Figure 6.

Interferon (IFN)-γ impairs osteoclastogenesis. Human CD14⁺ monocytes were cultured in the presence of Rh IFN-γ or endogenous IFN-γ from conditioned medium (CM) IFN-γ in equivalent concentration (0.5 ng/mL). Neutralizing anti–IFN-γ Ab was added when indicated (10 μg/mL). A: Tartrate-resistant acid phosphatase (TRAP) staining. B: Morphologic changes of osteoclasts using Rhodamine Phalloidin staining, nuclei were counterstained with DAPI. C: Resorption pits (white) on black-and-white binary images (images were converted by ImageJ) in each treatment group. Representative areas of the cultures are shown for each condition. D: Numbers of osteoclasts with more than three nuclei per cell were counted and plotted in each group. E: Quantification of resorption pit areas (white), assessed as a percentage of the whole area. F–K: Relative gene expression data for TRAP (F), Cathepsin K (G), RANK (H), TRAF6 (I), CD80 (J), and CD86 (K). X-fold changes were calculated relative to M+R-treated wells. n = 6 human monocyte donors (A–K). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Scale bars: 200 μm (A–C). Ab, antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, macrophage colony-stimulating factor at 25 ng/mL; R, receptor activator of nuclear factor-κB ligand at 30 ng/mL; Rh, recombinant human; TRAF, tumor necrosis factor receptor-associated factor.