Fig. 5

Fig. 5 Metformin- and temozolomide-mediated GBM cytotoxicity. (a) Efficiency of AMPKα activation by metformin. U3031MG cells were transfected with the indicated siRNAs for 48 h and treated with DMSO (-) or 5 mM metformin for another 24 h, before cell lysis and immunoblotting for the indicated proteins. Densitometric quantification of LKB1, phosphorylated (activation loop) AMPKα (pAMPKα) and total AMPKα protein levels relative to GAPDH, with the basal expression levels of LKB1, pAMPKα, and AMPKα in U3031MG siC (lane 1) arbitrarily set to 1.00. (b) Extreme limiting dilution assay of U3031MG cells transfected with siC (black, red symbols) or siLKB1 (green, blue symbols; siRNA pool), treated with DMSO (black, green symbols) or 5 mM metformin (red, blue symbols), and cultured for 7 days. The number of sphere-containing wells per plating density is plotted in the table below. (c) Cell viability assay of U3031MG cells transfected with siC or siLKB1, treated with DMSO, 5 mM metformin or increasing concentrations of TMZ, and cultured for 24 h. The % of viable cells is plotted relative to the siC treated only with DMSO. An arrow shows 125 µM TMZ used in panel (d). (d) Cell viability assay of U3031MG cells transfected with siC or siLKB1 (pool), treated with DMSO, 5 or 10 mM metformin with or without 125 µM TMZ, and cultured for 24 h. The percentage of viable cells is plotted relative to the siC treated only with DMSO. (e) The lowest dose of TMZ (µM) required for 50% inhibition of viability (IC₅₀) of U3031MG cells transfected with siC or siLKB1, co-treated with DMSO or 5 mM metformin and cultured for 24 h. (f) Cell death assay measuring the percent of annexin-V-positive cells using flow cytometry of U3031MG cells transfected with siC or siLKB1 (pool) and co-treated with DMSO, 5 or 10 mM metformin with or without 125 µM TMZ and cultured for 24 h. The results (b–f) are mean ± SEM of at least two independent experiments and significant differences at *p < 0.05, **p < 0.01 by one-way analysis of variance. GBM, glioblastoma; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LKB1, liver kinase B1; siRNA, small interfering RNA; TMZ, temozolomide