Figure 7

LPS induced PI-3K/Akt activation and PI-3K inhibitor (wortmannin) suppressed this in a time- and dose-dependent manner. (A-B) Primary human chondrocytes in monolayer culture were either left untreated (controls), treated with lipopolysaccharides (LPS) (0, 0.1, 1, 10, 100 and 1000 ng/ml) for 50 min or stimulated with 100 ng/ml LPS for the indicated times. Nuclear extracts were prepared and assayed for Akt activation by western blotting as described in Materials and methods. (C-D) Primary human chondrocytes in monolayer cultures were either left untreated or were prestimulated with wortmannin for the indicated concentrations and treated with 100 ng/ml LPS for 50 min, or prestimulated with 20 nM wortmannin for 12 h followed by co-treatment with 100 ng/ml LPS for the indicated times. Nuclear extracts were prepared and assayed for Akt activation by western blot analysis as described in Materials and methods. Housekeeping protein β-actin served as a loading control and remained unaffected. PI-3K, phosphatidylinositol 3-kinase.