Figure 8.

NCG stimulates osteoblast differentiation via ER signalling. (A) MOBs (2000 cells) in 96-well plates were treated with vehicle, E2 (10 nM) or NCG (100 nM) in the presence or absence of ICI 182780 (1 nM) for 48 h. ALP activity was determined as described in Figure 1A. RNA was isolated after 48 h of treatment in the presence or absence of ICI182780 and qPCR was performed for BMP2 (B), RUNX2 (C), OPG (D) and RANKL (E). (F) OPG-to-RANKL ratio based on their mRNA levels. (G) Protein blots of ERα and ERβ. Osteoblasts were treated with or without E2 (10 nM) or NCG (100 nM) for 24 h. Proteins extracted from cell lysates were transblotted onto a membrane and probed with anti-ERα and anti-ERβ primary antibodies followed by the corresponding secondary antibodies. (H and I) Relative intensity of chemiluminescence was measured and individual ER to β-actin ratio was calculated. (J and K) For measuring ERE dependent activity, Huh7 cells were co-transfected with 50 ng ERα or ERβ plasmid, 200 ng ERE-containing luciferase reporter plasmid, and 50 ng pEGFP internal reporter plasmid using the Lipofectamine LTS reagent according to the manufacturer's instructions. Transfected cells were treated with vehicle, E2 (10 nM) or NCG (100 nM) for 24 h. The ERE firefly luciferase activities were normalized for pEGFP values. Results were obtained from three independent experiments in triplicate and data were expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 versus control.