Inhibition of RANKL responses by Nox1 siRNAs in BMM cells. (A) RT-PCR analysis of the expression of Nox isoforms in BMM cells. Expected product sizes of Nox 1 to 4 are 238 base pair (bp), 159 bp, 209 bp, and 220 bp, respectively. Expression of Nox1 and Nox2 mRNAs was indicated with arrows. n.s or Nox1-specific siRNAs were synthesized and transfected into BMM cells (B-E). (B) Specific reduction of Nox1 mRNA was shown by RT-PCR. (C) BMM cells transfected with nonspecific (n.s) or Nox1 siRNAs were treated with RANKL or interleukin 1β (IL-1β) for 10 minutes, and ROS levels were determined as in Figure 1A. (D) On day 5, cells were fixed and stained for TRAP. Formation of TRAP-positive MNCs was severely suppressed by Nox1 siRNAs (original magnification, × 100). (E) As in panel C, except that whole-cell extracts were detected with phosphopeptide-specific Abs as in Figure 3A. *P < .05; **P < .005. Data represent means ± SDs of 3 independent experiments, each performed in duplicate (C-E).