Figure 5
Effect of Ccn2-deficient osteocytes on osteoclastogenesis of GST-RANKL-treated RAW264.7 cells. (A) Illustration of co-culture system of collagen gel-embedded WT and KO osteocyte-enriched population of cells and RANKL-treated RAW264.7 cells. (B) WT and KO mice at 1 month of age were injected with tamoxifen, and after 1 month, femurs were collected. Then, osteocyte-enriched cell population was isolated from WT and KO mice and cultured for 2 days. Thereafter, WT and KO osteocyte-enriched populations were embedded into collagen gel at a density of 5 × 104/well in 24-well plates. After having been cultivated for 2 days, RAW264.7 cells were inoculated onto the gel and treated with GST-RANKL for 2 days. Then, cell lysates were prepared; and Western blot analysis was performed by using anti-NFATc1, CTSK and β-actin antibodies. (C) The amounts of NFATc1 and CTSK were determined densitometrically and were normalized to the amount of β-actin. Graph shows the relative density of NFATc1 (upper) and CTSK (lower) in WT (n = 3) or KO cells (n = 3). In these graphs, the ordinate indicates the relative ratio with respect to the sample from WT cells (ratio = 1.0), and bars represent mean and standard deviation. The data were analyzed by Student t-test, and p < 0.05 (*) was considered significant.
