Fig 1: Metformin activates apoptotic cell death; (A) MDA-MB-231 cells were plated in 96 well plate and then either left untreated or treated with millimolar (upper graphs) or micromolar (lower graphs) doses of metformin. ADP/ATP ratio was determined as indicated in Material and Methods; (B) MDA-MB-231 cells were seeded on glass coverslip and then left untreated or treated with 30 µM metformin. At the end of the treatment, cells were stained with 10 µg/mL JC-1 as indicated under Material and Methods. Mitochondria fluorescence was observed by confocal microscopy. First row: Red fluorescence representing JC1 aggregates. Second row: Green fluorescence representing JC1 monomers. Third row: Merging of the first two rows. Alternatively, cells were treated with 30 µM metformin and stained with JC-1 as above. Red (FL2H) and green (FL1H) fluorescence intensity was quantified by Flow Cytometry; (C)upper panel: MDA-MB-231 cells were kept in the presence or absence of metformin for the time indicated and then processed to obtain mitochondrial fractions. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. PHB and CDK4 were used as loading and purity control, respectively. Lower panel: MDA-MB-231 cells were kept in the presence or absence of metformin for the time indicated and then processed to obtain whole cellular extracts. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. CDK4 and PHB were used as loading control. Right panel: MCF-7 cells were kept in the presence or absence of metformin for 20 days and then processed to obtain mitochondrial or whole cellular extracts. BAX and CYCS expression levels were determined by Western blot as indicated under Material and Methods. Densitometric analysis of the gels was performed as indicated under Material and Methods. PHB and CDK4 were used as purity and loading controls; (D) MDA-MB-231 cells were kept in the presence or absence of metformin for 20 days. At the end of the treatment cells were harvested and percentage of sub-G1 (M1) cells was determined by propidium iodide staining as described in the Material and Methods section. All experiments in this figure were repeated three times. * Significantly different from control untreated cells.