Figure 1.

GILZ is downregulated in AMs upon MyD88-dependent and MyD88-independent TLR activation. (A) Responsiveness of AMs toward the TLR1/2 ligand Pam₃CSK₄ (Pam, 100 ng/mL, 6 h) and the TLR3 ligand Poly(I:C) (PIC, 10 μg/mL, 6 h). Cytokine production was determined by Luminex bead assay (n = 4, triplicates; p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with Co; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with Pam-treated AMs). (B) AMs were left untreated or treated with LPS (1 μg/mL), Pam (1 μg/mL) or PIC (10 μg/mL) for 2 h and GILZ mRNA was quantified by qRT-PCR using ACTB as a housekeeping gene (n = 4, duplicates; ^***p < 0.001 compared with Co). (C–E): AMs were treated with Pam (C,E; 1 μg/mL) or PIC (D,E; 10 μg/mL) for the indicated time points and GILZ levels were determined by Western blot. Tubulin served as a loading control. (C,D) Representative blots. (E) GILZ signal intensities were quantified and normalized to tubulin values [n = 3; ^*,^#p < 0.05 compared with 0 h for Pam (^*) and PIC (^#)-treated cells].