Fig. 1: Construction and confirmation of an acquired TMZ-resistant GBM xenograft model.

A Schematic process of the acquired TMZ-resistant GBM tumors and cells. B Tumor growth assay of cells derived from indicated xenografts. The lower panel showing the tumors collected at the final day. C Tumor growth assay of indicated xenografts after the first TMZ injection. Mice were subcutaneously injected with indicated cells and subsequently received intragastric administration of TMZ (20 mg/kg) every two days after the tumors reached 100 mm³. Tumor volume was monitored as indicated the day from the 1st TMZ injection. Xenografts were collected on the 12nd day post TMZ-treatment. D Tumor growth assay of xenografts received the indicated treatment. Mice were subcutaneously injected with indicated cells and subsequently received intragastric administration of normal saline (NS) or TMZ (20 mg/kg) every two days after the tumors reached 100 mm³. Tumor volume was monitored at the indicated day from the 1st treatment. Tumors were collected on the 12nd day post TMZ-treatment. E IC50 assay indicating different drug response between TMZ-S and TMZ-R cells (n = 3, **p < 0.01). F qRT–PCR assay measuring the expression of ABCB1 and ABCC3 mRNA in the indicated cells and tumors (n = 3, **p < 0.01). G Flow cytometry (FC) assay (left) and the statistical analysis of mean fluorescence intensity (MFI; right) showing the uptake of DOX by the indicated cells, the shift in fluorescence intensity of a population of cells (n = 3, **p < 0.01). H Analysis of MGMT promoter methylation in U87 cells and derived indicated tumors by methylation-specific PCR (MSP). M and U indicating methylated (M) and unmethylated (U) status of the promoter respectively; + and − representing the bisulfite-converted methylated and unmethylated DNA respectively. I RT-PCR measurement showing the expression of MGMT in indicated cells. PC-3 cells served as a MGMT-positive control.