Effects of N-acetylcysteine (NAC) on RANKL-induced ROS production, MAP kinase activation, and osteoclastogenesis. (A) BMM cells were treated with exogenous H₂O₂ in the indicated concentrations. Phosphorylated forms of ERK1/2 (P-ERK1/2), JNK1/2 (P-JNK1/2), and p38 MAP kinase (MAPK; P-p38) in whole-cell extracts were detected with phosphopeptide-specific Abs. The membranes were striped and probed with Abs against ERK1/2, JNK1/2, and p38 MAPK as indicated to confirm that similar amounts of whole-cell extracts were analyzed. Protein bands were quantified by densitometry, and levels of phosphorylated MAPK were normalized to levels of MAPK, respectively. The fold increase in the stimulated cells compared with untreated cells is shown. Data represent the means ± SDs of at least 3 independent experiments. *P < .05. (B) RAW264.7 cells were pretreated with increasing doses of NAC as indicated for 60 minutes. Cells were then treated with 50 ng/mL RANKL and further incubated for 10 minutes, and ROS was assayed as in Figure 1A. (C) As in panel A, except that NAC was used instead of H₂O₂. *P < .05; **P < .005. (D) Inhibitory effects of NAC on osteoclastogenesis in BMM cells. BMM cells were incubated with RANKL and M-CSF in the absence or presence of NAC (30 mM). On day 5, cells were fixed and stained for TRAP. TRAP-positive cells appeared as red cells (original magnification, × 100). Formation of TRAP-positive multinucleated cells (MNCs) was inhibited by NAC treatment.