FIGURE 8.
Structure/function analysis of osteoclast VCL. A, WT and mutated VCL constructs. B and C, HA-tagged WT and mutated VCL constructs were retrovirally transduced into LysM-VCL splenic macrophages. The cells were maintained in M-CSF and RANKL for 4 days. B, expression of each transductant and osteoclast differentiation marker was determined by immunoblot. Empty vector (vector) transduced into WT and LysM-VCL splenic macrophages served as a control. C, expression of HA-tagged VCL811–1066 was determined by immunoblot with anti-HA antibody. D, left panels, transduced osteoclasts, generated on plastic for 3 days, stained for TRAP activity. Middle panels, transduced splenic macrophages cultured with M-CSF and RANKL on bone slices. After 5 days, the cells were stained with Alexa-Fluor-546-phallodin to visualize the actin cytoskeleton. Right panels, following removal of the transduced osteoclasts, resorption pits were visualized by wheat germ agglutinin-lectin staining. E and F, histomorphometric analysis of actin ring area/field (E) and pit area/total area (F). ****, p < 0.0001; ***, p < 0.001; **, p < 0.01 compared with LysM VCL vector. Scale bars, 50 μm. Error bars, S.D.