Figure 3.

Runt-related transcription factor 2 (RUNX2) knockdown augments sulforaphane (SFN)-mediated inhibition of osteoclast differentiation. A, Representative microscopic images (×20 objective magnification) of osteoclasts from culture of bone marrow monocytes (BMM) with conditioned media from T47D-shRUNX2^Dox cells treated with dimethyl sulfoxide (DMSO) or SFN in the absence or presence of doxycycline (Dox). B, Western blot showing the expression of RUNX2 protein in T47D-shRUNX2^Dox cells treated with DMSO or SFN in the absence or presence of Dox. The values below the blot indicate the relative expression of RUNX2 compared to control. C, Quantitation of number of osteoclasts shown in panel A. Results shown are mean ± SD (n=6). Statistical significance of difference was analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. Quantitation of secreted levels of receptor activator of nuclear factor-κB ligand (RANKL, D), Cathepsin K (E), and tumor necrosis factor-α (TNFα, F) in cell culture supernatant of MDA-MB-231 cells after 24- and 48-hour of treatment with different doses of SFN. Results shown are mean ± SD (n=3). Statistical significance of difference was analyzed by ANOVA with Dunnett’s adjustment. Consistent results were obtained from repeated experiments. G, Nuclear factor-κB (NF-κB)-dependent luciferase activity in MDA-MB-231 cells after DMSO or SFN treatment for 24 hours. Results shown are mean ± SD (n=3). Statistical significance of difference was analyzed by ANOVA with Dunnett’s adjustment. Consistent results were obtained from repeated experiments. H, Luciferase activity of NF-κB in T47D-shRUNX2^Dox cells after treatment with DMSO or SFN in the absence or presence of Dox for 24 hours. Results shown are mean ± SD (n=3). Statistical significance of difference analyzed by ANOVA with Bonferroni’s multiple comparison test. Consistent results were obtained from repeated experiments.