To identify which downstream effectors of AKT signaling were involved in mediating KU0060648 enhanced TMZ efficacy, we extracted total proteins from U87 and M059K cells after treatment with control solvent, 1 or 10 μM KU0060648, 100 μM TMZ or their combination. KU0060648 dose-dependently inhibited AKT signaling downstream effectors, such as pro-proliferative factor c-Myc, anti-apoptotic proteins survivin or Mcl-1, invasion related molecule MMP-9 and angiogenesis promoting factor VEGF (Figure 6A). Besides, activity of GSK-3β, the acknowledged direct substrates of AKT, was also suppressed by KU0060648 treatment (Figure 6A). It was noteworthy that TMZ alone did increase, although not enormously, the activities of DNA-PKcs and its downstream AKT, leading to elevate expression of pro-invasion and pro-angiogenesis effectors. Significantly, combination of KU0060648 enhanced the inhibitory effect on the DNA-PKcs/AKT signaling compared with each agent alone (Figure 6A), which suggested an underlying machinery of synergy between KU0060648 and TMZ. Similar trends were also obtained in mRNA level using qPCR (Figure 6B).