Fig. 3.

Metformin and everolimus do not influence autophagy and ROS production. a MCF-7 and MDA-MB-231 cells stably expressing LC3-GFP cultured in 11 mM and 2.75 mM glucose-containing medium were treated with indicated concentrations of 10 nM everolimus and/ or 5 mM metformin for 48 h. 20 μM bafilomycin was added 1 h prior to evaluation of the assay. Fluorescent GFP-LC-3 foci per individual cell were counted. Data are presented as mean ± SD of three different experiments. b MCF-7 and MDA-MB-231 cells cultured in 11 mM and 2.75 mM glucose-containing medium were treated with everolimus (10 nM) or metformin (5 mM) for 48 h. Western Blotting for LC-3 revealed that there is no increased LC-3 cleavage after either treatment. c MCF-7 cells were treated with 250 μM H₂O₂ for 1 h. Hydrogenperoxide induces ROS production in MCF-7 cells, as demonstrated by increase in cellular H₂DCF fluorescence. d MCF-7 and MDA-MB-231 cultured in 11 mM or 2.75 mM glucose-containing medium were treated with indicated concentrations of everolimus or metformin for 48 h, and stained with 10 μM H₂DCF. Everolimus and metformin do not induce significant changes in ROS levels compared to untreated cells. Data are presented as mean ± SD of three different experiments