Figure 6

AAT dose-dependently inhibits osteoclast function. (A) Percentage of pit area resorbed by osteoclast cells derived from bMM cells. Osteoclast cells were generated on 96-well osteo assay surface plate using M-CSF (d 0–7) and RANKL (d 4–7). Different concentrations of AAT as indicated were added from d 0–7 according to Exp-1 (which is diagrammatically mentioned in Figure 1A). b Percentage of pit area resorbed by osteoclast cells derived from RAW 264.7 cells. Cells were treated with RANKL (d 0–6) on 96-well osteo assay surface plate. Different concentration of AAT as indicated were added from d 0–6. (C) Percentage of pit area resorbed by osteoclast cells derived from bMM cells. Here, bMM cells were first treated with M-CSF (d 0–6) and RANKL (d 4–6) on tissue culture plate without AAT. At d 6, generated osteoclast cells were harvested, plated on 96-well osteo assay surface plate and treated with M-CSF, RANKL and different concentrations of AAT for an additional 3 d. (D) Percentage of pit area resorbed by osteoclast cells from RAW 264.7 cells. Cells were treated with RANKL (d 0–6) on tissue culture plate without AAT. Generated osteoclasts were harvested at d 6, plated on 96-well osteo assay surface plate and treated with RANKL and different concentrations of AAT for an additional 3 d. (E–H) Representative images of pits documented in (C): (E) RANKL + M-CSF (considered as positive control); (F) RANKL + M-CSF + AAT (0.5 mg/mL); (G) RANKL + M-CSF + AAT (1 mg/mL); (H) RANKL + M-CSF + AAT (2 mg/mL). Values are means ± SEM of at least triplicate samples. Data were analyzed using one-way analysis of variance followed by Dunnett’s multiple comparison test. *P <0.05, **P <0.001, ***P <0.0001. Scale bar = 500 µm; yellow arrow indicates resorption pit.