Figure 7.

Relationship between CREB and PCNA expression and cell proliferation. A: Osteoblasts were treated with melatonin (2 mM; indicated by “+”) or vehicle for 24 or 48 h. The expression of proliferating cell nuclear antigen (PCNA) was assessed by Western blotting. Protein levels were normalized to the respective GAPDH band, and data are displayed as the percent optical density relative to the corresponding control (24 or 48 h). The analysis revealed that cells treated with melatonin exhibited weaker PCNA expression. B: Osteoblasts were treated with melatonin (2 mM; indicated by “+”) or vehicle and transfected with either lentiviral CREB shRNA or a control shRNA. The expression levels of p-CREB and PCNA were assessed by Western blotting. Protein levels were analyzed and normalized to the corresponding GAPDH band, and the results are shown as the percent optical density relative to the respective control (untreated) for each protein. Transfection with lentiviral CREB shRNAs decreased the expression of PCNA, and this effect was more pronounced by treatment with melatonin. C: Osteoblast cells were pre-treated with melatonin (2 mM) and transfected with either lentiviral CREB shRNA or a control shRNA. Cell proliferation was assessed with a MTT assay, and the results are displayed as the percent absorbance at 490 nm relative to controls (untreated). All experiments were performed in triplicate and data are shown as the mean ± S.E.M. *P < 0.05 or **P < 0.01, compared to controls.