Fig. 5: ATM-mediated Phosphorylation of AATF allows recruitment of XRCC4 to DSB sites.
a 4121 GSCs were transduced with shAATFs in combination with Flag-AATFWt, Flag-AATFS189A, or Flag-AATFS189D. 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.
