Figure 7

Echinalkamide inhibits the bone resorbing activity of mature osteoclasts. BMMs were stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of echinalkamide on mineralized matrix Osteo Assay Surface 96-well plates for 7 days. After that, (A) the cells were removed and photographed under a light microscope at the indicated magnification (50×). (B) The resorption areas (%) were quantified using the Image J program. (C, D) The mRNA expression levels of MMP-9 and cathepsin K were analyzed by quantitative real-time PCR. GAPDH was used as the internal control. (E) The protein levels of MMP-9 and cathepsin K were analyzed by western blot analysis. Actin was used as the internal control. Full blots are provided in Supplementary Fig. S02. All the data were confirmed by technical replicated (n = 3). The results are presented as the mean ± SD. Values with different letters (a, b, c, d, e) are significantly different one from another (one-way ANOVA followed by Newman–Keuls multiple range test, p < 0.05).