Fig. 4: DNA damage induces AATF phosphorylation and causes dissociation of the AATF-XRCC4 complex. | Nature Communications

Fig. 4: DNA damage induces AATF phosphorylation and causes dissociation of the AATF-XRCC4 complex.

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

Fig. 4

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-AATFS189A 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-AATFWt, Flag-AATFS189A or Flag-AATFS189D 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.

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