Val-083 cytotoxicity is exerted independently of MMR or MGMT expression. A, Viability of parental U-251 MG, T98G, U-87 MG, LN-18, and SF268 cell lines upon exposure to different concentrations of Val-083 measured by MTT. Values are represented as percentage of cell viability compared with DMSO control. B, Cell-cycle profile of U-251 MG or T98G cells treated with different doses of TMZ or Val-083 for 48 hours. C, Competition assay of U-251 MG cells expressing shRNAs targeting MSH6 (shMSH6.3908), PMS2 (shPMS2.946), and MSH2 (shMSH2.357) after treatment with TMZ or VAL-083. The plotted value is the fold enrichment of the shRNA-expressing cells (eGFP+) compared with the control cells (mCherry+) and normalized to the DMSO control. Statistical test: Two-way ANOVA (P values: *P < 0.05, **P < 0.01, compared with DMSO treated). D, CFA of U-251 MG cells expressing control shRNA or shMSH6 after treatment with TMZ or Val-083 for 9 days. E, Cell-cycle profile of U-251 MG cells expressing control shRNA (shRen.660) or shMSH6.3908 after treatment with TMZ or Val-083 for 48 hours. F, Quantification of mean γH2AX fluorescence in nuclei of cells treated with vehicle, TMZ, or Val-083 for 48 hours. Values are normalized to the DMSO control, and fold increase in H2AX signal is plotted. Statistical test: Two-way ANOVA (P values: ***, P < 0.001; ****, P < 0.0001, compared with DMSO treated).