Journal: bioRxiv

Article Title: An ATM/Wip1-dependent timer controls the minimal duration of a DNA-damage mediated cell cycle arrest

doi: 10.1101/042119

Figure Lengend Snippet: ( A ) U2OS cells transfected with GAPDH or Wip1 siRNA were treated with NCS (5 nM) and collected after 2, 6 and 20 h. Chromatin fractions were probed with indicated antibodies. ( B ) RPE cells transfected with Wip1 siRNA were microirradiated, fixed 1 or 24 h later, and stained with the indicated antibodies. ( C) HA-KAP1-WT or-S824A were immunopurified from cells exposed to NCS, incubated with His-Wip1 and probed with pS824-KAP1 or KAP1 antibody. (D) U2OS cells were fixed 1 h after treatment with NCS, incubated with His-Wip1 (0-5ng/ul) and probed for gH2AX and pS824-KAP1. Plot shows mean nuclear fluorescence intensity, error bars indicate SD. (E ) Kap1 is dephosphorylated before Plk1 activation. RPE cells transfected with TP53 siRNA and U2OS cells were followed as in and stained for pS824-Kap1. For RPE cells, the times were modified as indicated. (F) RPE cells transfected with GAPDH or TP53 siRNA were synchronized by HU, released to fresh media for 5 h (R5) and treated with NCS for indicated times. Nocodazole (NZ) was added 1 h after NCS. Where indicated, cells were incubated in the presence of BI2536. Whole cell lysates were probed with indicated antibodies. ( G ) Overexpression of Kap1-S824A phenocopies ATM inhibition. Cumulative mitotic entry of ≥300 U2OS cells expressing inducible HA-tagged Kap1-wt (red) or Kap1-S824A (green) after treatment with NCS (4 nM) and subsequent treatment after 1h with ATRi or ATRi + ATMi.

Article Snippet: Antibodies against pSMC3-S1083 (#IHC00070), pKap1-S824 (#A300-767A and GTX63711), Kap1 (#A300-274A and GTX62973), pS966-SMC1 (A300-050A), and PP4C (GTX114659) were from Bethyl Lab and GeneTex.

Techniques: Transfection, Staining, Incubation, Fluorescence, Activation Assay, Modification, Over Expression, Inhibition, Expressing

Journal: bioRxiv

Article Title: An ATM/Wip1-dependent timer controls the minimal duration of a DNA-damage mediated cell cycle arrest

doi: 10.1101/042119

Figure Lengend Snippet: (A) U2OS or RPE cells were transfected with Wip1 siRNA, microirradiated, and stained after 1 or 24 h with the indicated antibodies. (B) U2OS and RPE cells were microirradiated in the presence of indicated inhibitors. After 1 h, cells were stained with the indicated antibodies. (C) EGFP or EGFP-Wip1 was immunoprecipitated from HEK293 cells using GFP-Trap. Endogenous KAP1 and p53 were probed with antibodies. (D) U2OS cells transfected with GAPDH or Wip1 siRNA were treated with NCS in combination with DMSO or ATMi for indicated times. Whole cell lysates were probed with indicated antibodies. (E) U2OS cells transfected with GAPDH, Wip1 or PP4C siRNA were treated with NCS for indicated times. Soluble and chromatin fractions were probed with indicated antibodies. (F) RPE cells transfected with GAPDH, Wip1 or PP4C siRNA were treated with NCS for indicated times. Whole cell lysates were probed with indicated antibodies.

Article Snippet: Antibodies against pSMC3-S1083 (#IHC00070), pKap1-S824 (#A300-767A and GTX63711), Kap1 (#A300-274A and GTX62973), pS966-SMC1 (A300-050A), and PP4C (GTX114659) were from Bethyl Lab and GeneTex.

Techniques: Transfection, Staining, Immunoprecipitation

Journal: Journal of Virology

Article Title: A Mechanism-Based Targeted Screen To Identify Epstein-Barr Virus-Directed Antiviral Agents

doi: 10.1128/jvi.01179-20

Figure Lengend Snippet: Figure 2. Validating 30 candidate compounds by measuring their effects on KAP1 432

Article Snippet: 348 349 Antibodies 350 Antibodies included mouse anti-FLAG Ab (Sigma F3165), goat anti-KAP1 Ab (Bethyl 351 Laboratories A303-838A), rabbit anti-KAP1 Ab (Bethyl Laboratories A300-274A), rabbit anti-p-352 S824-KAP1 Ab (Bethyl Laboratories A300-767A), mouse anti-EA-D Ab (EMD MAB8186), 353 IRDye 800CW-conjugated donkey anti-rabbit IgG (LI-COR Bioscience 926-32213), and 354 IRDye 680RD-conjugated donkey anti-goat IgG (LI-COR Bioscience 926-68047).

Techniques:

Journal: Journal of Virology

Article Title: A Mechanism-Based Targeted Screen To Identify Epstein-Barr Virus-Directed Antiviral Agents

doi: 10.1128/jvi.01179-20

Figure Lengend Snippet: Figure 9. Selected candidate compounds inhibit phosphorylation of KAP1 in an independent 487

Article Snippet: 348 349 Antibodies 350 Antibodies included mouse anti-FLAG Ab (Sigma F3165), goat anti-KAP1 Ab (Bethyl 351 Laboratories A303-838A), rabbit anti-KAP1 Ab (Bethyl Laboratories A300-274A), rabbit anti-p-352 S824-KAP1 Ab (Bethyl Laboratories A300-767A), mouse anti-EA-D Ab (EMD MAB8186), 353 IRDye 800CW-conjugated donkey anti-rabbit IgG (LI-COR Bioscience 926-32213), and 354 IRDye 680RD-conjugated donkey anti-goat IgG (LI-COR Bioscience 926-68047).

Techniques: Phospho-proteomics

Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in immortalized MEFs. (A) Cells (WT, MJI-53; KO, MJI-1) were treated for 24 h using 0 or 3 μM aphidicolin. (B) Cells (lane 1–3, WT, MJI-91; lanes 4–6, KO, MJI-186; lanes 7–9, WT, MJI-105; lanes 1–12, KO, MJI-84) were treated for 24 h using 0, 1 or 3 μM aphidicolin. Actin and 53BP1 serve as loading controls. DYNLL1 is used as a surrogate marker for loss of ASCIZ. (C) Quantification of western blot band intensities. An arbitrary unit of 100 represents the average band intensity for the respective phospho-protein in the 3 μM aphidicolin-treated wildtype samples on each membrane. Graphs indicate the mean ± standard error, n = 3. Additional loading controls for total KAP1, p53 and H2AX are shown in Supplementary Fig. S1A and B.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Marker, Western Blot, Membrane

Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in primary MEFs. Freshly prepared primary MEFs from a wildtype and an Asciz −/− embryo were treated for 24 h with 0, 1 or 3 μM aphidicolin. For comparison, cultures from the same embryos were also irradiated with 2-Gy and allowed to recover for 30 min. Similar results for another independent pair of wildtype and Asciz −/− primary MEFs are shown in Supplementary Fig. S1C, and additional loading controls for total KAP1, p53 and H2AX are shown in Supplementary Fig. S1D.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Comparison, Irradiation

Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in human ASCIZ/ATMIN knockout cells. (A, B) BL30 cells were cultured as indicated in the presence or absence of doxycycline to induce a non-targeting control guide RNA or the guide RNA against ASCIZ , and aphidicolin to elicit prolonged DNA replication stress as indicated. For comparison, control guide RNA transduced cells were also irradiated for canonical ATM activation without doxycycline or aphidicolin treatment in panel A. Panel B shows a blot of the same samples except the irradiated control. Additional loading controls for total ATM, KAP1 and p53 are shown in Supplementary Fig. S1E.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Knock-Out, Cell Culture, Control, Comparison, Irradiation, Activation Assay

Journal: BMC cancer

Article Title: Functional deficiency of NBN, the Nijmegen breakage syndrome protein, in a p.R215W mutant breast cancer cell line.

doi: 10.1186/1471-2407-14-434

Figure Lengend Snippet: Figure 2 Assessment of cellular radiosensitivity in the colony formation assay. A. Cellular radiosensitivity of p.R215W mutant cells (HCC1395) compared with wild type breast epithelial cells (MCF10A) as measured by the colony formation assay after irradiation at doses of 0, 0.1, 0.25, 0.5 or 1Gy. The surviving fraction is presented as the mean value with SEM from at least 3 independent experiments. B. Induction of PARP1 cleavage after irradiation with 2 Gy in p.R215W mutant cells (HCC1395) compared with wild type breast epithelial cells (MCF10A) as measured by immunoblotting of cleaved PARP1 (89 kDa) and total PARP1 (116 kDa). C. Immunoblot analysis of radiation-induced ATM signalling in HCC1395 cells compared with MCF10A. Cells were untreated or irradiated with 0.5, 1, 2, 4 or 6 Gy as indicated. Protein extracts were prepared 30 min after irradiation and were analysed through Western blotting for their immunoreactivity towards the phosphorylated forms of SMC1 (pSer966, top panel), KAP1 (p824, middle panel), and CHEK2 (pSer19, bottom panel). β-actin served as the loading control in each experiment.

Article Snippet: Antibodies to NBN, SMC1pS966, KAP1-pS824 were obtained from Novus Biologicals (rabbit polyclonal); anti CHEK2-pS19 from Cell Signaling (rabbit polyclonal); anti RAD50 (mouse monoclonal) from Abcam; anti MRE11 (mouse monoclonal 12D7) from GeneTex; PARP1 and cleaved PARP1 (rabbit polyclonal) from Cell Signaling, and anti β-Actin (mouse monoclonal) from Sigma.

Techniques: Colony Assay, Mutagenesis, Irradiation, Western Blot, Control