Journal: Nature Communications

Article Title: EP300 deficiency leads to chronic replication stress mediated by defective replication fork protection

doi: 10.1038/s41467-025-67171-z

Figure Lengend Snippet: A Schematic model depicting replication fork stalling, fork protection, and nucleolytic degradation, (Created in BioRender. Madireddy, A. (2025), is licensed under CC BY 4.0, https://BioRender.com/xtm4wme ). B DNA fiber analysis of 50uM hydroxyurea (HU) (60 min) treated EP300WT (NA1, NA4), EP300Mut (NA2, NA3) and EP300WT NA-ATLL (NA1, NA4) cells treated with negative control (S, S) stereoisomer or with JQAD1 (500 nM for 48 h) treatment to assess replication fork stalling. The fork rate (CldU/IdU ratio) is indicated. N = 3. C DNA fiber analysis measuring nucleolytic degradation after 4 mM HU treatment in EP300WT, EP300Mut NA-ATLL cells. The fork rate (CldU/IdU ratio) is indicated, N = 3. D Analysis of the number of single-stranded gaps (ssDNA)/Iododeoxyuridine (IdU) foci (green) per cell nuclei (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA2, NA3) cells exposed to 2 mM HU (4 h), and EP300WT (NA4) cells treated with JQAD1 (500 nM). ssDNA gaps are measured by IdU incorporation under non-denaturing conditions. Representative images are shown on the right, N = 3. E Analysis of number of pRPA Ser4/8 foci (red) per cell nuclei (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA2, NA3) cells exposed to 2 mM HU (4 h), and EP300WT (NA4) cells treated with 500 nM JQAD1. Representative images are shown on the right, N = 3. F Protein expression of phospho-RPA Ser4/8 in the presence or absence of EP300, presented as a fold change quantified from the immunoblot in Supp. Fig. S . G Analysis of the number of single-stranded gaps/breaks (ssDNA)/Iododeoxyuridine (IdU) foci per cell nucleus (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA3) cells exposed to 2 mM HU (4 h), in the presence or absence of 50uM Mirin (4 h) (Mre11 nuclease inhibitor), N = 3. H Analysis of the number of single-stranded gaps/breaks (ssDNA)/ Iododeoxyuridine (IdU) foci per cell nucleus (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA3) cells exposed to 2 mM HU (4 h), in the presence or absence of 20uM C5 (24 h) (DNA2 nuclease inhibitor), N = 3. For all experiments, data are presented as mean values +/− SD. P -values were determined by a two-tailed Student T-test. P -values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. Scale bar 10 μm. N represents three experimental replicates from independent cultures of cells. Source data are provided as a Source Data file.

Article Snippet: Exponentially growing cells were labelled with 50 uM IdU for 48 hours before treatment with MRE11 inhibitor- Mirin, DNA2 inhibitor- C5 (HY-128729, MedChem express) and 2 mM hydroxyurea in the last 4 h prior to collection.

Techniques: Negative Control, Expressing, Western Blot, Two Tailed Test

Journal: Cell Death & Disease

Article Title: HAT1 functions as a lactyltransferase and mediates RPA1 lactylation to promote DNA repair and radioresistance in lung adenocarcinoma

doi: 10.1038/s41419-025-08113-x

Figure Lengend Snippet: A The level of lactylated RPA1 in mutant cells with RPA1 K88R, K163R, K167R, and K267R. B The level of RPA1 interacting ssDNA in mutant cells with RPA1 K88R, K163R, K167R and K267R. C The level of RPA1 interacted with HR factors, including BLM, MRE11, RAD51, and NBS1, in mutant cells with RPA1 K88R, K163R, K167R, and K267R. D Relative HR efficiency in mutant cells with RPA1 K88R, K163R, K167R and K267R. All data were analyzed using two-way analysis of variance (ANOVA), ** P < 0.01.

Article Snippet: Antibodies against HAT1 (#11432-1-AP), β-actin (#66009-1-Ig), GAPDH (#60004-1-Ig), FLAG (#66008-4-Ig), HA (#51064-2-AP), BLM (#30254-1-AP), MRE11 (#10744-1-AP), NBS1 (#55025-1-AP) were obtained from Proteintech Group.

Techniques: Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: MRE11 orchestrates porcine oocyte meiotic progression by modulating the spindle assembly checkpoint

doi: 10.3389/fcell.2025.1635110

Figure Lengend Snippet: Subcellular localization and protein expression of Mre11 during porcine meiotic maturation. (A) Representative immunofluorescence images showing the localization of endogenous Mre11 in porcine oocytes at different developmental stages. Oocytes were immunostained with an anti-Mre11 antibody and counterstained with Hoechst. Scale bar, 2.5 μm. (B) Representative image showing the co-localization of Mre11 with kinetochores in porcine oocytes at the pro-metaphase I (Pro-M I) stage. Oocytes were immunostained with anti-Mre11 and CREST antibodies, and counterstained with Hoechst. Scale bar, 2.5 μm.

Article Snippet: Rabbit polyclonal anti-MRE11 antibody was purchased from Proteintech Group (Rosemont, IL, United States; Cat# 16370-1-AP); rabbit monoclonal anti-BUBR1 antibody was purchased from Abcam (Cambridge, United Kingdom, United States; Cat# ab133699); Mouse anti-α-tubulin-FITC antibody (Cat# F2168), mouse monoclonal anti-acetylation-α-tubulin antibody (T7451), peanut agglutinin (PNA)-FITC (L7381) and DAPI (D9542) were purchased from Sigma (Aldrich, St Louis, MO, United States); Rabbit polyclonal anti-γ-H2AX antibody (Cat# ab26350) was purchased from Abcam (Cambridge, United Kingdom); TRITC Phalloidin TRITC antibody (Cat# MX4405) was purchased from Maokang Biotechnology (Shanghai, China); Furthermore, goat anti-mouse IgG Alexa Fluor 488 antibody (Cat# A11029), donkey anti-Sheep IgG Alexa Fluor 594 antibody (Cat# A11016) were purchased from Thermo Fisher (Waltham, MA, United States). human anti-centromere antibody was purchased from Antibodies Incorporated (Davis, CA, United States; Cat# CA95617); rabbit monoclonal anti-GAPDH antibody was purchased from Cell Signaling Technology (Danvers, MA, United States; Cat# 2118).

Techniques: Expressing, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: MRE11 orchestrates porcine oocyte meiotic progression by modulating the spindle assembly checkpoint

doi: 10.3389/fcell.2025.1635110

Figure Lengend Snippet: Effect of Mre11 inhibition on porcine oocyte meiotic progression. (A) Representative images of cumulus cell expansion and polar body extrusion in control and Mre11-inhibited oocytes cultured in vitro for 44 h. Oocytes were denuded after culture to observe polar body extrusion. COCs: cumulus-oocyte complexes; DOs: denuded oocytes. Scale bars, 500 μm (a, e); 200 μm (b, f); 250 μm (c, g); 30 μm (d, h). (B) The rate of polar body extrusion was recorded in control and Mre11-inhibited groups treated with different concentrations (20 μM, 40 μM, 60 μM, and 100 μM) after 44 h of culture. (C) Representative images showing chromosome morphology at different developmental stages of oocyte maturation. DNA was counterstained with propidium iodide (PI). Scale bar, 5 μm. (D) The percentage of oocytes at different developmental stages was quantified in control and Mre11-inhibited groups. Data in panels (B) and (D) are presented as mean percentages (mean ± SEM) from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Rabbit polyclonal anti-MRE11 antibody was purchased from Proteintech Group (Rosemont, IL, United States; Cat# 16370-1-AP); rabbit monoclonal anti-BUBR1 antibody was purchased from Abcam (Cambridge, United Kingdom, United States; Cat# ab133699); Mouse anti-α-tubulin-FITC antibody (Cat# F2168), mouse monoclonal anti-acetylation-α-tubulin antibody (T7451), peanut agglutinin (PNA)-FITC (L7381) and DAPI (D9542) were purchased from Sigma (Aldrich, St Louis, MO, United States); Rabbit polyclonal anti-γ-H2AX antibody (Cat# ab26350) was purchased from Abcam (Cambridge, United Kingdom); TRITC Phalloidin TRITC antibody (Cat# MX4405) was purchased from Maokang Biotechnology (Shanghai, China); Furthermore, goat anti-mouse IgG Alexa Fluor 488 antibody (Cat# A11029), donkey anti-Sheep IgG Alexa Fluor 594 antibody (Cat# A11016) were purchased from Thermo Fisher (Waltham, MA, United States). human anti-centromere antibody was purchased from Antibodies Incorporated (Davis, CA, United States; Cat# CA95617); rabbit monoclonal anti-GAPDH antibody was purchased from Cell Signaling Technology (Danvers, MA, United States; Cat# 2118).

Techniques: Inhibition, Control, Cell Culture, In Vitro

Journal: Frontiers in Cell and Developmental Biology

Article Title: MRE11 orchestrates porcine oocyte meiotic progression by modulating the spindle assembly checkpoint

doi: 10.3389/fcell.2025.1635110

Figure Lengend Snippet: Effect of Mre11 inhibition on BubR1 localization in porcine oocytes. (A) Representative images showing the localization of BubR1 at the metaphase I stage in control and Mre11-inhibited oocytes. Scale bar, 5 μm. (B) Quantitative analysis of BubR1 fluorescence intensity in control and Mre11-inhibited oocytes. Data are presented as the mean percentage (mean ± SEM) from at least three independent experiments. **P < 0.001.

Article Snippet: Rabbit polyclonal anti-MRE11 antibody was purchased from Proteintech Group (Rosemont, IL, United States; Cat# 16370-1-AP); rabbit monoclonal anti-BUBR1 antibody was purchased from Abcam (Cambridge, United Kingdom, United States; Cat# ab133699); Mouse anti-α-tubulin-FITC antibody (Cat# F2168), mouse monoclonal anti-acetylation-α-tubulin antibody (T7451), peanut agglutinin (PNA)-FITC (L7381) and DAPI (D9542) were purchased from Sigma (Aldrich, St Louis, MO, United States); Rabbit polyclonal anti-γ-H2AX antibody (Cat# ab26350) was purchased from Abcam (Cambridge, United Kingdom); TRITC Phalloidin TRITC antibody (Cat# MX4405) was purchased from Maokang Biotechnology (Shanghai, China); Furthermore, goat anti-mouse IgG Alexa Fluor 488 antibody (Cat# A11029), donkey anti-Sheep IgG Alexa Fluor 594 antibody (Cat# A11016) were purchased from Thermo Fisher (Waltham, MA, United States). human anti-centromere antibody was purchased from Antibodies Incorporated (Davis, CA, United States; Cat# CA95617); rabbit monoclonal anti-GAPDH antibody was purchased from Cell Signaling Technology (Danvers, MA, United States; Cat# 2118).

Techniques: Inhibition, Control, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: MRE11 orchestrates porcine oocyte meiotic progression by modulating the spindle assembly checkpoint

doi: 10.3389/fcell.2025.1635110

Figure Lengend Snippet: Effect of Mre11 inhibition on spindle assembly and chromosome alignment in porcine oocytes. (A) Representative images depicting spindle morphologies and chromosome alignment in control and Mre11-inhibited oocytes. Metaphase I (M I) oocytes were immunostained with anti-α-tubulin-FITC antibody to visualize spindles and counterstained with propidium iodide (PI) to visualize chromosomes. Scale bar, 5 μm. (B) The frequency of aberrant spindles was recorded in control and Mre11-inhibited oocytes. Data in panel (B) are presented as mean percentages (mean ± SEM) from at least three independent experiments.

Article Snippet: Rabbit polyclonal anti-MRE11 antibody was purchased from Proteintech Group (Rosemont, IL, United States; Cat# 16370-1-AP); rabbit monoclonal anti-BUBR1 antibody was purchased from Abcam (Cambridge, United Kingdom, United States; Cat# ab133699); Mouse anti-α-tubulin-FITC antibody (Cat# F2168), mouse monoclonal anti-acetylation-α-tubulin antibody (T7451), peanut agglutinin (PNA)-FITC (L7381) and DAPI (D9542) were purchased from Sigma (Aldrich, St Louis, MO, United States); Rabbit polyclonal anti-γ-H2AX antibody (Cat# ab26350) was purchased from Abcam (Cambridge, United Kingdom); TRITC Phalloidin TRITC antibody (Cat# MX4405) was purchased from Maokang Biotechnology (Shanghai, China); Furthermore, goat anti-mouse IgG Alexa Fluor 488 antibody (Cat# A11029), donkey anti-Sheep IgG Alexa Fluor 594 antibody (Cat# A11016) were purchased from Thermo Fisher (Waltham, MA, United States). human anti-centromere antibody was purchased from Antibodies Incorporated (Davis, CA, United States; Cat# CA95617); rabbit monoclonal anti-GAPDH antibody was purchased from Cell Signaling Technology (Danvers, MA, United States; Cat# 2118).

Techniques: Inhibition, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: MRE11 orchestrates porcine oocyte meiotic progression by modulating the spindle assembly checkpoint

doi: 10.3389/fcell.2025.1635110

Figure Lengend Snippet: Effect of Mre11 inhibition on the acetylation level of α-tubulin in porcine oocytes. (A) Representative images of microtubule fibers before and after nocodazole treatment in control and Mre11-inhibited oocytes. Oocytes were immunostained with an anti-α-tubulin-FITC antibody to visualize microtubules and counterstained with propidium iodide (PI) to visualize chromosomes. Scale bar, 10 μm. (B) Representative images showing the acetylation of α-tubulin in control and Mre11-inhibited oocytes. Oocytes were immunostained with an anti-acetyl-α-tubulin (Lys-40) antibody to assess the acetylation level of α-tubulin. Scale bar, 5 μm. (C) The fluorescence intensity of acetylated α-tubulin was quantified in control and Mre11-inhibited oocytes. Data in panel (C) are presented as mean percentages (mean ± SEM) from at least three independent experiments. ***P < 0.001.

Article Snippet: Rabbit polyclonal anti-MRE11 antibody was purchased from Proteintech Group (Rosemont, IL, United States; Cat# 16370-1-AP); rabbit monoclonal anti-BUBR1 antibody was purchased from Abcam (Cambridge, United Kingdom, United States; Cat# ab133699); Mouse anti-α-tubulin-FITC antibody (Cat# F2168), mouse monoclonal anti-acetylation-α-tubulin antibody (T7451), peanut agglutinin (PNA)-FITC (L7381) and DAPI (D9542) were purchased from Sigma (Aldrich, St Louis, MO, United States); Rabbit polyclonal anti-γ-H2AX antibody (Cat# ab26350) was purchased from Abcam (Cambridge, United Kingdom); TRITC Phalloidin TRITC antibody (Cat# MX4405) was purchased from Maokang Biotechnology (Shanghai, China); Furthermore, goat anti-mouse IgG Alexa Fluor 488 antibody (Cat# A11029), donkey anti-Sheep IgG Alexa Fluor 594 antibody (Cat# A11016) were purchased from Thermo Fisher (Waltham, MA, United States). human anti-centromere antibody was purchased from Antibodies Incorporated (Davis, CA, United States; Cat# CA95617); rabbit monoclonal anti-GAPDH antibody was purchased from Cell Signaling Technology (Danvers, MA, United States; Cat# 2118).

Techniques: Inhibition, Control, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: MRE11 orchestrates porcine oocyte meiotic progression by modulating the spindle assembly checkpoint

doi: 10.3389/fcell.2025.1635110

Figure Lengend Snippet: Effect of Mre11 inhibition on cytoplasmic actin assembly in porcine oocytes. (A) Representative images showing the distribution of actin filaments at the oocyte cortex and cytoplasm in the control and Mre11-inhibited groups. Red indicates F-actin. Scale bar, 20 μm. (B) Quantification of fluorescence intensity of F-actin at the cortex in the control and Mre11-inhibited groups. (C) Quantification of fluorescence intensity of F-actin in the cytoplasm in the control and Mre11-inhibited groups. (D) Quantitative analysis of F-actin fluorescence intensities at the cortex and in the cytoplasm. Data in panels (B–D) are presented as mean percentages (mean ± SEM) from at least three independent experiments. ***P < 0.001.

Article Snippet: Rabbit polyclonal anti-MRE11 antibody was purchased from Proteintech Group (Rosemont, IL, United States; Cat# 16370-1-AP); rabbit monoclonal anti-BUBR1 antibody was purchased from Abcam (Cambridge, United Kingdom, United States; Cat# ab133699); Mouse anti-α-tubulin-FITC antibody (Cat# F2168), mouse monoclonal anti-acetylation-α-tubulin antibody (T7451), peanut agglutinin (PNA)-FITC (L7381) and DAPI (D9542) were purchased from Sigma (Aldrich, St Louis, MO, United States); Rabbit polyclonal anti-γ-H2AX antibody (Cat# ab26350) was purchased from Abcam (Cambridge, United Kingdom); TRITC Phalloidin TRITC antibody (Cat# MX4405) was purchased from Maokang Biotechnology (Shanghai, China); Furthermore, goat anti-mouse IgG Alexa Fluor 488 antibody (Cat# A11029), donkey anti-Sheep IgG Alexa Fluor 594 antibody (Cat# A11016) were purchased from Thermo Fisher (Waltham, MA, United States). human anti-centromere antibody was purchased from Antibodies Incorporated (Davis, CA, United States; Cat# CA95617); rabbit monoclonal anti-GAPDH antibody was purchased from Cell Signaling Technology (Danvers, MA, United States; Cat# 2118).

Techniques: Inhibition, Control, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: MRE11 orchestrates porcine oocyte meiotic progression by modulating the spindle assembly checkpoint

doi: 10.3389/fcell.2025.1635110

Figure Lengend Snippet: Effect of Mre11 inhibition on meiotic spindle positioning and the formation of actin caps in porcine oocytes. (A) Representative images showing spindle positioning in control and Mre11-inhibited oocytes. (B) To quantify spindle position after Mre11 inhibition, D is defined as the distance from the proximal end of the spindle pole to the cortex, while L is defined as the distance from the distal end of the spindle pole to the cortex. (C) The D/L ratio in the Mre11-inhibited group was significantly higher than that in the control group. (D) Formation of actin caps and CGFDs. After Mre11 inhibition, the actin cap over a chromosome was disrupted, and Mre11 inhibition also affected CGFD formation. Red indicates actin; green indicates CGs; blue indicates chromatin. Scale bar, 20 μm.

Article Snippet: Rabbit polyclonal anti-MRE11 antibody was purchased from Proteintech Group (Rosemont, IL, United States; Cat# 16370-1-AP); rabbit monoclonal anti-BUBR1 antibody was purchased from Abcam (Cambridge, United Kingdom, United States; Cat# ab133699); Mouse anti-α-tubulin-FITC antibody (Cat# F2168), mouse monoclonal anti-acetylation-α-tubulin antibody (T7451), peanut agglutinin (PNA)-FITC (L7381) and DAPI (D9542) were purchased from Sigma (Aldrich, St Louis, MO, United States); Rabbit polyclonal anti-γ-H2AX antibody (Cat# ab26350) was purchased from Abcam (Cambridge, United Kingdom); TRITC Phalloidin TRITC antibody (Cat# MX4405) was purchased from Maokang Biotechnology (Shanghai, China); Furthermore, goat anti-mouse IgG Alexa Fluor 488 antibody (Cat# A11029), donkey anti-Sheep IgG Alexa Fluor 594 antibody (Cat# A11016) were purchased from Thermo Fisher (Waltham, MA, United States). human anti-centromere antibody was purchased from Antibodies Incorporated (Davis, CA, United States; Cat# CA95617); rabbit monoclonal anti-GAPDH antibody was purchased from Cell Signaling Technology (Danvers, MA, United States; Cat# 2118).

Techniques: Inhibition, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: MRE11 orchestrates porcine oocyte meiotic progression by modulating the spindle assembly checkpoint

doi: 10.3389/fcell.2025.1635110

Figure Lengend Snippet: Effect of Mre11 inhibition on DNA damage levels in porcine oocytes. (A) Representative images showing DNA damage in control and Mre11-inhibited oocytes. Oocytes were immunostained with an anti-γH2A.X antibody and counterstained with Hoechst. Scale bar, 5 μm. (B) Quantification of γH2A.X fluorescence intensity in control and Mre11-inhibited oocytes. Data are presented as mean percentages (mean ± SEM) from at least three independent experiments. ***P < 0.001.

Article Snippet: Rabbit polyclonal anti-MRE11 antibody was purchased from Proteintech Group (Rosemont, IL, United States; Cat# 16370-1-AP); rabbit monoclonal anti-BUBR1 antibody was purchased from Abcam (Cambridge, United Kingdom, United States; Cat# ab133699); Mouse anti-α-tubulin-FITC antibody (Cat# F2168), mouse monoclonal anti-acetylation-α-tubulin antibody (T7451), peanut agglutinin (PNA)-FITC (L7381) and DAPI (D9542) were purchased from Sigma (Aldrich, St Louis, MO, United States); Rabbit polyclonal anti-γ-H2AX antibody (Cat# ab26350) was purchased from Abcam (Cambridge, United Kingdom); TRITC Phalloidin TRITC antibody (Cat# MX4405) was purchased from Maokang Biotechnology (Shanghai, China); Furthermore, goat anti-mouse IgG Alexa Fluor 488 antibody (Cat# A11029), donkey anti-Sheep IgG Alexa Fluor 594 antibody (Cat# A11016) were purchased from Thermo Fisher (Waltham, MA, United States). human anti-centromere antibody was purchased from Antibodies Incorporated (Davis, CA, United States; Cat# CA95617); rabbit monoclonal anti-GAPDH antibody was purchased from Cell Signaling Technology (Danvers, MA, United States; Cat# 2118).

Techniques: Inhibition, Control, Fluorescence

Journal: Communications Biology

Article Title: βTrCP facilitates MRN complex localization on chromatin to enhance DNA repair

doi: 10.1038/s42003-025-08462-5

Figure Lengend Snippet: A Cytosolic (S100) and nuclear (NE) fraction from transiently transfected Cos-7 cells with pCS2HA βTrCP was used to immunoprecipitate HA βTrCP. Immunocomplexes were analyzed with the indicated antibodies. IgG: immunoprecipitation using normal mouse serum as a control. The input lane (Inp) was loaded with 1/20 of the extract used in each assay. B Nuclear extracts (NE) from Cos-7 cells transfected with pCS2HA βTrCP were immunoprecipitated with anti-MRE11, anti-NBS1 or anti-RAD50 and analyzed by western blot. Immunoprecipitations using normal mouse or rabbit serum (IgG) were used as controls. Inp: input of nuclear extracts. C Similar to ( A ) and ( B ), but using NP40 extracts from non-transfected Cos-7 cells, treated with DNase. IP IgG immunoprecipitation with normal rabbit serum as a control. The immunoprecipitation efficiency of βTrCP was also tested with anti-β catenin, a known partner of this F-box protein. D Ubiquitination assay of in vitro-transcribed and translated MRE11, NBS1 or RAD50 labeled with [ 35 S] was carried out in the presence or absence of the following products: recombinant SCF (βTrCP) complex produced in Sf21 insect cells, E1 (His 6 -E1), E2 (His 6 -UbcH3 and UbcH5a), and ubiquitin (Ub). The samples were incubated at 30 °C for 1 h and analyzed by SDS-PAGE and autoradiography. Polyubiquitylated proteins appear as a smear.

Article Snippet: The pCMV6 Flag MRE11 was obtained from Origene (Rockville, MD, USA).

Techniques: Transfection, Immunoprecipitation, Control, Western Blot, Ubiquitin Proteomics, In Vitro, Labeling, Recombinant, Produced, Incubation, SDS Page, Autoradiography

Journal: Communications Biology

Article Title: βTrCP facilitates MRN complex localization on chromatin to enhance DNA repair

doi: 10.1038/s42003-025-08462-5

Figure Lengend Snippet: A In silico identification of potential consensus binding motifs for βTrCP in human MRE11 (residues 596–603), NBS1 (residues 272–278) and RAD50 (residues 783–790). Arrows indicate the amino-acid changes (in red) introduced to generate mutants in these motifs. B βTrCP was immunoprecipitated from NP40 extracts of Cos-7 transfected cells with pCMV6 Flag MRE11 or pCMV6 Flag MRE11Δβ, and coimmunoprecipitation of Flag MRE11 or Flag MRE11Δβ was assessed using anti-Flag. The presence of RAD50 and NBS1 in the immunocomplexes was also analyzed. Anti-PLK1 and anti-βTrCP were used as controls of βTrCP immunoprecipitation. IP IgG: immunoprecipitation with normal rabbit serum as a control. Inp: NP40 extracts. C NP40 extracts from Cos-7 cells transfected with pCMV6 Flag MRE11 or pCMV6 Flag MRE11Δβ were used to immunoprecipitate Flag-tagged proteins and analyze their association with RAD50 and NBS1. IP IgG: immunoprecipitation with normal mouse serum as a control. Inp: NP40 extracts. Anti-α tubulin was used as a loading control.

Article Snippet: The pCMV6 Flag MRE11 was obtained from Origene (Rockville, MD, USA).

Techniques: In Silico, Binding Assay, Immunoprecipitation, Transfection, Control

Journal: Communications Biology

Article Title: βTrCP facilitates MRN complex localization on chromatin to enhance DNA repair

doi: 10.1038/s42003-025-08462-5

Figure Lengend Snippet: A U2OS Flp In T-REx HA βTrCPΔF cells, induced or not with doxycycline (DOX), were irradiated (10 Gy) and harvested 2, 4, 6 or 24 h later. Western blot analysis was performed to assess DNA damage repair, indicated by the progressive decrease in γH2AX levels. Anti-β actin was used as a loading control. B The graph shows the quantification of γH2AX levels from ( A ). Individual data points were represented as square markers. Error bars represent the SD ( n = 3). C U2OS cells were transfected with either pCMV6 Flag MRE11Δβ or empty vector, irradiated (10 Gy), and harvested at the indicated time points. γH2AX levels were used as a marker of DNA damage repair. Anti-β actin was used as a loading control D . Quantification of γH2AX levels from ( C ). Individual data points were represented as square markers in the graph. Error bars represent the SD ( n = 3). E U2OS Flp In T-REx HA βTrCPΔF cells were cultured on pre-sterilized glass coverslips with or without DOX, irradiated (10 Gy) or not, and treated 6 h later with CSK buffer for 10 min to remove soluble fractions. The remaining insoluble chromatin-associated fraction was analyzed by light microscopy. The scale bar represents 10 μm. F Quantification of MRE11 levels in chromatin in cells expressing HA βTrCPΔF or not, and irradiated (10 Gy) or not (25 cells per condition). G U2OS Flp In T-REx HA βTrCPΔF cells, induced or not with DOX, were irradiated (10 Gy) or not, and analyzed 1 h later for DNA damage using the comet assay. Each dot represents the comet-tail length of an individual nucleus (50 cells per condition). The line indicates the average tail length for each condition. H Representative images of each condition from the comet assay described in ( G ). Scale bar: 10 μm. ns non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test).

Article Snippet: The pCMV6 Flag MRE11 was obtained from Origene (Rockville, MD, USA).

Techniques: Irradiation, Western Blot, Control, Transfection, Plasmid Preparation, Marker, Cell Culture, Light Microscopy, Expressing, Single Cell Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Genome rearrangements induced by the stimulation of end-joining of DNA double strand breaks through multiple phosphorylation of MRE11 by the kinase PKB/AKT1

doi: 10.1093/nar/gkaf468

Figure Lengend Snippet: Stimulation of EJ-mediated deletion by PKB involves the activation of MRN/ATM signaling. ( A ) Frequency of EJ-mediated deletions (CD4+ cells) in GC92 cells with or without transfection of the PKB expression plasmid and with or without treatment with the DNA-PK inhibitor NU7026 (10 μM, left panel), ATM inhibitor KU55933 (10 μM, middle panel), or MRE11 inhibitor Mirin (10 μM, right panel) for 24 h after I-SceI transfection. The histograms show the quantitative data (means ± SEMs) from six (ATMi KU55933) and four (DNA PK inhibitor NU7026 and Mirin) independent experiments. Significant differences between experimental groups were analyzed by Kolmogorov–Smirnov test. ( B ) Western blot analysis of KAP1 phosphorylation at Ser824 in GC92 cells with or without transfection of PKB after etoposide treatment (1 μM, 2 h) where indicated. ( C ) pATM Ser1981 and pCHK2 Thr68 foci formation in GC92 cells expressing PKB or its kinase-dead form (PKB kd) with or without exposure to 5 Gy IR. Upper panel: representative images acquired 4 h after irradiation. Lower panel: quantitative data (means ± SEMs) from two to four independent experiments. Significant differences between experimental groups were analyzed by Kruskal–Wallis test. ( D ) PLA with probes targeting MRE11 and NBS1 in GC92 cells with or without exposure to 5 Gy IR. Upper panel: representative images acquired 1 h after irradiation. Lower panel: quantitative data (means ± SEMs) of puncta in three to nine independent experiments. Significant differences between experimental groups were analyzed by Kruskal–Wallis test.

Article Snippet: In vitro phosphorylation was performed with recombinant Mre11 (Origen TP309414) and recombinant active PKB (Millipore 14-276) in the presence of 1× kinase buffer (Cell Signaling, 9802), 5 μM ATP (Cell Signaling, 9804), and 10 μCi ATP [γ- 32 P] (Perkin Elmer, BLU0021 100UC) at 30°C for 15 min.

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Phospho-proteomics, Irradiation

Journal: Nucleic Acids Research

Article Title: Genome rearrangements induced by the stimulation of end-joining of DNA double strand breaks through multiple phosphorylation of MRE11 by the kinase PKB/AKT1

doi: 10.1093/nar/gkaf468

Figure Lengend Snippet: PKB phosphorylates MRE11 in vitro and interacts with MRE11 in cells. ( A ) In silico identification of MRE11 residues as potential targets of PKB kinase activity. ( B ) In vitro phosphorylation of MRE11 by PKB. The kinase activity of recombinant PKB toward recombinant MRE11 was evaluated by a radioactive assay in the presence of ATP [γ- 32 P]. In the first lane, pGSK3, a specific substrate of PKB, served as a positive control for PKB activity. In the second lane, no substrate was added. In the following lanes, 1 μg of MRE11 was incubated with increasing quantities of PKB. ( C ) Phosphorylated peptides of MRE11 identified by mass spectrometry. ( D ) HA-PKB coimmunoprecipitated with FLAG-MRE11. HA-PKB and FLAG-MRE11 were overexpressed in GC92 cells. Immunoprecipitation of FLAG-MRE11 was performed with an anti-FLAG antibody, and HA-PKB was detected in the coimmunoprecipitates by western blotting with an anti-HA antibody. ( E ) Western blot showing the level of MRE11 and PKB proteins in GC92 cells transfected with control or PKB expression plasmids and treated with the indicated times with cycloheximide (50 μg/ml). ( F ) PLA with probes targeting MRE11 and PKB in GC92 cells with or without exposure to 5 Gy IR. Left panel: representative images of unirradiated cells or cells irradiated with 5 Gy (after 1 h and 4 h). Right panel: quantitative data (means ± SEMs) of PLA puncta per cell (left histogram) or per nucleus (right histogram). Each value is presented as the average from 3 to 10 independent experiments. Significant differences between experimental groups were analyzed by Kruskal–Wallis test.

Article Snippet: In vitro phosphorylation was performed with recombinant Mre11 (Origen TP309414) and recombinant active PKB (Millipore 14-276) in the presence of 1× kinase buffer (Cell Signaling, 9802), 5 μM ATP (Cell Signaling, 9804), and 10 μCi ATP [γ- 32 P] (Perkin Elmer, BLU0021 100UC) at 30°C for 15 min.

Techniques: In Vitro, In Silico, Activity Assay, Phospho-proteomics, Recombinant, Radioactivity, Positive Control, Incubation, Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Control, Expressing, Irradiation

Journal: Nucleic Acids Research

Article Title: Genome rearrangements induced by the stimulation of end-joining of DNA double strand breaks through multiple phosphorylation of MRE11 by the kinase PKB/AKT1

doi: 10.1093/nar/gkaf468

Figure Lengend Snippet: Phosphorylation of Mre11 by MRE11 is required for stimulation of genomic rearrangements. ( A ) Frequency of EJ-mediated deletions (CD4+ cells) in GC92 cells transfected with WT-MRE11 in presence of absence of a PKB inhibitor (inh IV, 2 μM). Histograms represent values from three to five individual experiments (means ± SEMs). Significant differences between experimental groups were analyzed by Kruskal–Wallis test. ( B ) Frequency of EJ-mediated deletions (CD4+ cells) in GC92 cells transfected with PKB and/or WT-MRE11 or TM-MRE11. Histograms represent values from three to five individual experiments (means ± SEMs). Significant differences between experimental groups were analyzed by Kruskal–Wallis test. ( C ) Chromosomal fusions in metaphase spreads from GC92 cells transfected with the control or PKB expression plasmid and/or the WT-MRE11 or TM-MRE11 expression plasmid. Quantification of fusion events (means ± SEMs) is shown on the Right panel. Representative images of metaphase spreads stained with DAPI and detected with a centromeric PNA probe are shown on the Left panel. A minimum of 75 metaphases were analyzed for each condition. Significant differences between experimental groups were analyzed by Kruskal–Wallis test.

Article Snippet: In vitro phosphorylation was performed with recombinant Mre11 (Origen TP309414) and recombinant active PKB (Millipore 14-276) in the presence of 1× kinase buffer (Cell Signaling, 9802), 5 μM ATP (Cell Signaling, 9804), and 10 μCi ATP [γ- 32 P] (Perkin Elmer, BLU0021 100UC) at 30°C for 15 min.

Techniques: Phospho-proteomics, Transfection, Control, Expressing, Plasmid Preparation, Staining