Figure 5

Both p50 and p65 NFκB are required for the IL-33-mediated inhibition of MCP-1 expression in human macrophages. Knockdown using siRNA against p50/p65 NFκB or negative control sequence (Neg) was carried out as Materials and Methods. THP-1 macrophages were then incubated for 12 h in the presence of vehicle (−) or 25 ng/ml IL-33 (+). Expression of mRNA for p50 (A) or p65 (B) or MCP-1 (E) was analyzed by RT-qPCR. Data represents mean ± SEM from three independent experiments. The expression of p50 or p65 protein levels was determined by Western blot analysis using β-actin as a control. A representative image with signal from immunoreactive p50, p65 or β-actin is shown (see Supplementary Figs 8 and 9 for corresponding full-length image for panels (C,D) respectively) with the histogram below it indicating p50 or p65 protein expression normalized to β-actin. The knockdown of p50/p65 in vehicle- or IL-33 treated cells was determined in relation to the negative control, which was arbitrarily assigned as 1 (A–D). The IL-33-mediated changes in MCP-1 expression in the negative control was compared to that following knockdown of p50/p65 (E) with values from cells transfected with negative control siRNA or p50/p65 siRNA and then treated with vehicle alone given an arbitrary value of 1. Statistical analysis was carried out using an unpaired Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).