Effects of KU-55933 and metformin on metabolism in MCF-7 cells.

<p>Cells were exposed to KU (10 µM) or metformin (5 mM) for 72 hrs. (<b>A</b>) The effect of KU-55933 or metformin on viable cell number was measured by counting cells able to exclude Trypan blue. Cell number was significantly reduced by KU-55933 (*<i>P</i> = 0.0042) and by metformin (**<i>P</i> = 0.0011). (<b>B</b>) Lactate production was significantly increased in cells treated with KU-55933 (*<i>P</i> = 0.0218) or metformin (**<i>P</i> = 0.0012). (<b>C</b>) Glucose consumption was increased with exposure to either KU-55933 (*<i>P</i> = 0.0463) or metformin (**<i>P</i> = 0.0058) treated cells. (<b>D</b>) Both KU-55933 and metformin decreased ATP levels in MCF-7 cells. Results are the mean ± S.E (<i>n</i> = 4). (KU-55933 compared to control *<i>P</i> = 0.0015 and metformin compared to control ** <i>P</i> = 0.0005). (<b>E</b>) Cells were incubated with JC-1 (2 µM), or H₂O₂ (100 µM, used to activate ATM by oxidative stress), or rotenone (1 µM), or FCCP (1 µM). Mitochondrial membrane potential was probed with JC-1 and visualized by flow cytometry. Loss of mitochondrial membrane potential (ΔΨ) is indicated by a decrease in FL2/FL1 fluorescence intensity ratio <b>(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049513#pone.0049513.s001" target="_blank">Figure S1</a> for flow cytometry data set)</b>. Results are expressed as mean ± S.E.M. (<i>n</i> = 4). KU-55933 (*<i>P</i> = 0.0003) and metformin (** <i>P</i><0.0001) both significantly decreased ΔΨ. (<b>F</b>) Total cellular respiration (black bars, left y-axis) of MCF-7 cells treated with KU-55933 or metformin was compared with untreated cells. Results are the mean ± S.E.M. (KU-55933 compared to control *<i>P</i> = 0.0045, and metformin compared to control ** <i>P</i> = 0.0496). Uncoupled respiration was determined in the presence of oligomycin. The percentage of uncoupled respiration was calculated as: (uncoupled respiration/total mitochondrial respiration), and is shown by hatched bars, right y-axis. (<b>G</b>) KU-55933 or metformin treatment increased cell death (<b>see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049513#pone.0049513.s002" target="_blank">Figure S2</a> for flow cytometry data set)</b>. Bars represent percentage of necrotic cells. Results are expressed as the mean ± S.E.M. (<i>n</i> = 3) in duplicate (KU-55933 compared to control *<i>P</i> = 0.0005, and metformin compared to control **<i>P</i> = 0.0299). (<b>H</b>) KU-55933 or metformin treatment resulted in increased apoptosis <b>(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049513#pone.0049513.s002" target="_blank">Figure S2</a> for flow cytometry data set).</b> Bars represent percentage of apoptotic cells. Results are expressed as the mean ± S.E.M. (n = 3) in duplicate (KU-55933 compared to control *<i>P</i><0.0001, and metformin compared to control **<i>P</i> = 0.0458).