Figure 1(A) Quantitative RT-PCR analysis of M.tb RNA in EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. ND, not detected. Scale bar, 20 μM. Data shown in (A) are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). Quantitative RT-PCR analysis for IFN-β mRNA (B) levels in wild type BMMs 5 and 24 hr post-treatment with EVs respectively (1 μg/ml equals approximately 3 x 108 vesicles/ml). Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed). IFN-β protein (C) levels in wild type BMMs 5 and 24 hr post-treatment with EVs respectively (1 μg/ml equals approximately 3 x 108 vesicles/ml). Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed). (D) IFN-β mRNA level was quantified in wild type BMMs treated with EVs at a concentration of 10μg/ml for the times indicated. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed). Quantitative RT-PCR analysis for IFN-β mRNA (E) levels in either WT, MAVS- and/or RIG-I-knockdown BMMs treated for 4 hr (E) with 10µg/ml EVs from uninfected or M.tb-infected macrophages. (Control) negative control siRNA. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed). IFN-β protein (F) levels in either WT, MAVS- and/or RIG-I-knockdown BMMs treated for 24 hr (F) with 10µg/ml EVs from uninfected or M.tb-infected macrophages. (Control) negative control siRNA. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed). (G) Similar to above except using WT and Mavs -/- BMMs. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed). (H) Similar to above except using WT and Mavs -/- BMMs. Scale bar, 20 μM. Data shown in (H) are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed). (I) Schematic of transwell assay for measuring IFN-β induction. BMMs were uninfected or infected with WT M.tb at a MOI of 5. Twenty-four hr post-infection, cells were transferred into the top chamber and co-cultured with naïve BMMs (Bottom chamber). The IFN-β mRNA levels in BMMs (bottom) were quantified by qRT-PCR after 24 hr. (J) Top chamber: WT BMMs; Bottom chamber: WT or Mavs -/- BMMs. Data shown in (J) are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed). (K) Western blot analysis for phospho-TBK1 (Ser172) in WT and MAVS-knockdown BMMs treated for 4 hr with EVs from uninfected or M.tb-infected macrophages. β-actin served as a loading control. Densitometry of the western blots for (K) are shown. (L) Western blot analysis of IRF3 under the conditions described above for TBK1. Histone H3 (H3) and β-actin were used as loading controls for nuclear fraction and whole-cell lysate (WCL) respectively. Densitometry of the western blots are shown. (M) Fluorescence microscopy analysis for nuclear translocation of IRF3 in wild type BMMs untreated (Mock) or treated with EVs from uninfected or M.tb-infected macrophages. Scale bar, 20 μM. (N) IRF3 nuclear translocation was analyzed in RIG-I- or MAVS-knockdown BMMs. Scale bar, 20 μM.