Inhibition of G-CSF-induced intracellular ROS production. Ba/F3GR cells were pretreated with the Src kinase inhibitor PP1, PI3-kinase inhibitor LY294002, Akt inhibitor A838450, or ERK1/2 inhibitor MEKI for 1 hour and then stimulated with 100 ng/mL G-CSF for 30 minutes (green) or not (purple). The increased levels of intracellular ROSs were determined by flow cytometry. (A) Inhibition of ROS production by kinase inhibitors. Results from a representative experiment. (B) Effects of kinase inhibitors on ROS production due to G-CSF (mean ± SD). (C) Effect of siRNA knock-down of Akt on ROS production. Ba/F3GR cells were treated for 24 hours with 200 nM siRNA to murine Akt1, scrambled sequence, or mock nucleofection. Cell lysates were analyzed for Akt and actin protein expression. Cells were also stimulated with 100 ng/mL G-CSF for 60 minutes or not, stained with DHR, and then analyzed for ROS production by flow cytometry. Data represented the mean ± SD. (Bottom) Western blot of nucleofected Ba/F3GR cells. Lane 1, untransfected cells; lane 2, mock nucleofection of cells; lane 3, nucleofection with Akt1 siRNA; lane 4, scrambled siRNA. (D) Neutrophils from bone marrow of wild-type and Lyn^-/- mice. The results showed that more than 95% of cells are neutrophils. Images obtained as in Figure 1C. (E) G-CSF-induced ROS production was impaired in neutrophils from Lyn^-/- mice. Neutrophils from bone marrow of wild-type and Lyn^-/- mice were loaded with 5 μM DHR with (green) or without (purple) 100 ng/mL G-CSF for 60 minutes. One representative is shown in the left panel; quantitative results (mean ± SD) were shown in the right panel.