Figure 5

NOD1-dependent induction of autophagy contributes to IRGM and LC3 recruitment to Mtb-containing vesicles. AMs were infected with M. tuberculosis H37Rv using an infection ratio of 5 bacteria/macrophage for 1 h. Non-phagocytized bacteria were washed away. Then, the macrophages were treated with 5 μg/ml of Tri-DAP for 24 h. Cells were pre-incubated with Rip2/p38 inhibitor SB203580 (SB) or PI3K inhibitor LY294002 (LY) for 30 min prior to Tri-DAP stimulation which blocks NOD1- or autophagy-mediated responses, as indicated. The subcellular localization of autophagy proteins was observed in untreated (A, C) and Tri-DAP treated cells (B, D) using anti-IRGM and anti-LC3 antibodies, which were detected using a secondary antibody coupled to 5 nm gold particles (indicated with arrowheads; TEM X 62,000). Gold particles colocalizing with bacteria were manually counted in 10 macrophages of each condition (E, F) and the differences between the treatments are indicated: ***p < 0.01 and *p < 0.05 vs. Mtb, ++p < 0.01 vs. Tri-DAP, Wilcoxon Rank test. Box plots indicate median and quartiles.