Fig. 1.

REV-ERBα negatively regulates Bmal1 expression in MC3T3-E1 cells. (A) A representation of the expression of Nr1d1 in femurs (cancellous and cortical bone) from C57BL/6J mice under light/dark cycle conditions. Bone was obtained from C57BL/6J mice every 4 h. Total RNA was isolated, and the level of mRNA was determined by real time qRT-PCR using specific primers for Nr1d1. Relative mRNA levels were normalized to Gapdh levels. Data represent the mean±s.e.m, n=6-8 animals. A representative result of three individual experiments is shown. White boxes, light period; black boxes, dark period. (B) REV-ERBα negatively regulates Bmal1 expression in MC3T3-E1 cells. Bmal1 mRNA was down-regulated by GSK4112 in a concentration-dependent manner in MC3T3-E1 cells. Cells were treated with GSK4112 at 3 to 30 µM for 12 h, harvested and processed for real time qRT-PCR. Each value represents the mean±s.e.m. of five separate experiments. *P<0.05, significantly different from each control value obtained in MC3T3-E1 cells cultured in the presence of DMSO (vehicle control). (C) Cells were incubated for 12 h in the presence of GSK4112 with SR8278 at a concentration of 10 µM, followed by the determination of Bmal1 levels by real time qRT-PCR. Each value represents the mean±s.e.m. of three or four separate experiments. *P<0.05, significantly different from each control value. NS, not significant.