N-acetyl-L-cysteine on the ROS production and cell proliferation. (A) Effect of NAC on the activation of Akt and ERK1/2. Ba/F3GR and Ba/F3GRprox cells were pretreated without or with 1 mM or 10 mM NAC, respectively, for 1 hour, followed by stimulation with or without100 ng/mL G-CSF for 10 minutes. Cell lysates were prepared and blotted with anti-phosphor-Akt S473 or anti-phospho-ERK1/2 antibody and reprobed with Akt or ERK1 antibody. (B) G-CSF-induced ROS production was diminished by the administration of NAC. Ba/F3GR and Ba/F3GRprox cells were pretreated with or without1 mM or 10 mM NAC, respectively, for 1 hour, then loaded with 5 μM DHR123 and stimulated with or without 100 ng/mL G-CSF for 30 minutes. The relative ROS level induced by G-CSF was determined by monitoring the increased fluorescence in the cells (mean ± SD). Blue bars indicate BaF3GR; red bars, BaF3GRprox. (C) NAC inhibited the proliferation of Ba/F3GR and GRprox. Ba/F3GR and Ba/F3GRprox cells growing in IL-3 were washed extensively with PBS, 105/mL cells for each sample was plated in G-CSF containing medium, the cells were untreated or treated with 1 mM or 10 mM NAC, and the cells were counted at 0, 24, 48, and 72 hours by trypan blue exclusive assay. The results represent 3 independent experiments. (D) Effect of NAC on cell-cycle progression. Ba/F3GR and Ba/F3GRprox cells were treated with 10 mM NAC for 48 hours in the presence of 100 ng/mL G-CSF. The cells were subjected to cell-cycle analysis by flow cytometry (Modfit LT). (E) Effect of H2O2 on cell growth. Trypan blue counting of Ba/F3GR cells grown in IL-3 containing medium wit or without varying doses of H2O2 was performed daily.
