Figure 6.

sLZIP promotes osteogenesis via enhancement of Runx2 transcriptional activity. (a) 293T cells were transfected with six copies of the Runx2 response element (OSE-Luc), β-galactosidase, Runx2, and various amounts of sLZIP (0.1, 0.25, and 0.5 μg). (b) 293T cells were transfected with OSE-Luc, β-galactosidase, Runx2, and various amounts of si-sLZIP (10, 50, and 100 nM). (c) 293T cells were transfected with OSE-Luc, β-galactosidase, PPARγ₂, Runx2, and sLZIP. (d) 293T cells were transfected with OSE-Luc, β-galactosidase, Runx2, HDAC3, and various amounts of sLZIP (0.25 and 0.5 μg). Promoter activity was determined by a luciferase assay. Luciferase activities were normalized to the β-galactosidase activity and presented as the relative activity. *P<0.01; **P<0.005. (e) Primary human MSCs and C3H10T1/2 cells were infected with sLZIP-expressing adenovirus or sLZIP shRNA expressing lentivirus, and differentiated into osteoblasts for 8 days. Scale bar, 50 μm. (f) MEF cells isolated from WT and sLZIP TG mice were differentiated into osteoblasts for 14 days. Cells were stained with Alizarin Red solution, and the number of nodules was counted. The number of bone nodules was counted in four randomly selected fields for each well, and data represent the mean of three independent experiments±S.E.M. *P<0.01. Scale bar, 50 μm. (g) C3H10T1/2 cells were infected with sLZIP-expressing adenovirus and differentiated into osteoblasts for 8 days. Total RNA was extracted and the mRNA expression level of osteoblast marker genes (APL, OC, and BSP) were determined using real-time PCR. GAPDH was used as an internal control. *P<0.001, **P<0.005. All experiments were performed in triplicate and the bar represents the mean±S.D.