Effects of MSH6 knockdown on GSC sensitivity to TMZ, ATM activation, and synergy. A) GBM4 cells were lentivirally transduced with MSH6 or nontargeting shRNA, with associated decrease in MSH6 protein by western blot. B) Dose–response curves of TMZ sensitivity after 5-day incubation. Error bars indicate 95% confidence intervals. Dotted line indicates 50% cell viability relative to mock-treated cells. C) Nontargeting (left) or MSH6 (right) shRNA-expressing cells were treated with indicated doses of TMZ [determined from dose–response curves in (B)] or PBS (mock) and 2.5 days later harvested and processed for western blotting. D) Effect of MSH6 shRNA on TMZ and G47Δ interaction. Data are shown as Fraction affected–Combination Index (CI) plot. CI < 1, CI = 1, CI > 1 represent synergistic, additive, and antagonistic interactions, respectively. The experiments were repeated at least three times for each of the conditions, each time in triplicate. ATM = ataxia telangiectasia mutated; ATR = ataxia telangiectasia and Rad3-related; GSC = glioblastoma stem cell; MSH6 = mutS homolog 6; PBS = phosphate buffered saline; p-ATM = phosphorylated ATM (Ser1981); p-ATR = phosphorylated ATR (Ser428); TMZ = temozolomide.