Journal: Nature Communications

Article Title: Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

doi: 10.1038/s41467-026-72034-2

Figure Lengend Snippet: a, b Surface plasmon resonance (SPR) experiment showing direct binding of a C-terminal Ku80 fragment (aa 384 – 732) to surface-coupled EndoG. a Sensorgram of a representative single-cycle measurement with EndoG as surface-coupled ligand and the C-terminal Ku80 fragment as analyte, applied in increasing concentrations (10, 20, 40, 80, 160, and 320 nM) (red line). The maximal response at equilibrium was marked for each concentration (black x). b Response-concentration plot from (a) shows the equilibrium response of each analyte concentration (black circles). A one-site binding fit (red line) was used to determine the dissociation constant K D . The measurement was done in triplicate, resulting in a mean K D and standard deviation of 72.7 ± 13.2 nM. c Structure of Ku80 variants analyzed. Ku80-wt comprises the N-terminal Von Willebrand A domain , the central DNA end-binding core (aa 210–531) , which further encompasses aa 449–477 heterodimerizing with Ku70 and is followed by a nuclear localization signal (NLS) . The structurally defined C-terminal domain (CTD, red) is predicted to form α-helices at aa 594–705 and an unstructured tail, which binds DNA-PKcs at the PIKK (PI3 kinase-like kinase) interaction motif (aa 718-732) , . For expression of Ku80-Ct, devoid of DNA, Ku70 and DNA-PKcs binding sites, a truncated CTD (aa 592-709) was fused to the NLS (aa 562–568). d Principle of MLL bcr rearrangement assay. Recombination measurements rely on quantification of EGFP-positivities post doxorubicin-treatment of K562 cells with chromosomally integrated reporter construct for recombination between differently mutated EGFP genes encompassing the 0.4 kbp therapy-related MLL bcr , . e Ku80 variant expression in K562. Proteins were extracted 24 h post-electroporation of K562 cells with expression constructs for Ku80 variants specified in (a) or empty vector (ctrl). Western blot analysis was performed using antibodies for total Ku80 and the N-terminal DDK/Flag-tag of ectopically expressed variants (representative from 2 independent experiments). Uncropped Western blots in Source Data (uncropped images). f , g Recombination measurements post-chemotherapeutic treatment. K562 reporter cells, ectopically expressing Ku80 variants for 24 h, were treated with 2.0 μM doxorubicin (blue) for 4 h followed by 72 h drug-free culture, with solvent (DMSO, white) or 10 µM etoposide (gray) for 4 h followed by 72 h drug-free culture or for 72 h with etoposide and FACS analysis. Recombination (rec.) frequencies of EGFP-positive living cells in the population were normalized to the mean of ctrl values set to 100% per experiment (average for doxorubicin: 2 × 10 −5 ; for etoposide, 4 h: 6 × 10 −5 and 72 h: 5 × 10 −4 ) to calculate relative rec. frequencies. Data are presented as mean + SEM. Significances were calculated by the Kruskal-Wallis H-test followed by a two-tailed Mann-Whitney-U test and values of p < 0.05 indicated, gating strategies in Supplementary Fig. , d. f Recombination measurements post doxorubicin treatment ( n = 6 samples from 3 independent experiments). g Recombination measurements post etoposide treatment. DMSO and etoposide (4 h): n = 6 samples from 3 independent experiments. Etoposide (72 h): n = 15 samples from 5 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For analysis of Ku80-wt, Ku80-Ct or Ku80(E720A, E721A) the following expression plasmids with a pCMV6-AN-DDK backbone were generated (OriGene, Rockville, Maryland, USA): pCMV6-AN-DDK-Ku80 expressing Ku80-wt (SC112985 ORF, OriGene) with N-terminal Flag-tag and pCMV6-AN-DDK-Ku80 C-terminus expressing Ku80-Ct through fusion of the Ku80-NLS motif P562-K568 (encoded by 5 ́-CCTACAGCTAAAAAATTAAAG-3 ́).

Techniques: SPR Assay, Binding Assay, Concentration Assay, Standard Deviation, Expressing, Construct, Variant Assay, Electroporation, Plasmid Preparation, Western Blot, FLAG-tag, Solvent, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

doi: 10.1038/s41467-026-72034-2

Figure Lengend Snippet: HeLa cells were transfected with expression constructs for Ku80-wt, Ku80-Ct or empty vector (ctrl) 24 h before experimental analysis. a Ku80 variant expression in HeLa. Protein extracts were subjected to immunoblotting to detect Ku80 and Flag-tagged variants (representative from 2 independent experiments). b Formation of EndoG foci in the nucleus. Immunofluorescence microscopy was performed after 4 h of treatment with H 2 O (white) or 0.5 μM doxorubicin (blue). Mean values + SEM are presented (H 2 O, ctrl: n = 160; H 2 O, Ku80-wt: n = 230; H 2 O, Ku80-Ct n = 250; doxorubicin, ctrl: n = 135; doxorubicin, Ku80-wt: n = 169; doxorubicin, Ku80-Ct n = 179 nuclei from 2 independent experiments). Significances were calculated by the Kruskal-Wallis H-test followed by a two-tailed Mann-Whitney-U test and **** p < 0.0001 indicated. Insets display the highlighted regions at two-fold magnification. Scale bars indicate 10 µm. c Chromatin immunoprecipitation (ChIP) in doxorubicin-treated HeLa reporter cells. Sonified DNA bound by proteins was immunoprecipitated with antibodies specific for EndoG, γH2AX or incubated with control IgG. After DNA isolation, PCR amplifying a 0.3 kbp DNA fragment adjacent to the MLL bcr (EGFP/MLLbcr, MLLbcr-2) was performed, band intensities evaluated, normalized to Ku80-wt (100%) and shown as mean + SD from 3 independent ChIP-EndoG and 4 independent ChIP-γH2AX experiments. Note that previous work already showed increased EndoG-binding to the MLL bcr in doxorubicin-treated cells . Antibody specificities were high as visualized in Source Data (uncropped images), though it remains possible that additional low-abundance bands are present but not visible at the selected exposure. d Genomic PCR. Genomic DNA isolated from doxorubicin-treated HeLa reporter cells, such as during ChIP experiments resembling input DNA, was subjected to PCR analysis as in ( c ). MLL bcr band intensities were corrected by the intensities of the MLL control PCR (ctrl), amplifying a 0.2 kbp fragment 8 kbp downstream of the MLL bcr (Intron20 in the genomic MLL ) and normalized to the mean of ctrl, Ku80-wt and Ku80-ct values (100%) each. Data from 6 independent experiments are presented as mean + SEM. Significances were calculated by the Kruskal-Wallis H-test followed by a two-tailed Mann-Whitney-U test and p < 0.05 indicated. PCR products were separated on SeaKem® LE Agarose (Lonza, Basel, Switzerland). Source data are provided as a Source Data file.

Article Snippet: For analysis of Ku80-wt, Ku80-Ct or Ku80(E720A, E721A) the following expression plasmids with a pCMV6-AN-DDK backbone were generated (OriGene, Rockville, Maryland, USA): pCMV6-AN-DDK-Ku80 expressing Ku80-wt (SC112985 ORF, OriGene) with N-terminal Flag-tag and pCMV6-AN-DDK-Ku80 C-terminus expressing Ku80-Ct through fusion of the Ku80-NLS motif P562-K568 (encoded by 5 ́-CCTACAGCTAAAAAATTAAAG-3 ́).

Techniques: Transfection, Expressing, Construct, Plasmid Preparation, Variant Assay, Western Blot, Immunofluorescence, Microscopy, Two Tailed Test, MANN-WHITNEY, Chromatin Immunoprecipitation, Immunoprecipitation, Incubation, Control, DNA Extraction, Binding Assay, Isolation

Journal: Nature Communications

Article Title: Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

doi: 10.1038/s41467-026-72034-2

Figure Lengend Snippet: a Scheme illustrating the positions of Ku peptides within Ku80-Ct. Nuclear localization signal (NLS); C-terminal domain (CTD, red). b Evaluation of peptide Ku3 in the EGFP -based reporter assay in K562. K562 reporter cells were nucleofected with increasing amounts of Ku3 peptide, cultivated for 24 h, treated for 4 h with 2 μM doxorubicin (blue) or H 2 O (white), released in drug-free medium for 72 h, recombination frequencies determined flow cytometrically and doxorubicin-treated cells without Ku3 set to 100% per experiment (average: 3 × 10 −5 ). Data are presented as mean + SEM; H 2 O/doxorubicin, 0: n = 6/6; 25: n = 4/3; 50: n = 6/6; 75: n = 4/4; 100: n = 6/6; 125: n = 4/4; 150: n = 4/4; 175: n = 4/4; 200: n = 6/6 samples from 2 (25, 75, 125, 150, 175) or 3 (0, 50, 100, 200) independent experiments. Significances were calculated for mock- and doxorubicin-treated cells separately by Kruskal-Wallis H-test, followed by the two-tailed Mann-Whitney-U test and p < 0.05 indicated. For viabilities according to SSC/FSC gating, see Supplementary Fig. . c Ku3 peptide evaluation in HeLa reporter cells. Increasing concentrations of the Ku3 peptide were nucleofected into cells, which were recultivated for 24 h, treated with 0.5 μM doxorubicin for 4 h and released in drug-free medium for 72 h. Recombination (rec.) frequencies were determined, presented, and statistics calculated as in ( b ); 0: n = 9; 25: n = 7; 50: n = 9; 75: n = 9 samples from 4 (25) or 5 (0, 50, 75) independent experiments. For viabilities according to SSC/FSC gating, see Supplementary Fig. . d Cell entry of Ku3 peptide. Brightfield images of HeLa cells nucleofected with H 2 0 or TAMRA-labeled Ku3 (left) and spinning disc confocal microscopy images at 532 nm laser excitation (right). Representative fields of view of > 20 cells. Scale bar is 20 µm. Source data are provided as a Source Data file.

Article Snippet: For analysis of Ku80-wt, Ku80-Ct or Ku80(E720A, E721A) the following expression plasmids with a pCMV6-AN-DDK backbone were generated (OriGene, Rockville, Maryland, USA): pCMV6-AN-DDK-Ku80 expressing Ku80-wt (SC112985 ORF, OriGene) with N-terminal Flag-tag and pCMV6-AN-DDK-Ku80 C-terminus expressing Ku80-Ct through fusion of the Ku80-NLS motif P562-K568 (encoded by 5 ́-CCTACAGCTAAAAAATTAAAG-3 ́).

Techniques: Reporter Assay, Two Tailed Test, MANN-WHITNEY, Labeling, Confocal Microscopy

Journal: Nature Communications

Article Title: Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

doi: 10.1038/s41467-026-72034-2

Figure Lengend Snippet: a, b Surface plasmon resonance (SPR) experiment showing direct binding of a C-terminal Ku80 fragment (aa 384 – 732) to surface-coupled EndoG. a Sensorgram of a representative single-cycle measurement with EndoG as surface-coupled ligand and the C-terminal Ku80 fragment as analyte, applied in increasing concentrations (10, 20, 40, 80, 160, and 320 nM) (red line). The maximal response at equilibrium was marked for each concentration (black x). b Response-concentration plot from (a) shows the equilibrium response of each analyte concentration (black circles). A one-site binding fit (red line) was used to determine the dissociation constant K D . The measurement was done in triplicate, resulting in a mean K D and standard deviation of 72.7 ± 13.2 nM. c Structure of Ku80 variants analyzed. Ku80-wt comprises the N-terminal Von Willebrand A domain , the central DNA end-binding core (aa 210–531) , which further encompasses aa 449–477 heterodimerizing with Ku70 and is followed by a nuclear localization signal (NLS) . The structurally defined C-terminal domain (CTD, red) is predicted to form α-helices at aa 594–705 and an unstructured tail, which binds DNA-PKcs at the PIKK (PI3 kinase-like kinase) interaction motif (aa 718-732) , . For expression of Ku80-Ct, devoid of DNA, Ku70 and DNA-PKcs binding sites, a truncated CTD (aa 592-709) was fused to the NLS (aa 562–568). d Principle of MLL bcr rearrangement assay. Recombination measurements rely on quantification of EGFP-positivities post doxorubicin-treatment of K562 cells with chromosomally integrated reporter construct for recombination between differently mutated EGFP genes encompassing the 0.4 kbp therapy-related MLL bcr , . e Ku80 variant expression in K562. Proteins were extracted 24 h post-electroporation of K562 cells with expression constructs for Ku80 variants specified in (a) or empty vector (ctrl). Western blot analysis was performed using antibodies for total Ku80 and the N-terminal DDK/Flag-tag of ectopically expressed variants (representative from 2 independent experiments). Uncropped Western blots in Source Data (uncropped images). f , g Recombination measurements post-chemotherapeutic treatment. K562 reporter cells, ectopically expressing Ku80 variants for 24 h, were treated with 2.0 μM doxorubicin (blue) for 4 h followed by 72 h drug-free culture, with solvent (DMSO, white) or 10 µM etoposide (gray) for 4 h followed by 72 h drug-free culture or for 72 h with etoposide and FACS analysis. Recombination (rec.) frequencies of EGFP-positive living cells in the population were normalized to the mean of ctrl values set to 100% per experiment (average for doxorubicin: 2 × 10 −5 ; for etoposide, 4 h: 6 × 10 −5 and 72 h: 5 × 10 −4 ) to calculate relative rec. frequencies. Data are presented as mean + SEM. Significances were calculated by the Kruskal-Wallis H-test followed by a two-tailed Mann-Whitney-U test and values of p < 0.05 indicated, gating strategies in Supplementary Fig. , d. f Recombination measurements post doxorubicin treatment ( n = 6 samples from 3 independent experiments). g Recombination measurements post etoposide treatment. DMSO and etoposide (4 h): n = 6 samples from 3 independent experiments. Etoposide (72 h): n = 15 samples from 5 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For analysis of Ku80-wt, Ku80-Ct or Ku80(E720A, E721A) the following expression plasmids with a pCMV6-AN-DDK backbone were generated (OriGene, Rockville, Maryland, USA): pCMV6-AN-DDK-Ku80 expressing Ku80-wt (SC112985 ORF, OriGene) with N-terminal Flag-tag and pCMV6-AN-DDK-Ku80 C-terminus expressing Ku80-Ct through fusion of the Ku80-NLS motif P562-K568 (encoded by 5 ́-CCTACAGCTAAAAAATTAAAG-3 ́).

Techniques: SPR Assay, Binding Assay, Concentration Assay, Standard Deviation, Expressing, Construct, Variant Assay, Electroporation, Plasmid Preparation, Western Blot, FLAG-tag, Solvent, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

doi: 10.1038/s41467-026-72034-2

Figure Lengend Snippet: HeLa cells were transfected with expression constructs for Ku80-wt, Ku80-Ct or empty vector (ctrl) 24 h before experimental analysis. a Ku80 variant expression in HeLa. Protein extracts were subjected to immunoblotting to detect Ku80 and Flag-tagged variants (representative from 2 independent experiments). b Formation of EndoG foci in the nucleus. Immunofluorescence microscopy was performed after 4 h of treatment with H 2 O (white) or 0.5 μM doxorubicin (blue). Mean values + SEM are presented (H 2 O, ctrl: n = 160; H 2 O, Ku80-wt: n = 230; H 2 O, Ku80-Ct n = 250; doxorubicin, ctrl: n = 135; doxorubicin, Ku80-wt: n = 169; doxorubicin, Ku80-Ct n = 179 nuclei from 2 independent experiments). Significances were calculated by the Kruskal-Wallis H-test followed by a two-tailed Mann-Whitney-U test and **** p < 0.0001 indicated. Insets display the highlighted regions at two-fold magnification. Scale bars indicate 10 µm. c Chromatin immunoprecipitation (ChIP) in doxorubicin-treated HeLa reporter cells. Sonified DNA bound by proteins was immunoprecipitated with antibodies specific for EndoG, γH2AX or incubated with control IgG. After DNA isolation, PCR amplifying a 0.3 kbp DNA fragment adjacent to the MLL bcr (EGFP/MLLbcr, MLLbcr-2) was performed, band intensities evaluated, normalized to Ku80-wt (100%) and shown as mean + SD from 3 independent ChIP-EndoG and 4 independent ChIP-γH2AX experiments. Note that previous work already showed increased EndoG-binding to the MLL bcr in doxorubicin-treated cells . Antibody specificities were high as visualized in Source Data (uncropped images), though it remains possible that additional low-abundance bands are present but not visible at the selected exposure. d Genomic PCR. Genomic DNA isolated from doxorubicin-treated HeLa reporter cells, such as during ChIP experiments resembling input DNA, was subjected to PCR analysis as in ( c ). MLL bcr band intensities were corrected by the intensities of the MLL control PCR (ctrl), amplifying a 0.2 kbp fragment 8 kbp downstream of the MLL bcr (Intron20 in the genomic MLL ) and normalized to the mean of ctrl, Ku80-wt and Ku80-ct values (100%) each. Data from 6 independent experiments are presented as mean + SEM. Significances were calculated by the Kruskal-Wallis H-test followed by a two-tailed Mann-Whitney-U test and p < 0.05 indicated. PCR products were separated on SeaKem® LE Agarose (Lonza, Basel, Switzerland). Source data are provided as a Source Data file.

Article Snippet: For analysis of Ku80-wt, Ku80-Ct or Ku80(E720A, E721A) the following expression plasmids with a pCMV6-AN-DDK backbone were generated (OriGene, Rockville, Maryland, USA): pCMV6-AN-DDK-Ku80 expressing Ku80-wt (SC112985 ORF, OriGene) with N-terminal Flag-tag and pCMV6-AN-DDK-Ku80 C-terminus expressing Ku80-Ct through fusion of the Ku80-NLS motif P562-K568 (encoded by 5 ́-CCTACAGCTAAAAAATTAAAG-3 ́).

Techniques: Transfection, Expressing, Construct, Plasmid Preparation, Variant Assay, Western Blot, Immunofluorescence, Microscopy, Two Tailed Test, MANN-WHITNEY, Chromatin Immunoprecipitation, Immunoprecipitation, Incubation, Control, DNA Extraction, Binding Assay, Isolation

Journal: Nature Communications

Article Title: Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

doi: 10.1038/s41467-026-72034-2

Figure Lengend Snippet: a Top: Complex of EndoG (cyan)/EndoGI (red) from Drosophila melanogaster (PDB ID 3IMS). Gray sphere: Mg 2+ ion. Bottom: Structure of the Ku80 fragment (light blue, residues 590–709, PDB ID: 1Q2Z), superposed on the EndoGI (transparent cartoon representation), bound to EndoG (cyan). Orange sticks: Fragment 687-693 (sequence TKEEASG) of Ku80, corresponding to the most conserved sequence motif among the extracted peptides. b Predicted docked complex of the Ku3 peptide (orange) bound to EndoG (cyan).

Article Snippet: For analysis of Ku80-wt, Ku80-Ct or Ku80(E720A, E721A) the following expression plasmids with a pCMV6-AN-DDK backbone were generated (OriGene, Rockville, Maryland, USA): pCMV6-AN-DDK-Ku80 expressing Ku80-wt (SC112985 ORF, OriGene) with N-terminal Flag-tag and pCMV6-AN-DDK-Ku80 C-terminus expressing Ku80-Ct through fusion of the Ku80-NLS motif P562-K568 (encoded by 5 ́-CCTACAGCTAAAAAATTAAAG-3 ́).

Techniques: Sequencing

Journal: Nature Communications

Article Title: Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

doi: 10.1038/s41467-026-72034-2

Figure Lengend Snippet: a Scheme illustrating the positions of Ku peptides within Ku80-Ct. Nuclear localization signal (NLS); C-terminal domain (CTD, red). b Evaluation of peptide Ku3 in the EGFP -based reporter assay in K562. K562 reporter cells were nucleofected with increasing amounts of Ku3 peptide, cultivated for 24 h, treated for 4 h with 2 μM doxorubicin (blue) or H 2 O (white), released in drug-free medium for 72 h, recombination frequencies determined flow cytometrically and doxorubicin-treated cells without Ku3 set to 100% per experiment (average: 3 × 10 −5 ). Data are presented as mean + SEM; H 2 O/doxorubicin, 0: n = 6/6; 25: n = 4/3; 50: n = 6/6; 75: n = 4/4; 100: n = 6/6; 125: n = 4/4; 150: n = 4/4; 175: n = 4/4; 200: n = 6/6 samples from 2 (25, 75, 125, 150, 175) or 3 (0, 50, 100, 200) independent experiments. Significances were calculated for mock- and doxorubicin-treated cells separately by Kruskal-Wallis H-test, followed by the two-tailed Mann-Whitney-U test and p < 0.05 indicated. For viabilities according to SSC/FSC gating, see Supplementary Fig. . c Ku3 peptide evaluation in HeLa reporter cells. Increasing concentrations of the Ku3 peptide were nucleofected into cells, which were recultivated for 24 h, treated with 0.5 μM doxorubicin for 4 h and released in drug-free medium for 72 h. Recombination (rec.) frequencies were determined, presented, and statistics calculated as in ( b ); 0: n = 9; 25: n = 7; 50: n = 9; 75: n = 9 samples from 4 (25) or 5 (0, 50, 75) independent experiments. For viabilities according to SSC/FSC gating, see Supplementary Fig. . d Cell entry of Ku3 peptide. Brightfield images of HeLa cells nucleofected with H 2 0 or TAMRA-labeled Ku3 (left) and spinning disc confocal microscopy images at 532 nm laser excitation (right). Representative fields of view of > 20 cells. Scale bar is 20 µm. Source data are provided as a Source Data file.

Article Snippet: For analysis of Ku80-wt, Ku80-Ct or Ku80(E720A, E721A) the following expression plasmids with a pCMV6-AN-DDK backbone were generated (OriGene, Rockville, Maryland, USA): pCMV6-AN-DDK-Ku80 expressing Ku80-wt (SC112985 ORF, OriGene) with N-terminal Flag-tag and pCMV6-AN-DDK-Ku80 C-terminus expressing Ku80-Ct through fusion of the Ku80-NLS motif P562-K568 (encoded by 5 ́-CCTACAGCTAAAAAATTAAAG-3 ́).

Techniques: Reporter Assay, Two Tailed Test, MANN-WHITNEY, Labeling, Confocal Microscopy

Journal: Nature Communications

Article Title: Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

doi: 10.1038/s41467-026-72034-2

Figure Lengend Snippet: Proximity ligation assay (PLA) was performed on fixed HeLa cells using antibodies against Ku80 and EndoG to detect Ku80-EndoG complex formation. a PLA of Ku80-EndoG complexes after doxorubicin treatment. HeLa cells were mock-transfected, cultured for 24 h, and treated with either doxorubicin (0.5 μM, 4 h, blue) or H₂O (white). Foci numbers were normalized to the means of mock-transfected, doxorubicin-treated cells and set to 100% per experiment. Data are presented as mean + SEM from n = 824 nuclei for H 2 O and n = 1200 nuclei for doxorubicin from 9 independent experiments. Statistical significance was determined using the Kruskal-Wallis H-test followed by the two-tailed Mann-Whitney U test (**** p < 0.0001). b Sequence-specific effect of Ku3 on Ku80-EndoG interactions after doxorubicin treatment. HeLa cells were nucleofected with Ku3, scrambled Ku3, or Ku1 peptide, cultured for 24 h, and treated with doxorubicin (0.5 μM, 4 h). PLA analysis was performed, and foci numbers were normalized to the means of control cells (no peptide transfected) and set to 100% per experiment (average: 2 foci/nucleus). Data are presented as mean + SEM from n = 596 nuclei for ctrl, n = 761 nuclei for Ku3, n = 659 nuclei for Scrambled and n = 445 nuclei for Ku1 from 4 independent experiments. Statistical significance was determined using the Kruskal-Wallis H-test followed by the two-tailed Mann-Whitney U test (**** p < 0.0001). c Ku3, but not its mutated versions, affects Ku80-EndoG interactions after doxorubicin treatment. HeLa cells were nucleofected with Ku3 or two mutated versions of Ku3 (Mut1 and Mut2), cultured for 24 h, and treated with doxorubicin (0.5 μM, 4 h). PLA analysis was performed, and foci numbers were normalized to the means of control cells (ctrl, no peptide transfected) and set to 100% per experiment. Data are presented as mean + SEM from n = 401 nuclei for ctrl, n = 536 nuclei for Ku3, n = 404 nuclei for Mut1 and n = 401 nuclei for Mut2 from 3 independent experiments. Statistical significance was determined using the Kruskal-Wallis H-test followed by the two-tailed Mann-Whitney U test (**** p < 0.0001). d PLA image gallery. As PLA controls, we included primary antibodies against Ku80 or EndoG (negative controls) or antibodies against Ku80 and Ku70 (positive control). Representative PLA images were derived from 4 and 3 independent experiments in ( b ) and ( c ), respectively. Margins of DAPI-stained nuclei are indicated by white stippled lines, and the scale bar represents 10 μm. Insets display highlighted regions at two-fold magnification. Source data are provided as a Source Data file.

Article Snippet: For analysis of Ku80-wt, Ku80-Ct or Ku80(E720A, E721A) the following expression plasmids with a pCMV6-AN-DDK backbone were generated (OriGene, Rockville, Maryland, USA): pCMV6-AN-DDK-Ku80 expressing Ku80-wt (SC112985 ORF, OriGene) with N-terminal Flag-tag and pCMV6-AN-DDK-Ku80 C-terminus expressing Ku80-Ct through fusion of the Ku80-NLS motif P562-K568 (encoded by 5 ́-CCTACAGCTAAAAAATTAAAG-3 ́).

Techniques: Proximity Ligation Assay, Transfection, Cell Culture, Two Tailed Test, MANN-WHITNEY, Sequencing, Control, Positive Control, Derivative Assay, Staining

Journal: Nature Communications

Article Title: Discovery of an Endonuclease G-inhibitory Ku80-peptide protecting against leukemogenic rearrangements at the MLL breakpoint cluster

doi: 10.1038/s41467-026-72034-2

Figure Lengend Snippet: a Upper panel: scheme of illumination pattern for EndoG bound fraction analysis. Lower panel: example tracks of EndoG wt recorded at 85 Hz, overlaid with an example frame of the corresponding movie. Scale bar is 2 µm. b Jump distance distributions of EndoG variants. Inset: Sketch of EndoG-HT diffusion and DNA binding. c Scheme of illumination pattern in time-lapse measurements with indicated frame cycle times. d Example tracks of EndoG wt recorded with 51.7 ms frame cycle time, overlaid with an example frame of the corresponding movie, and kymographs of indicated molecules. Scale bar is 2 µm. e Survival time distributions of EndoG variants at time-lapse conditions shown on top. f State spectra of dissociation rates of EndoG variants obtained with GRID using all data (red). As an error estimation, GRID was run 500 times, with each run using 80 % of the data (black circles). The dotted line indicates the dissociation rate of 0.01 s −1 . g Long residence times (inverse of the weighted average of dissociation rates below 0.01 s −1 in (f)) of EndoG variants and EndoG wt in the presence of a transfection control plasmid (tcr), Ku80 C-terminus, and Ku3-derived peptides. Error bars denote SD of the resampled data. h Long bound fractions of EndoG variants, and EndoG wt in the presence of tcr, Ku80 C-terminus and Ku3 derived peptides calculated from amplitudes in ( f ) and Supplementary Fig. and bound fractions in Supplementary Fig. . Data represented as value ± SD from Gaussian error propagation. The error bars are smaller than the data markers and are therefore not visible. Source data are provided as a Source Data file. Measurement statistics are given in Supplementary Table .

Article Snippet: For analysis of Ku80-wt, Ku80-Ct or Ku80(E720A, E721A) the following expression plasmids with a pCMV6-AN-DDK backbone were generated (OriGene, Rockville, Maryland, USA): pCMV6-AN-DDK-Ku80 expressing Ku80-wt (SC112985 ORF, OriGene) with N-terminal Flag-tag and pCMV6-AN-DDK-Ku80 C-terminus expressing Ku80-Ct through fusion of the Ku80-NLS motif P562-K568 (encoded by 5 ́-CCTACAGCTAAAAAATTAAAG-3 ́).

Techniques: Diffusion-based Assay, Binding Assay, Transfection, Control, Plasmid Preparation, Derivative Assay

Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: FOXN3 and the KU70/KU80/SREBP-1 complex colocalize at the promoters of SREBP-1 response genes. ( A ) Mass spectrometry analysis of HEK293T cells transfected with Flag-tagged FOXN3 reveals the number of unique peptides associated with FOXN3. The representative peptide sequences of KU70 and KU80, which were immunoprecipitated by Flag-tagged FOXN3, are presented. ( B ) An anti-Flag Co-IP assay was performed in HEK293T cells transfected with the specified plasmids to investigate the association of Flag-tagged FOXN3 with the KU70/KU80/SREBP-1 complex (P: precursor of SREBP-1). ( C ) Anti-FOXN3 Co-IP analysis was performed in HepG2 cells to examine the endogenous association between FOXN3 and KU70/KU80/SREBP-1 complex. (P: precursor of SREBP-1; N: nuclear form of SREBP-1). ( D ) Anti-Flag Co-IP assays were performed in HepG2 cells to detect the association of Flag-tagged FOXN3 with the KU70/KU80/SREBP-1 complex following treatment with FFA (400 μM) at the indicated time points. The cells were treated with MG-132 (20 μM, 4 h) prior to collection (P: precursor of SREBP-1; N: nuclear form of SREBP-1). ( E ) Venn diagrams showing the overlapping peaks identified by anti-KU80, anti-FOXN3, and anti-SREBP-1 CUT&Tag analyses in HepG2 cells treated with FFA (400 μM, 24 h). ( F ) Venn diagrams displaying the number of genes co-targeted by KU80, FOXN3, and SREBP-1 based on CUT&Tag analyses in HepG2 cells treated with FFA (400 μM, 24 h). ( G ) Density distributions (normalized read densities) of the mapped reads from anti-KU80, anti-FOXN3, and anti-SREBP-1 CUT&Tag analyses at the transcription start site (TSS) are shown. ( H ) Genomic distribution of the transcriptional targets of KU80, FOXN3, and SREBP-1 in HepG2 cells treated with FFA (400 μM, 24 h), as determined via CUT&Tag data analysis. ( I ) The CUT&Tag data analysis in HepG2 cells treated with FFA (400 μM, 24 h) displays the binding profiles of KU80, FOXN3, and SREBP-1 to representative SREBP-1 response genes. The graphs represent the proportion of reads enriched in the peak region relative to every one million total reads. The blotting data D was quantified as the mean fold change from two independent experiments using ImageJ software and was analyzed using two-tailed Student’s t -tests; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies utilized were as follows: anti-SREBP-1 (14088-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-His (66005-1-Ig, Proteintech), anti-KU70 (10000-0-AP, Proteintech), anti-FOXN3 (25399-1-AP, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-KU70 (10723-1-AP, Proteintech), anti-KU80 (16389-1-AP, Proteintech), anti-β-actin (20536-1-AP, Cell Signaling Technology), anti-FASN (10624-2-AP, Proteintech), anti-SCD1 (28678-1-AP, Proteintech), anti-LDLR (10785-1-AP, Proteintech), anti-HMGCS1 (17643-1-AP, Proteintech), anti-SREBP-1 (66875-1-Ig, Proteintech, for IP), anti-rabbit IgG (30000-0-AP, Proteintech), and anti-FOXN3 (ab129453, Abcam).

Techniques: Mass Spectrometry, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Software, Two Tailed Test