Effect of GILZ on TNF-α regulation of Osx transcription.

<p>(A, B) Transient transfection and luciferase-reporter assays. (A) C3H10T1/2 cells were transfected with Osx-Luc promoter reporter and treated either with TNF-α (10 ng/ml) or an indicated kinase inhibitor alone, or with TNF-α plus an inhibitor for 24 hr and then harvested for luciferase assays. The concentration of TNF-α used in this experiment is based on the study by Lu et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031717#pone.0031717-Lu1" target="_blank">[14]</a> as the promoter reporter construct is same. (B) C3H10T1/2 cells were cotransfected with Osx-Luc reporter and a GILZ expression plasmid and treated with TNF-α and ERK inhibitor PD98059 as indicated. Luciferase activity was measured as in A. these experiments were performed 3 times in triplicate. (C) Real-time qRT-PCR analysis. MSCs were first cultured in OS medium for 5 days (to initiate the osteogenic differentiation program) and then were treated continuously with PD98059 for 17 hr, or treated with PD98059 for 5 hr prior to TNF-α (0.5 or 2 ng/ml, T.5 and T2, respectively) treatment for 12 hr before harvesting for RNA isolation and qRT-PCR analysis. Data were normalized to β-actin and expressed as fold changes relative to the mRNA level of untreated (GM) Cells. Experiments were performed 2 times in duplicates with the same results. Error bars indicate SD. a, <i>p</i><0.01 (n = 2).