Fig. 3. Independence of HA inhibition of osteoclastogenesis on CD44. (A) HA does not suppress CD44 expression. BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4 days in the presence or absence of HA (1 µg/ml). At each day, cells were collected and surface expression of CD44 was examined by flow cytometry. Red line, -HA; blue line, + HA; black line, secondary (2°) Ab control. MFI, mean fluorescence intensity. (B) CD44 independence of osteoclastogenesis inhibition by HA. BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 6 days. Anti-CD44 Ab (1 µg/ml) and HA (0.5-1 µg/ml) were included for the whole culture period where indicated. Cells were stained for TRAP, and TRAP⁺ MNCs were counted. NS, no significant difference. (C) Effectiveness of the anti-CD44 Ab to neutralize the CD44-OPN-dependent response of BMMs. BMMs were placed on the top chamber of a transwell plate. Anti-CD44 Ab (1 µg/ml) and HA (1 µg/ml) were added to the top chamber and OPN (a CD44 ligand, 2 µg/ml) was added to the bottom chamber. The plate was incubated for 8 hours. The number of migrated cells was determined after hematoxylin staining. ^**P<0.01 compared with OPN-treated control.