Knockdown of Tusc2 in bone marrow-derived macrophage-like cells (BMMs) attenuates RANKL-induced osteoclast differentiation. (A) BMMs were transfected with control or Tusc2 siRNAs. The mRNA levels of Tusc2 were assessed by quantitative real-time PCR. Data represent the mean ± SD of triplicate samples. **P < 0.01 versus the control. (B) Transfected BMMs were cultured in the presence of M-CSF and various concentrations of RANKL for 3 days. Cultured cells were stained for TRAP (left panel). The number of TRAP-positive multinucleated cells (MNCs) per well were counted (right panel). **P < 0.01 versus the control. (C, D) Transfected BMMs were cultured in the presence of M-CSF and RANKL for the indicated times. (C) The mRNA levels of c-fos, NFATc1, and TRAP were assessed by quantitative real-time PCR. Data represent the mean ± standard deviation (SD) of triplicate samples. *P < 0.05 and **P < 0.01 versus the control. (D) Cell lysates were harvested from cultured cells and immunoblotted with the indicated antibodies.