Fig. 4. Involvement of p38/PI3K/Akt signaling cascade in visfatin-stimulated ROS production in endothelial cells. Cells were pre-incubated for 1 h in the absence or presence of the ERK1/2 inhibitor PD98059 (PD, 30 μM), PI3K inhibitor LY294002 (LY, 30 μM), p38 MAPK inhibitor SB203580 (SB, 20 μM), or ROS scavenger NAC (5 mM). Cells were then incubated in the absence or presence of visfatin (100 ng/ml) in the continued absence or presence of the inhibitor for a further (A and B) 30 min or (C) 24 h. The phosphorylation of p85 subunit of PI3K and Akt was analyzed by western blot with antibodies against phosphorylated human p85 subunit of PI3K and Akt or total human p85 subunit of PI3K and Akt (A and B) and the production of ROS were detected by fluorescence intensity (C). The results are the mean ± SEM for three separate experiments, each in triplicate. *P<0.05 compared with the untreated control; ^#P<0.05 compared with the visfatin alone group.