Fig. 7. Requirement for TBK-1 in IL-1β mediated autophagic killing of mycobacteria.

A. RAW264.7 macrophages were knocked down for Atg7 (by Atg7 siRNA transfection 48 h prior to infection), infected with M. tuberculosis H37Rv for 1h, washed and then left untreated or treated with 10 ng/ml recombinant murine IL-1β for 2 h after which they were lysed and plated for CFU determination, and survival expressed relative to sample transfected with control scrambled siRNA and not treated with IL-1β. Immunoblots, Atg7 knockdown and levels of Atg5-Atg12 complexes. B. BCG survival, % CFU of M. tuberculosis var. bovis BCG recovered from bone marrow macrophages derived from Atg7^fl/fl LysM-Cre⁻ and Atg7^fl/fl LysM-Cre⁺ mice treated with 50 ng/ml IL-1β (16 h preinfection + 4 h post-infection). Data, means ± se (n=3, *, p<0.05; t-test. C. BCG survival, % CFU recovered from RAW264.7 macrophages treated with IL-1β with and without 10 nM BX795. D. BCG survival in infected RAW264.7 (and knocked down or not for TBK-1) macrophages stimulated with IL-1β. E,F. RAW264.7 macrophages were incubated in full medium (Control) or induced by adding IL-1β to full medium. Cells were pretreated with 10 nM BX795 where indicated. Macrophages were treated with or without bafilomycin A1 (BafA1) to inhibit autophagic degradation of LC3-II, and cellular extracts analyzed by immunoblotting. Graphs, densitometric analyses of LC3-II levels normalized to actin levels (LC3-II/Actin). G,H. Cells, treatments and analysis as in E and F but in the absence of bafilomycin A1. Data, means ± se (n=3; †, p≥0.05; **, p<0.01; ANOVA).