Figure 8.

Drug resistance after high-dose TMZ plus O6BG treatment. (A) Treatment schedule was as follows: 3 × 10⁶ A2058 cells were seeded into a 25-cm plate and treated with 100 µM TMZ in the presence of O6BG. After about 6 weeks, one colony of surviving cells (called clone 61) emerged and was cultured for further analysis by WB and CFA. (B) Cell lysate was prepared from clone 61 and analyzed by WB for MGMT and MSH6 protein levels. Lysates from A375 and A2058 cells were included as MGMT-positive and -negative controls, respectively; lysates from HCT116 and DLD1 cells were included as MSH6-positive and -negative controls, respectively. (C) To confirm drug sensitivity in correlation with MMR deficiency, clone 61 was treated with increasing concentrations of TMZ in the presence or absence of O6BG, and CFA was performed 12 days later (n = 3, ±SE). (D) Clone 61 was treated with increasing concentrations of TMZ (single dose, as per schedule listed in Figure 4). After complete recovery, mass cultures were analyzed for MGMT protein levels by WB analysis with actin as the loading control.