Protective effect of Met against Dox induced cytotoxicity depends on AMPK activity.

<p>(A) H9c2 cardiomyoblasts were cultured with reduced FBS (1%) for 72 hours, and then cell lysates were isolated with RIPA buffer supplemented with appropriate phosphatase inhibitors. AMP-activated protein kinase activities in the cell lysates were measured using an AMPK Kinase Assay Kit (Cyclex). (B) H9c2 cell lysates from 72 hour-culture were subject to western blotting using antibodies against phosphorylated or total AMPKα and acetyl-CoA carboxylase (ACC). The histograms show the densitometric scanning results. (C) H9c2 cells were cultured for 72 hours with reduced FBS (1%) in 96-well culture plates under indicated conditions with or without compound-C (10 µM). Cell viabilities were evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104888#s2" target="_blank">Materials and Methods</a>. (D) H9c2 cells stably over expressed wild type (WT), dominant negative (DN) or constitutively active (CA) AMPKα cDNA were cultured for 72 hours with reduced FBS (1%) under the indicated conditions. The cell viabilities were evaluated as described. Values represent mean ± S.D. from at least ten (A, C, D) or quadruplicate (B) samples for each treatment at varying treatment conditions. * p<0.05 vs CTR; # p<0.05 Dox 10 nM vs Dox 10 nM + Met 0.1 mM; $ p<0.05 Met 0.1 mM vs Met 1.0 mM (A), * p<0.05 (B, C, D).