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. 2015 Apr 13;10(4):e0123721. doi: 10.1371/journal.pone.0123721

Fig 3. Metformin increases GB cell death and induces autophagic processes.

Fig 3

(A) Quantification of apoptotic and necrotic cell death, using flow cytometry and Annexin-V/PI staining, 12hrs, 24hrs and 48hrs after treatment. Metformin significantly increases the number of AV positive cells (*p<0.05, **p<0.01 compared to control, n = 3). (B-C) Quantification of vesicle acidification, using flow cytometry and Acridine Orange staining, following 12hrs, 24hrs or 48hrs treatment in presence or not of 10mM metformin (B) and/or 10μM bafilomycin (C). Metformin increases vesicle acidification for U251, LN18 and SF767 cells. This effect can be reversed using the autophagy inhibitor, bafilomycin (NS: Non-Significant, *p<0.05, **p<0.01, *** p<0.001 n = 5). (C) Western Blot analyses for LC3b, Beclin-1 and p62 and respective quantifications showing induction of autophagy in response to metformin treatment (*p<0.05, **p < 0.01 compared to control, n = 3).