Figure 3

After stimulation with the NOD1 ligand, macrophages increased their expression of autophagy proteins Atg9, LC3 and IRGM. Alveolar macrophages (AMs), monocyte-derived macrophages (MDMs) and monocytes (MNs) were incubated in the presence of 5 μg/ml of Tri-DAP for 24 h. Cells were pre-incubated with Rip2/p38 inhibitor SB203580 (SB) for 30 min prior to Tri-DAP stimulation to block NOD1-mediated responses, as indicated. Atg9 and LC3 proteins were measured in the cytosolic fractions by western blot analysis (A). The fold increases relative to the unstimulated cells were calculated after being normalized to tubulin, mean ± SE is depicted, n = 3 (B, C). The up-regulation of IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using the Taqman system and ΔΔCT method for relative quantification. The fold changes in gene expression relative to the unstimulated cells of at least 6 subjects are depicted; bold lines indicate medians, *p < 0.05 (D). IRGM protein was measured in the cytosolic fractions of three subjects by western blot, and the fold increases relative to the unstimulated cells are indicated after being normalized to tubulin; mean ± SE is depicted, n = 3 (E, F).