Figure 4

LPS induced the phosphorylation and translocation of p65 and the IKK inhibitor BMS-345541 or 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 30 min (A) or stimulated with 100 ng/ml LPS for the indicated times (B). Nuclear extracts were prepared and assayed for nuclear factor-κB (NF-κB) (p65) activation by western blot analysis as described in Materials and methods. (C-F) Primary human chondrocytes in monolayer cultures were either left alone or pretreated with BMS-345541 or with wortmannin for the indicated concentrations followed by treatment with 100 ng/ml LPS for 30 min (C and E), or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h and co-treated with 100 ng/ml LPS for the indicated times (D and F). Nuclear extracts were prepared and assayed for NF-κB (p65) activation by western blot analysis as described in Materials and methods. Synthesis of poly(ADP-ribose) polymerase (PARP) remained unaffected in nuclear extracts. IKK, IκB kinase; PI-3K, phosphatidylinositol 3-kinase.