Figure 2

LPS induces VCAM-1 expression via NADPH oxidase/ROS. (A) Cells were pretreated with MCI-186 (MCI, 100 μM), APO (100 μM), or DPI (10 μM), and then incubated with LPS for 8 h. The levels of VCAM-1 protein were determined. (B) Cells were pretreated with MCI-186 (MCI), APO, or DPI, and then incubated with LPS for 4 h or 6 h. The mRNA levels and promoter activity of VCAM-1 were determined. (C, D, E) Cells were transfected with p47^phox, Nox2, Nox4, or scrambled siRNA, and then incubated with LPS for 8 h. The protein levels of p47^phox, Nox2, Nox4, and VCAM-1 were determined. (F) Cells were stimulated with LPS for the indicated times. NADPH oxidase and ROS generation were measured. (G) DHE fluorescence image after 30 min of stimulation with LPS. (H) Cells were pretreated with MCI-186 (MCI), APO, or DPI, and then treated with LPS for 30 min. ROS generation was measured. (I) Cells were stimulated with LPS for the indicated times. The membrane and cytosolic fractions were prepared and subjected to Western blot using an anti-p47^phox antibody. Gsα and GAPDH were used as a marker protein for membrane and cytosolic fractions, respectively. All figures are representative of three independent experiments, performed in duplicate. Data are expressed as means ± SEM. *P < 0.05; ^#P < 0.01, as compared with the cells exposed to LPS alone (A, B, H), scrambled siRNA alone (upper panels of C, D, E), LPS + scrambled siRNA (lower panels of C, D, E), or vehicle alone (F, I).