Effect of MIG on the migration of TRAP-positive osteoclasts. (A) Osteoclast precursors were cultured in the presence 50 ng/mL M-CSF plus 100 ng/mL RANKL for 60 to 72 hours. Cells were washed with PBS, suspended in serum-free α-MEM, and loaded to the upper well of transwell chambers. The lower well contained serum-free medium with the indicated concentration of MIG. After 6 to 8 hours, cells migrated onto the lower well were fixed and stained with hematoxylin. ^*Significant difference from the medium control (P < .01). (B) Conditioned media were obtained from wild-type and STAT1-deficient osteoclast precursors incubated with RANKL for 24 to 30 hours. Conditioned medium was added to the lower well, and osteoclasts were loaded to the upper well. After 6 to 8 hours, cells that had migrated to the lower well were counted. ^*Significant difference between the indicated groups (P < .01). (C) The lower well of transwell chambers contained conditioned medium from RANKL-treated cells and either a MIG neutralizing antibody or the control immunoglobulin G (IgG). Migrated cells were counted as described. ^*Significant difference between the indicated groups (P < .01). (D) Conditioned media were obtained from osteoclast precursors treated for 24 hours with 100 ng/mL RANKL in the presence or absence of SB203580. Osteoclasts were added to the upper well and were allowed to migrate toward the conditioned medium added to the lower well. Migrated cells were scored after 8-hour incubation. ^*Significant difference between the indicated groups (P < .01).