C2C12 myotubes were incubated with adenovirus encoding GFP or AMPD3+GFP for 24 h. During the final hour, myotubes were treated with either 0.6 mM 2,4 dinitrophenol (DNP) or vehicle (methanol). (A) Representative Western blot images for pAMPKα(Thr172), AMPKα, pACC(Ser79), ACC, phosphorylation of pAMPK targeted residues, PGC-1α, and OXPHOS proteins. (B-D) Densitometry analysis of Western blots demonstrating AMPD3 overexpression does not increase pAMPK(Thr172) expression, pACC(Ser79), or pAMPK targeted residues. (E-F) Densitometry analysis of PGC-1α and OXPHOS proteins expression. (G) Luciferase activity in C2C12 myotubes transfected with PGC-1α-promoter luciferase reporter plasmid. Mean±SD, n=6/condition, two-way ANOVA with Tukey’s multiple comparisons or two-tailed unpaired t-test when comparing fewer than four groups. # p<0.05 main effect of DNP, ** p<0.01 vs GFP.