Fig 2: Activation of autophagy by rapamycin abolished the effect of acacetin on osteoclast differentiation and bone resorption in vitro. BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (25 ng/mL) in the presence of acacetin (25 µM) and rapamycin (50 nmol/L) for 6 h. Protein expression of LC3 (A) was detected by western blotting analysis. BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (25 ng/mL) in the presence of acacetin (25 µM) and rapamycin (50 nmol/L) for 3 days. Protein expression of c-Fos and NFATc1 (B) was detected by western blotting analysis. BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (25 ng/mL) in the presence of acacetin (25 µM) and rapamycin (50 nmol/L) for 7 days. TRAP-positive multinucleated osteoclasts (=3 nuclei) were counted using TRAP assay (C,D). BMMs were seeded in Osteo Assay Surface plate and stimulated with RANKL (50 ng/mL) and M-CSF (25 ng/mL). After immature osteoclasts were formed, acacetin (25 µM) and rapamycin (50 nmol/L) were added for 3 days. Bone resorption area was quantified (E,F). * p < 0.05 versus the vehicle-treated group; # p < 0.05 versus the RANKL+M-CSF group; ^ p < 0.05 versus the RANKL+M-CSF+acacetin group.

Fig 3: BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (25 ng/mL) in the presence of acacetin (25 µM) for 6 h. Western blotting results and responding quantification of PI3K phosphorylation, AKT phosphorylation, mTOR phosphorylation, and LC3II expression were shown. * p < 0.05 versus the vehicle-treated group; # p < 0.05 versus the RANKL+M-CSF group, n = 3.