Overexpression of Tusc2 in bone marrow-derived macrophage-like cells (BMMs) enhances RANKL-induced osteoclast differentiation. (A) BMMs were cultured with M-CSF and RANKL for the indicated times. The mRNA levels of Tusc2, NFATc1, and TRAP were assessed by quantitative real-time PCR. **P < 0.01, and ***P < 0.001 versus the control. (B) BMMs were transduced with pMX-IRES-EGFP (control) or Tusc2 retrovirus, and cultured in the presence of M-CSF and various concentrations of RANKL for 3 days. Cultured cells were stained for TRAP (left panel). The TRAP-positive multinucleated cells (MNCs) per well were counted (right panel). **P < 0.01, and ***P < 0.001 versus the control. (C, D) BMMs were transduced with pMX-IRES-EGFP (control) or Tusc2 retrovirus and cultured in the presence of M-CSF and RANKL for the indicated times. (C) The mRNA levels of c-Fos, NFATc1, TRAP, and OSCAR were assessed by quantitative real-time PCR. Data represent the mean ± standard deviation (SD) of triplicate samples. **P < 0.01 and ***P < 0.001 versus the control. (D) Cell lysates were harvested from cultured cells and immunoblotted with the indicated antibodies. (E) Transfected BMMs were cultured with M-CSF alone or M-CSF and RANKL on inorganic crystalline calcium phosphate plates. Resorption lacunae were visualized by bright-field microscopy. **P < 0.01 versus the control.