Curcumol sensitized TMZ-resistant glioma cells to TMZ. (A) Scheme for establishment of TMZ-resistant glioma cells. Briefly, the parental U251/A172 cells were cultured in DMEM medium containing increasing concentrations of TMZ until they acquired resistance to TMZ. (B) MTT assay was performed to compare cell viability against TMZ in resistant cells and their respective parental cells. (C) Isobologram analysis determined the effects of curcumol combined with TMZ on cell proliferation of TMZ-resistant glioma cells. (D) Colony formation assay was used to assess the clonogenicity of TMZ-resistant glioma cells following treatment with different concentrations of curcumol and/or TMZ for 14 days. (E) MS-PCR analysis of the MGMT promoter methylation status and the western blotting analysis of MGMT protein level in TMZ-resistant glioma cells following treatment with different concentrations of curcumol and/or TMZ. The density of each band was quantified using imaging analysis and the relative band density values were calculated as the ratio of methylated MGMT to that of methylated plus un-methylated MGMT. MGMT unmethylated: 92 bp; MGMT methylated: 8l bp; U: unmethylated; M: methylated; MM: molecular marker. *P < 0.05, **P < 0.01.