Sealing zone dynamics and osteoclast activity in digestion.

<p><b>A</b> Osteoclasts were treated with siRNAs targeting the indicated genes and then plated onto osteological discs. After 48 hours, the digested areas of osteological discs (seen in white whereas the surface appears in grey) were visualized by microscopy. <b>B</b> Bone marrow osteoclast precursors isolated from long bones of Cre-ERT2+/+,PSTPIP1+/+ and Cre-ERT2-/-,PSTPIP1+/+ mice were treated with M-CSF and RANKL and the resulting osteoclasts were further treated with tamoxifen as indicated in Materials and Methods. Sealing zones of osteoclasts plated on osteological discs were stained with phalloidin and observed by confocal microscopy. <b>C</b> PSTPIP1 and PSTPIP2 expression was determined by western blotting. <b>D</b> The activity of these osteoclasts in resorption was determined as indicated above and as described in Materials and Methods. Scale bars 50 μm. The figures presented are representative of at least 3 independent experiments. <b>E</b> Quantification of resorption pit assays were perfomed as indicated in materials and methods. Quantifications of areas from 3 different experiments are plotted in chart (mean ± SD * represents p<0.05 and ** p<0.01, significance was calculated using <i>t</i>-test). Knockdown efficiencies (>90%) determined by western blotting were as presented in previous figures. <b>F</b> The change in relative sealing zone diameter per minute was plotted for each condition; controls of individual experiments were taken together. Statistical significance was tested using students t-test for each KD. (Mean ± SD, **** p< 0.0001).