FIG. 1.

Overexpression of NFAT2 rescues the defect in RANKL-dependent osteoclast differentiation of PLCγ2^−/− cells but not bone resorption. (A to E) WT and PLCγ2^−/− BMDM were retrovirally infected with pMX (empty vector) (A, C), pMX-NFAT2 (B, D), or pMX-PLCγ2 (E). Cells were grown in the presence of 10 ng/ml M-CSF and 100 ng/ml RANKL for 5 to 7 days, fixed, and TRAP stained. TRAP⁺ osteoclasts were observed using a light microscope with a 10× objective. (F) The number of TRAP⁺ osteoclasts per well was determined and graphed for each sample. (G) WT and PLCγ2^−/− BMDM were retrovirally infected with empty vector (pMX) or NFAT2 and grown in the presence of 10 ng/ml M-CSF and 100 ng/ml RANKL. After 3 days, cells were lysed and Western blotted (WB) for the indicated protein. (H) WT and PLCγ2^−/− BMDM retrovirally infected with empty vector (pMX), NFAT2 (pMX-NFAT2), or PLCγ2 (pMX-PLCγ2) were cultured on dentin for 10 days with RANKL and M-CSF, fixed, and stained with fluorescein isothiocyanate-phalloidin to detect actin rings (top row; 10× objective). Inserts were enlarged fourfold. Cells were removed with 2 N NaOH and resorption pits visualized by staining with peroxidase-conjugated wheat germ agglutinin. Pits were analyzed by light microscopy with a 10× objective. Black lines delineate the resorbed areas (bottom row). (I) Area of bone resorption per field was determined using Image J software and graphed for each sample.