Figure 4

ERK1/2 is required for the IL-33-mediated inhibition of MCP-1 and ICAM-1 expression in human macrophages. Knockdown using adenovirus encoding shRNA against ERK1 or scramble (Scr) sequence or siRNA against ERK2 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 ERK1 (A), ERK2 (B), MCP-1 (E,G) and ICAM-1 (F,H) was analyzed by RT-qPCR. Data represents mean ± SEM from three to five independent experiments. The expression of ERK1/2 protein levels was determined by Western blot analysis using β-actin as a control (C,D). A representative image with signal from immunoreactive ERK1/2 or β-actin is shown (see Supplementary Figs 6 and 7 for corresponding full-length image for panels (C,D) respectively) with the histogram below it indicating ERK-1 or -2 protein expression normalized to β-actin from three to four independent experiments. The knockdown of ERK1/2 in vehicle- or IL-33 treated cells was determined in relation to the scramble/negative control, which was arbitrarily assigned as 1 (A–D). The IL-33-mediated changes in MCP-1 and ICAM-1 expression in the scramble/negative control was compared to that following knockdown of ERK1/2 (E–H) with values from cells treated with scramble/negative control or ERK1 shRNA/ERK2 siRNA and then incubated with vehicle alone given an arbitrary value of 1. Statistical analysis was carried out using an unpaired Student’s t-test (A,E,F) or Man Whitney U test (B–D,G,H) (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).