Journal: Frontiers in Oncology

Article Title: Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

doi: 10.3389/fonc.2021.612354

Figure Lengend Snippet: HSP90i by NW457 leads to downregulation of DNA damage response factors, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in human glioblastoma cells. (A) Transcriptomic profiling of regulators of the DNA damage response (DDR) in LN229 and T98G cells. mRNA expression levels were determined by qRT-PCR, normalized to a matrix of 3 reference genes (18S rRNA, δ-amino-laevulinate-synthase, and β2-microglobulin), and calibrated to the results of untransformed human astrocytes. For both cell lines, three replicates were analyzed and are displayed as x-fold log2-values. (B) Time course analysis of DDR regulator protein expression in LN229 and T98G cells upon HSP90i by 10 nM NW457. Arrowheads indicate the bands that were used for quantification. Protein levels were normalized to a matrix comprising vinculin and α-tubulin and are depicted as x-fold log2-values compared to the 0 h controls. (C) Immunofluorescence microscopy of γH2AX and 53BP1 DNA damage repair foci in LN229 and T98G cells upon irradiation at 2 Gy ± HSP90i by NW457. Cells were treated with NW457 (10 nM) or DMSO for 24 h, irradiated, and fixed at the indicated times. Cells were stained for γH2AX, 53BP1, and DNA and subjected to deconvolution immunofluorescence microscopy. Scale bar depicts 10 µm. (D) Quantification of DNA damage repair kinetics from (C) . γH2AX and 53BP1 double-positive foci in at least 20 randomly picked nuclei were counted by hand. Individual data points with superimposed means ± 95% confidence intervals are displayed, and overall curve comparison was performed by two-way ANOVA. (E) Clonogenic survival of LN229 and T98G cells upon irradiation at 0–8 Gy ± HSP90i by NW457. Cells were pre-treated with 10 nM NW457 or DMSO for 24 h followed by irradiation at the indicated doses, and colony formation was allowed for 13 d ± continuous NW457 treatment. Individual data points of 4 independent experiments are shown, linear-quadratic regression lines are superimposed, and overall curve comparison was performed by two-way ANOVA.

Article Snippet: Cells were stained with monoclonal mouse anti-γH2AX (Merck Millipore) and polyclonal rabbit anti-53BP1 (Bio-Techne, Wiesbaden, Germany) antibodies diluted in 3% isotonic bovine serum albumin and 0.1% Triton X-100 for 2 h at room temperature.

Techniques: Irradiation, Expressing, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining, Comparison

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

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: Cells

Article Title: WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

doi: 10.3390/cells8101258

Figure Lengend Snippet: WIP1 plays role in DNA double-strand break repair in S-phase cells. ( A ) Quantification of 53BP1 foci in replicating (EdU+) cells after irradiation. U2OS parental cell lines with or without combined treatment with WIP1i and two independent WIP1 knockout cell lines were pulse-labeled with EdU for 30 min before irradiation. Cells were fixed after pre-extraction at indicated time-points and stained with 53BP1 antibody. Click chemistry was used to visualize EdU. Mean of median foci number +/- SD is plotted (n ≥ 3). Statistical significance evaluated by two tailed t -test. ( B ) Quantification of 53BP1 foci in non-replicating (EdU-) cells after irradiation. As in A. ( C ) Quantification of 53BP1 foci in replicating (EdU+) cells after irradiation. U2OS parental, WIP1 knockout and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were irradiated and analyzed as in A. ( D ) Quantification of 53BP1 foci in non-replicating (EdU-) cells after irradiation. U2OS parental, WIP1 knockout and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were irradiated and analyzed as in A. ( E ) Traffic light reporter assay in U2OS cells after transfection with indicated siRNA. Cells were transfected with ISceI together with BFP-donor vector with or without pretreatment with 1 μM WIP1i 2 days after siRNA transfection. Efficiency of repair was analyzed by FACS 3 days after ISceI and BFP-donor transfection. Plotted is mean +/− SD. Statistical significance evaluated by two-tailed t -test. ( F ) Efficiency of repair by HR and NHEJ in Traffic light reporter assay as in E. ( G ) Representative plots from Traffic light reporter assay in E.

Article Snippet: Following antibodies were used: WIP1 antibody (clone F-10, sc-376257), p21 (sc-397), p53 (clone D01, sc-126), BRCA1 (sc-6954), rabbit-53BP1 (sc-22760), RAD51 (sc-6862) and TFIIH (sc-293, used as loading control) from Santa Cruz Biotechnology (Dallas, TX, USA); phoshpo-Thr543-53BP1 (#3428), phospho-S15-p53 (#9284) from Cell Signaling Technology (Danvers, MA, USA); RPA2 (clone 9H8, ab2175), and phospho-Ser1524-BRCA1 (ab2401) from Abcam (Cambridge, UK), γH2AX (05-636), and mouse monoclonal 53BP1 (MAB3802) from Merck Millipore (Burlington, MA, USA); phospho-S824-KAP1 (GTX63711), KAP1 (GTX62973) and PP4C (GTX114659) from Genetex (Irvine, CA, USA); secondary Alexa Fluor conjugated antibodies from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Irradiation, Knock-Out, Labeling, Extraction, Staining, Two Tailed Test, Mutagenesis, Reporter Assay, Transfection, Plasmid Preparation

Journal: Cells

Article Title: WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

doi: 10.3390/cells8101258

Figure Lengend Snippet: WIP1 delays recruitment of BRCA1 and dephosphorylation of 53BP1 at T543. ( A ) Co-immunoprecipitation of WIP1 and 53BP1. HEK293 cells were transfected with either empty GFP or GFP-WIP1, subjected to immunoprecipitation using GFP-Trap 24 h after transfection and by Western blotting with 53BP1 antibody. Ponceau staining with indicated positions of GFP (empty arrowhead) and GFP-WIP1 (full arrowhead) are shown. ( B ) HEK293 cells transfected with EGFP or EGFP-WIP1 were exposed to 3 Gy of IR, collected at indicated times and proteins were immunoprecipitated by GFP Trap. ( C ) Quantification of 53BP1 pT543 signal intensity in replicating (EdU+) cells after irradiation. U2OS parental and WIP1 knockout cell lines were pulse-labeled with EdU for 30 min before irradiation. Cells were fixed after pre-extraction at indicated time-points after IR and stained with p53BP1 T543 antibody. Click chemistry was used to visualize EdU. Mean of median total intensity +/− SD is plotted. ( D ) Western blot analysis of whole cell lysates of U2OS cells transfected with GAPDH or PP4C siRNA in response to irradiation and/or WIP1 inhibitor. ( E ) Quantification of 53BP1 pT543 signal intensity in replicating (EdU+) cells after irradiation. U2OS parental and WIP1 knockout cell lines were transfected with control or PP4C siRNA 2 days before irradiation. Cells were processed and analyzed as in C. ( F ) Quantification of RPA2 foci in replicating (EdU+) cells after irradiation. U2OS parental cell lines with or without combined treatment with WIP1i were pulse-labeled with EdU for 30 minutes before irradiation. Cells were fixed after pre-extraction at indicated time-points and stained with RPA2 and RAD51 antibodies. Click chemistry was used to visualize EdU. Mean of median foci number +/− SD is plotted. ( G ) Quantification of RAD51 foci in replicating (EdU+) cells after irradiation as in F.

Article Snippet: Following antibodies were used: WIP1 antibody (clone F-10, sc-376257), p21 (sc-397), p53 (clone D01, sc-126), BRCA1 (sc-6954), rabbit-53BP1 (sc-22760), RAD51 (sc-6862) and TFIIH (sc-293, used as loading control) from Santa Cruz Biotechnology (Dallas, TX, USA); phoshpo-Thr543-53BP1 (#3428), phospho-S15-p53 (#9284) from Cell Signaling Technology (Danvers, MA, USA); RPA2 (clone 9H8, ab2175), and phospho-Ser1524-BRCA1 (ab2401) from Abcam (Cambridge, UK), γH2AX (05-636), and mouse monoclonal 53BP1 (MAB3802) from Merck Millipore (Burlington, MA, USA); phospho-S824-KAP1 (GTX63711), KAP1 (GTX62973) and PP4C (GTX114659) from Genetex (Irvine, CA, USA); secondary Alexa Fluor conjugated antibodies from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Transfection, Western Blot, Staining, Irradiation, Knock-Out, Labeling, Extraction, Control

Journal: Cells

Article Title: WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

doi: 10.3390/cells8101258

Figure Lengend Snippet: WIP1 deficient cells are more sensitive to PARP inhibition. ( A ) Cell survival of parental U2OS, two independent U2OS-WIP1-KO cell lines with or without combined treatment with WIP1i was evaluated 7 days after treatment with indicated doses of olaparib using resazurin viability assay. Plotted is mean +/− SD, n ≥ 3. Statistical significance evaluated by two-way ANOVA. ( B ) Cell survival of parental U2OS, U2OS-WIP1-KO cells and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 in response to 5 μM olaparib as in A. Statistical significance evaluated by two-tailed t -test (n ≥ 3). ( C ) Percentage of dead cells was evaluated by Hoechst 33258 staining and FACS analysis 7 days after treatment with 5 μM olaparib in U2OS cell line with or without combined treatment with WIP1i. Plotted is mean +/− SD. ( D ) Cell survival of RPE and MCF7 cell lines with or without combined treatment with WIP1i was evaluated 7 days after treatment with indicated doses of olaparib using resazurin viability assay. Plotted is mean +/− SD. N ≥ 3. Statistical significance evaluated by two-way ANOVA. ( E ) Cells were transfected with control siRNA (siNC) or siRNA to PP4C (siPP4C). Cell survival was evaluated after 7 days of treatment with olaparib and DMSO or WIP inhibitor. Statistical significance evaluated by two-tailed t -test (n = 3). ( F ) Quantification of 53BP1 foci number 3 days after treatment with olaparib. U2OS cells were treated with indicated doses of olaparib together with or without WIP1i for 3 days, fixed, stained with 53BP1 antibody and percentage of cells having 0–3, 3–10 and >10 foci were quantified. Mean +/− SD is plotted, n ≥ 3. ( G ) Quantification of 53BP1 foci after treatment with olaparib. U2OS-WIP1-KO cells and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were treated with WIP1i and olaparib for 3 days, fixed after pre-extraction and stained with 53BP1 antibody. Number of 53BP1 foci in S/G2 cells was evaluated using DAPI content of >2 n to gate S-G2 cells. Mean of median foci number +/− SD is plotted, n ≥ 3. Statistical significance evaluated by two-tailed t -test. ( H ) Response of U2OS and U2OS-WIP1-KO cell lines to treatment with 5 μM olaparib for 24–72h was analyzed by Western blotting using indicated antibodies. I ) Quantification of 53BP1 foci 3 days after treatment with olaparib. MCF7 cells were transfected with indicated siRNAs and treated after 2 days with WIP1i and olaparib alone or combined for further 3 days. Cells were fixed after pre-extraction and stained with 53BP1 antibody. Number of 53BP1 foci in S/G2 cells was evaluated using DAPI content of >2 n to gate S-G2 cells. Mean of median foci number +/− SD is plotted. Statistical significance evaluated by two-tailed t -test.

Article Snippet: Following antibodies were used: WIP1 antibody (clone F-10, sc-376257), p21 (sc-397), p53 (clone D01, sc-126), BRCA1 (sc-6954), rabbit-53BP1 (sc-22760), RAD51 (sc-6862) and TFIIH (sc-293, used as loading control) from Santa Cruz Biotechnology (Dallas, TX, USA); phoshpo-Thr543-53BP1 (#3428), phospho-S15-p53 (#9284) from Cell Signaling Technology (Danvers, MA, USA); RPA2 (clone 9H8, ab2175), and phospho-Ser1524-BRCA1 (ab2401) from Abcam (Cambridge, UK), γH2AX (05-636), and mouse monoclonal 53BP1 (MAB3802) from Merck Millipore (Burlington, MA, USA); phospho-S824-KAP1 (GTX63711), KAP1 (GTX62973) and PP4C (GTX114659) from Genetex (Irvine, CA, USA); secondary Alexa Fluor conjugated antibodies from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Inhibition, Viability Assay, Mutagenesis, Two Tailed Test, Staining, Transfection, Control, Extraction, Western Blot

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