In order to determine whether KU0060648 inhibited glioma growth and sensitized temozolomide through suppression of DNA damage repair, we detected γH2AX foci, a frequently used assay visualizing the occurrence of DSBs, in KU0060648 single agent group or co-administrated with TMZ (Supplementary Figure S6). Indeed, there was undetectable level of background foci formation per cell in DMSO-treated control group, which was not increased by sole KU0060648 (data not shown). Such a finding prompted that KU0060648 exerted its anti-malignancy effects on glioma cells via a DNA damage repair independent manner, which consisted with the effect of NU7441, another well-known DNA-PKcs specific inhibitor, in a previous study . Exposure of 100 μM temozolomide alone did form γH2AX foci in both U87 and M059K cells. However, it declined rapidly, as only 11% (U87) and 16% (M059K) remained respectively after 120 hours. Additional KU0060648 did not affect the level of TMZ-induced foci formation but significantly retarded the loss of γH2AX foci. On the basis of these results, we postulated that suppressing glioma malignancies and enhancing TMZ efficacy by DNA-PKcs inhibitor might not merely depend on inhibition of DSBs repair, and thus, DNA-PKcs inhibitors can be used as single or synergized with TMZ against glioma.