Salinomycin-induced AMPK activation requires ROS production in osteoblastoma cells.

<p>U2OS cells were treated with vehicle or 10 μM of salinomycin (Sali) for 6, 12 and 24 hours, cellular ROS level was analyzed, quantified results were shown (A). The effect of NAC (500 μM, 3 hours pretreatment) on salinomycin (Sali, 10 μM, 12 hours) or H₂O₂ (250 μM, 2 hours)-induced AMPK activation in U2OS cells was tested by western blots (B). AMPK phosphorylation was quantified (C). U2OS and MG-63 cells were treated with salinomycin (Sali, 10 μM) in the presence or absence of NAC (500 μM, 3 hours pretreatment), cell viability, Annexin V percentage, and LC3B puncta were also detected (D and F). The effect of NAC (250/500 μM, 3 hours pretreatment) on salinomycin (Sali, 10 μM, 12 hours)-induced AMPK activation in MG-63 cells was also tested (E). (G) The proposed signaling pathways of this study: in osteoblastoma cells, salinomycin induces ROS production, which is required for both AMPK activation and apoptosis. Activated AMPK promotes autophagy activation, which is anti-apoptosis and anti-cell death. Experiments in this figure were repeated three times. ^{<b><i>#</i></b>}<i>p</i><0.05 vs. Vehicle group. *<i>p</i><0.05.