TACE silencing restores M-CSFR expression in monocytes and OC formation and activation in the presence of GM-CSF and IL-4 in combination. (A) Monocytes seeded at 106/mL in 24-well plates were transfected with TACE or scrambled siRNA and cultured in the presence or absence of GM-CSF and IL-4 in combination. After culturing for 48 hours, cell lysates were harvested. M-CSFR immunoreactivity was analyzed by immunoblotting with antibodies against M-CSFR extracellular domain. β-Actin was used as a loading control. (B) The monocytes transfected with TACE or scrambled siRNA were cultured in quadruplicate in 96-well plates for 2 days. Soluble M-CSFR levels in the culture supernatants were measured by ELISA. Data are expressed as means ± SD; *P < .05. (C) The monocytes transfected with TACE or scrambled siRNA were cultured in the presence of M-CSF and sRANKL. GM-CSF and IL-4 in combination and a soluble M-CSF receptor-Fc fusion protein (10 μg/mL) were added to the indicated wells. After culturing for 21 days, the cells were stained for TRAP.
