FIGURE 2.

PI3K/Akt and MAPKs are involved in thrombin-mediated IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity in A549 cells. A, cells were transfected with 0.5 μg of pcDNA (mock) or with 0.5 μg of AktDN for 24 h or were pretreated with an equivalent vehicle control (DMSO), 10 μm LY294002, or 1 μm of the Akt inhibitor (Akt inh) for 30 min and then incubated with 10 units/ml thrombin for another 24 h. IL-8/CXCL8 levels were determined. Data are presented as the mean ± S.E. of three experiments performed in duplicate. *, p < 0.05, as compared with thrombin treatment. B, A549 cells were transfected with 0.2 μg of IL-8/CXCL8 WT-Luc and 0.5 μg of pBK-CMV-LacZ or cotransfected with 0.5 μg of pcDNA (mock) or with 0.5 μg of AktDN for 24 h or were pretreated with an equivalent vehicle control (DMSO) or 10 μm LY294002 for 30 min and then incubated with 10 units/ml thrombin for another 24 h. Cells were harvested for the luciferase assay as described in the legend for Fig. 1. Data are presented as the mean ± S.E. of three experiments performed in duplicate. *, p < 0.05, as compared with thrombin treatment. C, cells were pretreated with an equivalent vehicle control (DMSO), 10 μm PD98059, 3 μm SB203580, or 10 μm SP600125 for 30 min and then incubated with an equivant vehicle control (double-distilled H₂O (−)) or 10 units/ml thrombin for another 24 h. IL-8/CXCL8 levels were determined. Data are presented as the mean ± S.E. of three experiments performed in duplicate. *, p < 0.05, as compared with thrombin treatment.