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Figure 6: The DBT-induced osteogenic effects and gene expressions are blocked by specific inhibitors. (a) MG-63 cultures stably transfected with pERE-Luc were pre-treated with buffer (0.1% DMSO; control), ICI 182 780 (an ER blocker; 0.1 μM) and U0126 (Erk inhibitor; 10 μM) for 3 h before the addition of DBT (1 mg mL−1) for 24 h to determine the luciferase activity driven by ERE activation (left panel) and Erk1/2 phosphorylation at 5 min (right panel). (b) Cultures were treated for 48 h as in (a) to determine cell number (by cell counting), cell proliferation (by MTT assay) and alkaline phosphatase (ALP) activity. (c) To investigate the signaling mechanisms of DBT-induced gene expressions, MG-63 cells were pre-treated with inhibitors and then DBT as in (a) for 24 h to measure the change of CCL-2, CCL-7, CCL-8, and galectin-9 mRNA expressions by real-time PCR analysis. Values are expressed in percentage of increase as compared with control cultures (without herbal extract), and in the ratio to basal reading where control (without herbal extract) equals to 1, mean ± SEM, where n = 5, each with triplicate samples. **P < .01, ***P < .001.