Fig. 2.

DKK1 expressions were regulated by the interactions between miRNAs and 3 ′UTR sequences in a stage- and cell-specific way. (A) C3H10T1/2 cells were treated with 50 μg/mL of ascorbic acid (AA) and were transfected with DKK1 3′ UTR construct (DKK1) or pMIR-REPORT (Control) at different time points. pMIR-β-gal was cotransfected to determine the transfection efficiency. Forty-eight hours after transfection, luciferase and β-gal levels were determined, and luciferase activity was normalized to β-galactosidase activity. Three independent experiments were performed in triplicates, and data are represented as mean ± SEM. *p < .05 versuscontrol group. (B) C3H10T1/2, MC3T3-E1, MLO-A5, MLO-Y4, and NIH3T3 cells were transfected with pMIR-REPORT (Control) or pMIR-REPORT-DKK1 3′ UTR (DKK1). Lucifearse and β-gal levels were determined as stated in panel A. Three independent experiments were performed in triplicate, and data are represented as mean ± SEM. *p < .01 versus control group. (C) Primary calvarial osteoblasts were treated with 50 μg/mL of ascorbic acid (AA) for 14 days and were transiently transfected with DKK1 3′ UTR construct (DKK1) or pMIR-REPORT (Control) at different time points. Lucifearse and β-gal levels were determined as stated in panel A. Three independent experiments were performed in triplicates, and data are represented as mean ± SEM. *p < .05 versuscontrol group.