Estrogenic activities of kaempferol in cultured osteoblasts. A: The chemical structure of kaempferol. B: Three repeats of ERE were tagged with a luciferase reporter gene to form pERE-Luc (upper panel). The DNA construct pERE-Luc was stably transfected into cultured osteoblasts, which were then treated with kaempferol at various concentrations or 17β-estradiol (1 nM) with or without an hour of pre-treatment with ICI 182,780 (100 nM). After 48 hours of treatment, luciferase activity was determined. As compared to DMSO control, statistically significant results include the effects of 17β-estradiol (P = 0.041), 10 μM (P = 0.0425) and 50 μM (P = 0.038). C: Cultured osteoblasts were treated with kaempferol (10 μM) and 17β-estradiol (1 nM) with or without pre-treatment with ICI 182,780 (100 nM) for 1 hour. Lysates were subjected to Western blot analysis to determine the phosphorylation of ERα at serine 118 and total ERα. D: The signals were quantified from the blots in (C) by calibrated densitometry. Values in all panels are expressed as the fold increase from basal reading (control culture; 0.02% DMSO) and are in mean ± SD, n = 5, each with triplicate samples.