B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45 + cells . ( G ) Representative FACS plots depicting gating strategies for defining Treg cell populations in Panels E and

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B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. (G) Representative FACS plots depicting gating strategies for defining Treg cell populations in Panels E and F


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( A ) CD4 + and CD8 + T cells were stimulated in vitro with ionomycin / PMA before intracellular staining for IFNγ , IL - 17 and IL - 9 and FACS analysis .

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Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (A) CD4+ and CD8+ T cells were stimulated in vitro with ionomycin/PMA before intracellular staining for IFNγ, IL-17 and IL-9 and FACS analysis.


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( B , C ) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to

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Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (B,C) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to D


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . CD4 + and CD8 + T cells were stained with CD44 , CD73 , FR4 and PD1 to monitor the formation of anergic / exhausted T cells . ( D ) Percentages of each population .

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Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (D) Percentages of each population.


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . CD4 + and CD8 + T cells were stained with CD44 , CD73 , FR4 and PD1 to monitor the formation of anergic / exhausted T cells . ( E ) Gating strategies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (E) Gating strategies.


Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( F ) FR4 and PD1 levels in WT vs . EGR4 - / - T cells .

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Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (F) FR4 and PD1 levels in WT vs. EGR4-/- T cells.


( 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 ) .

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(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 ) .

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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 ) .

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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 ) .

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(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 ) .

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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 ) .

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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 ) .

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(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 ) .

(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).


( 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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(N) IRF3 nuclear translocation was analyzed in RIG-I- or MAVS-knockdown BMMs. Scale bar, 20 μM.


( C ) Yield of purified EVs from BMMs infected with various M . tb CDC 1551 strains based on Nanosight analysis . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

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(C) Yield of purified EVs from BMMs infected with various M.tb CDC 1551 strains based on Nanosight analysis. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


( B ) EVs from cells infected with WT , ∆ esxA or esxA - complemented ( ∆ esxA - C ) M . tb Erdman strains .

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(B) EVs from cells infected with WT, ∆esxA or esxA-complemented (∆esxA-C) M.tb Erdman strains.


( D ) EVs from cells infected with WT , ∆ esxA or esxA - complemented ( ∆ esxA - C ) M . tb Erdman strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

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(D) EVs from cells infected with WT, ∆esxA or esxA-complemented (∆esxA-C) M.tb Erdman strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


qRT - PCR analysis for M . tb RNA in EVs from BMMs infected with various M . tb CDC 1551 strains ( E ) ND , not detected . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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qRT-PCR analysis for M.tb RNA in EVs from BMMs infected with various M.tb CDC 1551 strains (E) ND, not detected. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


qRT - PCR analysis for M . tb RNA in EVs from BMMs infected with various M . tb Erdman strains ( F ) . ND , not detected . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

qRT-PCR analysis for M.tb RNA in EVs from BMMs infected with various M.tb Erdman strains (F). ND, not detected. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


qRT - PCR analysis for IFN - β mRNA ( G ) in WT BMMs treated with EVs for 4 hr and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb CDC 1551 strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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qRT-PCR analysis for IFN-β mRNA (G) in WT BMMs treated with EVs for 4 hr and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb CDC 1551 strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


IFN - β protein ( H ) levels in WT BMMs treated with EVs for 4 hr and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb CDC 1551 strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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IFN-β protein (H) levels in WT BMMs treated with EVs for 4 hr and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb CDC 1551 strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


( I ) ELISA analysis for IFN - β secreted by BMMs treated with EVs plus liposomes containing M . tb RNA . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

(I) ELISA analysis for IFN-β secreted by BMMs treated with EVs plus liposomes containing M.tb RNA. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


qRT - PCR analysis for IFN - β mRNA ( J ) in WT BMMs treated with EVs for 4 and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb Erdman strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

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qRT-PCR analysis for IFN-β mRNA (J) in WT BMMs treated with EVs for 4 and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb Erdman strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


IFN - β protein ( K ) levels in WT BMMs treated with EVs for 4 and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb Erdman strains . Data shown in K ) are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

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IFN-β protein (K) levels in WT BMMs treated with EVs for 4 and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb Erdman strains. Data shown in K) are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


M . tb CFU in WT mouse BMMs pre - treated with EVs minus co - treatment with IFN - ɣ . BMMs were treated with EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) cells for 0 , 24 , 48 and 72 hr following a 1 hr M . tb infection . Mock , no EV treatment . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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M.tb CFU in WT mouse BMMs pre-treated with EVs minus co-treatment with IFN-ɣ. BMMs were treated with EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) cells for 0, 24, 48 and 72 hr following a 1 hr M.tb infection. Mock, no EV treatment. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. ; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


M . tb CFU in WT mouse BMMs pre - treated with EVs plus ( B ) co - treatment with IFN - ɣ . BMMs were treated with EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) cells for 0 , 24 , 48 and 72 hr following a 1 hr M . tb infection . Mock , no EV treatment . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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M.tb CFU in WT mouse BMMs pre-treated with EVs plus (B) co-treatment with IFN-ɣ. BMMs were treated with EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) cells for 0, 24, 48 and 72 hr following a 1 hr M.tb infection. Mock, no EV treatment. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. ; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy analysis for colocalization of M . tb ( GFP ) with lysosome marker Lamp - 1 in WT mouse BMMs . Cells were pre - treated for 5 hr with EVs from uninfected or M . tb - infected macrophages plus IFN - γ , and then infected with GFP - expressing M . tb for 24 hr prior to immunostaining . quantitative analysis of M . tb colocalization with Lamp - 1 in WT mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence microscopy analysis for colocalization of M.tb (GFP) with lysosome marker Lamp-1 in WT mouse BMMs. Cells were pre-treated for 5 hr with EVs from uninfected or M.tb-infected macrophages plus IFN-γ, and then infected with GFP-expressing M.tb for 24 hr prior to immunostaining. quantitative analysis of M.tb colocalization with Lamp-1 in WT mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy analysis for colocalization of M . tb ( GFP ) with lysosome marker Lamp - 1 in Mavs - / - ( E and F ) mouse BMMs . Cells were pre - treated for 5 hr with EVs from uninfected or M . tb - infected macrophages plus IFN - γ , and then infected with GFP - expressing M . tb for 24 hr prior to immunostaining . quantitative analysis of M . tb colocalization with Lamp - 1 in Mavs - / - mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence microscopy analysis for colocalization of M.tb (GFP) with lysosome marker Lamp-1 in Mavs-/- (E and F) mouse BMMs. Cells were pre-treated for 5 hr with EVs from uninfected or M.tb-infected macrophages plus IFN-γ, and then infected with GFP-expressing M.tb for 24 hr prior to immunostaining. quantitative analysis of M.tb colocalization with Lamp-1 in Mavs-/- mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy as described above except using control siRNA - treated ( G and H ) BMMs . quantitative analysis of M . tb colocalization with Lamp - 1 in control siRNA - treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence microscopy as described above except using control siRNA-treated (G and H) BMMs. quantitative analysis of M.tb colocalization with Lamp-1 in control siRNA-treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy as described above except using RIG - I siRNA - treated ( I and J ) BMMs . quantitative analysis of M . tb colocalization with Lamp - 1 in RIG - I siRNA - treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence microscopy as described above except using RIG-I siRNA-treated (I and J) BMMs. quantitative analysis of M.tb colocalization with Lamp-1 in RIG-I siRNA-treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


M . tb CFU in infected Mavs - / - ( K ) mouse BMMs pre - treated with IFN - ɣ plus EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) BMMs . CFU was determined immediately after the 1 hr infection or 24 and 72 hr post - infection . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M.tb CFU in infected Mavs-/- (K) mouse BMMs pre-treated with IFN-ɣ plus EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. CFU was determined immediately after the 1 hr infection or 24 and 72 hr post-infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. ; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


M . tb CFU in control siRNA - treated ( L ) or RIG - I siRNA - treated ( M ) mouse BMMs pre - treated with IFN - ɣ plus EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) BMMs . CFU was determined immediately after the 1 hr infection or 24 and 72 hr post - infection . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

M.tb CFU in control siRNA-treated (L) or RIG-I siRNA-treated (M) mouse BMMs pre-treated with IFN-ɣ plus EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. CFU was determined immediately after the 1 hr infection or 24 and 72 hr post-infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


( A ) Immunofluorescence microscopy analysis for colocalization of M . tb with Lamp - 1 in WT BMMs pre - treated with EVs from macrophages infected with WT , ∆ secA2 or secA2 - complemented ( ∆ secA2 - C ) M . tb CDC 1551 strains . The cells were pre - treated with EVs supplemented with recombinant mouse IFN - γ for 5 hr and subsequently infected for 24 hr with GFP - expressing M . tb . ( B ) quantitative analysis for the colocalization of M . tb with Lamp - 1 . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM ; * p < 0 . 05 and * * p < 0 . 01 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Immunofluorescence microscopy analysis for colocalization of M.tb with Lamp-1 in WT BMMs pre-treated with EVs from macrophages infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. The cells were pre-treated with EVs supplemented with recombinant mouse IFN-γ for 5 hr and subsequently infected for 24 hr with GFP-expressing M.tb. (B) quantitative analysis for the colocalization of M.tb with Lamp-1. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM ; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.


( C ) M . tb CFU in WT BMMs pre - pretreated with recombinant mouse IFN - γ and EVs from macrophages infected with WT , ∆ secA2 or secA2 - complemented ( ∆ secA2 - C ) M . tb CDC 1551 strains . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . n . s . , not significant ; * p < 0 . 05 and * * p < 0 . 01 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) M.tb CFU in WT BMMs pre-pretreated with recombinant mouse IFN-γ and EVs from macrophages infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. n.s., not significant; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.


( D ) and ( E ) liposomes containing RNA isolated from EVs released from BMMs infected with WT M . tb were also added to cells 5 hr prior to infection . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not

(D) and (E) liposomes containing RNA isolated from EVs released from BMMs infected with WT M.tb were also added to cells 5 hr prior to infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant


( F ) liposome - encapsulated RNA isolated from EVs released from BMMs infected with WT M . tb was included during EV and IFN - γ pre - treatment . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . n . s . , not significant ; * p < 0 . 05 and * * p < 0 . 01 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) liposome-encapsulated RNA isolated from EVs released from BMMs infected with WT M.tb was included during EV and IFN-γ pre-treatment. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. n.s., not significant; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.


Immunofluorescence microscopy and quantitative analysis for colocalization of M . tb with autophagosome marker LC3 in control siRNA - treated BMMs that were untreated or pre - treated for 5 hr with recombinant mouse IFN - γ and EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) macrophages followed by a 24 hr infection with GFP - expressing M . tb . Mock , untreated . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

Immunofluorescence microscopy and quantitative analysis for colocalization of M.tb with autophagosome marker LC3 in control siRNA -treated BMMs that were untreated or pre-treated for 5 hr with recombinant mouse IFN-γ and EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) macrophages followed by a 24 hr infection with GFP-expressing M.tb. Mock, untreated. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy and quantitative analysis for colocalization of M . tb with autophagosome marker LC3 ( C and D ) in RIG - I siRNA - treated BMMs . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

Immunofluorescence microscopy and quantitative analysis for colocalization of M.tb with autophagosome marker LC3 (C and D) in RIG-I siRNA-treated BMMs. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


( E and F ) Immunofluorescence microscopy and quantitative analysis for colocalization of M . tb with markers LC3 in WT BMMs that were left untreated or pre - treated for 5 hr with recombinant mouse IFN - γ and EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) macrophages , followed by a 24 hr infection with GFP - expressing M . tb . Mock , untreated . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

(E and F) Immunofluorescence microscopy and quantitative analysis for colocalization of M.tb with markers LC3 in WT BMMs that were left untreated or pre-treated for 5 hr with recombinant mouse IFN-γ and EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) macrophages, followed by a 24 hr infection with GFP-expressing M.tb. Mock, untreated. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy and quantitative analysis for colocalization of M . tb with markers LC3 in Mavs - / - BMMs . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence microscopy and quantitative analysis for colocalization of M.tb with markers LC3 in Mavs -/- BMMs. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy quantitative analysis for colocalization of M . tb with NOX2 ( I and J ) as described above in WT BMMs . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence microscopy quantitative analysis for colocalization of M.tb with NOX2 (I and J) as described above in WT BMMs. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy quantitative analysis for colocalization of M . tb with p47phox ( K and L ) as described above in WT BMMs . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

Immunofluorescence microscopy quantitative analysis for colocalization of M.tb with p47phox (K and L) as described above in WT BMMs. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy quantitative analysis for colocalization of M . tb similar to ( I - L ) , but using Mavs - / - BMMs . NOX2 ( M and N ) Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence microscopy quantitative analysis for colocalization of M.tb similar to ( I -L), but using Mavs -/- BMMs. NOX2 (M and N) Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy quantitative analysis for colocalization of M . tb similar to ( I - L ) , but using Mavs - / - BMMs . p47phox ( O and P ) Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

Immunofluorescence microscopy quantitative analysis for colocalization of M.tb similar to ( I -L), but using Mavs -/- BMMs. p47phox (O and P) Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


( A and B ) Quantitative analysis of immunofluorescence microscopy images for colocalization of M . tb with Lamp - 1 in WT ( A ) and Mavs - / - ( B ) BMMs infected with M . tb for 24 hr and then treated for an additional 24 hr with moxifloxacin ( MFX ) and / or EVs from M . tb - infected BMMs ( EVs + MFX ) in the presence of recombinant mouse IFN - ɣ . Mock , no EVs or moxifloxacin treatment ; EVs _ Control , EVs from uninfected BMMs . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A and B) Quantitative analysis of immunofluorescence microscopy images for colocalization of M.tb with Lamp-1 in WT (A) and Mavs -/- (B) BMMs infected with M.tb for 24 hr and then treated for an additional 24 hr with moxifloxacin (MFX) and/or EVs from M.tb-infected BMMs (EVs+MFX) in the presence of recombinant mouse IFN-ɣ. Mock, no EVs or moxifloxacin treatment; EVs_Control, EVs from uninfected BMMs. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


LC3 ( C ) , colocalization with M . tb were analyzed . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC3 (C), colocalization with M.tb were analyzed. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


NOX2 ( D ) colocalization with M . tb were analyzed . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

NOX2 (D) colocalization with M.tb were analyzed. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


p47phox ( E ) colocalization with M . tb were analyzed . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

p47phox (E) colocalization with M.tb were analyzed. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


Mavs - / - BMMs were used and colocalization of M . tb with LC3 ( F ) were quantified . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mavs -/- BMMs were used and colocalization of M.tb with LC3 (F) were quantified. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


Mavs - / - BMMs were used and colocalization of M . tb with NOX2 ( G ) were quantified . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

Mavs -/- BMMs were used and colocalization of M.tb with NOX2 (G) were quantified. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


Mavs - / - BMMs were used and colocalization of M . tb with p47phox ( H ) were quantified . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

Mavs -/- BMMs were used and colocalization of M.tb with p47phox (H) were quantified. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


( I and J ) M . tb CFU analysis in WT ( I ) and Mavs - / - ( J ) BMMs infected with M . tb for 24 hr followed by treatment with EVs , moxifloxacin or combination in the presence of IFN - ɣ for 24 and 72 hr . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I and J) M.tb CFU analysis in WT (I) and Mavs -/- (J) BMMs infected with M.tb for 24 hr followed by treatment with EVs, moxifloxacin or combination in the presence of IFN-ɣ for 24 and 72 hr. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


Representative histopathological analysis for lung sections of WT ( B ) mice that were infected with M . tb and subsequent left untreated ( Mock ) or treated with EVs from uninfected BMMs ( EVs _ Control ) , EVs from M . tb - infected BMMs ( EVs ) , moxifloxacin ( MXF ) , or combination of EVs and MXF ( EVs + MXF ) . Data shown is representative of two independent experiments . The results are the mean ± SD ( n = 4 mice per group ) . Scale bar , 100

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative histopathological analysis for lung sections of WT (B) mice that were infected with M.tb and subsequent left untreated (Mock) or treated with EVs from uninfected BMMs (EVs_Control), EVs from M.tb-infected BMMs (EVs), moxifloxacin (MXF), or combination of EVs and MXF (EVs+MXF). Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). Scale bar, 100 μM


M . tb CFU in the lung and spleen of WT ( C ) mice treated with EVs , MXF or combination of both . Data shown is representative of two independent experiments . The results are the mean ± SD ( n = 4 mice per group ) . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M.tb CFU in the lung and spleen of WT (C) mice treated with EVs, MXF or combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


Representative histopathological analysis for lung sections of Mavs - / - ( D ) mice that were infected with M . tb and subsequent left untreated ( Mock ) or treated with EVs from uninfected BMMs ( EVs _ Control ) , EVs from M . tb - infected BMMs ( EVs ) , moxifloxacin ( MXF ) , or combination of EVs and MXF ( EVs + MXF ) . Data shown is representative of two independent experiments . The results are the mean ± SD ( n = 4 mice per group ) . Scale bar , 100

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative histopathological analysis for lung sections of Mavs -/- (D) mice that were infected with M.tb and subsequent left untreated (Mock) or treated with EVs from uninfected BMMs (EVs_Control), EVs from M.tb-infected BMMs (EVs), moxifloxacin (MXF), or combination of EVs and MXF (EVs+MXF). Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). Scale bar, 100 μM


M . tb CFU in the lung and spleen of Mavs - / - ( E ) mice treated with EVs , MXF or combination of both . Data shown is representative of two independent experiments . The results are the mean ± SD ( n = 4 mice per group ) . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M.tb CFU in the lung and spleen of Mavs -/- (E) mice treated with EVs, MXF or combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


ELISA analysis for , IFN - β , TNF - α , IL - 1β and IFN - γ protein level in serum isolate from M . tb - infected WT ( F ) or Mavs - / - ( G ) mice treated with EVs , MXF or a combination of both . Data shown is representative of two independent experiments . The results are the mean ± SD ( n = 4 mice per group ) . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ELISA analysis for, IFN-β, TNF-α, IL-1β and IFN-γ protein level in serum isolate from M.tb-infected WT (F) or Mavs -/- (G) mice treated with EVs, MXF or a combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


( a ) FLAG - AMBRA1 was transfected into HEK293 cells together with ULK1WT or ULK1K46I Myc - tagged proteins . Protein extracts were immunoprecipitated ( IP ) using anti - FLAG antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) FLAG-AMBRA1 was transfected into HEK293 cells together with ULK1WT or ULK1K46I Myc-tagged proteins. Protein extracts were immunoprecipitated (IP) using anti-FLAG antibody.


( b ) HEK293 cells were grown either in normal or in EBS medium for 1 h ; protein extracts were immunoprecipitated using anti - ULK1 antibody or rabbit immunoglobulin as a negative control ( IgG ) . Autophagy induction is shown by p62 degradation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) HEK293 cells were grown either in normal or in EBS medium for 1 h; protein extracts were immunoprecipitated using anti-ULK1 antibody or rabbit immunoglobulin as a negative control (IgG). Autophagy induction is shown by p62 degradation.


( c ) HEK293 cells were treated as in b , in the presence of leupeptin and stained by anti - AMBRA1 ( red ) and anti - ULK1 ( green ) antibodies . The merge of the two signals and DAPI ( blue ) for nuclear staining is shown in the right panels . Scale bar , 6 μm . The outlined areas are shown at a higher magnification in the lower panels . White arrows point to the co - localization puncta . Measurements by Metamorph indicate a co - localization ratio of 46 % between ULK1 and AMBRA1 on autophagy induction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) HEK293 cells were treated as in b, in the presence of leupeptin and stained by anti-AMBRA1 (red) and anti-ULK1 (green) antibodies. The merge of the two signals and DAPI (blue) for nuclear staining is shown in the right panels. Scale bar, 6 μm. The outlined areas are shown at a higher magnification in the lower panels. White arrows point to the co-localization puncta. Measurements by Metamorph indicate a co-localization ratio of 46% between ULK1 and AMBRA1 on autophagy induction.


( d , e ) ULK1 and AMBRA1 constructs are illustrated . Asterisks mark nonspecific bands corresponding to immunoglobulins . ( d ) HEK293 cells were co - transfected with vectors encoding AMBRA1 together with ULK1 - Myc FL ( full length ) or its deletion mutants encoding the kinase and the proline - rich domains ( Δ829 - 1 , 051 ) , and the ULK1 carboxy - terminal domain ( Δ1 - 828 ) . Protein extracts were immunoprecipitated using anti - Myc antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (d) HEK293 cells were co-transfected with vectors encoding AMBRA1 together with ULK1-Myc FL (full length) or its deletion mutants encoding the kinase and the proline-rich domains (Δ829-1,051), and the ULK1 carboxy-terminal domain (Δ1-828). Protein extracts were immunoprecipitated using anti-Myc antibody.


( d , e ) ULK1 and AMBRA1 constructs are illustrated . Asterisks mark nonspecific bands corresponding to immunoglobulins . ( e ) HEK293 cells were co - transfected with vectors encoding ULK1 - HA together with β - galactosidase - Myc ( βgal ) , AMBRA1 full - length ( FL ) , and F1 , F2 or F3 Myc - tagged proteins . Protein extracts were immunoprecipitated using anti - HA antibody . The relative amounts of co - immunoprecipitatedULK1 and F1 or F3 , respectively , are the same ( see upper and lower left panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (e) HEK293 cells were co-transfected with vectors encoding ULK1-HA together with β-galactosidase-Myc (βgal), AMBRA1 full-length (FL), and F1, F2 or F3 Myc-tagged proteins. Protein extracts were immunoprecipitated using anti-HA antibody. The relative amounts of co-immunoprecipitatedULK1 and F1 or F3, respectively, are the same (see upper and lower left panel).


( f ) HEK293 cells were co - transfected with vectors encoding AMBRA1 , ULK1 - Myc and ATG13 - FLAG . Protein extracts were immunoprecipitated using anti - FLAG antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) HEK293 cells were co-transfected with vectors encoding AMBRA1, ULK1-Myc and ATG13-FLAG. Protein extracts were immunoprecipitated using anti-FLAG antibody.


( g ) HEK293 cells were transfected with scramble siRNA or ATG13 siRNA . Protein extracts were immunoprecipitated using ULK1 antibody or IgG . The band density ratio of immunoprecipitated AMBRA1 relative to immunoprecipitated ULK1 is analysed , with the control ratio arbitrarily defined as 1 . 00 . n = 3 extracts prepared from independent experiments ; data are presented as means ± s . d . and significance is P = 0 . 04 . Uncropped images of blots / gels are shown in Supplementary Fig . S6 . Source data of statistical analysis are shown in Supplementary Table S1 .

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(g) HEK293 cells were transfected with scramble siRNA or ATG13 siRNA. Protein extracts were immunoprecipitated using ULK1 antibody or IgG. The band density ratio of immunoprecipitated AMBRA1 relative to immunoprecipitated ULK1 is analysed, with the control ratio arbitrarily defined as 1.00. n = 3 extracts prepared from independent experiments; data are presented as means±s.d. and significance is P = 0.04. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.


( a ) HEK293 cells were transfected with AMBRA1 or the empty vector , as a control . ULK1 , AMBRA1 and actin were analysed by western blotting .

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(a) HEK293 cells were transfected with AMBRA1 or the empty vector, as a control. ULK1, AMBRA1 and actin were analysed by western blotting.


( b ) Protein extracts from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos for the Ambra1 gene - trap mutation at developmental day 14 . 5 were analysed by western blotting using anti - Ulk1 or anti - Hsp90 ( loading control ) antibodies . Quantification is shown on the right . n = 6 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 037 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Protein extracts from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos for the Ambra1 gene-trap mutation at developmental day 14.5 were analysed by western blotting using anti-Ulk1 or anti-Hsp90 (loading control) antibodies. Quantification is shown on the right. n = 6 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.


( c ) AMBRA1 , ULK1 and actin ( as a protein loading control ) were analysed by western blotting in HeLa cells transiently transfected with scramble siRNA or AMBRA1 siRNA . Quantification is shown on the right . n = 5 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 007 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) AMBRA1, ULK1 and actin (as a protein loading control) were analysed by western blotting in HeLa cells transiently transfected with scramble siRNA or AMBRA1 siRNA. Quantification is shown on the right. n = 5 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.007.


( d ) HeLa cells stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector ; AMBRA1 , ULK1 , ATG13 , p - ATG13 and actin levels were analysed by western blotting . Quantification of ULK1 and pATG13 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 006 for ULK1 and P = 0 . 044 and P = 0 . 011 for pATG13 .

(d) HeLa cells stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector; AMBRA1, ULK1, ATG13, p-ATG13 and actin levels were analysed by western blotting. Quantification of ULK1 and pATG13 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.006 for ULK1 and P = 0.044 and P = 0.011 for pATG13.


( e ) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide ( CHX ) for 6 and 8 h before extraction . ULK1 , actin and AMBRA1 were analysed by western blotting . Quantification is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 049 and P = 0 . 02 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide (CHX) for 6 and 8 h before extraction. ULK1, actin and AMBRA1 were analysed by western blotting. Quantification is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.049 and P = 0.02.


( f ) HEK293 cells were transfected as in a ; ATG13 , pATG13 , ULK1 , AMBRA1 and actin were analysed by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) HEK293 cells were transfected as in a; ATG13, pATG13, ULK1, AMBRA1 and actin were analysed by western blotting.


( g ) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected with β - galactosidase or AMBRA1 plasmid respectively using an anti - ULK1 antibody . As a control , rabbit IgG was used . The resulting precipitates were subjected to ULK1kinase assay using MBP as the substrate . Purified complexes were analysed by autoradiography and by western blotting using an anti - ULK1 antibody . Total extracts were analysed using an anti - AMBRA1 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected with β-galactosidase or AMBRA1 plasmid respectively using an anti-ULK1 antibody. As a control, rabbit IgG was used. The resulting precipitates were subjected to ULK1kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody. Total extracts were analysed using an anti-AMBRA1 antibody.


( a ) Protein extracts from HeLa cells were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control and analysed by western blotting using anti - Lys63 specific ubiquitin and anti - ULK1 antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Protein extracts from HeLa cells were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control and analysed by western blotting using anti-Lys63 specific ubiquitin and anti-ULK1 antibodies.


( b ) HeLa cells were co - transfected with ULK1 - Myc alone or in combination with wild - type ubiquitin or its mutant ( K48R or K63R ) HA - tagged constructs . Protein extracts were immunoprecipitated using an anti - Myc antibody . Purified complexes and total extracts were analysed by western blotting using anti - Myc and anti - HA antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) HeLa cells were co-transfected with ULK1-Myc alone or in combination with wild-type ubiquitin or its mutant (K48R or K63R) HA-tagged constructs. Protein extracts were immunoprecipitated using an anti-Myc antibody. Purified complexes and total extracts were analysed by western blotting using anti-Myc and anti-HA antibodies.


( c ) In HeLa cells , AMBRA1 was downregulated by RNAi . Protein extracts were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control . Purified complexes and total extracts were analysed by western blotting using anti - ULK1 , anti - Lys - 63 ubiquitin and anti - AMBRA1 antibodies . Of note , ULK1 levels in the co - immunoprecipitation experiment do not change in this case in the AMBRA1 siRNA lane , owing to saturation of the anti - ULK1 antibody ( see also Fig . 4h ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) In HeLa cells, AMBRA1 was downregulated by RNAi. Protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting using anti-ULK1, anti-Lys-63 ubiquitin and anti-AMBRA1 antibodies. Of note, ULK1 levels in the co-immunoprecipitation experiment do not change in this case in the AMBRA1 siRNA lane, owing to saturation of the anti-ULK1 antibody (see also Fig. 4h).


( d ) HeLa cells were co - transfected with vectors encoding for ubiquitin Lys - 63 - HA alone or in combination with AMBRA1 . Protein extracts were immunoprecipitated using an anti - ULK1 antibody or with IgG as a negative control and Ub - Lys - 63 - HA , ULK1 and AMBRA1 were analysed by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) HeLa cells were co-transfected with vectors encoding for ubiquitin Lys-63-HA alone or in combination with AMBRA1. Protein extracts were immunoprecipitated using an anti-ULK1 antibody or with IgG as a negative control and Ub-Lys-63-HA, ULK1 and AMBRA1 were analysed by western blotting.


( e , f ) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control . Purified complexes and total extracts were analysed by western blotting as in a . Quantifications are shown on the right . In both panels , n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 003 and P = 0 . 0015 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e,f) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting as in a. Quantifications are shown on the right. In both panels, n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.003 and P = 0.0015, respectively.


( a ) HeLa cells were transfected with FLAG - TRAF6 or grown in starvation medium for 1 h . Some of them were treated with chloroquine . LC3 , p62 and actin were analysed by western blotting . Quantification of LC3II and p62 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 017 for LC3 and P = 0 . 01 and P = 0 . 04 for p62 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) HeLa cells were transfected with FLAG-TRAF6 or grown in starvation medium for 1 h. Some of them were treated with chloroquine. LC3, p62 and actin were analysed by western blotting. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.017 for LC3 and P = 0.01 and P = 0.04 for p62.


( b ) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG - TRAF6 . Quantification of LC3II and p62 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 001 and P = 0 . 0018 for LC3 and P = 0 . 004 for p62 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG-TRAF6. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001 and P = 0.0018 for LC3 and P = 0.004 for p62.


( c ) Count of LC3 puncta in HeLa cells transduced as in b . Five different confocal microscopy images for each experiment were randomly chosen and GFP - positive dots were examined on the images with identical brightness and contrast settings . The results are represented as the average number of LC3 dots with 100 cells analysed per sample . Confocal images are shown in Supplementary Fig . S3b .

(c) Count of LC3 puncta in HeLa cells transduced as in b. Five different confocal microscopy images for each experiment were randomly chosen and GFP-positive dots were examined on the images with identical brightness and contrast settings. The results are represented as the average number of LC3 dots with 100 cells analysed per sample. Confocal images are shown in Supplementary Fig. S3b.


( d ) HEK293 cells were transfected with AMBRA1 and FLAG - TRAF6 as indicated . Protein extracts were immunoprecipitated using an anti - FLAG antibody .

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(d) HEK293 cells were transfected with AMBRA1 and FLAG-TRAF6 as indicated. Protein extracts were immunoprecipitated using an anti-FLAG antibody.


( e ) HeLa cells were transfected as in b and FLAG - TRAF6 was purified as in d . Quantification of immunoprecipitated TRAF6 is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 0003 .

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(e) HeLa cells were transfected as in b and FLAG-TRAF6 was purified as in d. Quantification of immunoprecipitated TRAF6 is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.0003.


( f ) In HeLa cells , TRAF6 was downregulated by RNAi . AMBRA1 , ULK1 , TRAF6 , ATG13 and actin were analysed by western blotting .

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(f) In HeLa cells, TRAF6 was downregulated by RNAi. AMBRA1, ULK1, TRAF6, ATG13 and actin were analysed by western blotting.


( g ) HeLa cells were co - transfected with vectors encoding ULK1 - Myc , FLAG - TRAF6 and Ub - Lys - 63 - HA , as indicated . Protein extracts were immunoprecipitated using an anti - Myc antibody .

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(g) HeLa cells were co-transfected with vectors encoding ULK1-Myc, FLAG-TRAF6 and Ub-Lys-63-HA, as indicated. Protein extracts were immunoprecipitated using an anti-Myc antibody.


( h ) HeLa cells stably interfered for AMBRA1 were transfected with FLAG - TRAF6 and Ub - Lys - 63 - HA as indicated . Protein extracts were immunoprecipitated using anti - ULK1 antibody .

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(h) HeLa cells stably interfered for AMBRA1 were transfected with FLAG-TRAF6 and Ub-Lys-63-HA as indicated. Protein extracts were immunoprecipitated using anti-ULK1 antibody.


( i ) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b . The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate . Purified complexes were analysed by autoradiography and by western blotting using an anti - ULK1 antibody .

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(i) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b. The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody.


( a - c ) Two canonical TRAF6 - binding sites in AMBRA1 are essential for its interaction with TRAF6 . HEK293 cells were co - transfected with vectors encoding FLAG - TRAF6 together with AMBRA1WT and mutant constructs . Protein extracts were immunoprecipitated using an anti - FLAG antibody . AMBRA1 and FLAG - TRAF6 were analysed by western blotting .

(a-c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6. HEK293 cells were co-transfected with vectors encoding FLAG-TRAF6 together with AMBRA1WT and mutant constructs. Protein extracts were immunoprecipitated using an anti-FLAG antibody. AMBRA1 and FLAG-TRAF6 were analysed by western blotting.


( d ) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide ( siRNA AMBRA1 ) . Then , cells were co - transfected with vectors encoding ubiquitin - HA and AMBRA1WT or AMBRA1AA , respectively . Protein extracts were immunoprecipitated using anti - ULK1 antibody ; ubiquitin - HA , ULK1 and AMBRA1 were analysed by western blotting .

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(d) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide (siRNA AMBRA1). Then, cells were co-transfected with vectors encoding ubiquitin-HA and AMBRA1WT or AMBRA1AA, respectively. Protein extracts were immunoprecipitated using anti-ULK1 antibody; ubiquitin-HA, ULK1 and AMBRA1 were analysed by western blotting.


( e ) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β - galactosidase ( negative control ) AMBRA1WT or AMBRA1AA respectively ; protein extracts were analysed by western blotting using anti - ULK1 , anti - AMBRA1 anti - ATG13 , anti - pATG13 and anti - actin antibodies .

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(e) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β-galactosidase (negative control) AMBRA1WT or AMBRA1AA respectively; protein extracts were analysed by western blotting using anti-ULK1, anti-AMBRA1 anti-ATG13, anti-pATG13 and anti-actin antibodies.


( f ) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h . LC3 , p62 , AMBRA1 and actin were analysed by western blotting . Quantification of LC3II and p62 are shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 03 for LC3 and P = 0 . 045 for p62 .

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(f) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h. LC3, p62, AMBRA1 and actin were analysed by western blotting. Quantification of LC3II and p62 are shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.03 for LC3 and P = 0.045 for p62.


( g ) GFP - LC3 MEF cells from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right . Bars represent mean ± s . e . m . of triplicate samples with 50 cells analysed per sample ; n = 3 independent experiments . Significance is P = 0 . 001 and P = 0 . 003 ( n = 3 ) . Scale bar , 10 μm .

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(g) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right. Bars represent mean±s.e.m. of triplicate samples with 50 cells analysed per sample; n = 3 independent experiments. Significance is P = 0.001 and P = 0.003 (n = 3). Scale bar, 10 μm.


( a ) HEK293 cells were co - transfected with vectors encoding ULK1 - HA and ULK1 - Myc and then grown either in normal ( Ctrl ) or in starvation ( EBS ) medium for 1 h . Protein extracts were immunoprecipitated using an anti - Myc antibody , and analysed by western blotting using anti - HA , and anti - Myc antibodies . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 001 .

(a) HEK293 cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and then grown either in normal (Ctrl) or in starvation (EBS) medium for 1 h. Protein extracts were immunoprecipitated using an anti-Myc antibody, and analysed by western blotting using anti-HA, and anti-Myc antibodies. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001.


( b ) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide ( TRAF6 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , cells were co - transfected with vectors encoding ULK1 - HA and ULK1 - Myc and protein complexes were immunoprecipitated using anti - Myc antibody . Protein extracts were analysed by western blotting using anti - HA , anti - Myc and anti - TRAF6 antibodies . Quantification of ULK1 - HA relative to ULK1 - Myc is reported on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 037 .

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(b) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide (TRAF6 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and protein complexes were immunoprecipitated using anti-Myc antibody. Protein extracts were analysed by western blotting using anti-HA, anti-Myc and anti-TRAF6 antibodies. Quantification of ULK1-HA relative to ULK1-Myc is reported on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.


( c ) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide ( AMBRA1 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , cells were co - transfected and analysed as in a . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 044 .

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(c) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide (AMBRA1 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.044.


( d ) HEK293 cells were co - transfected with vectors encoding for ULK1 - HA , ULK1 - Myc and AMBRA1WT or AMBRA1AA respectively . Protein complexes were immunoprecipitated and analysed as in a . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 014 .

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(d) HEK293 cells were co-transfected with vectors encoding for ULK1-HA, ULK1-Myc and AMBRA1WT or AMBRA1AA respectively. Protein complexes were immunoprecipitated and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.014.


( a ) Highly purified mTOR was incubated with radiolabelled ATP without substrate , with purified FLAG - AMBRA1 and purified FLAG - AMBRA1S52A and analysed by autoradiography . The lower panels show a FLAG and mTOR western blot ( WB ) .

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(a) Highly purified mTOR was incubated with radiolabelled ATP without substrate, with purified FLAG-AMBRA1 and purified FLAG-AMBRA1S52A and analysed by autoradiography. The lower panels show a FLAG and mTOR western blot (WB).


( b ) HEK293 cells expressing FLAG - RAPTOR were subjected to FLAG immunoprecipitation . Protein complexes were analysed by western blotting using anti - FLAG and anti - AMBRA1 antibodies .

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(b) HEK293 cells expressing FLAG-RAPTOR were subjected to FLAG immunoprecipitation. Protein complexes were analysed by western blotting using anti-FLAG and anti-AMBRA1 antibodies.


( c ) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti - pSer - 52 and anti - AMBRA1 antibodies .

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(c) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti-pSer-52 and anti-AMBRA1 antibodies.


( d ) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β - galactosidase , AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h . Protein extracts were analysed by western blotting using anti - LC3 , anti - p62 and anti - actin antibodies . Quantification of LC3II and p62 bands is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 038 .

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(d) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β-galactosidase, AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h. Protein extracts were analysed by western blotting using anti-LC3, anti-p62 and anti-actin antibodies. Quantification of LC3II and p62 bands is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.038.


( e ) GFP - LC3 MEF cells from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right . Bars represent mean ± s . e . m . of triplicate samples and 50 cells analysed per sample . n = 3 independent experiments . Significance is P = 0 . 001 and P = 0 . 003 . Scale bar , 10

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(e) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right. Bars represent mean±s.e.m. of triplicate samples and 50 cells analysed per sample. n = 3 independent experiments. Significance is P = 0.001 and P = 0.003. Scale bar, 10 μm


( f ) HeLa cells stably interfered for AMBRA1 were co - transfected with vectors encoding Ub - Lys - 63 - HA and AMBRA1WT or AMBRA1S52A , respectively . Protein extracts were immunoprecipitated using an anti - ULK1 antibody and ubiquitin Lys - 63 - HA , ULK1 , AMBRA1 and actin were analysed by western blotting .

(f) HeLa cells stably interfered for AMBRA1 were co-transfected with vectors encoding Ub-Lys-63-HA and AMBRA1WT or AMBRA1S52A, respectively. Protein extracts were immunoprecipitated using an anti-ULK1 antibody and ubiquitin Lys-63-HA, ULK1, AMBRA1 and actin were analysed by western blotting.


( g ) HeLa cells stably interfered for AMBRA1 were transduced as in d ; protein extracts were analysed by western blotting using anti - AMBRA1 , anti - ATG13 , anti - pATG13 and anti - actin antibodies . Quantification of the p - ATG13 band is shown below .

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(g) HeLa cells stably interfered for AMBRA1 were transduced as in d; protein extracts were analysed by western blotting using anti-AMBRA1, anti-ATG13, anti-pATG13 and anti-actin antibodies. Quantification of the p-ATG13 band is shown below.


( h ) HeLa cells transfected with ULK1 - HA , ULK1 - Myc and different AMBRA1 constructs , respectively . Protein complexes were immunoprecipitated using anti - HA antibody and analysed by western blotting using anti - HA , anti - Myc and anti - AMBRA1 antibodies .

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(h) HeLa cells transfected with ULK1-HA, ULK1-Myc and different AMBRA1 constructs, respectively. Protein complexes were immunoprecipitated using anti-HA antibody and analysed by western blotting using anti-HA, anti-Myc and anti-AMBRA1 antibodies.


A MOAP - 1 co - localizes with p62 in the cytoplasmic inclusion bodies upon proteasome inhibition . HCT116 cells were transfected with plasmid encoding GFP - MOAP - 1 or GFP only control . 14 hours later , cells were treated with the proteasome inhibitor MG132 ( 5 µM ) for 8 hours and analyzed by immunofluorescence ( IF ) with anti - GFP ( in green ) and anti - p62 ( in red ) antibodies . Data information : , nuclei were counterstained with DAPI ( blue )

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A MOAP-1 co-localizes with p62 in the cytoplasmic inclusion bodies upon proteasome inhibition. HCT116 cells were transfected with plasmid encoding GFP-MOAP-1 or GFP only control. 14 hours later, cells were treated with the proteasome inhibitor MG132 (5 µM) for 8 hours and analyzed by immunofluorescence (IF) with anti-GFP (in green) and anti-p62 (in red) antibodies. Data information: , nuclei were counterstained with DAPI (blue)


B Endogenous MOAP - 1 resides at the p62 bodies upon proteasome inhibition . HCT116 cells were treated with MG132 ( 5 µM ) for 8 hours and analyzed by IF with anti - MOAP - 1 ( in green ) and anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue )

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B Endogenous MOAP-1 resides at the p62 bodies upon proteasome inhibition. HCT116 cells were treated with MG132 (5 µM) for 8 hours and analyzed by IF with anti-MOAP-1 (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)


C MOAP - 1 is recruited to the p62 bodies upon exposure to cellular stresses . LO2 hepatocytes were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , cells were then treated with MG132 ( 5 µM ) , arsenic trioxide ( As2O3 , 10 µM ) or diethylnitrosamine ( DEN , 200 µM ) for 8 hours each . Cells were then processed for IF analysis with anti - Myc ( in green ) and anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue )

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C MOAP-1 is recruited to the p62 bodies upon exposure to cellular stresses. LO2 hepatocytes were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were then treated with MG132 (5 µM), arsenic trioxide (As2O3, 10 µM) or diethylnitrosamine (DEN, 200 µM) for 8 hours each. Cells were then processed for IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)


D MOAP - 1 spontaneously localizes to the p62 bodies at resting state in the liver cancer cell lines , HepG2 , Huh - 1 , JHH5 and JHH7 . The liver cancer cell lines were transfected with the plasmid encoding Myc - MOAP - 1 . 14 hours later , transfected cells were subjected to IF analysis with anti - Myc ( in green ) and anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue )

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D MOAP-1 spontaneously localizes to the p62 bodies at resting state in the liver cancer cell lines, HepG2, Huh-1, JHH5 and JHH7. The liver cancer cell lines were transfected with the plasmid encoding Myc-MOAP-1. 14 hours later, transfected cells were subjected to IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)


E Absence of the aggregated patterns of MOAP - 1 in the p62 deficient cells . WT and p62 KO HepG2 cells were transfected with plasmid encoding Myc - MOAP - 1 for 14 hours and the cells were subjected to IF analysis as in ( D ) . Data information : , nuclei were counterstained with DAPI ( blue )

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E Absence of the aggregated patterns of MOAP-1 in the p62 deficient cells. WT and p62 KO HepG2 cells were transfected with plasmid encoding Myc-MOAP-1 for 14 hours and the cells were subjected to IF analysis as in (D). Data information: , nuclei were counterstained with DAPI (blue)


F Western blotting analysis of p62 and Myc - MOAP - 1 protein levels in the WT and p62 KO HepG2 cells as described in ( E ) . Actin as loading control .

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F Western blotting analysis of p62 and Myc-MOAP-1 protein levels in the WT and p62 KO HepG2 cells as described in (E). Actin as loading control.


A MOAP - 1 downregulates the levels of the diethylnitrosamine ( DEN ) - induced p62 bodies in a time - dependent manner . LO2 cells were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , cells were treated with DEN ( 200 µM ) for the indicated period of times , before being subjected to IF with anti - Myc ( in green ) and anti - p62 ( in red ) antibodies . Cells expressing Myc - MOAP - 1 were marked by dashed lines . Data information : nuclei were counterstained with DAPI ( blue )

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A MOAP-1 downregulates the levels of the diethylnitrosamine (DEN)-induced p62 bodies in a time-dependent manner. LO2 cells were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were treated with DEN (200 µM) for the indicated period of times, before being subjected to IF with anti-Myc (in green) and anti-p62 (in red) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. Data information: nuclei were counterstained with DAPI (blue)


B MOAP - 1 downregulates the p62 bodies in the resting HepG2 liver cancer cells . HepG2 were transfected with plasmid encoding Myc - MOAP - 1 . At the indicated time post - transfection , transfected cells were subjected to IF as in ( A ) . Data information : nuclei were counterstained with DAPI ( blue )

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B MOAP-1 downregulates the p62 bodies in the resting HepG2 liver cancer cells. HepG2 were transfected with plasmid encoding Myc-MOAP-1. At the indicated time post-transfection, transfected cells were subjected to IF as in (A). Data information: nuclei were counterstained with DAPI (blue)