Journal: Nucleic Acids Research

Article Title: Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility

doi: 10.1093/nar/gkad128

Figure Lengend Snippet: NHEJ repair was impaired by Cd ions through prolonged DNA-PKcs phosphorylation, and subsequently promoted dissociation of DNA-PKcs from DNA ends. ( A ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of Ku70 and Ku80 on the chromatin fraction. ( B ) U2OS cells were transfected with NHEJ reporter and/or I-SceI plasmids for 4 h, followed by treatment with or without CdCl 2 (100 μM) for 3 h, and ChIP assay was performed using anti-Ku80 antibody, followed by PCR to detect the Ku80 bound DSB fragment. ( C ) Immunoblotting of whole cell extracts from GC-2 cells treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) analyzed at the indicated times. ( D ) Immunoblotting of whole cell extracts from GC-2 cells treated by X-rays (2 Gy) with or without CdCl 2 (20 μM) analyzed at the indicated times. ( E ) U2OS cells were transfected with NHEJ reporter and/or I-SceI plasmids for 4 h, followed by treatment with or without CdCl 2 (100 μM) for 3 h, and ChIP assay was performed using anti-DNA-PKcs antibody, followed by PCR to detect DNA-PKcs bound DSB fragment. ( F ) U2OS cells were treated by H 2 O 2 (1 mM) with or without CdCl 2 (100 μM) for 1 h, followed by IP, with Ku80 antibody associated DNA-PKcs analyzed by western blot. ‘*’ indicates nonspecific band. ( G ) Quantitative analysis of DNA-PKcs binding with Ku80 by immunocoprecipitation in (F). ( H ) Immunoblotting of chromatin fraction from GC-2 cells treated by X-rays (2 Gy) with or without CdCl 2 (20 μM) analyzed at the indicated times. ( I ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of Artemis on the chromatin fraction. ** P < 0.01; *** P < 0.001; ns indicates non-significant difference. Data presented as mean ± SD from three independent experiments.

Article Snippet: The antibodies used in this study were: phospho-H2AX (Ser139) (2577), phospho-H2AX (Ser139) (80312), H2AX (7631), phospho-CHK1 (Ser317) (12302), CHK1 (2360), CHK2 (6334), phospho-CHK2 (Thr68) (2197), Ku80 (2180), Ku70 (4588), phospho-DNA-PKcs (Ser2056) (68716), DNA-PKcs (38168), GAPDH (5174), PP2A (2038), Artemis (13381), ATM (2873), PP5 (2289), Histone H4 (13919), Phospho-ATM (Ser1981) (5883) and 53BP1 (88439), purchased from Cell Signaling Technology, USA; RAD51 (ab133534) and PP1 (ab134947), purchased from Abcam, USA; Phospho-DNA-PKcs (Thr2609) (NBP1-02456), purchased from NOVUS, USA.

Techniques: Phospho-proteomics, Western Blot, Transfection, Binding Assay

Journal: Nucleic Acids Research

Article Title: Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility

doi: 10.1093/nar/gkad128

Figure Lengend Snippet: PP5 inactivation induced by Cd ions mediated the prolonged phosphorylation of DNA-PKcs. ( A ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of phosphorylation of ATM in whole cells. ( B ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, and/or pretreated with 200nM OA for 30 min, followed by immunoblot analysis of indicated protein in whole cells. ( C ) After siPP5 for 72 h, GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of indicated protein in whole cells. ( D ) Quantitative analysis of pDNA-PKcs s2056 by immunoblot analysis in (C). ( E ) U2OS cells were transfected with PP5, PP2A or PP1 siRNA for 72 h, then transfected with NHEJ reporter and/or I-SceI 4 h later, followed by treatment with or without CdCl 2 (100 μM) for 3 h; ChIP assay was performed using anti-DNA-PKcs antibody, followed by PCR to detect DNA-PKcs bound DSB fragment. ( F ) GC-2 cells stably expressing the PP5 wildtype (WT) or H304A mutant were treated by X-rays (2 Gy) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of the indicated protein in whole cells. * P < 0.05; ** P < 0.01; *** P < 0.001; ns indicates non-significant difference. Data presented as mean ± SD from three independent experiments.

Article Snippet: The antibodies used in this study were: phospho-H2AX (Ser139) (2577), phospho-H2AX (Ser139) (80312), H2AX (7631), phospho-CHK1 (Ser317) (12302), CHK1 (2360), CHK2 (6334), phospho-CHK2 (Thr68) (2197), Ku80 (2180), Ku70 (4588), phospho-DNA-PKcs (Ser2056) (68716), DNA-PKcs (38168), GAPDH (5174), PP2A (2038), Artemis (13381), ATM (2873), PP5 (2289), Histone H4 (13919), Phospho-ATM (Ser1981) (5883) and 53BP1 (88439), purchased from Cell Signaling Technology, USA; RAD51 (ab133534) and PP1 (ab134947), purchased from Abcam, USA; Phospho-DNA-PKcs (Thr2609) (NBP1-02456), purchased from NOVUS, USA.

Techniques: Phospho-proteomics, Western Blot, Transfection, Stable Transfection, Expressing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility

doi: 10.1093/nar/gkad128

Figure Lengend Snippet: PP5 activity was impaired by Cd ions in a competitive pattern. ( A ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of PP5 on the chromatin fraction. ( B ) The dose effects of CdCl 2 on the activity of PP5 in vitro with Mn ions (0.2 μM) and Cd ions (0, 0.02, 0.04, 0.08, 0.2 μM). ( C ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) for 1 h, whole cell extracts were immunoprecipitated by PP5 antibody, and the activity of PP5 was determined in vitro . ( D ) The activity of PP5 inhibited by Cd ions (0.04 μM) was reversed by MnCl 2 (0, 0.04, 0.2, 0.4 μM), Zn (0, 0.04, 0.2, 0.4 μM) or Ca ions (0, 0.04, 0.2, 0.4 μM) in vitro . ( E ) GC-2 cells were treated by H 2 O 2 (40 μM) and CdCl 2 (20 μM) with or without MnCl 2 (10 μM) at the indicated times, followed by immunoblot analysis of indicated protein in whole cells. ( F ) U2OS cells were transfected with NHEJ reporter and I-SceI 4 h later, followed by treatment with CdCl 2 (100 μM) and with or without MnCl 2 (20 μM) for 3 h; ChIP assay was performed using anti-DNA-PKcs antibody, followed by PCR to detect DNA-PKcs bound DSB fragment. ( G ) GC-2 cells after transient cotransfection with the NHEJ reporter and/ or I-SceI, after which MnCl 2 (10 μM) was added 30 min before CdCl 2 (20 μM) treatment; the efficiency of NHEJ repair was determined by flow cytometry. n > 10 000 cells. ( H ) Representative images of the comet assay that was utilized to analyze the level of DNA damage in GC-2 cells, which were treated by H 2 O 2 (40 μM) with or without Cd ions (20 μM) and/or Mn ions (10 μM) at the indicated times. Scale bar, 100 μm. (I) Quantitative analysis of the DNA tail in cells treated as in (H). n > 100 cells.* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Data presented as mean ± SD.

Article Snippet: The antibodies used in this study were: phospho-H2AX (Ser139) (2577), phospho-H2AX (Ser139) (80312), H2AX (7631), phospho-CHK1 (Ser317) (12302), CHK1 (2360), CHK2 (6334), phospho-CHK2 (Thr68) (2197), Ku80 (2180), Ku70 (4588), phospho-DNA-PKcs (Ser2056) (68716), DNA-PKcs (38168), GAPDH (5174), PP2A (2038), Artemis (13381), ATM (2873), PP5 (2289), Histone H4 (13919), Phospho-ATM (Ser1981) (5883) and 53BP1 (88439), purchased from Cell Signaling Technology, USA; RAD51 (ab133534) and PP1 (ab134947), purchased from Abcam, USA; Phospho-DNA-PKcs (Thr2609) (NBP1-02456), purchased from NOVUS, USA.

Techniques: Activity Assay, Western Blot, In Vitro, Immunoprecipitation, Transfection, Cotransfection, Flow Cytometry, Single Cell Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility

doi: 10.1093/nar/gkad128

Figure Lengend Snippet: Cd ions induced male reproductive dysfunction in a mouse model was reversed by Mn ions. ( A ) Representative images of the GC-2 cells treated by Cd ions (20 μM) for 24 h with or without Mn ions (10 μM), colony formation was determined. ( B ) Quantitative analysis of relative colony formation as in A, normalized to untreated group. ( C ) GC-2 cells were treated by Cd ions (20 μM) for 24 h, pretreated with or without Mn ions (10 μM), the cell viability was determined and normalized to the untreated group. ( D ) The percentage of motile sperm was determined after male mice intraperitoneal injection of Cd ions (2 mg/kg) with or without Mn ions (20 mg/kg). n = 8. ( E ) The percentage of abnormal sperm was determined after male mice were treated with Cd ions with or without Mn ions. n = 8. ( F ) Representative images of the immunohistochemistry of p-DNA-PKcs S2056 and γ-H2AX in testes of mice following intraperitoneal injection of Cd ions as pretreatment, with or without Mn ions. n = 8. Scale bar, 50 μm. ( G ) Quantitative analysis of p-DNA-PKcs S2056 expression in testis as indicated in (F). ( H ) Quantitative analysis of γ-H2AX expression in testis as indicated in (F). ** P < 0.01; *** P < 0.001. Data presented as mean ± SD.

Article Snippet: The antibodies used in this study were: phospho-H2AX (Ser139) (2577), phospho-H2AX (Ser139) (80312), H2AX (7631), phospho-CHK1 (Ser317) (12302), CHK1 (2360), CHK2 (6334), phospho-CHK2 (Thr68) (2197), Ku80 (2180), Ku70 (4588), phospho-DNA-PKcs (Ser2056) (68716), DNA-PKcs (38168), GAPDH (5174), PP2A (2038), Artemis (13381), ATM (2873), PP5 (2289), Histone H4 (13919), Phospho-ATM (Ser1981) (5883) and 53BP1 (88439), purchased from Cell Signaling Technology, USA; RAD51 (ab133534) and PP1 (ab134947), purchased from Abcam, USA; Phospho-DNA-PKcs (Thr2609) (NBP1-02456), purchased from NOVUS, USA.

Techniques: Injection, Immunohistochemistry, Expressing

Journal: Nucleic Acids Research

Article Title: Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility

doi: 10.1093/nar/gkad128

Figure Lengend Snippet: A model illustrating the process of Cd ions inhibited NHEJ repair pathway. In this model, Cd ions competitively replace activating ions (Mn) to inhibit the activity of PP5, which results in decreased dephosphorylation of DNA-PKcs at S2056 in its PQR cluster. Hyper-phosphorylation of DNA-PKcs leads to its disassociation from DNA ends and the Ku complex, impairing the followed DNA ends ligation.

Article Snippet: The antibodies used in this study were: phospho-H2AX (Ser139) (2577), phospho-H2AX (Ser139) (80312), H2AX (7631), phospho-CHK1 (Ser317) (12302), CHK1 (2360), CHK2 (6334), phospho-CHK2 (Thr68) (2197), Ku80 (2180), Ku70 (4588), phospho-DNA-PKcs (Ser2056) (68716), DNA-PKcs (38168), GAPDH (5174), PP2A (2038), Artemis (13381), ATM (2873), PP5 (2289), Histone H4 (13919), Phospho-ATM (Ser1981) (5883) and 53BP1 (88439), purchased from Cell Signaling Technology, USA; RAD51 (ab133534) and PP1 (ab134947), purchased from Abcam, USA; Phospho-DNA-PKcs (Thr2609) (NBP1-02456), purchased from NOVUS, USA.

Techniques: Activity Assay, De-Phosphorylation Assay, Phospho-proteomics, Ligation

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Astragaloside IV alleviates lung inflammation in Klebsiella pneumonia rats by suppressing TGF-β1/Smad pathway

doi: 10.1590/1414-431X2023e12203

Figure Lengend Snippet: A , Expression of transforming growth factor (TGF)-β1 in lung tissues in K. pneumoniae rat model treated with astragaloside IV (AS-IV) or TGF-β1 inhibitor SB-431542 detected by immunohistochemistry (scale bar 50 μm); B , Expression levels of proteins related with TGF-β1/Smad, p-Smad2/Smad2, and p-Smad3/Smad3 pathways in lung determined by western blot. Data are reported as means±SD (n=3). *P<0.05, **P<0.01 compared with Control group; # P<0.05, ## P<0.01 compared with K. pneumoniae group (ANOVA).

Article Snippet: The primary antibodies were: TGF-β1 polyclonal antibodies (1:800, SAB4502954, Sigma-Aldrich), p-Smad2 polyclonal antibodies (1:100, orb451161, Biorbyt, China), Smad2 polyclonal antibodies (1:100, orb507656, Biorbyt), p-Smad3 polyclonal antibodies (1:100, orb313112, Biorbyt), Smad3 polyclonal antibodies (1:100, orb94696, Biorbyt), NF-κB p-p65 polyclonal antibodies (1:100, orb501839, Biorbyt), NF-κB p65 polyclonal antibodies (1:100, orb344389, Biorbyt), IκBα polyclonal antibodies (1:1000, 4812, Cell Signaling Technology, China), p-IκBα polyclonal antibodies (1:800, 2859, Cell Signaling Technology), and GAPDH polyclonal antibodies (1:1000,5174, Cell Signaling Technology).

Techniques: Expressing, Immunohistochemistry, Western Blot, Control

Journal: Journal of toxicology and environmental health. Part A

Article Title: Triphlorethol-a improves the non-homologous end joining and base-excision repair capacity impaired by formaldehyde.

doi: 10.1080/15287394.2011.567957

Figure Lengend Snippet: FIGURE 2. Effect of triphlorethol-A on HCHO-induced DNA strand breaks. (A) The cell lysates were electrophoresed and the phospho- histone H2A.X was detected using a specific antibody. (B) Representative images and % cellular DNA damage detected by comet assay. Asterisk indicates significantly different from control (p < .05); ∗∗significantly different from HCHO-treated cells (p < .05). (C) Ku 70 and phospho DNA-PKcs proteins were detected using specific antibodies (color figure available online).

Article Snippet: Ku 70, phospho DNA-PKcs, phospho Akt, Akt, and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Single Cell Gel Electrophoresis, Control

Journal: Annals of hematology

Article Title: Expression analysis of proteins involved in the non homologous end joining DNA repair mechanism, in the bone marrow of adult de novo myelodysplastic syndromes.

doi: 10.1007/s00277-009-0823-6

Figure Lengend Snippet: Fig. 2 Expression analysis οf enzymes DNAPKcs and Ku80 by Western blotting. M: marker Pageruler Prestained Protein Ladder (Fermentas) N: normal control, 3-8: protein samples from MDS patients

Article Snippet: Then, membranes were incubated overnight at 4°C in antibody buffer (non fat dry milk in TBS-T) containing primary antibody: Ku70 clone 2C3.11, 1:1000 (Abcam, Cambridge, UK), Ku80, Ab-7, 1:2000 (Cell Signaling), DNAPKcs, ab230, 1:3000 (Abcam), XRCC4, 1:6000 (Serotec, Oxford, UK), Ligase IV, ab6145, 1:2000 (Abcam) and Actin clone AC-15, 1:1000 (Sigma, Gillingham, UK).

Techniques: Expressing, Western Blot, Marker, Control

Journal: Cell Death Discovery

Article Title: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

doi: 10.1038/cddiscovery.2017.72

Figure Lengend Snippet: Overexpression of Akt isoforms and DNA-PKcs in A549 or HEK293T cells. Expression of Akt and DNA-PKcs fusion proteins. ( a ) Schematic overview of AKT1, AKT2 and AKT3 constructs fused to mCherry as well as the constructs expressing different subdomains of DNA-PKs fused to eGFP. ( b ) A549 cells were transfected with the indicated constructs. Twenty-four hours after the transfection, fluorescent images were acquired. Cells expressing mCherry or eGFP alone served as controls. Scale bars: 20 μm. ( c ) HEK293T cells were transfected with the indicated constructs. Twenty-four hours after the transfection, the cells were lysed and eGFP fusion proteins were precipitated using the GFP-Trap. Whole-cell lysates (mCherry) and the bound fractions (eGFP) were subjected to SDS-PAGE followed by western blot analysis using antibodies specific for mCherry or eGFP.

Article Snippet: Prior to western blot analysis, the eGFP-tagged DNA-PKcs domains were enriched by IP with GFP-Trap (ChromoTek GmbH) according to the manufacturer’s protocol.

Techniques: Over Expression, Expressing, Construct, Transfection, SDS Page, Western Blot

Journal: Cell Death Discovery

Article Title: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

doi: 10.1038/cddiscovery.2017.72

Figure Lengend Snippet: Akt1 and Akt3 but not Ak2 interact with DNA-PKcs. K-RAS-mutated A549 cells were transfected with the indicated plasmids. Forty-eight hours after the transfection, cells were irradiated with 4 Gy and lysed 10 min post-irradiation. IP of eGFP was performed using GFP-Trap. The co-IPs for Akt1 ( a ) Akt2 ( b ) and Akt3 ( c ) were analyzed by western blotting using Akt-specific antibodies. The densitometry values represent the ratios of Akt1/eGFP ( a ), Akt2/eGFP ( b ) and Akt3/eGFP ( c ). Cells were transfected with eGFP-DNA-PKcs-N and were either mock irradiated or irradiated with 4 Gy. Subsequently, the cells were lysed at the indicated times post-irradiation and the eGFP-tagged proteins were precipitated from the soluble protein fraction. The bound fractions were subjected to SDS-PAGE and immunoblot analysis using anti-eGFP ( d , e ), antibodies against the Akt1 and Ak2 isoforms ( d ) or against the Akt2 and Akt3 isoforms ( e ). Ponseau staining was shown for locating protein bands on immunoblots.

Article Snippet: Prior to western blot analysis, the eGFP-tagged DNA-PKcs domains were enriched by IP with GFP-Trap (ChromoTek GmbH) according to the manufacturer’s protocol.

Techniques: Transfection, Irradiation, Western Blot, SDS Page, Staining

Journal: Cell Death Discovery

Article Title: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

doi: 10.1038/cddiscovery.2017.72

Figure Lengend Snippet: Targeting Akt inhibits Akt/DNA-PKcs complex formation. K-RAS-mutated A549 cells were transfected with eGFP-PKcs (a.a. 1-421) and mCherry-Akt1. ( a ) Forty-eight hours after transfection, the cells were treated with DMSO (D) or 10 μ M of MK2206 (MK) for 1 h and then irradiated with 4 Gy. The cells were lysed 10 min post-IR and eGFP-DNA-PKcs-N was precipitated as described. The input and bound fractions were subjected to SDS-PAGE and immunoblot analysis, and eGFP was detected in the inputs and under the IP conditions as the loading control. The experiment was performed in two biological replicates. The results from one experiment are shown. ( b ) The cells were lysed 10 min past-IR and immunoblot analysis was subjected. The phosphorylation of Akt (Ser-473) and PRAS40 (Thr-246) was analyzed by immunoblotting in the whole-cell lysates using phospho-specific antibodies. The blots were stripped and re-probed with the antibodies against Akt1 and PRAS40. Data indicates mean P-Akt (Ser-473)±S.E.M. from three independent experiments. The asterisks indicate a significant inhibition of Akt phosphorylation following pretreatment with MK2206 (5 μ M) for 2 h followed by irradiation with 4 Gy (* P <0.05, *** P <0.001, Student's t -test).

Article Snippet: Prior to western blot analysis, the eGFP-tagged DNA-PKcs domains were enriched by IP with GFP-Trap (ChromoTek GmbH) according to the manufacturer’s protocol.

Techniques: Transfection, Irradiation, SDS Page, Western Blot, Inhibition

Journal: Cell Death Discovery

Article Title: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

doi: 10.1038/cddiscovery.2017.72

Figure Lengend Snippet: Effect of Akt isoforms and DNA-PKcs on post-irradiation cell survival of K-RAS-mutated A549 cells. ( a ) Forty-eight hours after the transfections with the indicated siRNAs, the cells were plated in six-well plates, the colonies were stained after about 10 days and the plating efficiencies were calculated by dividing the number of colonies formed to the number of cells seeded. The data presented are the mean plating efficiencies (PE)±S.E.M. of 12 replicates from two independent experiments. ( b ) Transfected cells with indicated siRNA were plated and X-ray irradiated 24 h later and then incubated for 10 days. Thereafter, the colonies were stained, and the survival fractions (SF) were calculated as described in the Materials and Methods section. The data presented are the mean survival fraction±S.E.M. of 12 replicates from two independent experiments. ( c ) Confluent A549 cells were treated with the vehicle (DMSO) or the DNA-PKcs inhibitor NU7026 at indicated concentrations for 1 h and then irradiated with 4 Gy. Protein samples were isolated 30 min after irradiation, and levels of P-DNA-PKcs (Ser-2056) and P-DNA-PKcs (Thr-2609) were determined by immunoblotting. The blots were then stripped and incubated with the DNA-PKcs antibody. ( d ) A549 cells were plated in six-well plates and 24 h later were treated with the vehicle (DMSO) or the indicated concentrations of the DNA-PKcs inhibitor NU7026 for 1 h. The cultures were then irradiated and incubated for 10 days. Thereafter, the colonies were stained, and the clonogenic fractions were calculated as described in Materials and Methods section. The data presented are the mean survival fraction±S.E.M. of six replicates from the parallel experiments. The asterisks indicate a statistically significant inhibition of plating efficiency ( a ) and radiosensitization after knockdown of Akt1 or Akt3 ( b ) (* P <0.05; ** P <0.01; *** P <0.001).

Article Snippet: Prior to western blot analysis, the eGFP-tagged DNA-PKcs domains were enriched by IP with GFP-Trap (ChromoTek GmbH) according to the manufacturer’s protocol.

Techniques: Irradiation, Transfection, Staining, Incubation, Isolation, Western Blot, Inhibition

Journal: Cell Death Discovery

Article Title: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

doi: 10.1038/cddiscovery.2017.72

Figure Lengend Snippet: Knockdown of Akt1 and Akt3 but not Akt2 inhibits proliferation and tumor growth in K-RAS-mutated MDA-MB-231 cells. ( a ) Cells (3×10 4 ) were plated in 6 cm culture dishes. At the indicated days after seeding, cells were counted and graphed. The data points represent the mean cell counts±S.E.M. of eight parallel experiments from two independent experiments. Asterisks indicate significant prolongation of PDT after knockdown of Akt1 and Akt3 compared with scramble-shRNA (scr-shRNA) (* P <0.05, *** P <0.001). ( b ) Protein samples were isolated from the cells counted on day 7 and expression of Akt isoforms was tested by immunoblotting. ( c ) Indicated cells (3×10 4 ) were plated for 24 h and treated with DNA-PKcs inhibitor NU7441 (10 μ M). Cells were count on day 6 after treatment and graphed. Data present mean cells numbers of eight data±S.E.M. obtained from two independent experiments. ( d ) Nude mice were injected with indicates cells (2×10 6 cells) in both dorsal flank and tumor growth assay was performed as described in Materials and Methods section. Data present mean tumor volume±S.E.M. of 14 tumors (seven mice) inoculated with MDA-MB-231-expressing scr-shRNA and of 12 tumors from six animals inoculated with MDA-MB-231 cells expressing Akt1-, Akt2- or Akt3-shRNA. Asterisks indicate a significant tumor growth delay by knockdown of Akt1 as well as Akt3 (*** P <0.001) and increased in tumor volume by knockdown of Akt2 (* P <0.05), measured 6 weeks after inoculation. ( e ) Representative images of tumors following inoculation of MDA-MB-231 cells expressing scr-shRNA as well as shRNA against the Akt isoforms.

Article Snippet: Prior to western blot analysis, the eGFP-tagged DNA-PKcs domains were enriched by IP with GFP-Trap (ChromoTek GmbH) according to the manufacturer’s protocol.

Techniques: shRNA, Isolation, Expressing, Western Blot, Injection, Growth Assay

Journal: Communications Biology

Article Title: ATM Inhibition Enhances Knock-in Efficiency by Suppressing AAV-Induced Activation of Apoptotic Pathways

doi: 10.1038/s42003-026-09604-z

Figure Lengend Snippet: a Western blotting results showing that treatment with the ATM inhibitor suppressed AAV-induced activation of DNA-PKcs, leading to reduced DSB repair via c-NHEJ. Protein was harvested 2 h after genome editing. b Quantification of Western blot results. The signal intensities were normalized to GAPDH. Fold changes are shown relative to the negative control. Data are presented as the mean ± SD. ( n = 3 independent replicates). Tukey’s multiple comparison test was conducted.

Article Snippet: 12 , DNA-PKcs Polyclonal antibody , Proteintech , 28534-1-AP , 1:100 , -.

Techniques: Western Blot, Activation Assay, Negative Control, Comparison

Journal: Gynecologic oncology

Article Title: 53BP1 as a Potential Predictor of Response in PARP Inhibitor-Treated Homologous Recombination-Deficient Ovarian Cancer

doi: 10.1016/j.ygyno.2019.01.015

Figure Lengend Snippet: A, relationship between Myriad HRD score and largest percent change in tumor cross sectional area from baseline in target lesions by RECIST [18] in the subset of ovarian cancers for which a numerical HRD score was available (n=13). B-D, IHC staining for 53BP1 (B), KU80 (C), and DNA-PKCS (D) was evaluated to give a modified H-score, which was compared to percent change in tumor cross-sectional area from baseline [18]. HR gene alterations are denoted as mutations (white) or methylation (blue) of BRCA1 (squares), BRCA2 (triangle), or RAD51C (diamond). Symbols that represent two separate patients but are not separable because of overlapping data points are denoted in grey. Ovarian cancers that were stained were sorted into those that were HR deficient (left panels) and those that were not HR deficient (right panels). Because not all ovarian cancers had sufficient sample to assess all antigens, the number of samples analyzed was: 53BP1 (B), HR-deficient n=16, HR-proficient n=16; KU80 and DNA-PKCS (C, D), HR-deficient n=15, HR-proficient n=10. Heat map analysis (E) reflects the H-score for all proteins analyzed, sorted by HR-deficiency and percent change in tumor cross-sectional area from baseline.

Article Snippet: Antibodies to DNA-PK CS (1:600; IHC00044, Bethyl Labs), KU80 (1:2000, clone EPR3467, Abcam), KU70 (1:500, clone N3H10, Abcam), XRCC4 (1:8000, clone 4/XRCC4, BD Biosciences), PARP1 (1:300, clone E102, Abcam), RAD51 (1:2000, clone EPR4030(3), Abcam), and 53BP1 (mouse monoclonal [ 30 ], a kind gift from Thanos Halazonetis, University of Geneva, Switzerland) were diluted in Background Reducing Diluent (Dako) and incubated for 15 min. A Leica Polymer Refine Detection System was used according to the supplier’s instructions.

Techniques: Immunohistochemistry, Modification, Methylation, Staining

Journal: Gynecologic oncology

Article Title: 53BP1 as a Potential Predictor of Response in PARP Inhibitor-Treated Homologous Recombination-Deficient Ovarian Cancer

doi: 10.1016/j.ygyno.2019.01.015

Figure Lengend Snippet: Univariate analysis of response with clinical parameters and H-scores

Article Snippet: Antibodies to DNA-PK CS (1:600; IHC00044, Bethyl Labs), KU80 (1:2000, clone EPR3467, Abcam), KU70 (1:500, clone N3H10, Abcam), XRCC4 (1:8000, clone 4/XRCC4, BD Biosciences), PARP1 (1:300, clone E102, Abcam), RAD51 (1:2000, clone EPR4030(3), Abcam), and 53BP1 (mouse monoclonal [ 30 ], a kind gift from Thanos Halazonetis, University of Geneva, Switzerland) were diluted in Background Reducing Diluent (Dako) and incubated for 15 min. A Leica Polymer Refine Detection System was used according to the supplier’s instructions.

Techniques: Mutagenesis

Journal: iScience

Article Title: Epidermal growth factor augments the self-renewal capacity of aged hematopoietic stem cells

doi: 10.1016/j.isci.2024.110306

Figure Lengend Snippet:

Article Snippet: rabbit anti-DNA-PKcs (p-Thr2609) primary antibody (1:100) , Novus Biological , NDP1-02456.

Techniques: Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Sequencing, Software