Suppressive effects of NADPH oxidase inhibitor DPI on ROS production, MAP kinase activation, and osteoclastogenesis in BMM cells. (A) BMM cells were pretreated with increasing doses of DPI as indicated for 60 minutes. Cells were then treated with 50 ng/mL RANKL and further incubated for 10 minutes, and ROS levels were determined as in Figure 1A. (B) As in Figure 3A, except that DPI was used instead of H₂O₂. *P < .05; **P < .005. (C) Suppressive effects of DPI on osteoclastogenesis in BMM cells. (Left) BMM cells were cultured with RANKL in the absence or presence of DPI for 5 days and stained for TRAP. (Right) TRAP-positive MNCs were counted. Formation of TRAP-positive MNCs was inhibited in a dose-dependent manner (original magnification, × 100). Data represent means ± SDs of 3 independent experiments, each performed in duplicate (A-C).