Journal: Nature Communications

Article Title: Elevated nonhomologous end-joining by AATF enables efficient DNA damage repair and therapeutic resistance in glioblastoma

doi: 10.1038/s41467-025-60228-z

Figure Lengend Snippet: a Co-immunofluorescent (co-IF) staining of γ-H2AX and SOX2 in orthotopic GB xenografts treated with IR (3 Gy) for 24 h. Nuclei were counterstained with Hoechst (blue). Left: representative images; scale bars, 20 μm. Middle: quantifications of γ-H2AX foci, each point represents a single cell ( n = 4, biologically independent samples). Right: the percentages of γ-H2AX + cells, each point represents a biologically independent sample. b IF staining of γ-H2AX in 4121 GSCs and matched DGCs treated with IR (3 Gy). Nuclei were counterstained with Hoechst (blue). Left: representative images; scale bars, 10 μm. Right: quantifications of γ-H2AX foci, each point represents a single cell ( n = 3, biologically independent experiments). c , d Schematic model for LC-MS/MS identifying differentially expressed proteins (DEPs) in GSCs and GB cells. Venn diagram ( c ) and heatmap analysis ( d ) of DEPs in GB cells vs. NHAs and GSCs vs. DGCs ( p < 0.01, fold change > 2.0). e Immunoblot (IB) analysis of γ-H2AX and AATF levels in GSCs and NHA. Cells transduced with shNT or shAATFs (independent sequences). f IF staining of γ-H2AX in 4121 GSCs transduced with shNT or shAATFs. Nuclei were counterstained with Hoechst (blue). Left: representative images; Scale bars, 10 μm. Right: quantifications of γ-H2AX foci, each point represents a single cell ( n = 3, biologically independent experiments). g , h IB analysis of expression of indicated proteins in GSCs and their matched DGCs ( g ), in GSCs, primary GB cells, NPCs, NSCs, and NHAs ( h ). i Tumor cells isolated from three GB patient samples. CD133+ and CD133- cells sorted using magnetic microbeads (left). IB analysis of AATF and XRCC4 in sorted cells (right). j , k Co-IF staining of AATF and SOX2 ( j ), or γ-H2AX ( k ) in 3 human GB specimens. Nuclei were counterstained with Hoechst (blue). Left: representative images; Scale bars, 20 μm. Right: quantifications of γ-H2AX or AATF intensity, each point represents a single cell ( n = 3, biologically independent samples). l Immunohistochemical (IHC) analysis of AATF and γ-H2AX in human GB specimens. Left: representative images; Scale bars, 50 μm. Right: Pearson correlation (two-sided) analysis of AATF and γ-H2AX IHC scores in 15 GB samples. Labels hGB#3, hGB#28, hGB#35, and hGB#68 refer to individual GB tumor specimens. For IB, protein levels were normalized to GAPDH or Tubulin. Independent experiments were performed three times with similar results ( e , g , h , i ). Data represent mean ± SD of at least three independent experiments ( a , b , f , j , k ). Unpaired two-sided Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: AATF (Proteintech Group for IB, 1:1000, Cat#10282-1-AP; for IF, 1:200, Cat#A301-032a; Santa Cruz for IHC, 1:200, Cat#sc-81225), XRCC4 (Santa Cruz for IB, 1:1000, for IHC 1:200, Cat#sc-365055; Proteintech Group for IF, 1:200,Cat# 15817-1-AP), γ-H2AX (Millipore for IB, 1:1000, Cat#05-636; Cell Signaling for IF 1:200, cat#9718), Flag (Sigma for IB, 1:2000, Cat#F7425), Cleaved-Caspase3 (Cell Signaling for IB, 1:1000; for IF, 1:100, Cat#9664 s), LIG4 (Proteintech Group for IB, 1:1000, Cat#12695-1-AP), DNA-PKcs (Cell Signaling for IB, 1:1000, Cat#4602), Ku80 (Proteintech Group for IB, 1:1000, Cat#16389-1-AP), ATM (Abcam for IB, 1:1000, Cat#ab32420), SOX2 (Santa Cruz for IB, 1:1000, cat#SC-365823; R&D for IF, 1:200,cat#AF2018), CD3 (Thermo Fisher for IF, 1:200, cat#14-0037-82), CD45 (Thermo Fisher for IF, 1:200, cat#58-0459-42), CD68 (Abcam for IF, 1:400, cat# ab955), GFAP (Cell Signaling for IF,1:400,cat#12389).

Techniques: Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transduction, Expressing, Isolation, Immunohistochemical staining

Journal: Nature Communications

Article Title: Elevated nonhomologous end-joining by AATF enables efficient DNA damage repair and therapeutic resistance in glioblastoma

doi: 10.1038/s41467-025-60228-z

Figure Lengend Snippet: a AATF-XRCC4 interaction was analyzed by IP with IgG or anti-AATF antibody in GSCs. IB for indicated proteins are shown. b Schematic of AATF domains (left). Co-IP analysis of AATF-XRCC4 interaction in 293 T cells expressing Flag-AATF mutants and mCherry-XRCC4 (right). c AATF-XRCC4 interaction analyzed by IP with anti-Flag M2 beads in 293 T cells expressing Flag-AATF mutants. d NHEJ repair activity in U2OS cells expressing shNT or shAATFs, transfected with linearized I-Scel NHEJ reporter (EJ5-GFP) (left). HR repair activity assessed using DR-GFP reporter (right). GFP-positive cells, indicating NHEJ and HR repair respectively, analyzed by flow cytometry. e Flag-AATF or Flag-AATF △331–380 mutant was expressed in U2OS cells transduced with shAATF#1. NHEJ activity was assessed as described in ( d ). f IB showing that full-length AATF, but not △331–380 mutant, decreases γ-H2AX accumulation in GSCs expressing shAATF. g IB showing expression of indicated proteins in GSCs transduced with shNT or shAATFs. h CHX chase analysis of protein degradation in 4121 GSCs transduced with shNT or shAATF#1. XRCC4 levels quantified and normalized to GAPDH. Data presented as mean ± SEM ( n = 3, biologically independent experiments). i IB analysis of protein expression in shNT or shAATF#1 GSCs treated with MG132 (10 μM) for 12 h. j IP with anti-XRCC4 antibody and IB with anti-Ub-k48 and anti-Ub antibodies in GSCs (shNT or shAATF#1) treated with MG132. The graph represents the relative levels of Ub (bottom). k Flag-vector or Flag-XRCC4 was expressed in 4121 GSCs transduced with shNT or shAATF#1. The expression of indicated proteins was assessed by IB. l Flag-vector or Flag-XRCC4 was expressed in U2OS transduced with shNT or shAATF#1. The NHEJ activity was assessed as described in ( d ). For IB, protein levels were normalized to immunoprecipitated AATF ( a–c ), GAPDH/Tubulin ( f , g , i , k ), or immunoprecipitated XRCC4 ( j ). Independent experiments were performed twice ( b ) or three times ( a , c , f – k ) with similar results. Data represent the mean ± SD of at least three independent experiments ( d , e , l , j ). Unpaired two-sided Student’s t -test ( d, e, h, j, l ). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: AATF (Proteintech Group for IB, 1:1000, Cat#10282-1-AP; for IF, 1:200, Cat#A301-032a; Santa Cruz for IHC, 1:200, Cat#sc-81225), XRCC4 (Santa Cruz for IB, 1:1000, for IHC 1:200, Cat#sc-365055; Proteintech Group for IF, 1:200,Cat# 15817-1-AP), γ-H2AX (Millipore for IB, 1:1000, Cat#05-636; Cell Signaling for IF 1:200, cat#9718), Flag (Sigma for IB, 1:2000, Cat#F7425), Cleaved-Caspase3 (Cell Signaling for IB, 1:1000; for IF, 1:100, Cat#9664 s), LIG4 (Proteintech Group for IB, 1:1000, Cat#12695-1-AP), DNA-PKcs (Cell Signaling for IB, 1:1000, Cat#4602), Ku80 (Proteintech Group for IB, 1:1000, Cat#16389-1-AP), ATM (Abcam for IB, 1:1000, Cat#ab32420), SOX2 (Santa Cruz for IB, 1:1000, cat#SC-365823; R&D for IF, 1:200,cat#AF2018), CD3 (Thermo Fisher for IF, 1:200, cat#14-0037-82), CD45 (Thermo Fisher for IF, 1:200, cat#58-0459-42), CD68 (Abcam for IF, 1:400, cat# ab955), GFAP (Cell Signaling for IF,1:400,cat#12389).

Techniques: Co-Immunoprecipitation Assay, Expressing, Activity Assay, Transfection, Flow Cytometry, Mutagenesis, Transduction, Plasmid Preparation, Immunoprecipitation

Journal: Nature Communications

Article Title: Elevated nonhomologous end-joining by AATF enables efficient DNA damage repair and therapeutic resistance in glioblastoma

doi: 10.1038/s41467-025-60228-z

Figure Lengend Snippet: a Live-cell imaging of the recruitment of mCherry-XRCC4 and GFP-AATF to laser damage tracks generated by laser micro-irradiation in 4121 GSCs. Left: representative images. Blue arrows indicate the irradiated regions. Right: quantifications of the fluorescence intensity of XRCC4 and AATF on the DNA damage tracks ( n = 4, biologically independent experiments; data are presented as mean ± SD; Two-way ANOVA). Scale bars, 5 μm. b , c 4121 GSCs ( b ), or 4121 GSCs expressing Flag-XRCC4 ( c ), were treated with IR (7.5 Gy) at indicated time points. The XRCC4-AATF interaction was analyzed by IP assay. d 293 T cells expressing Flag-AATF were pretreated with KU-55933 (ATM inhibitor) or NU7441 (DNA-PKcs inhibitor) for 1 h, and were then treated with IR (7.5 Gy, 30 min) as indicated. The XRCC4-AATF interaction was analyzed by IP with anti-Flag M2 beads. e 4121 GSCs were treated with IR (7.5 Gy) at the indicated time points. ATM-AATF interaction was analyzed by IP with IgG or anti-ATM antibody. f 4121 GSCs expressing Flag-AATF were treated with IR (7.5 Gy) at the indicated time points and collected for IP with anti-Flag M2 beads, and IB with p-SQ antibody. g 4121 GSCs were pretreated with KU-55933 for 1 h, and were then treated with IR (7.5 Gy, 15 min) as indicated. Cell lysates were collected for IP with anti-AATF antibody, and IB with p-SQ antibody. h 4121 GSCs expressing Flag-AATF or Flag-AATF S189A mutants were treated with IR (7.5 Gy, 15 min) and collected for IP with anti-Flag M2 beads, and IB with p-SQ antibody. i 4121 GSCs expressing Flag-AATF Wt , Flag-AATF S189A or Flag-AATF S189D mutants were treated with IR (7.5 Gy) for 30 min. The XRCC4-AATF interaction was analyzed by IP with anti-Flag M2 beads. For IB, protein levels were normalized to immunoprecipitated AATF ( b , d , f – i ), XRCC4 ( c ), or ATM ( e ) respectively. Independent experiments were performed three times with similar results. The graphs represent the relative levels of indicated proteins ( b , c , e , f , right, mean ± SD). Unpaired two-sided Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: AATF (Proteintech Group for IB, 1:1000, Cat#10282-1-AP; for IF, 1:200, Cat#A301-032a; Santa Cruz for IHC, 1:200, Cat#sc-81225), XRCC4 (Santa Cruz for IB, 1:1000, for IHC 1:200, Cat#sc-365055; Proteintech Group for IF, 1:200,Cat# 15817-1-AP), γ-H2AX (Millipore for IB, 1:1000, Cat#05-636; Cell Signaling for IF 1:200, cat#9718), Flag (Sigma for IB, 1:2000, Cat#F7425), Cleaved-Caspase3 (Cell Signaling for IB, 1:1000; for IF, 1:100, Cat#9664 s), LIG4 (Proteintech Group for IB, 1:1000, Cat#12695-1-AP), DNA-PKcs (Cell Signaling for IB, 1:1000, Cat#4602), Ku80 (Proteintech Group for IB, 1:1000, Cat#16389-1-AP), ATM (Abcam for IB, 1:1000, Cat#ab32420), SOX2 (Santa Cruz for IB, 1:1000, cat#SC-365823; R&D for IF, 1:200,cat#AF2018), CD3 (Thermo Fisher for IF, 1:200, cat#14-0037-82), CD45 (Thermo Fisher for IF, 1:200, cat#58-0459-42), CD68 (Abcam for IF, 1:400, cat# ab955), GFAP (Cell Signaling for IF,1:400,cat#12389).

Techniques: Live Cell Imaging, Generated, Irradiation, Fluorescence, Expressing, Immunoprecipitation

Journal: Nature Communications

Article Title: Elevated nonhomologous end-joining by AATF enables efficient DNA damage repair and therapeutic resistance in glioblastoma

doi: 10.1038/s41467-025-60228-z

Figure Lengend Snippet: a 4121 GSCs were transduced with shAATFs in combination with Flag-AATF Wt , Flag-AATF S189A , or Flag-AATF S189D . IB analysis of indicated proteins are shown. b , c 4121 GSCs, treated as in ( a ), were treated with IR (3 Gy) at indicated time points. IB ( b ) and IF ( c ) analyses of γ-H2AX. Nuclei were counterstained with Hoechst (blue). Left: representative images; scale bars, 10 μm. Right: quantifications of γ-H2AX ( b , n = 3 biologically independent experiments; mean ± SEM) ( c , each point represents a single cell; n = 5 biologically independent experiments; mean ± SD). d 4121 GSCs, treated as in ( a ), were treated with IR (7.5 Gy) at indicated time points. Ku80-XRCC4 interaction was analyzed by IP. Left: representative images; Right: quantifications of XRCC4 ( n = 3, biologically independent experiments; mean ± SEM). e Live-cell imaging of XRCC4 recruitment to laser micro-irradiation-induced DNA damage in GSCs treated as in ( a ). Left: representative images; scale bars, 5 μm. Blue arrows indicate irradiated regions. Right: quantifications of XRCC4 fluorescence intensity on DNA damage tracks ( n = 4 biologically independent experiments; mean ± SD). f NHEJ activity was assessed in U2OS cells transduced with shAATFs in combination with Flag-AATF ( n = 3 biologically independent experiments; mean ± SD). g 4121 GSCs, treated as in ( a ), were exposed to increasing IR dose for 48 h. Cell viability was assessed by the CellTiter-Glo assay ( n = 4 biologically independent experiments; mean ± SD). h–k , BALB/c nude mice intracranially implanted with 4121 GSCs (Luciferase). Mice were treated with IR (3 Gy, weekly, 3 times) from day 9 post-implantation ( h , top). GB xenografts were tracked by bioluminescence; representative images and quantifications are shown ( h , i , n = 6 mice for each group; mean ± SEM). Kaplan–Meier survival plots of mice ( j , Log-rank Mantel–Cox test). Co-IF staining of γ-H2AX and SOX2 in GB xenografts ( k , top). Intensity of γ-H2AX staining in SOX2+ cells ( k , bottom; each point represents a single cell, n = 3 biologically independent samples, mean ± SD). Scale bars, 20 μm. For IB, protein levels were normalized to GAPDH ( a , b ) or immunoprecipitated Ku80 ( d ). Independent experiments were performed three times with similar results ( a , b , d ). The curves represent relative levels of indicated proteins ( b , d , right). Unpaired two-sided Student’s t -test ( b–d , f , i , k ), or Two-way ANOVA ( e , g ). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: AATF (Proteintech Group for IB, 1:1000, Cat#10282-1-AP; for IF, 1:200, Cat#A301-032a; Santa Cruz for IHC, 1:200, Cat#sc-81225), XRCC4 (Santa Cruz for IB, 1:1000, for IHC 1:200, Cat#sc-365055; Proteintech Group for IF, 1:200,Cat# 15817-1-AP), γ-H2AX (Millipore for IB, 1:1000, Cat#05-636; Cell Signaling for IF 1:200, cat#9718), Flag (Sigma for IB, 1:2000, Cat#F7425), Cleaved-Caspase3 (Cell Signaling for IB, 1:1000; for IF, 1:100, Cat#9664 s), LIG4 (Proteintech Group for IB, 1:1000, Cat#12695-1-AP), DNA-PKcs (Cell Signaling for IB, 1:1000, Cat#4602), Ku80 (Proteintech Group for IB, 1:1000, Cat#16389-1-AP), ATM (Abcam for IB, 1:1000, Cat#ab32420), SOX2 (Santa Cruz for IB, 1:1000, cat#SC-365823; R&D for IF, 1:200,cat#AF2018), CD3 (Thermo Fisher for IF, 1:200, cat#14-0037-82), CD45 (Thermo Fisher for IF, 1:200, cat#58-0459-42), CD68 (Abcam for IF, 1:400, cat# ab955), GFAP (Cell Signaling for IF,1:400,cat#12389).

Techniques: Transduction, Live Cell Imaging, Irradiation, Fluorescence, Activity Assay, Glo Assay, Luciferase, Staining, Immunoprecipitation

Journal: Nature Communications

Article Title: Elevated nonhomologous end-joining by AATF enables efficient DNA damage repair and therapeutic resistance in glioblastoma

doi: 10.1038/s41467-025-60228-z

Figure Lengend Snippet: a Schematic drawing of the synthesized polypeptides of AATF. NLS, nuclear localization signal. b 4121 GSCs were treated with the indicated polypeptides for 6 h. Live-cell imaging of FITC-tagged polypeptides was performed in three biologically independent experiments, and representative images are shown. Scale bars, 5 μm. c 4121 GSCs were treated with the indicated polypeptides and MG132 (10 μM) for 6 h. AATF-XRCC4 interaction was analyzed by IP with IgG or anti-AATF antibody. d 4121 GSCs preincubated with the indicated polypeptides were treated with IR (3 Gy) (top), or TMZ (50 μM) (bottom) at the indicated time points. IB analysis of γ-H2AX and XRCC4 levels are shown. The curves represent the relative levels of γ-H2AX (right, mean ± SD; Unpaired two-sided Student’s t -test). e–g BALB/c nude mice were intracranially implanted with 4121 GSCs expressing Luciferase. Mice were treated with control, IR (3 Gy, once a week, 3 times), TMZ (40 mg/kg, every two days, 5 times), the mix of three polypeptides (10 mg/kg, every two days, 5 times), or the combined treatment from day 9 post-implantation. GB xenografts were tracked by bioluminescence and the representative images are shown ( e ). Bioluminescent quantifications of tumor growth are shown ( f , n = 6 mice for each group; mean ± SD; Unpaired two-sided Student’s t -test). Kaplan–Meier survival plots of mice are shown ( g , Log-rank Mantel–Cox test). For IB, protein levels were normalized to immunoprecipitated AATF ( c ), or GAPDH ( d ). Independent experiments were performed three times with similar results ( c , d ). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: AATF (Proteintech Group for IB, 1:1000, Cat#10282-1-AP; for IF, 1:200, Cat#A301-032a; Santa Cruz for IHC, 1:200, Cat#sc-81225), XRCC4 (Santa Cruz for IB, 1:1000, for IHC 1:200, Cat#sc-365055; Proteintech Group for IF, 1:200,Cat# 15817-1-AP), γ-H2AX (Millipore for IB, 1:1000, Cat#05-636; Cell Signaling for IF 1:200, cat#9718), Flag (Sigma for IB, 1:2000, Cat#F7425), Cleaved-Caspase3 (Cell Signaling for IB, 1:1000; for IF, 1:100, Cat#9664 s), LIG4 (Proteintech Group for IB, 1:1000, Cat#12695-1-AP), DNA-PKcs (Cell Signaling for IB, 1:1000, Cat#4602), Ku80 (Proteintech Group for IB, 1:1000, Cat#16389-1-AP), ATM (Abcam for IB, 1:1000, Cat#ab32420), SOX2 (Santa Cruz for IB, 1:1000, cat#SC-365823; R&D for IF, 1:200,cat#AF2018), CD3 (Thermo Fisher for IF, 1:200, cat#14-0037-82), CD45 (Thermo Fisher for IF, 1:200, cat#58-0459-42), CD68 (Abcam for IF, 1:400, cat# ab955), GFAP (Cell Signaling for IF,1:400,cat#12389).

Techniques: Synthesized, Live Cell Imaging, Expressing, Luciferase, Control, Immunoprecipitation

Journal: Nature Communications

Article Title: Elevated nonhomologous end-joining by AATF enables efficient DNA damage repair and therapeutic resistance in glioblastoma

doi: 10.1038/s41467-025-60228-z

Figure Lengend Snippet: a , b BALB/c nude mice intracranially implanted with 4121 GSCs (Luciferase) with indicated treatment. Mice were treated with control, IR (3 Gy, weekly, 3 times), Dox (1 mg/mL in water), or the combined treatment from day 9 post-implantation. GB xenografts were tracked by bioluminescence ( a , n = 6 mice for each group; mean ± SD). Kaplan–Meier survival plots of mice are shown ( b , Log-rank Mantel–Cox test). c Flag-vector or Flag-XRCC4 was expressed in 4121 GSCs transduced with shAATF#1. Cells were treated with IR (3 Gy) at indicated time points. IB analysis of indicated proteins shown. The curve represents relative levels of γ-H2AX (right, mean ± SD). d GSCs expressing shNT, shAATF#1 or shAATF#1 plus Flag-XRCC4 were treated with increasing IR dose for 48 h ( n = 5 biologically independent experiments; mean ± SD). Cell viability was assessed by the CellTiter-Glo assay. (Two-way ANOVA). e–g BALB/c nude mice intracranially implanted with 4121 GSCs (Luciferase). GB xenografts were tracked by bioluminescence ( e , n = 6 mice for each group; mean ± SD). Kaplan–Meier survival plots of mice are shown ( f , Log-rank Mantel–Cox test). IF staining of γ-H2AX or CCP3 in GB xenografts are shown ( g , n = 5 biologically independent samples, mean ± SD). Scale bars, 20 μm. h IB analysis of AATF and XRCC4 expression in resected human GB tissues and paired normal brains. i–m IHC analysis of AATF and XRCC4 expression in serial sections of glioma tissue microarrays. Representative images ( i ) and histoscore quantification ( j ) of AATF and XRCC4 are shown. Scale bars, 50 μm. (grade I-II, n = 89 independent tissue samples from patients; grade III-IV, n = 67 independent tissue samples from patients). Pearson correlation analysis (two-sided) between AATF and XRCC4 IHC scores is shown ( k ). Kaplan–Meier survival analysis of patients in high-grade gliomas ( l ) and all gliomas ( m ) are shown. (Log-rank Mantel–Cox test). For IB, protein levels were normalized to GAPDH ( c , h ). Independent experiments were performed three times with similar results ( c , h ). Unpaired two-sided Student’s t -test ( a , c , e , g , j ). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: AATF (Proteintech Group for IB, 1:1000, Cat#10282-1-AP; for IF, 1:200, Cat#A301-032a; Santa Cruz for IHC, 1:200, Cat#sc-81225), XRCC4 (Santa Cruz for IB, 1:1000, for IHC 1:200, Cat#sc-365055; Proteintech Group for IF, 1:200,Cat# 15817-1-AP), γ-H2AX (Millipore for IB, 1:1000, Cat#05-636; Cell Signaling for IF 1:200, cat#9718), Flag (Sigma for IB, 1:2000, Cat#F7425), Cleaved-Caspase3 (Cell Signaling for IB, 1:1000; for IF, 1:100, Cat#9664 s), LIG4 (Proteintech Group for IB, 1:1000, Cat#12695-1-AP), DNA-PKcs (Cell Signaling for IB, 1:1000, Cat#4602), Ku80 (Proteintech Group for IB, 1:1000, Cat#16389-1-AP), ATM (Abcam for IB, 1:1000, Cat#ab32420), SOX2 (Santa Cruz for IB, 1:1000, cat#SC-365823; R&D for IF, 1:200,cat#AF2018), CD3 (Thermo Fisher for IF, 1:200, cat#14-0037-82), CD45 (Thermo Fisher for IF, 1:200, cat#58-0459-42), CD68 (Abcam for IF, 1:400, cat# ab955), GFAP (Cell Signaling for IF,1:400,cat#12389).

Techniques: Luciferase, Control, Plasmid Preparation, Transduction, Expressing, Glo Assay, Staining

Journal: Nature Communications

Article Title: Elevated nonhomologous end-joining by AATF enables efficient DNA damage repair and therapeutic resistance in glioblastoma

doi: 10.1038/s41467-025-60228-z

Figure Lengend Snippet: Prior to DNA damage, AATF forms a complex with XRCC4; Upon DNA damage, ATM interacts with and phosphorylates AATF at its Ser189 residue, which drives AATF-XRCC4 complex dissociation, thereby allowing rapid recruitment of XRCC4 to DSB sites to drive NHEJ repair (left). Knockdown of AATF results in polyubiquitination and subsequent proteasomal degradation of XRCC4, thus inhibiting NHEJ repair (middle). The non-phosphorylatable AATF mutant variant blocks recruitment of XRCC4 to DSB sites and inhibits NHEJ repair (right).

Article Snippet: The following antibodies were used: AATF (Proteintech Group for IB, 1:1000, Cat#10282-1-AP; for IF, 1:200, Cat#A301-032a; Santa Cruz for IHC, 1:200, Cat#sc-81225), XRCC4 (Santa Cruz for IB, 1:1000, for IHC 1:200, Cat#sc-365055; Proteintech Group for IF, 1:200,Cat# 15817-1-AP), γ-H2AX (Millipore for IB, 1:1000, Cat#05-636; Cell Signaling for IF 1:200, cat#9718), Flag (Sigma for IB, 1:2000, Cat#F7425), Cleaved-Caspase3 (Cell Signaling for IB, 1:1000; for IF, 1:100, Cat#9664 s), LIG4 (Proteintech Group for IB, 1:1000, Cat#12695-1-AP), DNA-PKcs (Cell Signaling for IB, 1:1000, Cat#4602), Ku80 (Proteintech Group for IB, 1:1000, Cat#16389-1-AP), ATM (Abcam for IB, 1:1000, Cat#ab32420), SOX2 (Santa Cruz for IB, 1:1000, cat#SC-365823; R&D for IF, 1:200,cat#AF2018), CD3 (Thermo Fisher for IF, 1:200, cat#14-0037-82), CD45 (Thermo Fisher for IF, 1:200, cat#58-0459-42), CD68 (Abcam for IF, 1:400, cat# ab955), GFAP (Cell Signaling for IF,1:400,cat#12389).

Techniques: Residue, Knockdown, Mutagenesis, Variant Assay