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eji2213-sup-0001.ppt2643K

Supporting information. Figure 1. The recruitment of autophagy proteins to the site of bacteria localization was NOD2- dependent. Human primary alveolar macrophages were infected with M. tuberculosis H37Rv (Mtb) at an MOI of 5 for 1 h. Non-phagocytosed bacteria were washed away, and the macrophages were incubated for an additional hour in presence of 10 mM SB203580, a Rip2/p38 inhibitor, to block NOD2 signaling prior to MDP stimulation. Cells were then treated with 10 μg/ml of MDP for 24 h. The subcellular localization of autophagy proteins was ascertained using anti-IRGM, anti-LC3 and anti-ATG16L1 antibodies and detected with a secondary antibody coupled to 5 nm gold particles (indicated with arrowheads; bar represents 150 nm, TEM magnification x 80,000). Data were generated from one experiment.

Supporting information. Figure 2. MDP administered after infection induces LC3-II conversion. Human primary alveolar macrophages were infected with M. tuberculosis H37Rv at an infection ratio of 1-2 bacteria/20 macrophages for 1 h. Non-phagocytosed bacteria were washed away, and the macrophages were then treated with 10 μg/ml of MDP or left with medium alone. (A) LC3 I/II protein expression was assessed in cytoplasmic extracts by western blot; tubulin was used as loading control. (B) The LC3 II/I ratio and (C) the amount of LC3 II normalized to tubulin relative to that of infected-only cells was calculated by densitometry using the ImageJ software. Data are depicted as the means ± SEM of n=3 subjects.

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