Dual Actions of ROS on Insulin Signaling Figure 9. Effects of antioxidant N-acetyl-L-cysteine on H2O2-induced alterations of insulin signal transduction in H4IIEC hepatocytes. (A–B) H4IIEC cells were serum-starved and treated with 10 mM of N-acetyl-L-cysteine overnight. Then the cells were treated with the indicated concentrations of H2O2 for 3 h, and stimulated with insulin (1 ng/mL) for 15 min. Total cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed using the indicated antibodies. (C) Quantitative data from densitometric analysis of p-Akt signals from three independent experiments were normalized to the values for total Akt and are expressed as mean fold increases over control 6 S.E.M. . * p,0.05, versus treatment with insulin alone; ** p,0.01, versus treatment with insulin alone. doi:10.1371/journal.pone.0027401.g009 PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27401