Fig. 1: AATF is highly expressed and involved in DNA damage repair in GSCs.

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.