IFN - β ( F ) in either WT , MAVS - / 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 mean ± SD ( n = 3 wells per group ) data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by Student ' s t - ( two ) .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O

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 Mavs - / - BMMs . Data shown are mean ± SD ( n = 3 wells per group ) data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by Student ' s t - ( two ) .

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

(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 Mavs - / - BMMs . Scale bar , 20 μM . Data shown in ( H ) are mean ± SD ( n = 3 wells per group ) data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by Student ' s t - ( two ) .

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

(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 mean ± SD ( n = 3 wells per group ) data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by Student ' s t - ( two ) .

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

(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 for phospho - TBK1 ( Ser172 ) in WT 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 western blots for ( K ) are shown .

O O O O O O O O B-MEASURED_VAR 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

(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 of IRF3 under conditions described above for TBK1 . Histone H3 ( H3 ) β - actin were used as loading controls for fraction whole - lysate ( WCL ) respectively . Densitometry of 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 ) microscopy for 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 translocation was analyzed in RIG - I - or MAVS - knockdown BMMs . Scale bar , 20 μM .

O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O

(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 on Nanosight . Data shown are mean ± SD ( n = 3 per group ) data shown are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O 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) 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 infected with WT , ∆ esxA or esxA - complemented ( ∆ esxA - C ) M . tb Erdman strains .

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

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


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

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

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

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

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

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

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

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

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

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

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

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

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

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

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 ) for 0 , 24 , 48 72 hr following a 1 hr M . tb infection . Mock , no EV treatment . Data shown are mean ± SD of three independent infections data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by two - Student ' s t - .

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

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 ) for 0 , 24 , 48 72 hr following a 1 hr M . tb infection . Mock , no EV treatment . Data shown are mean ± SD of three independent infections data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by two - Student ' s t - .

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

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

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

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

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 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 H ) BMMs . quantitative of M . tb colocalization with Lamp - 1 in control siRNA - treated mouse BMMs Data shown are mean ± SD of three independent infections data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by two - Student ' s t - .

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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

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 J ) BMMs . quantitative of M . tb colocalization with Lamp - 1 in RIG - I siRNA - treated mouse BMMs Data shown are mean ± SD of three independent infections data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by two - Student ' s t - .

O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

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 1 hr infection or 24 72 hr post - infection . Data shown are mean ± SD of three independent infections data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by two - Student ' s t - .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 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 1 hr infection or 24 72 hr post - infection . Data shown are mean ± SD of three independent infections data shown are representative of at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by two - Student ' s t - .

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

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 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 . were pre - treated with EVs supplemented with recombinant mouse IFN - γ for 5 hr subsequently infected for 24 hr with GFP - expressing M . tb . ( B ) quantitative for colocalization of M . tb with Lamp - 1 . Data shown are mean ± SD of three independent infections data shown are representative of at least three independent experiments . Scale bar , 5 μM ; * p < 0 . 05 * * p < 0 . 01 by two - Student ' s t - .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

(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 EVs from macrophages infected with WT , ∆ secA2 or secA2 - complemented ( ∆ secA2 - C ) M . tb CDC 1551 strains . Data shown are mean ± SD of three independent infections data shown are representative of at least three independent experiments . n . s . , not significant ; * p < 0 . 05 * * p < 0 . 01 by two - Student ' s t - .

O O O 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 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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 quantitative 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 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 mean ± SD of three independent infections data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by two - Student ' s t - .

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

O O O O O O O O O O O O B-MEASURED_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

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 F ) Immunofluorescence microscopy quantitative 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 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 mean ± SD of three independent infections data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by two - Student ' s t - .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O

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

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

O O O O O O O O O O B-MEASURED_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

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

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

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 B-MEASURED_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

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

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 B ) Quantitative of immunofluorescence microscopy for colocalization of M . tb with Lamp - 1 in WT ( A ) Mavs - / - ( B ) BMMs infected with M . tb for 24 hr then treated for an additional 24 hr with moxifloxacin ( MFX ) / or EVs from M . tb - infected BMMs ( EVs + MFX ) in 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 . results are mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by Student ' s t - ( two ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(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 . results are mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by Student ' s t - ( two ) .

B-MEASURED_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

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 . results are mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by Student ' s t - ( two ) .

B-MEASURED_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

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 . results are mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by Student ' s t - ( two ) .

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

O O O O O O O O O O O O O B-MEASURED_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

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

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

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

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

(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 for lung sections of WT ( B ) mice that were infected with M . tb 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 MXF ( EVs + MXF ) . Data shown is representative of two independent experiments . results are 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

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

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

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 for lung sections of Mavs - / - ( D ) mice that were infected with M . tb 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 MXF ( EVs + MXF ) . Data shown is representative of two independent experiments . results are 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

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

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

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 for , IFN - β , TNF - α , IL - 1β IFN - γ 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 . results are mean ± SD ( n = 4 mice per group ) . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 by Student ' s t - ( two ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 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

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).


Representative of hematoxylin eosin - stained testicular sections from ( V : 3 ) a man diagnosed with obstructive azoospermia , serving as control . magnified view of boxed is shown in lower left corner of image from . blue arrow indicates unaligned chromosomes in representative metaphase . Scale bars , 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of hematoxylin and eosin-stained testicular sections from the patient (V:3) and a man diagnosed with obstructive azoospermia, serving as the control. The magnified view of the boxed area is shown in the lower left corner of the image from the patient. The blue arrow indicates unaligned chromosomes in the representative metaphase cells. Scale bars, 50 μm.


Immunofluorescence of testicular sections from control V : 3 with antibodies against H3S10p ( red ) , a marker of metaphase . nuclei were stained with Hoechst 33342 ( blue ) . magnified view of red box is shown in lower left corner of image from .

O O O O O O O O O O O O B-MEASURED_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

Immunofluorescence staining of testicular sections from control and patient V:3 with antibodies against H3S10p (red), a marker of metaphase cells. The nuclei were stained with Hoechst 33342 (blue). The magnified view of the red box is shown in the lower left corner of the image from the patient.


Sanger sequencing chromatograms showing homozygous M1AP splicing mutation in heterozygous mutation in their fertile brother . arrowhead indicates position of mutation .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

Sanger sequencing chromatograms showing the homozygous M1AP splicing mutation in patients and heterozygous mutation in their fertile brother. The arrowhead indicates the position of the mutation.


Western blot of M1AP expression in various tissues from adult wild - type ( WT ) mice . GAPDH served as loading control .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

Western blot analysis of M1AP expression in various tissues from adult wild-type (WT) mice. GAPDH served as the loading control.


Western blot of M1AP expression in testes from mice at different days postpartum ( dpp ) . GAPDH served as loading control .

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Western blot analysis of M1AP expression in testes from mice at different days postpartum (dpp). GAPDH served as the loading control.


Immunofluorescence of M1AP ( green ) SYCP3 ( red ) on spermatocyte spreads of WT or Spo11 - / - mice .

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Immunofluorescence staining of M1AP (green) and SYCP3 (red) on spermatocyte spreads of WT or Spo11-/- mice.


of M1AP foci in WT spermatocytes at indicated stages . L , leptotene ; E / MZ , early / mid zygotene ; LZ , late zygotene ; EP , early pachytene ; M / LP , mid / late pachytene ; Z - like , zygotene - like .

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Quantification of M1AP foci in WT spermatocytes at the indicated stages. L, leptotene; E/MZ, early/mid zygotene; LZ, late zygotene; EP, early pachytene; M/LP, mid/late pachytene; Z-like, zygotene-like.


Testicular histology from 8 - week - old control M1apKI / KI mice . magnified view of boxed is shown in lower left corner of image of M1apKI / KI mice . blue arrow indicates unaligned chromosomes in representative metaphase .

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Testicular histology from 8-week-old control and M1apKI/KI mice. The magnified view of the boxed area is shown in the lower left corner of the image of M1apKI/KI mice. The blue arrow indicates unaligned chromosomes in the representative metaphase cells.


Meiotic metaphase I ( MMI ) spermatocytes stained with Giemsa . arrows indicate univalent chromosomes .

Meiotic metaphase I (MMI) spermatocytes stained with Giemsa. The arrows indicate univalent chromosomes.


Frequencies of nuclei with univalents.


Frequencies of nuclei with only XY univalents , only autosome univalents , or both XY autosome univalents .

Frequencies of nuclei with only XY univalents, only autosome univalents, or both XY and autosome univalents.


Representative spread spermatocytes stained for SYCP3 ( green ) MLH1 ( red ) .

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Representative spread spermatocytes stained for SYCP3 (green) and MLH1 (red).


Frequencies of autosomal chromosomes with 0 , 1 , 2 or 3 MLH1 foci .

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Frequencies of autosomal chromosomes with 0, 1, and 2 or 3 MLH1 foci.


Frequencies of nuclei with an MLH1 focus at pseudoautosomal region ( PAR ) of XY chromosomes .

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Frequencies of nuclei with an MLH1 focus at the pseudoautosomal region (PAR) of XY chromosomes.


Representative spread spermatocytes stained for lateral element ( SYCP3 , green ) central element ( SIX6OS1 , red ) in early mid / late pachynema . Miniaturized H1t ( ) are shown in left lower corner of overlay . arrows indicate untouching XY chromosomes .

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Representative spread spermatocytes stained for the lateral element (SYCP3, green) and the central element (SIX6OS1, red) in early and mid/late pachynema. Miniaturized H1t staining (white) images are shown in the left lower corner of the overlay images. The arrows indicate the untouching XY chromosomes.


of nuclei with XY untouching in early mid / late pachynema .

Quantification of nuclei with XY untouching in early and mid/late pachynema.


Immunofluorescence with antibodies against SYCP3 ( red ) MSH4 ( green ) on spermatocyte spreads .

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Immunofluorescence staining with antibodies against SYCP3 (red) and MSH4 (green) on spermatocyte spreads.


mean number of MSH4 foci per nucleus in control M1apKI / KI spermatocytes at indicated stages .

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The mean number of MSH4 foci per nucleus in control and M1apKI/KI spermatocytes at the indicated stages.


Immunofluorescence with antibodies against SYCP3 ( red ) RAD51 ( green ) on spermatocyte spreads .

Immunofluorescence staining with antibodies against SYCP3 (red) and RAD51 (green) on spermatocyte spreads.


mean number of RAD51 foci per nucleus in control M1apKI / KI spermatocytes at indicated stages .

The mean number of RAD51 foci per nucleus in control and M1apKI/KI spermatocytes at the indicated stages.


Immunofluorescence with antibodies against SYCP3 ( red ) DMC1 ( green ) on spermatocyte spreads .

Immunofluorescence staining with antibodies against SYCP3 (red) and DMC1 (green) on spermatocyte spreads.


mean number of DMC1 foci per nucleus in control M1apKI / KI spermatocytes at indicated stages .

The mean number of DMC1 foci per nucleus in control and M1apKI/KI spermatocytes at the indicated stages.


Western blot of M1AP TEX11 expression in ovaries ( 16 . 5 days postcoitum ( dpc ) ) testes ( 14 dpp ) from WT mice . GAPDH served as loading control .

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Western blot analysis of M1AP and TEX11 expression in fetal ovaries (16.5 days postcoitum (dpc)) and testes (14 dpp) from WT mice. GAPDH served as the loading control.


Immunofluorescence with antibodies against SYCP3 ( red ) MLH3 ( green ) on oocyte spreads from control M1apKI / KI ( 18 . 5 dpc ) . Number of MLH3 foci per in control M1apKI / KI oocytes .

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Immunofluorescence staining with antibodies against SYCP3 (red) and MLH3 (green) on oocyte spreads from control and M1apKI/KI embryos (18.5 dpc). Number of MLH3 foci per cell in control and M1apKI/KI oocytes.


Representative haemotoxylin - stained ovarian sections of 5 - dpp - old control M1apKI / KI mice . Follicle total oocyte per ovary at 5 dpp .

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Representative haemotoxylin-stained ovarian sections of 5-dpp-old control and M1apKI/KI mice. Follicle counts and total oocyte counts per ovary at 5 dpp.


Co - IP using anti - M1AP antibody with whole - testis lysates of WT mice M1ap - / - mice , followed by western blot analyses of M1AP , SHOC1 , TEX11 .

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Co-IP using anti-M1AP antibody with whole-testis lysates of WT mice and M1ap-/- mice, followed by western blot analyses of M1AP, SHOC1, and TEX11.


Co - IP detecting interaction between M1AP ( fused with an N - terminal MYC tag ) each of ZZS components ( fused with an N - terminal GFP tag ) exogenously expressed in HEK - 293T .

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Co-IP detecting the interaction between M1AP (fused with an N-terminal MYC tag) and each of the ZZS complex components (fused with an N-terminal GFP tag) exogenously expressed in HEK-293T cells.


Immunofluorescence of M1AP ( green ) TEX11 ( red ) on spermatocyte spreads of WT mice . Ratios of M1AP - TEX11 co - localizing foci to M1AP total foci or TEX11 total foci at indicated meiotic prophase substages . Each dot represents calculated from one nucleus .

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Immunofluorescence staining of M1AP (green) and TEX11 (red) on the spermatocyte spreads of WT mice.   Ratios of M1AP-TEX11 co-localizing foci to the M1AP total foci or TEX11 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.


Immunofluorescence of M1AP ( green ) DMC1 ( red ) on spermatocyte spreads of WT mice . Ratios of M1AP - DMC1 co - localizing foci to M1AP total foci or DMC1 total foci at indicated meiotic prophase substages . Each dot represents calculated from one nucleus .

Immunofluorescence staining of M1AP (green) and DMC1 (red) on the spermatocyte spreads of WT mice.   Ratios of M1AP-DMC1 co-localizing foci to the M1AP total foci or DMC1 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.


Immunofluorescence of M1AP ( green ) RPA2 ( red ) on spermatocyte spreads of WT mice . Ratios of M1AP - RPA2 co - localizing foci to M1AP total foci or RPA2 total foci at indicated meiotic prophase substages . Each dot represents calculated from one nucleus .

Immunofluorescence staining of M1AP (green) and RPA2 (red) on the spermatocyte spreads of WT mice.   Ratios of M1AP-RPA2 co-localizing foci to the M1AP total foci or RPA2 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.


Immunofluorescence of TEX11 ( green ) SYCP3 ( red ) on spermatocyte spreads of control M1apKI / KI testes . mean number of TEX11 foci per in control M1apKI / KI spermatocytes . Frequencies of nuclei with TEX11 foci detected at PAR in spread early pachytene spermatocytes with touching XY chromosomes .

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Immunofluorescence staining of TEX11 (green) and SYCP3 (red) on the spermatocyte spreads of control and M1apKI/KI testes.   The mean number of TEX11 foci per cell in control and M1apKI/KI spermatocytes.   Frequencies of nuclei with TEX11 foci detected at the PAR in spread early pachytene spermatocytes with touching XY chromosomes.


A Speed of propidium iodide ( PI ) penetration into fibroblasts during 60 min of fixation with either 4 % PFA or 3 % glyoxal . N = 3 independent experiments . Glyoxal fixation enables PI to penetrate far more rapidly into .

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A Speed of propidium iodide (PI) penetration into fibroblasts during 60 min of fixation with either 4 % PFA or 3 % glyoxal. N = 3 independent experiments. Glyoxal fixation enables PI to penetrate far more rapidly into the cells.


B Speed of FM 1 - 43 penetration in similar experiments . arrowhead points to one example of ongoing endocytosis during PFA fixation . N = 3 - 4 independent experiments . general pattern of FM 1 - 43 entry was similar to that of propidium iodide . Only first 10 minutes are shown , to enable an optimal observation of kinetics of first stages of FM 1 - 43 entry . results parallel those obtained with PI : faster penetration during glyoxal fixation . Scale bar = 40 µm , * p < 0 . 05 , * * p < 0 .

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B Speed of FM 1-43 penetration in similar experiments. The arrowhead points to one example of ongoing endocytosis during PFA fixation. N = 3-4 independent experiments. The general pattern of FM 1-43 entry was similar to that of propidium iodide. Only the first 10 minutes are shown, to enable an optimal observation of the kinetics of the first stages of FM 1-43 entry. The results parallel those obtained with PI: faster penetration during glyoxal fixation. Scale bar = 40 µm, * p < 0.05, ** p < 0.01


Figure 2 A comparison of morphological changes taking place during fixation with PFA or glyoxal . changes were visualized by DIC taken at 5 minute intervals during fixation . graph shows correlation of each image to first frame . N = 50 ( PFA ) 54 ( glyoxal ) regions analyzed , from 3 independent experiments . higher correlation value indicates that glyoxal preserves initial with higher accuracy than PFA . Scale bar = 20 µm , * p < 0 . 05 , * * p < 0 . 01

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Figure 2 A comparison of morphological changes taking place during fixation with PFA or glyoxal. The changes were visualized by DIC images taken at 5 minute intervals during fixation. The graph shows the correlation of each image to the first frame. N = 50 (PFA) and 54 (glyoxal) cellular regions analyzed, from 3 independent experiments. The higher correlation value indicates that glyoxal preserves the initial cell morphology with higher accuracy than PFA. Scale bar = 20 µm, * p < 0.05, ** p < 0.01


A SDS - PAGE gel showing rat brain cytoplasm incubated for 60 min with different fixatives . graph shows summed of bands in each lane . Fixed either no longer run into gel or form only smears . To compare efficiency of fixation , bands that survive fixation were summed were expressed as % of an unfixed control . of PFA - fixed samples was significantly higher than that of glyoxal - fixed samples ( N = 5 independent experiments ) . Glut = 0 . 2 % glutaraldehyde .

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A SDS-PAGE gel showing rat brain cytoplasm incubated for 60 min with different fixatives. The graph shows the summed intensity of the bands in each lane. Fixed proteins either no longer run into the gel or form only smears. To compare the efficiency of fixation, the bands that survive fixation were summed and were expressed as % of an unfixed control. The intensity of PFA-fixed samples was significantly higher than that of glyoxal-fixed samples (N = 5 independent experiments). Glut = 0.2% glutaraldehyde.


B of nucleic after fixation . propidium iodide signal in fibroblasts was significantly higher for samples fixed with glyoxal pH 4 ( N = 6 - 8 ) . To whether fixed nucleic were still available for specific detection , we performed FISH for GAPDH in cultured neurons , using a standard protocol provided by company Affymetrix . signal of samples fixed with glyoxal ( pH 4 ) was significantly higher than for PFA - fixed samples ( N = 5 - 6 ) . Scale bar = 10 µm , * p < 0 . 05 , * * p < 0 .

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B Staining of nucleic acids after fixation. The propidium iodide signal in fibroblasts was significantly higher for samples fixed with glyoxal pH 4 (N = 6-8). To test whether the fixed nucleic acids were still available for specific detection, we performed FISH for GAPDH in cultured neurons, using a standard protocol provided by the company Affymetrix. The fluorescence signal of the samples fixed with glyoxal (pH 4) was significantly higher than for PFA-fixed samples (N = 5-6). Scale bar = 10 µm, * p < 0.05, ** p < 0.01


Figure 4 STED imaging of hippocampal neurons fixed with either PFA or glyoxal . Strong differences in labeling patterns can be observed . are brighter less " spotty " for glyoxal - fixed samples . Structures such as filaments or organelles are more easily detected . of signal ( fold over background ) shows that 16 out of 20 stainings are significantly brighter in glyoxal fixed samples compared to PFA fixed samples . N = 35 - 132 objects . Scale bar = 6 µm for β - actin α - tubulin , 3 µm for other . * * p < 0 . 01 , * * * p < 0 . 001

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Figure 4 STED imaging of primary hippocampal neurons fixed with either PFA or glyoxal. Strong differences in labeling patterns can be observed. The images are brighter and less "spotty" for the glyoxal-fixed samples. Structures such as filaments or organelles are more easily detected. Quantification of the fluorescence signal (fold over background) shows that 16 out of 20 stainings are significantly brighter in glyoxal fixed samples compared to the PFA fixed samples. N = 35 - 132 objects. Scale bar = 6 µm for β-actin and α-tubulin, and 3 µm for the other proteins. ** p < 0.01, *** p < 0.001


Figure 5 Comparison of immunostaining NUP160 after fixation with either PFA or glyoxal . HeLa were stained for nucleoporin NUP160 after fixation with either PFA , glyoxal pH 4 or glyoxal pH 5 . intensities ( fold over background ) were compared are shown in graph . of signals shows that glyoxal pH 4 fixation allows for significantly brighter stainings . N = 73 - 156 per condition analyzed . Scale bar = 10 µm . * * * p < 0 . 001

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Figure 5 Comparison of immunostaining NUP160 after fixation with either PFA or glyoxal. HeLa cells were stained for the nucleoporin complex protein NUP160 after fixation with either PFA, glyoxal pH 4 or glyoxal pH 5. Fluorescence intensities (fold over background) were compared and are shown in the graph. The quantification of fluorescence signals shows that glyoxal pH 4 fixation allows for significantly brighter stainings. N = 73 - 156 cells per condition analyzed. Scale bar = 10 µm. *** p < 0.001


Figure 6 Comparison of immunostained AtT20 after fixation with either PFA or glyoxal . AtT20 stained for SNARE syntaxin1 SNAP25 autophagy marker LC3B , were compared with regard to ( fold over background ) of stainings . of shows that glyoxal fixation allows for significantly brighter stainings of SNARE . LC3B is brighter in PFA fixed . N = 9 - 20 per condition . Scale bar = 5 µm . * p < 0 . 05 , * * * p < 0 . 001

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Figure 6 Comparison of immunostained AtT20 cells after fixation with either PFA or glyoxal. AtT20 cells stained for the SNARE proteins syntaxin1 and SNAP25 and the autophagy marker LC3B, were compared with regard to the fluorescence intensity (fold over background) of the stainings. Quantification of the intensity shows that glyoxal fixation allows for significantly brighter stainings of the membrane SNARE proteins. LC3B staining is brighter in PFA fixed cells. N = 9 - 20 cells per condition. Scale bar = 5 µm. * p < 0.05, *** p < 0.001


A hippocampal neurons were stained for Actin Ankyrin G . A comparison between PFA - glyoxal - fixed samples shows that actin with phalloidin works as least as well in both , showing prominent actin rings . Ankyrin G is brighter in PFA - fixed .

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A Primary hippocampal neurons were stained for Actin and Ankyrin G. A comparison between PFA- and glyoxal-fixed samples shows that actin staining with phalloidin works as least as well in both, showing the prominent actin rings. Ankyrin G staining is brighter in PFA-fixed cells.


B hippocampal neurons were stained for pan - Nav Kv7 . 2 . Both stainings seem to work at least as well for glyoxal fixed neurons as for PFA fixed neurons . of K - channels shows a slightly more regular pattern in glyoxal fixed neurons .

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B Primary hippocampal neurons were stained for pan-Nav and Kv7.2. Both stainings seem to work at least as well for glyoxal fixed neurons as for PFA fixed neurons. Staining of K-channels shows a slightly more regular pattern in glyoxal fixed neurons.


C hippocampal neurons were stained for Neurofilament L betaII spectrin . While spectrin seems to be equally well in both fixation conditions , neurofilament is brighter in glyoxal fixed .

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C Primary hippocampal neurons were stained for Neurofilament L and betaII spectrin. While the spectrin staining seems to be equally well in both fixation conditions, neurofilament staining is brighter in glyoxal fixed cells.


D Growth cones of hippocampal neurons were stained for actin βIII - tubulin after either glyoxal or PFA + glutaraldehyde fixation . latter is a standard fixation used for co - labeling of tubulin actin , is a stronger fixation than normal PFA fixation , which is incompatible with many organelle immunostainings ( unlike glyoxal fixation ) . filopodia lamellipodia of growth cones seem to be well stained for samples fixed with glyoxal , whereas samples fixed with PFA glutaraldehyde seem to have lost some of finer actin structures . Tubulin seems to be a bit better stained in samples fixed with PFA glutaraldehyde . Scale bars = 1 µm .

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D Growth cones of hippocampal neurons were stained for actin and βIII-tubulin after either glyoxal or PFA + glutaraldehyde fixation. The latter is a standard fixation used for the co-labeling of tubulin and actin, and is a stronger fixation than normal PFA fixation, which is incompatible with many organelle immunostainings (unlike glyoxal fixation). The filopodia and lamellipodia of the growth cones seem to be well stained for the samples fixed with glyoxal, whereas the samples fixed with PFA and glutaraldehyde seem to have lost some of the finer actin structures. Tubulin seems to be a bit better stained in samples fixed with PFA and glutaraldehyde. Scale bars = 1 µm.


Figure 8 Comparison of immunostained sepia fin after fixation with either PFA or glyoxal . Sepia fin was fixed with respective fixative stained for neuropeptide FMRFamide . A clear change in can be observed between samples fixed with PFA samples fixed with glyoxal . former appear broken swollen , while glyoxal - fixed ones appear complete . effect is presumably due to different speed of penetration into tissue / or fixation . Scale bar = 5 µm .

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Figure 8 Comparison of immunostained sepia fin after fixation with either PFA or glyoxal. Sepia fin was fixed with the respective fixative and stained for the neuropeptide FMRFamide. A clear change in morphology can be observed between samples fixed with PFA and samples fixed with glyoxal. The former appear broken and swollen, while the glyoxal-fixed ones appear complete. The effect is presumably due to the different speed of penetration into tissue and/or fixation. Scale bar = 5 µm.


Figure 9 Comparison of immunostained ventricular myocytes fixed 10 min with either PFA or glyoxal . Freshly isolated murine ventricular myocytes were either fixed with 4 % PFA or 3 % glyoxal immunostained for caveolin - 3 or ryanodine receptor type 2 . of of stainings show significantly brighter stainings for glyoxal fixed myocytes . graph shows mean values error bars represent standard deviations . N = 10 ( RyR2 ) 12 ( Cav3 ) myocytes per condition . Scale bar = 2 µm . * * * p < 0 . 001

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Figure 9 Comparison of immunostained ventricular myocytes fixed 10 min with either PFA or glyoxal. Freshly isolated murine ventricular myocytes were either fixed with 4% PFA or 3% glyoxal and immunostained for caveolin-3 or ryanodine receptor type 2. Quantification of the fluorescence intensity of the stainings show significantly brighter stainings for glyoxal fixed myocytes. The graph shows mean values and error bars represent standard deviations. N = 10 (RyR2) and 12 (Cav3) myocytes per condition. Scale bar = 2 µm. *** p < 0.001


Figure 10 Comparison of immunostained mouse inner hair after fixation with either PFA or glyoxal . Acutely dissected organs of corti were fixed in respective fixative immunostained for inner hair synaptic . of for each shows a significant increase in signal to noise for three target ( CtBP2 , Calretinin Homer1 ) fixed with glyoxal . None of stained shows a significant decrease in after glyoxal fixation . Representative show maximum projections from z - stacks of inner hair ribbon synapses . N = 5 independent stainings from two per condition ( PSD95 , CtBP2 , Otoferlin , Calretinin ) 10 - 15 per condition ( CaV1 . 3 Homer1 ) . Scale bar = 2 µm . * * p < 0 . 01 , * * * p < 0 . 001

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Figure 10 Comparison of immunostained mouse inner hair cells after fixation with either PFA or glyoxal. Acutely dissected organs of corti were fixed in the respective fixative and immunostained for inner hair cell proteins and synaptic proteins. The quantification of fluorescence intensity for each staining shows a significant increase in signal to noise ratio for three target proteins (CtBP2, Calretinin and Homer1) fixed with glyoxal. None of the stained proteins shows a significant decrease in fluorescence after glyoxal fixation. Representative images show maximum intensity projections from z-stacks of inner hair cell ribbon synapses. N = 5 independent stainings from two animals per condition (PSD95, CtBP2, Otoferlin, Calretinin) and 10 - 15 images per condition (CaV1.3 and Homer1). Scale bar = 2 µm. ** p < 0.01, *** p < 0.001


Figure 11 Comparison of stained vimentin α - synuclein in human neuroglioma after fixation with either PFA or glyoxal . were fixed with PFA or glyoxal for 10 minutes , were stained for endogenous vimentin , or for expressed alpha - synuclein . A of intensities indicates that glyoxal fixation allows for significantly brighter stainings for alpha - synuclein , but that PFA was superior for endogenous vimentin ( leftmost graph ) . of vimentin expressed with an mOrange2 tag was also analyzed after fixation with PFA or with glyoxal ; latter allowed more mOrange2 to be detected ( rightmost graph ) . N = 29 - 81 regions per condition . * p < 0 . 05 , * * * p < 0 . 001 Scale bar : 10 µm .

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Figure 11 Comparison of stained vimentin and α-synuclein in human neuroglioma cells after fixation with either PFA or glyoxal. Cells were fixed with PFA or glyoxal for 10 minutes, and were stained for endogenous vimentin, or for expressed alpha-synuclein. A quantification of the staining intensities indicates that glyoxal fixation allows for significantly brighter stainings for alpha-synuclein, but that PFA was superior for endogenous vimentin (leftmost graph). The fluorescence intensity of vimentin expressed with an mOrange2 tag was also analyzed after fixation with PFA or with glyoxal; the latter allowed more mOrange2 fluorescence to be detected (rightmost graph). N = 29 - 81 cell regions per condition. * p < 0.05, *** p < 0.001 Scale bar: 10 µm.


Figure 12 Comparison of immunostainings for mitochondrial after fixation with either PFA or glyoxal . were stained with MitoTracker Orange prior to fixation with respective fixative immunostained for mitochondrial ATP5B , COA6 , NDUFA9 TIM23 . of shows a significant increase of ( signal over background ) for 2 markers ( ATP5B Mitotracker ) after fixation with glyoxal , whereas for remaining 3 immunostainings seem to be more efficient after fixation with PFA , albeit differences are small . N = 18 - 128 per condition . Scale bar = 5 µm . * * * p < 0 . 001

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Figure 12 Comparison of immunostainings for mitochondrial proteins after fixation with either PFA or glyoxal. Cells were stained with MitoTracker Orange prior to fixation with the respective fixative and immunostained for the mitochondrial proteins ATP5B, COA6, NDUFA9 and TIM23. Quantification of the staining intensity shows a significant increase of fluorescence (signal over background) for 2 markers (ATP5B and Mitotracker) after fixation with glyoxal, whereas for the remaining 3 proteins immunostainings seem to be more efficient after fixation with PFA, albeit the differences are small. N = 18 - 128 cells per condition. Scale bar = 5 µm. *** p < 0.001


Figure 13 Comparison of immunostained HeLa after fixation with either PFA or glyoxal . of a variety of was compared between fixed with PFA or glyoxal . is shown as of signal derived from PFA fixed . 8 out of 18 target which were stained show significantly brighter signal when fixed with glyoxal . Only 4 show significantly reduced . For LC3B of less than 5 was quantified , therefore single data points were plotted in addition to bars . N = 3 - 44 per condition . Scale bar = 10 µm . * * p < 0 . 01 , * * * p < 0 . 001

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Figure 13 Comparison of immunostained HeLa cells after fixation with either PFA or glyoxal. The fluorescence intensity of a variety of proteins was compared between cells fixed with PFA or glyoxal. Quantification is shown as percentage of signal derived from PFA fixed cells. 8 out of the 18 target proteins which were stained show significantly brighter signal when fixed with glyoxal. Only 4 proteins show significantly reduced staining intensity. For LC3B the intensity of less than 5 cells was quantified, therefore single data points were plotted in addition to the bars. N = 3 - 44 cells per condition. Scale bar = 10 µm. ** p < 0.01, *** p < 0.001


Figure 14 Comparison of immunostained U2OS hippocampal neurons after either PFA or glyoxal fixation . Immunostaining of Na / K ATPase in hippocampal neurons shows a different distribution of between PFA fixed nucleus fixed samples . While in PFA - fixed neurons antibody falsely stains glyoxal as well as cytoplasm ( 100 % of 82 we analyzed ) , in nucleus - fixed neurons U2OS is devoid of signal , appears to be correctly labeled ( arrowheads ; 100 % of 60 we analyzed ) . mitochondria were immunostained for KDEL ( Tom20 ) ER ( EGFP - KDEL ) . fixation / or of mitochondria seems to be comparable in glyoxal in PFA fixed . of ER shows an improved signal to noise . signal appears de - localized from ER for multiple PFA - stained ( 25 % of 36 analyzed , see arrows ) , while this is rare for glyoxal - stained ( 3 . 4 % of 58 analyzed ) . Scale bar = 10 µm .

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Figure 14 Comparison of immunostained U2OS cells and primary hippocampal neurons after either PFA or glyoxal fixation. Immunostaining of the Na/K ATPase in primary hippocampal neurons shows a different distribution of the protein between PFA fixed and nucleus fixed samples. While in PFA-fixed neurons the antibody falsely stains the glyoxal as well as the cytoplasm (100% of the 82 cells we analyzed), in nucleus-fixed neurons the U2OS is devoid of signal, and the membrane appears to be correctly labeled (arrowheads; 100% of the 60 cells we analyzed). mitochondria cells were immunostained for KDEL (Tom20) and ER (EGFP-KDEL). The fixation and/or staining of mitochondria seems to be comparable in glyoxal and in PFA fixed cells. The staining of the ER shows an improved signal to noise ratio. The signal appears de-localized from the ER for multiple PFA-stained cells (25% of 36 analyzed cells, see arrows), while this is rare for the glyoxal-stained cells (3.4% of 58 analyzed cells). Scale bar = 10 µm.


A - B Confocal showing of Olfactory Marker ( OMP ) ß3 - tubulin along dorsal aspect of mouse olfactory epithelium . While sections from both types of fixative show OMP signal in olfactory sensory neuron somata , their dendrites , axons , axon bundles ( green arrow ) located above olfactory epithelium exemplify clear signal - to - noise benefits of glyoxal fixation versus that of PFA - fixative . Immunostaining with ß3 - tubulin antibody stains dendrites axons in both PFA glyoxal - fixed tissue , but strong of cilia can only be observed in glyoxal - fixed sections ( B ) .

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A - B Confocal images showing staining of Olfactory Marker Protein (OMP) and ß3-tubulin along the dorsal aspect of the mouse olfactory epithelium. While sections from both types of fixative show OMP signal in the olfactory sensory neuron somata, their dendrites, and axons, the axon bundles (green arrow) located above the olfactory epithelium exemplify the clear signal-to-noise ratio benefits of glyoxal fixation versus that of the PFA-fixative. Immunostaining with the ß3-tubulin antibody stains the dendrites and axons in both PFA and glyoxal-fixed tissue, but strong staining of the cilia can only be observed in the glyoxal-fixed sections (B).


C - D Confocal depicting bundles of axons belonging to olfactory sensory neurons on path toward olfactory bulb . Identities of axons are in part defined by neuropilin - 1 ( Nrp1 ) neuropilin - 2 ( Nrp2 ) expression , visualized here with antibodies raised against two . While complementary expression of two molecules can be seen in PFA - fixed sections ( C ) , glyoxal - fixed sections ( D ) exhibit profoundly improved signal - to - noise ratios for Nrp - 1 ( red arrows ) , in case of Nrp - 2 , also segmentation of axon bundle into varying of Nrp - 2 ( green arrows ) .

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C - D Confocal images depicting bundles of axons belonging to olfactory sensory neurons on the path toward the olfactory bulb. Identities of the axons are in part defined by the neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2) expression levels, visualized here with antibodies raised against the two proteins. While complementary expression of the two molecules can be seen in the PFA-fixed sections (C), the glyoxal-fixed sections (D) exhibit profoundly improved signal-to-noise ratios for Nrp-1 (red arrows), and in the case of Nrp-2, also the segmentation of the axon bundle into varying levels of Nrp-2 (green arrows).


E - F Confocal of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons . axons of olfactory sensory neurons can be readily visualized with PFA ( green ) in superficial olfactory nerve layer terminating in glomeruli located below ( green arrows ) . While sections fixed with either PFA or glyoxal display adequate , signal distribution of Immunostaining - fixed tissue appears more irregular ( E ) , seemingly lacking neurofilimentary that appears preserved in glyoxal - fixed sections ( F ) . glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral / tufted as well as PFA of periglomerular neurons . Immunostaining with Vesicular Glutamate Transporter 2 ( VGLUT2 ) allows visualization of these structures is easily seen in glyoxal - fixed section ( F ) , while it appears antigen was masked by PFA fixation as no signal above background can be seen in PFA - fixed panel ( red arrows in E ) . Note that a different polyclonal antibody for VGLUT2 from same provider does provide signal with glyoxal , albeit weaker versus Glyoxal ( inset ) . with ß3 - tubulin touts benefits of glyoxal both due to signal improvements in case of mild in axons of olfactory nerve layer that is only visible in glyoxal - fixed tissue , but also in preserving tissue as demonstrated by dendritic processes inside glomeruli ( blue arrows ) in external plexiform layer located below glomeruli .

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E - F Confocal images of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons. The axons of olfactory sensory neurons can be readily visualized with PFA staining (green) in the superficial olfactory nerve layer and terminating in glomeruli located below (green arrows). While sections fixed with either PFA or glyoxal display adequate staining levels, the signal distribution of the Immunostaining-fixed tissue appears more irregular (E), seemingly lacking the neurofilimentary morphology that appears preserved in the glyoxal-fixed sections (F). The glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral/tufted cells as well as PFA of periglomerular neurons. Immunostaining with Vesicular Glutamate Transporter 2 (VGLUT2) allows visualization of these structures and is easily seen in the glyoxal-fixed section (F), while it appears the antigen was masked by PFA fixation as no signal above background can be seen in the PFA-fixed panel (red arrows in E). Note that a different polyclonal antibody for VGLUT2 from the same provider does provide signal with glyoxal, albeit weaker versus Glyoxal (inset). Staining with ß3-tubulin touts the benefits of glyoxal both due to the signal improvements in the case of the mild staining in the axons of the olfactory nerve layer that is only visible in the glyoxal-fixed tissue, but also in preserving tissue morphology as demonstrated by the dendritic processes inside glomeruli (blue arrows) and in the external plexiform layer located below the glomeruli.


( A - C ) developing vasculature was imaged over time in an abdominal window chamber model with two - photon microscopy with ( A ) GFP - labeled , ( B ) TdTomato labeled TECs in cyan ( C ) perfusion . ( D ) Top : a representative image of MC38 vasculature , bottom : segmentation with skeletonization . ( E - N ) following parameters were quantified from segmented image each day : ( E ) vessel tortuosity , ( F ) directional coherence , ( G ) perfusion , ( H ) number of sprouts , ( I ) fraction of branches < 80 µm , ( J ) fraction of branches > 400 µm , ( K ) to diameter , ( L ) nodes per mm2 , ( M ) vessel , ( N ) vessel diameter .

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(A-C) The developing tumor vasculature was imaged over time in an abdominal window chamber model with two-photon microscopy with (A) GFP-labeled tumor cells, (B) TdTomato labeled TECs in cyan and (C) perfusion. (D) Top: a representative image of MC38 tumor vasculature, bottom: segmentation with skeletonization. (E-N) The following parameters were quantified from the segmented image each day: (E) vessel tortuosity, (F) directional coherence, (G) perfusion, (H) number of sprouts,(I) fraction of branches <80 µm, (J) fraction of branches >400 µm, (K) length to diameter ratio, (L) nodes per mm2, (M) vessel length, and (N) vessel diameter.


developing MC38 vasculature was imaged in an abdominal window chamber model with two - photon microscopy after IR at indicated times . ( A ) ( top ) Representative two - photon of a control MC38 vasculature ( cyan ) perfusion ( red ) at day 2 4 . ( Bottom ) zoom of same region from day 0 to day 4 of vasculature ( cyan ) perfusion ( red ) . ( B - C ) Single dose 15 Gy IR ( B ) 5 × 3 Gy ( C ) fractionated IR treated MC38 vasculature ( top ) at day 2 day 4 . ( Bottom ) zoom of same region from day 0 to day 4 of vasculature ( cyan ) perfusion ( red ) .

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The developing MC38 tumor vasculature was imaged in an abdominal window chamber model with two-photon microscopy after IR at the indicated times. (A) (top) Representative two-photon images of a control MC38 tumor vasculature (cyan) and perfusion (red) at day 2 and 4. (Bottom) zoom of the same tumor region from day 0 to day 4 of the tumor vasculature (cyan) and perfusion (red). (B-C) Single dose 15 Gy IR (B) and 5×3 Gy (C) fractionated IR treated MC38 tumor vasculature (top) at day 2 and day 4. (Bottom) zoom of the same tumor region from day 0 to day 4 of the tumor vasculature (cyan) and perfusion (red).


( E - L ) Quantified vascular parameters , for each day of imaging following parameters were quantified from segmented normalized to day one if indicated : ( E ) normalized node density , ( F ) normalized tortuosity , ( G ) normalized branch < 80μm , ( H ) normalized branch > 400μm , ( I ) vessel branch , ( J ) vessel diameter , ( K ) normalized to diameter , ( L ) normalized perfused vessels .

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(E-L) Quantified vascular parameters, for each day of imaging the following parameters were quantified from segmented images and normalized to day one if indicated: (E) normalized node density , (F) normalized tortuosity, (G) normalized branch length < 80μm, (H) normalized branch length > 400μm, (I) vessel branch length, (J) vessel diameter, (K) normalized length to diameter ratio, (L) normalized perfused vessels.


( M - N ) Subcutaneous growth measurements of MC38 ( M ) B16F10 ( N ) tumors .

(M-N) Subcutaneous tumor growth measurements of MC38 (M) and B16F10 (N) tumors.


( A - B ) Representative immunofluorescent of control treated vasculature from MC38 tumors . vessels ( anti - CD31 ) apoptosis ( anti - caspase 3 ) in ( A ) control ( B ) 48 hours after 15 Gy IR . Arrows indicate apoptotic TEC . ( C ) of apoptotic vessels after single dose 15 Gy IR from immunofluorescent of whole MC38 section ( n = 6 - 8 biological replicates per group ) . ( D ) of apoptotic vessels after fractionated 5 × 3 Gy IR from immunofluorescent of whole MC38 sections ( n = 6 - 8 biological replicates per group ) . ( E ) of apoptosis in TECs from B16F10 sections ( n = 4 - 6 biological replicates per group ) . ( F ) of apoptotic vessels according to on thin whole MC38 sections ( Ctrl n = 4 treatment n = 7 biological replicates ) . ( G ) of apoptotic vessels according to in B16F10 sections ( Ctrl n = 4 treatment n = 10 biological replicates ) . ( H - I ) ( H ) diameter ( I ) of apoptotic vessels vs vessels from segmented thick 80 μm MC38 sections ( n = 4 biological replicates ) .

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(A-B) Representative immunofluorescent micrographs of control and treated tumor vasculature from MC38 tumors. Tumor vessels (anti-CD31) and apoptosis (anti-cleaved caspase 3) in (A) control and (B) 48 hours after 15 Gy IR. Arrows indicate apoptotic TEC. (C) quantification of apoptotic vessels after single dose 15 Gy IR from immunofluorescent images of whole MC38 tumor section (n= 6-8 biological replicates per group). (D) Quantification of apoptotic vessels after fractionated 5×3 Gy IR from immunofluorescent images of whole MC38 tumor sections (n= 6-8 biological replicates per group). (E) Quantification of apoptosis in TECs from B16F10 tumor sections (n= 4-6 biological replicates per group). (F) Quantification of apoptotic vessels according to size on thin whole MC38 tumor sections (Ctrl n=4 and treatment n=7 biological replicates). (G) Quantification of apoptotic vessels according to size in B16F10 tumor sections (Ctrl n=4 and treatment n= 10 biological replicates). (H-I) Area (H) and diameter (I) of apoptotic tumor vessels vs all tumor vessels from segmented thick 80 μm MC38 tumor sections (n=4 biological replicates).