Effect of onalespib treatment on select DNA repair proteins in patient-derived glioma stem cell lines: (A) MGMT promoter methylation status of glioma PDX lines (M, methylated; UM, unmethylated). (B) Expression of ATM, CHK1, RAD51, MSH2 in GSC811, GSC11, GSC262, GSC23, GSC214, GSC231 and GSC267 cells. GAPDH was loaded as a control and the immunoblot is representative of three independent experiment. (C) Effect of onalespib treatment (0.4μM) on levels of ATM, CHK1, RAD51, MSH2 and γ-H2AX in GSC262 and GSC811 cells over time by immunoblotting. GAPDH and total H2AX were evaluated as loading controls. Figure is representative of four independent experiments. (D) Effect of onalespib treatment (0.4μM) over time on the activation of the ATR kinase as measured by levels of p-ATR, ATR, p-CHK1, and CHK1. GAPDH was measured as a loading control. Figure is representative of three independent experiments. (E) Quantitation of percent inhibition of HR in U2OS reporter cells treated with onalespib (0.1for 24h). Graph is representative of three independent experiments in triplicate (p<0.0001, two-tailed p test). (F) Effect of dose and time dependent exposure to onalespib on cell survival in GSC262 and GSC811 cells.