Fig. 5

PMT-stimulated mTOR activation is required for the full functional activity of the osteoclasts. (a) Lysates of cells stimulated for 4 days with PMT concentrations ranging from 10 pM to 5 nM were analyzed for activity of cathepsin K (cleavage of its specific substrate) by fluorescence detection. (b) RAW264.7 cells stimulated with PMT (5 nM) w or w/o rapamycin (10 ng/ml) were analyzed for cathepsin K activity by fluorescence detection. As controls, cells were treated with rapamycin, the solvent control DMSO or were left untreated. (c) Cells were plated and stimulated on fluoresceinated calcium phosphate plates for 6 days to allow osteoclast differentiation. A fluorescent dye released into the supernatant dependent on the degree of bone resorption was measured with a fluorometer. (d ) Osteoclasts were differentiated with RANKL and PMT, respectively, on bone slices and the pit area of resorbed bone was determined. (e) Bone resorption induced by RANKL, PMT or PMT in the presence of rapamycin was quantified by determining the pit area. (f) Cellular apoptosis was determined by performing a caspase-Glo 3/7 Assay. Cells were incubated for 48 h with PMT (5 nM), the solvent control DMSO, rapamycin (10 ng/ml) or left untreated. As a positive control for apoptosis, cells were incubated with 1 μM doxorubicin for 6 h. Cells were lysed and the luminescence signal was recorded. The indicated standard deviations of all figures were calculated from three independent experiments (mean ± SD; n = 3). Statistical analysis for all graphs was performed by unpaired Student’s t-test (*: p ≤ 0.05, **: p ≤0.005, ***: p ≤ 0.0005****: p ≤ 0.0001)