Fig 3.

Umb attenuates RANKL-induced osteoclast differentiation and bone resorption in vitro. (A) BMMs were cultured for 4 days in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) with the control (DMSO) or Umb. Cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, and stained with TRAP solution. (B) TRAP-positive MNCs counted as osteoclasts. (C) BMMs were cultured for 3 days at the indicated doses of Umb in the presence of M-CSF (30 ng/mL). Cell viability was determined by XTT assay. (D) Mature osteoclasts were seeded on dentin slices and treated for 48 h with Umb (200 μM). Thereafter, surviving osteoclasts were detected by TRAP staining (upper left) and TRAP-positive MNCs were counted (upper right). Attached cells were removed from the plates and photographed under a light microscope. Pit areas are lined in red (lower left). Pit areas were quantified using ImageJ (lower right). ^**P < 0.01, ^***P < 0.001 versus the control.