ZAS3 is essential for RANKL-mediated osteoclastogenesis of bone marrow precursors.

<p>(A) Western blot analysis of total protein lysates prepared from bone marrow macrophages (BMM) cultured without (−) or with (+) sRANKL (50 ng/ml) for 3 days. Bone marrow aspirates isolated from WT mice were cultured in complete medium with M-CSF (50 ng/ml) for 3 days, and non-adherent cells were harvested and further cultured with M-CSF (50 ng/ml) and sRANKL (50 ng/ml) for 3 days. (B) Immunohistochemical analysis of ZAS3 (red) in bone marrow macrophages (BMM) cultured with M-CSF and sRANKL (50 ng/ml each) at an early phase of osteoclastogenesis. White arrow indicates a mononucleated cell in which ZAS3 shows prominently nuclear localization. Yellow arrow highlights the membrane proximity of ZAS3 in a cell that contained two nuclei and therefore, had undergone the first cell fusion. Nuclei were stained with DAPI (blue). (C) TRAP staining of BMM cultured in the presence of M-CSF and sRANKL (50 ng/ml each). More TRAP+ (cells stained red) and OLC were observed from <i>ZAS3+/+</i> than <i>ZAS3−/−</i> BMM at day 4 (upper panels) and day 6 (lower panel). At day 6, most <i>ZAS3+/+</i> BMM had differentiated into large multinucleated TRAP positive cells while <i>ZAS3−/−</i> BMM was inefficient to do so and failed to form large spreading TRAP+ OLC. Scale bar, 100 µm. Data were representatives of 1 out of 3 independent experiments. (D) Bar charts showing the number of TRAP+ multinucleated OLC (more than 3 nuclei) formed in response to sRANKL and M-CSF stimulation (initially plated 5000 cells per well of an 8 well slide). Data were expressed as the mean ± SD from 3 independent experiments. (E) Western blot analysis of protein lysates of BMM cultured with M-CSF and sRANKL for 6 days with indicated antibodies. +/+ <i>ZAS3</i> WT mice, −/− <i>ZAS3</i> KO mice, and +/− heterozygous ZAS3 mice.