FIGURE 8.

MN1 knock-out osteoblasts have reduced ability to support 1,25(OH)₂D₃-stimulated osteoclastogenesis. A, osteoclastogenic co-culture analysis was performed as described under “Experimental Procedures” using WT and MN1 KO osteoblasts. Co-cultures were stained for TRAP, an osteoclast-selective marker. Representative images of TRAP-stained co-cultures are shown. Color images are shown in supplemental Fig. 1. B, TRAP-positive multinucleated cells (≥3 nuclei) were counted and compared. Data represent the mean ± S.D. (n = 3 in each group). The inset shows a high magnification image of TRAP positive, multinucleated osteoclasts. The scale bar represents 60 μm. C, MN1 WT and KO osteoblasts were differentiated for 15 days and treated with ethanol (−) or 10 nm 1,25(OH)₂D₃ (+) for 24 h. Northern blot analysis was performed to examine mRNA expression levels of OPG, RANKL, macrophage colony-stimulating factor (M-CSF), or 18 S RNA.