Fig. 3.

Effect of exogenous IL-32γ on osteoblast differentiation. a WT calvarial osteoblast (OB) precursor cells were cultured for 4 weeks with osteogenic medium in the absence (None) or presence of IL-32γ (100 ng/ml) to induce OB differentiation. The cells were fixed once a week and were used for measurement of alkaline phosphatase (ALP) or for staining with alizarin red (AR) or Von Kossa (VK). b WT calvarial cells were stimulated with IL-32γ (100 ng/ml) in the presence of osteogenic media (OM) for the indicated time and subjected to reverse transcription PCR analysis of the expression of genes that encode DKK-1, BMP-2, BMPRII, and LRP-5. c The DKK-1 protein level in the culture supernatant from the cells after 1 week of OB differentiation in the absence (None) or presence of IL-32γ was determined by ELISA. d The relative expressions of DKK-1 mRNA were evaluated in human OBs treated with IL-32γ (50 and 100 ng/ml). e WT calvarial cells were stimulated with IL-32γ (100 ng/ml) and Wnt3a (20 ng/ml) in osteogenic media for the indicated times and whole cell lysates obtained from cultured cells were analyzed by western blotting with antibodies specific for active β-catenin, total β-catenin or β-actin (loading control). The relative protein levels were quantified by densitometry. Data are expressed as the mean ± SD of triplicate experiments; ***p < 0.001, *p < 0.05, versus 0 h treated with IL-32γ; ^# p < 0.05, versus 0 h treated with Wnt3a. BMP bone morphogenetic protein, BMPRII bone morphogenetic protein receptor II, DKK-1 Dickkopf-1, IL interleukin, LRP-5 low-density lipoprotein receptor-related protein 5, W week