Role of the ROS-dependent–PI3K pathway on DNR-induced ERK1 stimulation.

(A) U937 cells were incubated with C2938 fluorescent probe for 1 hour followed by incubation in the absence or presence of 25 mM N-Ac for 2 hours followed by 0.1 μM or 1 μM DNR for 5 minutes. The cells were washed, and cell fluorescence was determined by using flow cytometry as described in “ Materials and methods.” Results are mean ± SEM of 3 independent determinations; *P < .01. (B) U937 cells were preincubated in the absence or in the presence of 25 mM N-Ac for 2 hours or 25 nM wortmannin for 30 minutes followed by 1 μM DNR for 5 minutes. PI3K kinase activity was assayed by thin-layer chromatography (TLC) as described in “ Materials and methods.” (Inset) Dose-effect inhibition of basal PI3K activity by wortmannin after 30 minutes of incubation. Results are representative of 3 independent experiments. ori indicates origin; PI3P, phosphatidylinositol-3-phosphate.