Induction of MIG mRNA expression by RANKL. (A) Time course of MIG mRNA induction by RANKL. Osteoclast precursors were stimulated with 50 ng/mL M-CSF or 50 ng/mL M-CSF plus 100 ng/mL RANKL for the indicated time. Total RNA was extracted from the treated cells. RNA was reverse transcribed and PCR amplified with MIG or GAPDH primers. (B) RANKL dose-dependent increase in MIG mRNA levels. Osteoclast precursors were treated with the indicated concentration of RANKL in the presence of 50 ng/mL M-CSF for 12 hours. (C) RANKL induction of MIG in the absence of M-CSF. Osteoclast precursors were stimulated with 100 ng/mL RANKL for the indicated time in the absence of M-CSF. (D) Lack of synergy between RANKL and M-CSF in MIG induction. Osteoclast precursors were incubated with 50 ng/mL M-CSF, 100 ng/mL RANKL, or both for 24 hours. (E) Inhibition of RANKL induction of MIG by OPG. Osteoclast precursors were pretreated with or without 500 ng/mL OPG and incubated with 50 ng/mL M-CSF and 100 ng/mL RANKL for 12 hours. (F) Effect of cycloheximide (CHX) on RANKL induction of MIG mRNA. Osteoclast precursor cells were pretreated with or without 1 μg/mL cycloheximide and were further stimulated with 50 ng/mL M-CSF plus 100 ng/mL RANKL for the indicated time. Total RNA was extracted from the treated cells. RNA was reverse transcribed and PCR amplified with MIG or GAPDH primers.