Figure 3. Effects of HL156A, TMZ, and combination treatment on cell viability.

A. HL156A and TMZ alone, at concentrations of 15 and 500uM, respectively, showed minimal effects on cell viability, enabling us to assess the cellular mechanism responsible for the effects of HL156A. B. ATP level was decreased on the treatment of HL156A, TMZ and HL156A+TMZ. C. ¹⁸F-FDG uptake was markedly decreased in GBM-TSs treated with HL156A, TMZ, or both. The decrease in FDG uptake was most prominent in the combination-treatment group, suggestive of a low metabolic status. D. Oxygen consumption rate was markedly decreased by treatment with HL156A or TMZ, and was prominently observed in the combination treatment group. Similar findings were observed with metformin plus TMZ treatment. E. Treatment with HL156A or HL156A+TMZ did not cause cell-cycle arrest or definite apoptotic cell death at the tested concentration. F. Effects of HL156A, TMZ, and combination treatment on AMPK and the mTOR pathway. Although biguanide is known to act as an AMPK agonist and inhibitor of mTOR signaling, neither AMPK activation nor subsequent mTOR inhibition was consistently observed, suggesting that AMPK-mTOR is not the major pathway by which HL156A and HL156A+TMZ act.