Figure 4

DEP regulate IL-1β-induced NF-κB activation in A549 cells. (A) A549 cells were stimulated with 50 pg/ml IL-1β for 1 h and treated with 100 μg/ml DEP for the indicated times. At each time point, the nuclear and cytosolic fractions were prepared and used for the analysis of p65 NF-κB, IκB-α and β-actin levels by Western blotting. (B) A549 cells were pretreated with MG132 (5 μM) or PDTC (100 μM) for 1 h and then stimulated with IL-1β (50 pg/ml) for 1 h followed by DEP treatment (100 μg/ml) for 5 min. The levels of p65 NF-κB, IκB-α and β-actin were analyzed by western blotting as mentioned above. (C) A549 cells were transfected with luciferase plasmid containing hBD-2 promoter for 20 h. The cells were pretreated with MG132 (5 μM) or PDTC (100 μM) for 1 h and then stimulated with IL-1β (50 pg/ml) for 1 h followed by DEP treatment (50 μg/ml) for 24 h. The cells were harvested and assayed for luciferase activity as described in Methods. Values are mean ± SD of four independent experiments. (D) A549 cells were pretreated with MG132 (5 μM) or PDTC (100 μM) for 1 h and then stimulated with IL-1β (50 pg/ml) for 1 h followed by DEP treatment (10 μg/ml) for 20 h. Total RNA was prepared and analyzed by RT-PCR using specific primers for hBD-2 and β-tubulin