Figure 4.

Effects of H₂O₂ boluses on subcellular redox state, AMPK activity, and ATP/ADP ratios in C2C12 myotubes. A–H, C2C12 myotubes were treated with serially diluted boluses of H₂O₂ (7.5, 75, 375, or 750 μm) in serum-free media for 10 or 30 min. A, Prx 2 dimerization and Prx-SO_2/3 formation were assessed by Western blotting. B–D, Western blots and quantification of AMPKα-phospho-Thr-172 and ACC-phospho-Ser-79 levels in H₂O₂-treated cells. Graphed values are mean ± S.D. of 3 biological replicates. Results at each time point were assessed by one-way ANOVA with Dunnett's multiple comparison post-test comparing all H₂O₂ treatments to untreated controls; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. E, AMPK-specific activity was measured as previously described. Graphed data are mean ± S.D. of 3 or mean ± range of 2 biological replicates, expressed as % untreated control. Statistical analysis was performed as described above. F, ATP/ADP ratios were measured as previously described. Graphed data are mean ± S.D. of 4 biological replicates, expressed as % untreated control. Statistical analysis was performed as described above. G and H, graphed data shows the relationship between Prx-SO_2/3 formation (Fig. 4A) and (E) AMPK-specific activity or (F) ATP/ADP ratios in response to H₂O₂ in cells. Values are mean ± S.E. of ≥3 or mean ± range of 2 biological replicates, expressed as % untreated control.