Fig. 4.

Effect of IKK-2 inhibitors on NF-κB activation and IL-8 and MCP-1 expression and release. A549 cells were pretreated with 1 μM [5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1) for 1 h or 20 μM SC-514 for 2 h prior to incubation with 10 μM methemoglobin. Cells were collected at the indicated times and nuclear extracts were prepared. Western blot analysis was performed to detect the presence of phosphorylated NF-κB p65 (ser536) and β-actin (loading control); representative blots are shown (A). Western blots were quantified by use of Image J software. The values for phosphorylated NF-κB p65 (ser536) normalized to β-actin are presented in Fig. 3B. A549 cells were pretreated with either TPCA-1 (left) for 1 h or SC-514 (right) for 2 h prior to the addition of methemoglobin. IL-8 and MCP-1 levels were determined in the cell supernatants after 24 h by ELISA (Fig. 3, C–F). IL-8 and MCP-1 mRNA levels relative to the housekeeping gene were determined after 2 h (G–J). In all panels data are presented as means ± SE of 4 independent experiments. Comparison between conditions was made by 1-way ANOVA with Bonferroni posttest, *P < 0.05, **P < 0.01, and ***P < 0.001. Asterisks on top of columns refer to comparison with the control (medium only); asterisks above lines refer to comparison between indicated conditions.