Figure 1
Gene expression of osteocyte markers and OPG production in MLO-Y4 cells embedded into collagen gel including rCCN2. (A) Illustration of the 3-D culture system of MLO-Y4 cells. (B) Fluorescence staining to visualize MLO-Y4 cells embedded into collagen gel with or without rCCN2. MLO-Y4 cells were embedded into collagen gel containing rCCN2 (300 ng/ml) and cultured for 2 days. Then, fluorescence staining was performed by using fluorescein phalloidin. MLO-Y4 cells with cell projections were detected, but CCN2 in the gel showed no effect. PBS was added to the gel as the vehicle. The bar represents 100 µm. (C) After MLO-Y4 cells had been embedded into collagen gel with rCCN2, they were cultured for 2 days. Total RNA was isolated and real-time RT-PCR analysis was performed by using specific primers. The amounts of osteocyte marker genes were normalized to that amount of Gapdh mRNA. Data show the value from independent samples of n = 6, and the graph presents the mean and standard deviation. The asterisk indicates a significant difference from PBS treatment (p < 0.05). (D) Western blot analysis was performed by using anti-OPG and GAPDH antibodies in MLO-Y4 cells embedded into the gel with or without rCCN2. (E) The amount of OPG was determined densitometrically, and these amounts were normalized to the amounts of GAPDH. Graph shows the relative density of OPG with (n = 3) or without rCCN2 (n = 3). Bars represent mean and standard deviation. The data were analyzed by Student t-test, and p < 0.05 (*) was considered significant.
