Fig. 2: Knockdown of AATF inhibits DNA damage repair in GSCs and sensitizes GB to chemoradiotherapy. | Nature Communications

Fig. 2: Knockdown of AATF inhibits DNA damage repair in GSCs and sensitizes GB to chemoradiotherapy.

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

Fig. 2

a–d 4121 GSCs transduced with shNT or shAATFs (a, b), or Flag-vector or Flag-AATF (c, d), were treated with IR (3 Gy) for indicated time points. IB (a, c) and IF (b, d) analyses of γ-H2AX are shown. Nuclei counterstained with Hoechst (blue). Left: representative images; Scale bars (b, d), 10 μm. Right: quantifications of γ-H2AX, each point represents a single cell (n = 3, biologically independent experiments). e 4121 GSCs transduced with shNT or shAATF#1 (red), or Flag-vector or Flag-AATF (green), were treated with increasing IR dose for 48 h or 72 h. Cell viability assessed by CellTiter-Glo assay. (Two-way ANOVA). f–j BALB/c nude mice intracranially implanted with GSCs transduced with Luciferase and Tet-on-inducible shNT or shAATF. Mice treated with control, IR (3 Gy, weekly, 3 times), Dox (1 mg/mL in water), or combined treatment from day 9 post-implantation (f, g, top). GB xenografts tracked by bioluminescence; representative images and quantifications are shown (f, g, n = 6, mean ± SD; Unpaired two-sided Student’s t-test). Kaplan–Meier survival plots of mice are shown (h, Log-rank Mantel–Cox test). Co-IF staining of SOX2 with γ-H2AX (i) or cleaved-caspase3 (CCP3) (j) in GB xenografts are shown. Left: representative images; Scale bars, 20 μm. Right: quantifications of γ-H2AX or CCP3 intensity, each point represents a single cell (n = 4, biologically independent samples). k 4121 GSCs transduced with shNT or shAATFs were treated with TMZ (50 μM) for indicated time points. IB analysis of indicated proteins are shown. l, m BALB/c nude mice intracranially implanted with GSCs (Luciferase) transduced with Tet-on-inducible-shNT or shAATF. Mice treated with control, TMZ (40 mg/kg, every 2 days, 5 times), Dox (1 mg/mL in water), or combined treatment from day 9 post-implantation (l, top). GB xenografts tracked by bioluminescence; representative images and quantifications are shown (l, n = 6 mice for each group; mean ± SD; Unpaired two-sided Student’s t-test). Kaplan–Meier survival plots of mice are shown (m, Log-rank Mantel–Cox test). For IB, protein levels were normalized to GAPDH. Independent experiments were performed three times with similar results. The curves represent the relative expression of γ-H2AX protein (a, c, k). Data represent mean ± SD of three independent experiments (a–e, i–k). Unpaired two-sided Student’s t-test. Source data are provided as a Source Data file.

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