Fig. 5. Involvement of p38/PI3K/Akt signaling cascade in visfatin-stimulated NOX activity and involvement of NOX in visfatin-stimulated ROS production, ICAM-1 and VCAM-1 expression, and monocyte adhesion in endothelial cells. (A) Cells were pre-incubated for 1 h in the absence or presence of the PI3K inhibitor LY294002 (LY, 30 μM), Akt inhibitor TCN (5 μM), p38 MAPK inhibitor SB203580 (SB, 20 μM), or NADPH oxidase inhibitor DPI (100 nM). 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 24 h. (A)The NADPH oxidase activity, (B) the protein expression of ICAM-1 and VCAM-1, and (C) the protein expression of NOX4 were measured. (D) Cells were transfected with iNOS shRNA or empty vector via lentiviral infection for 24 h, and incubated for a further 24 h in the absence or presence of visfatin (100 ng/ml). The expression of NOX4 and ICAM-1 was measured. 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.