Effect of Nutlin-3 and L-685,458, used alone or in combination, on osteoclastic differentiation. Adherent PBMCs were cultured in presence of M-CSF + RANKL for 12 days, in the absence or presence of Nutlin-3 and/or L-685,458. After 12 days, cultures were analyzed for osteoclastic differentiation. Representative fluorescence microscopy fields of osteoclastic cultures, treated as indicated, are shown in the left panel. DAPI-stained nuclei of polynucleated osteoclasts are circled. Original magnification: ×400; 40×/0.75 NA objective. In the right panel, osteoclastic differentiation was quantified by scoring the number of TRAP-positive multinucleated cells, containing 3 or more nuclei. Data represent the means ± SD of 4 different experiments.