On day 16, mice were sacrificed and the tumors were resected

wing H2O2 treatment, ARVM were lysed, scraped, and collected in cold lysis buffer, and total protein concentration was measured by Bradford 15155536 assay. In the AMPK experiments Compound C was purchased from Sigma Aldrich, St. Louis, MO. GFT505 Samples were prepared and subjected to SDS-PAGE and Western blotting. Membranes were probed for autophagic proteins with the following antibodies: polyclonal anti-LC3; anti-ATG5; anti-ATG7 , polyclonal anti-beclin-1 , and polyclonal phosphomTOR antibody and phospho-Akt . In addition, membranes were probed for phosphorylated and total ERK and phosphorylated and total AMPK using the following antibodies: monoclonal against p-p44/42 , polyclonal total p44/42 ERK, polyclonal p-AMPK , and polyclonal AMPK. AntiGAPDH monoclonal antibody was used as a loading control house keeping protein. Membranes were then probed with either goat anti-rabbit or goat anti-mouse horseradish peroxidase conjugated secondary antibodies . Blots were detected with ECLTM Western Blotting Statistical analysis All data is expressed as means SEM; differences among multiple conditions were determined by ANOVA followed by a paired t-test with the Bonferroni correction for multiple comparisons. p values <0.05 were considered significant. Results APN attenuated H2O2-mediated loss of cell viability The MTT assay was used to determine cardiomyocyte survival in response to pathophysiological oxidative stress. Isolated adult rat ventricular myocytes were exposed to increasing concentrations H2O2 for 30, 60 and 90 min and viability were assessed by the MTT assay. At the 90 min time point, there was no decrease in cell viability at H2O2 concentrations of 10, 50, and 100M. Cell viability decreased slightly at H2O2 concentrations of 500M, and was maximally at 1000M. Similar to other studies, the latter concentration was shown to induce cell death. Thus H2O2 decreased viability by 342%. Pretreatment with APN prior to H2O2 exposure partially improved cell viability , but did not completely rescue the 34% reduction in cell viability caused by H2O2 treatment. 3 Adiponectin Modulates Cardiac Myocyte Autophagy doi: 10.1371/journal.pone.0068697.g001 APN inhibited H2O2-induced LC3-II and p62 expression and autophagosome formation Autophagosome formation is indicative of autophagic activity. Microtubule-associated protein light chain 3 is involved in autophagy and exists in two forms: LC3-I is the free cytosolic form, while LC3-II is conjugated to phosphotidylethanolamine and is incorporated in the autophagosome membrane. LC3-II and LC3-I protein expression were measured by Western blot and the ratio of LC3-II to LC3-I protein expression was used as a measurement of autophagosome formation and as an indirect indication of autophagy. In ARVM, 1mM H2O2 increased the LC3-II/LC3-I ratio by a factor of 3.41.0. Pretreatment with APN abrogated this increased LC3-II/LC3-I ratio. To corroborate these findings, green fluorescent protein -labeled LC3 expressing ARVMs were treated with 1mM H2O2 in the presence or absence of APN and analyzed under 60x 22634634 oil magnification. H2O2 increased the number of GFP-LC3 puncta per cell by a factor of 2.70.2, and pretreatment with APN attenuated this increase. Although increased LC3-II/LC3I ratio suggests autophagosome accumulation, increased p62 expression suggests defects in the lysosomal end of the pathway, we thus measured p62 expression in H2O2 stimulated ARVM. H2O2 increased p62 expression and pretreatment with APN attenuated this inc