FIGURE 7.

Secondary screening for effect on osteoclast differentiation by KM91104. A secondary cellular assay was used to determine the effect of KM91104 on osteoclast differentiation and fusion after 5 days of continuous exposure. TRAP is a marker enzyme for osteoclast differentiation that converts p-nitrophenyl phosphate to p-nitrophenol under acidic conditions. The test cell line was RAW 264.7 cells differentiated in the presence of 100 ng/ml soluble recombinant RANKL. A, total solubilized TRAP assay was used to determine the effect of compound KM91104 on RANKL-mediated differentiation of RAW 264.7 cells (see “Experimental Procedures”). The concentration range of compound tested was 0.3–40 μm, as indicated. C indicates control, vehicle only added. Amount of p-nitrophenol released indicates the degree of differentiation after 5 days of growth with RANKL. Differentiation was not significantly affected up to 20 μm KM91104 concentration (n = 3, in duplicate; error bars are ± S.D.). B shows quantitative analysis of TRAP-stained fixed cultures (see “Experimental Procedures”). Cell counts for TRAP-positive (red) multinucleated cells with ≥2 nuclei after treatment with compound KM91104 are plotted. Controls are normalized to 100% (n = 3, in duplicate; error bars are ± S.D.). Inset panel shows primary culture mouse BMM cells (TRAP-stained, red) differentiated with M-CSF and RANKL and exposed continuously to 1.2 μm KM91104 for 5 days. Ability of primary BMM cells to form large osteoclasts (>20 nuclei) has not been impaired at this concentration.