Fig. 6

Hypoxia affects the efficacy of metformin only during short-term experiments. a MCF-7 and MDA-MB-231 cultured in 11 mM or 2.75 mM glucose-containing medium were treated with indicated concentrations of everolimus and metformin under normoxic or hypoxic conditions. Glucose was not replenished throughout the treatment. Cell viability was measured after 96 h using an MTT assay. Hypoxia alone already decreased cell viability to 50% of the viability in normoxia. Data are presented as mean ± SD of three different experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. b MCF-7 cells cultured in 11 mM or 2.75 mM glucose-containing medium were treated with indicated concentrations of everolimus and metformin under normoxic or hypoxic conditions for 48 h. Western Blotting was carried out for HIF1a, S6 and p-S6. A representative blot of 2 experiments is shown. c MCF-7 cells were plated in medium containing 11 mM or 2.75 mM glucose at a concentration of 500 cells/well. After 8 days of treatment, MCF7 colonies, consisting of at least 50 cells, were counted. MCF7 cells were treated with indicated concentrations of metformin +/− everolimus (10 nM). Data are presented as mean + SD of three different experiments