Caspase-2 regulates osteoclast numbers via apoptosis.

<p>(A) Primary cultures of <i>in vitro</i> differentiated <i>Casp2^+/+</i> and <i>Casp2^−/−</i> osteoclasts grown in 96-well plates were TRAP-stained. (B) TRAP-positive, multinucleated osteoclasts were counted (<i>*</i>, <i>p</i><0.05). (C) TRAP activity of in vitro cultured primary osteoclasts from <i>Casp2^+/+</i> and <i>Casp2^−/−</i> mice was measured and normalized to cell viability as determined by MTT assay (<i>*</i>, <i>p</i><0.0005). (D) Osteoclasts were cultured in the presence of a caspase-2-selective inhibitor, zVDVAD and, (E) Intact TRAP-positive osteoclasts were counted (<i>*</i>, <i>p</i><0.0001). (F) Osteoclasts cultured with or without zVDVAD were stained with TUNEL and counted (<i>*</i>, <i>p</i><0.001). (G) Overexpression of wild type caspase-2 but not its enzymatic mutant (Casp2-m) in the osteoclast precursor cell line, RAW264.7, significantly reduced TRAP activity (<i>*</i>, <i>p</i>< 0.005). Scale bar, 100 μm.