Extended Data Fig. 7: Lactylation of KU70 K317 enhances the NHEJ function and maintains stemness of GSC.
a, b, Two independent shRNAs targeting MCT1 decreased the self-renewal of GSCs compared with shCONT, as measured by sphere number quantification (n = 6) (a) and the extreme limiting dilution assays (b) in 3028 and MES28. c, d, Sphere number quantification (n = 6) (c) and the extreme limiting dilution assays (d) performed in 3028 and MES28 with the deletion of KU70 and rescued by KU70 WT and KU70 K317R. e, Cell viability of six patient-derived GSCs (MES28, 3028, 2907, GSC23, RKI and 3264) after 48 h treatment of vehicle control (DMSO) and various concentrations of NHEJ inhibitor NU7441 combined with the stimulation of lactate (n = 6). f, GST pull-down assay performed to detect the interaction between KU80 and KU70 WT or KU70 K317R in vitro. g, Immunoblot results showing the interaction between KU70 and KU80 with anti-KU70 antibody in 3028 and MES28 GSCs with or without P300-Flag (1287-1663AA) and the 10 mM lactate stimulated. h, Immunoblot analysis of GSCs γ-H2AX level and the interaction level between KU70 and KU80 under the stimulation of 10 mM lactate combined transduced with shCONT, shKU70 and rescued by KU70 (WT) or KU70 (E330A) or KU70 (K317R) or KU70 (K317R E330A) after KU70 knockdown. i, Effects of shCONT, shKU70 and rescued by KU70 (WT) or KU70 (E330A) or KU70 (K317R) or KU70 (K317R E330A) after KU70 knockdown on the efficiency of NHEJ in MES28 GSC with the stimulation of lactate (n = 3). j, Immunoblot analysis after KU70 K317-Peptide blockade in GSCs. The experiments were performed three times independently (f, g, j). Data are presented as mean ± SD (e, i), box plots show the median (centre), the 25th and 75th percentiles (bounds of box), and whiskers indicate the minimum and maximum values (a, c). Statistical significance was determined by a two-tailed Student’s t-test (a, c, i) and a likelihood ratio test (LRT) comparing the single-hit model to alternative models (two-sided). P-values were computed based on the χ² distribution of the likelihood ratio statistic (b, d).
