PBMC derived from representative HIV-1-infected patients or healthy donors (Co.). Immunoperoxidase stainings are shown. (d, e) Correlation between 53BP1S25P, viral status and HAART treatment. PBMCs from untreated HIV-1-infected patients were subjected to 53BP1S25P staining and the values were plotted against the number of viral particles in the serum. The P-values refer to the correlation coefficients (d). A cohort of HIV-1- donors, untreated HIV-1 carriers and HAART-treated subjects with undetectable viremia were analyzed for 53BP1S25P (e)

Article Snippet: Rabbit antibody against phospho-53BP1 (Novus Biologicals) and biotinylated goat anti-rabbit IgG were incubated with tissue sections.

Techniques: Phospho-proteomics, In Vivo, Infection, Immunohistochemistry, Control, Labeling, Staining, Derivative Assay

Journal: Nature Communications

Article Title: Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation

doi: 10.1038/s41467-019-13981-x

Figure Lengend Snippet: a γH2AX, 53BP1, RPA2, RAD51 and BRCA1 localization in control depleted (siCtrl) and Treacle-depleted (siTreacle) U2OS cells 2 h after I-Ppo1 transfection. b Quantification of the experiment in a , γH2AX: siCtrl ( n = 177) versus siTreacle ( n = 200), 53BP1: siCtrl ( n = 261) versus siTreacle ( n = 239), RPA2: siCtrl ( n = 152) versus siTreacle ( n = 168) BRCA1: siCtrl ( n = 525) versus siTreacle ( n = 246) and RAD51: siCtrl ( n = 525) versus siTreacle ( n = 246) cells (all datapoints are shown, bar represents mean, error bars represent S.D.) c γH2AX, 53BP1, RPA2, RAD51 and BRCA1 localization in control depleted (siCtrl) and TOPBP1-depleted (siTOPBP1) U2OS cells 2 h after I-Ppo1 transfection. d Quantification of the experiment in c , γH2AX: siCtrl ( n = 403) versus siTOPBP1 ( n = 205), 53BP1: siCtrl ( n = 239) versus siTOPBP1 ( n = 256), RPA2: siCtrl ( n = 649) versus siTOPBP1 ( n = 359) BRCA1: siCtrl ( n = 228) versus siTOPBP1 ( n = 348) and RAD51: siCtrl ( n = 228) versus siTOPBP1 ( n = 348) cells (all datapoints are shown, bar represents mean, error bars represent S.D.). e Clonogenic surivival analysis of I-Ppo1-transfected control (siCtrl), Treacle-depleted (siTreacle) and TOPBP1-depleted (siTOPBP) U2OS cells (red bars represent the mean). f Clonogenic surivival analysis of I-Ppo1-transfected WT and NBS1DN U2OS cells (red bars represent the mean). g Model (see text for details). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used at the indicated dilutions: Treacle (rabbit, Sigma Life Science, HPA038237, 1/100), TOPBP1 (rabbit, Bethyl, A300-111A, 1/500), Nucleophosmin (mouse, Thermofisher, MA5-17141, 1/300), BRCA1 (mouse, sc-6454, Santa Cruz, 1/100), RAD51 (rabbit, Santa Cruz, sc-8349, 1/100), γH2AX (mouse, Millpore, 05-636, 1/500), MDC1 (mouse, Abcam, Ab50003, 1/300), Cyclin A (mouse, BD biosciences, 611269, 1/100), RPA2 (mouse, Abcam, Ab2175, 1/250), RPA2 pS4/S8 (rabbit, Bethyl A300-245, 1/400), 53BP1 (mouse, gift from T. Halazonetis, 1/20) NBS1 (rabbit, Novus, NB100-143, 1/200), MRE11 (mouse, GeneTex, GTX70212, 1/1000), UBF (mouse, Santa Cruz, sc-13121, 1/100), ATM (rabbit, Abcam, ab32420, 1/250), ATR (rabbit, Cell Signaling E1S3S, 1/100), V5 (mouse, Abcam, ab27671, 1/500).

Techniques: Control, Transfection

Journal: Carcinogenesis

Article Title: Genotoxicity of alcohol is linked to DNA replication-associated damage and homologous recombination repair.

doi: 10.1093/carcin/bgs340

Figure Lengend Snippet: Fig. 6. (A) Percentage of γ-H2AX (black columns), RAD51 (white columns) and 53BP1 (gray columns) positive V79 cells fixed at 0, 4 and 8 h after 30 min of treatment with 60 mM AA. Bars represent standard error for three independent experiments. (B) Colocalization of RAD51/53BP1 and γH2AX foci in V79 cells fixed at 8 h after 30 min of treatment with 60 mM AA visualized by confocal microscopy. The statistical significance compared with untreated cells was determined by Student’s t-test (*P < 0.05, **P < 0.01,***P < 0.001). The standard error of three independent experiments with 200 cells counted for each experiment is depicted.

Article Snippet: Primary monoclonal mouse γH2AX antibody (clone JBW301) was obtained from Millipore (Stockholm, Sweden), primary monoclonal rabbit RAD51 antibody (clone PC130) was obtained from Calbiochem, primary monoclonal rabbit 53BP1 antibody was obtained from Bethyl Lab (Montgomery, TX); Click-IT EdU Alexa Fluor® Imagine kit, secondary antibody Alexa Fluor® 555 donkey antimouse IgG (2 mg/ml) and Alexa Fluor® 488 antirabbit IgG (2 mg/ ml), TOPRO-3 iodide and Prolong® Gold were purchased from Invitrogen (Stockholm, Sweden).

Techniques: Confocal Microscopy