Fig. 2.

The Ric-8B gene is repressed during osteoblast differentiation. (A) MC3T3 cells were cultured for 24 days in the presence (II and IV) or absence (I and III) of 50 μg/ml AA and 3 mM β-glycerophosphate. The cells were then photographed using an optical microscope (I and II) or stained with Alizarin Red to visualize mineralized nodules (III and IV). (B) MC3T3 cells were cultured for up to 24 days in medium supplemented with 50 μg/ml AA from day 3. The cells were collected at the indicated times, and Ric-8B mRNA levels were determined by QPCR, normalizing with respect to GAPDH mRNA expression. DIV, days of differentiation in vitro. Bars represent means ± the standard errors of the means of three independent experiments. Statistically significant differences were determined by an analysis of variance test. NS, not significant; **, P < 0.01; ***, P < 0.001. (C) To control for osteoblast differentiation, OC gene mRNA expression was determined by QPCR.