Figure 5

IL-1β induces MCP1 expression via both NF-κB-dependent and -independent pathways. (A) HASMCs were pretreated with the indicated concentrations of the NF-κB inhibitor, PDTC, for 30 min prior to IL-1β (5 ng/ml) treatment for 10 h. Total RNA was isolated, and RT-PCR analysis was performed using MCP1 gene-specific primers and the internal control gene, β-actin. Two additional experiments yielded similar results. A representative study is shown. (B) The amount of secreted MCP1 protein was determined in the supernatant after IL-1β treatment for 10 h using the human MCP1 ELISA kit. Data are presented as mean values obtained from three independent experiments, and bars represent standard deviations. The significance was determined by Student's t-test (^*P < 0.05 vs untreated control). (C) HASMCs were pretreated with PDTC at the indicated concentrations prior to IL-1β for 6 h. Nuclear extracts were prepared, and EMSA was conducted using [³²P]-labeled NF-κB-specific oligonucleotide. (D) HASMCs were seeded onto 8-well chamber slides and pretreated with several inhibitors prior to IL-1β treatment for 6 h. After fixation and permeabilization, cells were stained with FITC-labeled anti-p65 antibody. FITC fluorescence was visualized under a confocal microscope.