We further characterized if inhibition of DNA-PKcs could suppress glioma cell growth or enhance TMZ sensitivity using KU0060648, a novel DNA-PKcs inhibitor which was applied in previous researches [26, 27]. As shown in Figure 2C, KU0060648 inhibited the activation of DNA-PKcs (left panel of Figure 2C) and the proliferation of glioma cells (right panel of Figure 2C) in a dose-dependent manner. Moreover, KU0060648-mediated DNA-PKcs inhibition led to sensitization of TMZ in glioma cells. Addition of 1 or 10 μM of KU0060648 increased TMZ efficacy remarkably with the IC50 values dropping from 487 ± 24 μM to 154 ± 16 μM or 44 ± 12 μM, respectively in U87 (Figure 2D). Similar results were observed in M059K (Figure 2D). To better understand the effect of KU0060648 on TMZ sensitivity, glioma cell lines with lower levels of p-DNA-PKcs, namely H4 and U373, were also investigated. In contrast to U87 and M059K, KU0060648 only mildly restored the sensitivity to TMZ in H4 and U373 (Supplementary Figure S5), which supported the idea that KU0060648-sensitized TMZ depended on the suppression of p-DNA-PKcs levels.