Journal: Nature Communications

Article Title: hnRNP H/F drive RNA G-quadruplex-mediated translation linked to genomic instability and therapy resistance in glioblastoma

doi: 10.1038/s41467-020-16168-x

Figure Lengend Snippet: a Immunofluorescence experiments in LN18 cells using the γ-H2AX, 53BP1 antibodies and DAPI staining. Mean intensities of γ-H2AX and 53BP1 in 2322 cells were plotted; the bottom and top of the box present the first and third quartile, respectively; the band inside the box shows the mean and the whiskers show the upper and lower extremes. Statistical significance was performed on the full cell populations. n = 2322 cells examined. Shown is a single representative field from one experiment over n = 2 independent experiments. For γ-H2AX: *** P -value = 4.26e-10, for 53BP1: *** P < 2.2e-16 (two-sided Mann & Whitney test). b Western blot analysis of γ-H2AX in LN18 cells treated with dose scale of carboxypyridostatin (cPDS) for 24 h. Shown is a representative result from n = 3 independent experiments. c Quantification of the γ-H2AX levels in LN18 treated with cPDS normalized to GAPDH levels and plotted relatively to the untreated condition. Data are presented as mean values ± SEM of n = 3 independent experiments, P -value = 0.0157 and P -value = 0.0457 for the 2 µM and 10 µM cPDS treatment respectively (two-sided paired t -test). d Quantification of DNA repair kinetics by western blot analysis of γ-H2AX after 4 Gy γ-irradiation in LN18 cells treated with control (siCtr) or hnRNP H/F (siH/F) siRNAs. Shown is a representative result from n = 2 independent experiments. e Plating efficiency assays measuring the cell survival fraction in LN18 treated with siCtr or siF siRNAs and submitted to a radiation dose scale. Data are presented as mean values ± SEM of 6 wells, P -value = 0.0003 and P -value = 0.0006 for the 2 Gy and 4 Gy dose, respectively (two-sided paired t -test). f Quantification of DNA repair kinetics by western blot analysis of γ-H2AX after temozolomide (TMZ) treatment in LN18 cells transfected with an empty plasmid (pICE) or a plasmid expressing Flag-hnRNP H/F. Shown is a representative result from n = 2 independent experiments. For all panels, source data are provided as a Source Data file.

Article Snippet: The coverslips were then incubated with primary antibodies in 1% normal goat serum/PBS at room temperature for 1 hr using antibodies against γ-H2AX (JBW301 Millipore 05-636; 1:500) and 53BP1 (Cell Signaling 2675; 1:200).

Techniques: Immunofluorescence, Staining, MANN-WHITNEY, Western Blot, Irradiation, Transfection, Plasmid Preparation, Expressing

Journal: Cell Cycle

Article Title: Polo-like kinase 1 inhibits DNA damage response during mitosis

doi: 10.4161/15384101.2014.977067

Figure Lengend Snippet: 53BP1 is phosphorylated during mitosis by Cdk1 and Plk1. ( A ) U2OS cells were irradiated with 3 Gy, fixed after 1 h and probed for γH2AX, 53BP1 and DAPI and analyzed by confocal microscopy. Single focal plane is shown. Bar indicates 10 μm. ( B ) Endogenous 53BP1 was immunoprecipitated from exponentially grown cells (Asynch.) or from cells arrested in mitosis by NZ, separated on 3–8% Tris-Acetate gel and analyzed by immunoblotting. Where indicated immunopurified 53BP1 was incubated with λ-phosphatase for 15 min at 30°C. ( C ) U2OS cells were grown exponentially (Asynch.), treated for 12 h with RO-3306 to arrest them in G2, or with BI2536 or NZ to arrest them in mitosis. Mitotic cells treated with NZ were collected by shake-off and incubated for additional 60 min with DMSO or with RO-3306 or SB202190. Whole cell lysates were separated on 3–8% Tris-Acetate or 4–12% Bis-Tris gels and probed with indicated antibodies.

Article Snippet: Following antibodies were used in this study: Cyclin B (sc-245), Cyclin A (sc-53230), BRCA1 (sc-6954), TFIIH (sc-293) and 53BP1 (sc-22760; Santa Cruz Biotechnology); pS10-histone H3 (06–570; Millipore-Upstate); anti-FLAG (F1804; Sigma); pS15-p53 (#9284S), γH2AX (#2577), pT210-Plk1 (#9062, clone D5H7) and pS1618–53BP1 (#6209, clone D4H11; Cell Signaling Technology); CREST (Cortex Biochem); anti-α-tubulin (clone TU-01) and anti-gamma-tubulin (clone TU-32, Exbio Praha).

Techniques: Irradiation, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation

Journal: Cell Cycle

Article Title: Polo-like kinase 1 inhibits DNA damage response during mitosis

doi: 10.4161/15384101.2014.977067

Figure Lengend Snippet: (See previous page). Plk1 phosphorylates 53BP1 in the UDR domain. ( A ) Purified GST or GST-53BP1-C-term were incubated with His-Plk1 in the presence of 32 P-γ-ATP and then separated on SDS-PAGE. Phosphorylation was detected by autoradiography or by immunoblotting with pS1618–53BP1 antibody. ( B ) Purified GST, GST-53BP1-C-term-WT or -S1618A were incubated with His-Plk1 and Phosphorylation was detected by autoradiography or by immunoblotting. ( C ) Unsynchronized cells (Asynch.) or cells arrested in mitosis by nocodazole or by Plk1 inhibitor (BI2536) were lyzed and probed with indicated antibodies. ( D ) U2OS cells were transfected with GAPDH or 53BP1 siRNA and grown asynchronically or arrested in mitosis by nocodazole. Arrowhead indicates the same position on the gel ( E ) U2OS cells were transfected by siRNA targeting GAPDH or Plk1. Nocodazole was added to cells transfected with GAPDH siRNA. Cells depleted of Plk1 spontaneously arrested in mitosis. Mitotic cells were collected by mitotic shake-off and analyzed by immunoblotting. ( F ) HeLa or U2OS cells were synchronized at G1/S transition by a double thymidine block, released to fresh media with nocodazole and collected in 2 h intervals. Media without nocodazole was used as control for cells that progressed to the following G1. ( G ) hRPE-TERT cells were grown exponentially or arrested in mitosis by nocodazole or BI2536 for 16 h and collected by mitotic shake-off. ( H ) Mitotic U2OS cells (NZ) were released to the fresh media and collected in 1 h intervals.

Article Snippet: Following antibodies were used in this study: Cyclin B (sc-245), Cyclin A (sc-53230), BRCA1 (sc-6954), TFIIH (sc-293) and 53BP1 (sc-22760; Santa Cruz Biotechnology); pS10-histone H3 (06–570; Millipore-Upstate); anti-FLAG (F1804; Sigma); pS15-p53 (#9284S), γH2AX (#2577), pT210-Plk1 (#9062, clone D5H7) and pS1618–53BP1 (#6209, clone D4H11; Cell Signaling Technology); CREST (Cortex Biochem); anti-α-tubulin (clone TU-01) and anti-gamma-tubulin (clone TU-32, Exbio Praha).

Techniques: Purification, Incubation, SDS Page, Autoradiography, Western Blot, Transfection, Blocking Assay

Journal: Cell Cycle

Article Title: Polo-like kinase 1 inhibits DNA damage response during mitosis

doi: 10.4161/15384101.2014.977067

Figure Lengend Snippet: 53BP1 phosphorylated at S1618 colocalize with Plk1. ( A ) U2OS cells were transfected with GAPDH or 53BP1 siRNA, fixed after 48 h and probed for endogenous 53BP1 and CREST (marker of centromeres) using confocal microscopy. Images represent single focal planes. Insets show magnified regions of the same image. Bar indicates 10 μm or 1 μm in the insets. ( B ) Mitotic cells expressing EGFP-Plk1 were probed with 53BP1 or pS1618–53BP1 and CREST and analyzed by confocal microscopy as in A. ( C ) U2OS cells transfected with control, 53BP1 or Plk1 siRNA were analyzed after 48 h by confocal microscopy. Alternatively cells were treated with BI2536 (10 nM, 3 h). Images represent single focal plane and bar indicates 10 μm. ( D ) Exponentially growing U2OS cells were fixed and stained with pS1618–53BP1 antibody. Bar indicates 10 μm. Total cell fluorescence was quantified in interphase and mitotic cells. Each dot represents one cell. Error bars indicate mean and SD. ( E ) U2OS cells were transfected with GAPDH or 53BP1 siRNA, fixed and probed for tubulin and DAPI. Morphology of mitotic spindles was scored as bipolar or aberrant (monopolar and multipolar; n=3, error bars indicate SD). ( F ) U2OS stably expressing EGFP-53BP1 were grown exponentially (Asynch.), synchronized in G2 by RO-3306 or in mitosis by nocodazole and 53BP1 was immunoprecipitated by GFP-Trap. Bound proteins were analyzed by immunoblotting.

Article Snippet: Following antibodies were used in this study: Cyclin B (sc-245), Cyclin A (sc-53230), BRCA1 (sc-6954), TFIIH (sc-293) and 53BP1 (sc-22760; Santa Cruz Biotechnology); pS10-histone H3 (06–570; Millipore-Upstate); anti-FLAG (F1804; Sigma); pS15-p53 (#9284S), γH2AX (#2577), pT210-Plk1 (#9062, clone D5H7) and pS1618–53BP1 (#6209, clone D4H11; Cell Signaling Technology); CREST (Cortex Biochem); anti-α-tubulin (clone TU-01) and anti-gamma-tubulin (clone TU-32, Exbio Praha).

Techniques: Transfection, Marker, Confocal Microscopy, Expressing, Staining, Fluorescence, Stable Transfection, Immunoprecipitation, Western Blot

Journal: Cell Cycle

Article Title: Polo-like kinase 1 inhibits DNA damage response during mitosis

doi: 10.4161/15384101.2014.977067

Figure Lengend Snippet: 53BP1 is phosphorylated by Cdk1/cyclin B. ( A ) Purified GST or GST-53BP1-C-term was phosphorylated in vitro by active Cdk1/cyclin B or p38a and phosphorylation was detected by autoradiography for 30 min or 5 h. ( B ) Various alanine mutants of GST-53BP1-C-term were phosphorylated in vitro by Cdk1/cyclin B.

Article Snippet: Following antibodies were used in this study: Cyclin B (sc-245), Cyclin A (sc-53230), BRCA1 (sc-6954), TFIIH (sc-293) and 53BP1 (sc-22760; Santa Cruz Biotechnology); pS10-histone H3 (06–570; Millipore-Upstate); anti-FLAG (F1804; Sigma); pS15-p53 (#9284S), γH2AX (#2577), pT210-Plk1 (#9062, clone D5H7) and pS1618–53BP1 (#6209, clone D4H11; Cell Signaling Technology); CREST (Cortex Biochem); anti-α-tubulin (clone TU-01) and anti-gamma-tubulin (clone TU-32, Exbio Praha).

Techniques: Purification, In Vitro, Autoradiography

Journal: Cell Cycle

Article Title: Polo-like kinase 1 inhibits DNA damage response during mitosis

doi: 10.4161/15384101.2014.977067

Figure Lengend Snippet: Phosphorylation of 53BP1 by Plk1 and Cdk1 inhibits its binding to ubiquitinated histones. ( A ) Purified GST, GST-53BP1-C-WT , GST-53BP1-C-S1618D or GST-53BP1-C-S1618D-S1609D were incubated with extract from U2OS-FLAG-ubiqiuitin cells treated with etoposide. Pull down was done by glutathione sepharose and bound proteins were analyzed by immunoblotting. ( B ) Purified GST-53BP1-C-WT was phosphorylated in vitro by Plk1 or Plk1 and Cdk1/cyclin B or mock phosphorylated and incubated with extract from U2OS-FLAG-ubiqiuitin cells treated with etoposide as in A. ( C ) U2OS cells transfected with EGFP-53BP1-WT, -S1618D or –S1609D-S1618D were pre-treated with BrdU, laser micro-irradiated and recruitment of EGFP-tagged proteins to irradiated area was assayed by life imaging. ( D ) Cells from ( C ) were fixed 3 h after exposure to ionizing radiation (3 Gy) and DNA damage foci were analyzed by automated high-content microscopy and spot detection module. Percentage of cells with more than 5 53BP1 foci is shown. (n=3, error bars indicate SD) ( E ) U2OS cells transfected with EGFP-53BP1-WT, -S1618D or -S1609D-S1618D were grown exponentially and analyzed 48 h after transfection by automated high-content microscopy. Average number of 53BP1 foci was quantified in G1 cells gated by the intensity of the DAPI. (n=3, error bars indicate SD) ( F ) Exponentially growing U2OS cells were fixed 3 h after irradiation with 3 Gy and probed with antibodies against 53BP1 and γ-tubulin. Note no difference in 53BP1 foci formation in late G2 cell with separated centrosomes.

Article Snippet: Following antibodies were used in this study: Cyclin B (sc-245), Cyclin A (sc-53230), BRCA1 (sc-6954), TFIIH (sc-293) and 53BP1 (sc-22760; Santa Cruz Biotechnology); pS10-histone H3 (06–570; Millipore-Upstate); anti-FLAG (F1804; Sigma); pS15-p53 (#9284S), γH2AX (#2577), pT210-Plk1 (#9062, clone D5H7) and pS1618–53BP1 (#6209, clone D4H11; Cell Signaling Technology); CREST (Cortex Biochem); anti-α-tubulin (clone TU-01) and anti-gamma-tubulin (clone TU-32, Exbio Praha).

Techniques: Binding Assay, Purification, Incubation, Western Blot, In Vitro, Transfection, Irradiation, Imaging, Microscopy

Journal: Cell Cycle

Article Title: Polo-like kinase 1 inhibits DNA damage response during mitosis

doi: 10.4161/15384101.2014.977067

Figure Lengend Snippet: Inhibition of Plk1 increases DNA repair capacity in mitotic cells. ( A ) U2OS cells were synchronized in mitosis by NZ (16 h), incubated for additional 2 h with DMSO or BI2536 (50 nM) and treated with NCS (2 nM) for indicated times. DNA lesions were quantified by neutral comet assay. Plotted is average amount of DNA in tails, error bars indicate SD. Circles and triangles indicate individual cells. ( B ) Cells were treated as in (A), fixed at indicated times and γH2AX levels were measured by FACS (at least 10 4 cells per condition, n=4, error bars indicate SD). ( C ) U2OS cells were transfected with siRNA targeting coding region or 3-UTR region of 53BP1 and knock down was evaluated by immunoblotting. ( D ) U2OS-TR cells stably transfected with EGFP-53BP1-WT, -S1618D or –S1609D-S1618D were transfected with siRNA targeting 3-UTR region of 53BP1. After 48h expression of EGFP-53BP1 was induced by tetracycline for 12h, cells were irradiated with 3 Gy and fixed 8h afterwards. BRCA1 foci were analyzed by automated high-content microscopy. Average number of 53BP1 foci was quantified in G1 cells gated by the intensity of the DAPI and negative Cyclin A signal (n=3, error bars indicate SD). ( E ) RPE cells stably expressing EGFP-53BP1-WT or –S1609D-S1618D were treated and analyzed as in ( D ). (n=4, error bars indicate SD). ( F ) RPE cells stably expressing EGFP-53BP1-WT or –S1609D-S1618D were treated as in ( D ) and γH2AX-positive foci were analyzed by automated high-content microscopy. Average number of γH2AX- foci was quantified in G1 cells gated by the intensity of the DAPI and negative Cyclin A signal (n=3, error bars indicate SD).

Article Snippet: Following antibodies were used in this study: Cyclin B (sc-245), Cyclin A (sc-53230), BRCA1 (sc-6954), TFIIH (sc-293) and 53BP1 (sc-22760; Santa Cruz Biotechnology); pS10-histone H3 (06–570; Millipore-Upstate); anti-FLAG (F1804; Sigma); pS15-p53 (#9284S), γH2AX (#2577), pT210-Plk1 (#9062, clone D5H7) and pS1618–53BP1 (#6209, clone D4H11; Cell Signaling Technology); CREST (Cortex Biochem); anti-α-tubulin (clone TU-01) and anti-gamma-tubulin (clone TU-32, Exbio Praha).

Techniques: Inhibition, Incubation, Neutral Comet Assay, Transfection, Western Blot, Stable Transfection, Expressing, Irradiation, Microscopy

Journal: Cell Cycle

Article Title: Polo-like kinase 1 inhibits DNA damage response during mitosis

doi: 10.4161/15384101.2014.977067

Figure Lengend Snippet: Model of 53BP1 inhibition by Plk1 and Cdk1 phosphorylation in mitosis. Following exposure of interphase cells to genotoxic stress, activation of ATM eventually leads to monoubiquitination of H2A by RNF168. Together with constitutive H4K20-me2 modification this allows recruitment of 53BP1 to DNA damage foci and its function in NHEJ repair. In mitosis, Cdk1 phosphorylates 53BP1 in the N-terminal part to generate a docking site for Plk1. In turn, Plk1 phosphorylates 53BP1 at S1618 within the UDR domain and disables binding of 53BP1 to H2A-Ub. In addition, Cdk1 phosphorylates S1609 and S1678 further inhibiting the ability of 53BP1 to bind to H2A-Ub. Mitotic 53BP1 is not phosphorylated by ATM in mitosis and its role in NHEJ is blocked.

Article Snippet: Following antibodies were used in this study: Cyclin B (sc-245), Cyclin A (sc-53230), BRCA1 (sc-6954), TFIIH (sc-293) and 53BP1 (sc-22760; Santa Cruz Biotechnology); pS10-histone H3 (06–570; Millipore-Upstate); anti-FLAG (F1804; Sigma); pS15-p53 (#9284S), γH2AX (#2577), pT210-Plk1 (#9062, clone D5H7) and pS1618–53BP1 (#6209, clone D4H11; Cell Signaling Technology); CREST (Cortex Biochem); anti-α-tubulin (clone TU-01) and anti-gamma-tubulin (clone TU-32, Exbio Praha).

Techniques: Inhibition, Activation Assay, Modification, Binding Assay

Journal: Molecular cell

Article Title: BRCA1 mutational complementation induces synthetic viability

doi: 10.1016/j.molcel.2020.04.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used: RAD51 ([N1C2] GeneTex), RPA32 (19/NA18 CalBiochem), RPA32 (4E4 Cell Signaling), CtIP (271339 Santa Cruz), RIF1 (A300–569A Bethyl), 53BP1 (3802 Millipore), phospho53BP1 (3428 Cell Signaling), BRCA1 (6954 Santa Cruz) BRCA1 (07–434 Millipore), PCNA (sc-56 Santa Cruz) and, HA tag (Cell Signaling 1:500).

Techniques: Recombinant, Plasmid Preparation, Staining, Mutagenesis, Modification, Software