Skip to main content
. Author manuscript; available in PMC: 2018 Feb 14.
Published in final edited form as: J Cell Physiol. 2015 Sep 18;231(5):1001–1014. doi: 10.1002/jcp.25188

Fig. 1. Runx2 promoter activity controls Runx2 mRNA accumulation prior to mitosis in MC3T3 osteoblasts.

Fig. 1

Runx2 protein and mRNA levels as well as Runx2 gene promoter activity were assessed during progression through the cell cycle in MC3T3-E1 osteoblasts to determine specific transition stages when Runx2 mRNA levels are modulated. Cells were synchronized by incubation for 24 h with mimosine to generate a late G1 phase block. (A) Cells were then released from late G1 phase arrest and stimulated to progress through the cell cycle by the addition of fresh culture medium without mimosine and harvested after 0, 6, 12, 18, 24, 30 and 36 h. Progression through successive cell cycle phases (G1 late, S, G2/M and G1 early) was monitored by flow cytometry. (B) Graphic representation of cell cycle stage data presented in panel A. (C) Expression of cell cycle markers was evaluated by western blot analysis (cyclins D, A and Cdk4) and RT-PCR (cyclins D, E, A and B). (D) Cell cycle-dependent modulations in Runx2 protein and mRNA levels were assessed by western blot and RT-PCR. (E) Graphic representation of cell cycle-related changes in Runx2 protein (between: 0 h & 12 h, P = 0.017; 0 h & 24 h, P = 0.018) and mRNA levels (between: 0 h & 6 h, P = 0.049; 18 h & 24 h, P = 0.001; 24 h & 36 h, P = 0.014). Datapoints represent the averages and standard deviation of multiple experiments. Protein and mRNA values were normalized to actin and GAPDH, respectively. (F) Relative promoter activity of Runx2 promoter/luciferase reporter gene construct (mouse 0.6 kb/LUC) is shown. Luciferase values were normalized to SV40/Ranilla construct activity. Values are means of three single experiments. Cell cycle phases as determined by flow cytometry are indicated at the bottom of the gels and graphs.