( E ) Quantification of HA ‐ ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild ‐ type HA ‐ ULK1 or the DFP mutant . Cells were treated as above , and the spot number counted using Imaris software . A representative immunofluorescence image is shown in Supplementary Figure S3F . * * * and * indicate P ‐ values of 0 . 0001 or 0 . 0158 , respectively . Figure source data can be found with the Supplementary data .

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

(E) Quantification of HA‐ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild‐type HA‐ULK1 or the DFP mutant. Cells were treated as above, and the spot number counted using Imaris software. A representative immunofluorescence image is shown in Supplementary Figure S3F. *** and * indicate P‐values of 0.0001 or 0.0158, respectively. Figure source data can be found with the Supplementary data.


( A ) atg1Δ cells containing either wild ‐ type Atg1 or the kinase ‐ dead Atg1 ‐ T226A mutant tagged with GFP were grown to mid log phase followed by starvation for 24 h in SD ‐ N . Samples were taken at indicated time points and GFP cleavage was analysed by western blotting . Processing of endogenous Ape1 under starvation conditions monitors the autophagic flux .

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 B-MEASURED_VAR O O O O O O O O

(A) atg1Δ cells containing either wild‐type Atg1 or the kinase‐dead Atg1‐T226A mutant tagged with GFP were grown to mid log phase followed by starvation for 24 h in SD‐N. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting. Processing of endogenous Ape1 under starvation conditions monitors the autophagic flux.


( B ) atg1Δ cells containing either GFP ‐ tagged wild ‐ type Atg1 or the Atg1 ‐ VE mutant were grown and analysed as in ( A ) .

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

(B) atg1Δ cells containing either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown and analysed as in (A).


( C ) atg1Δpep4Δ cells containing YFP ‐ tagged wild ‐ type Atg1 or the Atg1 ‐ VE mutant were grown to log phase and starved in SD ‐ N . Cells were monitored by time ‐ lapse microscopy and the vacuolar accumulation of Atg1 was quantified as described in ' Materials and methods ' . Data are plotted as the mean ± s . e . m . from at least 90 quantified cells . Additional images are shown in Supplementary Figure S3H . Bar = 5 μm .

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

(C) atg1Δpep4Δ cells containing YFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown to log phase and starved in SD‐N. Cells were monitored by time‐lapse microscopy and the vacuolar accumulation of Atg1 was quantified as described in 'Materials and methods'. Data are plotted as the mean±s.e.m. from at least 90 quantified cells. Additional images are shown in Supplementary Figure S3H. Bar=5 μm.


( D ) atg1Δatg13Δ cells expressing GFP ‐ tagged wild ‐ type Atg13 , and either wild ‐ type Atg1 or the Atg1 ‐ VE mutant ( left panel ) and wild ‐ type cells containing Atg13 ‐ GFP and either wild ‐ type Atg13 or Atg13 ‐ FV were grown to mid log phase followed by starvation for 6 h in SD ‐ N media . GFP cleavage was analysed by western blotting . Figure source data can be found with the Supplementary data .

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

(D) atg1Δatg13Δ cells expressing GFP‐tagged wild‐type Atg13, and either wild‐type Atg1 or the Atg1‐VE mutant (left panel) and wild‐type cells containing Atg13‐GFP and either wild‐type Atg13 or Atg13‐FV were grown to mid log phase followed by starvation for 6 h in SD‐N media. GFP cleavage was analysed by western blotting. Figure source data can be found with the Supplementary data.


A , B Representative images of OG immunohistochemistry on paraffin - embedded colon sections from control , UC and CD patients ( n = 4 ) . Intensities of staining were scored on the right . Scale bars = 50 μm . ( one - way ANOVA , OGT : Healthy vs UC P = 0 . 0002 , Healthy vs CD P = 0 . 003 ; O - GlcNAc : Healthy vs UC P = 0 . 0365 , Healthy vs CD P = 0 .

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A, B Representative images of OG immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125


A , B Representative images o O - GlcNAc ( B ) immunohistochemistry on paraffin - embedded colon sections from control , UC and CD patients ( n = 4 ) . Intensities of staining were scored on the right . Scale bars = 50 μm . ( one - way ANOVA , OGT : Healthy vs UC P = 0 . 0002 , Healthy vs CD P = 0 . 003 ; O - GlcNAc : Healthy vs UC P = 0 . 0365 , Healthy vs CD P = 0 .

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 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

A, B Representative images o O-GlcNAc (B) immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125


C Levels of intestinal epithelial OGT and O - GlcNAcylation in a second cohort of control , UC , and CD patients . ( one - way ANOVA , OGT : Control n = 10 , UC n = 9 , CD n = 6 ; Control vs UC P = 0 . 035 , Control vs CD P = 0 . 003 ; O - GlcNAc : Control n = 10 , UC n = 8 , CD n = 8 ; Control vs UC P = 0 . 0027 , Control vs CD P = 0 . 008

O O O O O O O O B-MEASURED_VAR 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 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 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

C Levels of intestinal epithelial OGT and O-GlcNAcylation in a second cohort of control, UC, and CD patients. (one-way ANOVA, OGT: Control n=10, UC n=9, CD n=6; Control vs UC P=0.035, Control vs CD P=0.003; O-GlcNAc: Control n=10, UC n=8, CD n=8; Control vs UC P=0.0027, Control vs CD P=0.008


D , E Regression plots of average immune - staining scores of OGT and O - GlcNAcylation against histological scores in UC ( D ) and CD ( E ) ( D : OGT n = 19 , O - GlcNAc n = 18 ; E : OGT n = 15 , O - GlcNAc n = 16 )

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

D, E Regression plots of average immune-staining scores of OGT and O-GlcNAcylation against histological scores in UC (D) and CD (E) (D: OGT n=19, O-GlcNAc n=18; E: OGT n=15, O-GlcNAc n=16)


A Body weight of male wildtype and Vil - Ogt KO mice at 6 weeks of age ( n = 6 , P = 0 . 000015 )

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A Body weight of male wildtype and Vil-Ogt KO mice at 6 weeks of age (n = 6, P = 0.000015)


B Incidence of rectal prolapse in male wildtype and Vil - Ogt KO mice ( WT n = 17 , KO n = 9 , P < 0 . 0001 )

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

B Incidence of rectal prolapse in male wildtype and Vil-Ogt KO mice (WT n = 17, KO n = 9, P < 0.0001)


C Representative colonoscopy images of male wildtype and Vil - Ogt KO mice

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C Representative colonoscopy images of male wildtype and Vil-Ogt KO mice


D Body weight of female wildtype and Vil - Ogt KO mice at 9 weeks of age ( WT n = 11 , heterozygous KO n = 4 , homozygous KO n = 5 , P < 0 . 0001 )

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D Body weight of female wildtype and Vil-Ogt KO mice at 9 weeks of age (WT n = 11, heterozygous KO n = 4, homozygous KO n = 5, P < 0.0001)


E Incidence of rectal prolapse in female wildtype and Vil - Ogt KO mice ( WT n = 9 , heterozygous KO n = 8 , homozygous KO n = 6 , P = 0 . 0439 )

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

E Incidence of rectal prolapse in female wildtype and Vil-Ogt KO mice (WT n = 9, heterozygous KO n = 8, homozygous KO n = 6, P = 0.0439)


F H & E staining of duodenum , jejunum , ileum , colon , and rectum of 10 - week - old male wildtype and Vil - Ogt KO mice . Scale bars = 100 μm

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F H&E staining of duodenum, jejunum, ileum, colon, and rectum of 10-week-old male wildtype and Vil-Ogt KO mice. Scale bars = 100 μm


A Numbers of Paneth cells in the ileum of wildtype and Vil - Ogt KO mice ( n = 6 , P = 0 . 0048 )

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

A Numbers of Paneth cells in the ileum of wildtype and Vil-Ogt KO mice (n = 6, P = 0.0048)


B Representative electron microscopic images of Paneth cells . Scale bars = 1 μm

B Representative electron microscopic images of Paneth cells. Scale bars = 1 μm


C Expression of AMP genes in ileum tissues of wildtype and Vil - Ogt KO mice ( n = 6 , Pan Defesin P = 0 . 031 , Defa3 P = 0 . 049 , Defa4 P = 0 . 013 , Defa5 P = 0 . 023 , Defa6 P = 0 . 034 , Ang4 P = 0 . 031 )

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

C Expression of AMP genes in ileum tissues of wildtype and Vil-Ogt KO mice (n = 6, Pan Defesin P = 0.031, Defa3 P = 0.049, Defa4 P = 0.013, Defa5 P = 0.023, Defa6 P = 0.034, Ang4 P = 0.031)


D Immunoblotting of LC3 in IEC cells isolated from WT and Vil - Ogt KO mice

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

D Immunoblotting of LC3 in IEC cells isolated from WT and Vil-Ogt KO mice


E Representative images of O - GlcNAc staining in Paneth cells of male wildtype and Defa6 - Ogt KO mice . Stars indicate lysozyme - positive Paneth cells

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E Representative images of O-GlcNAc staining in Paneth cells of male wildtype and Defa6-Ogt KO mice. Stars indicate lysozyme-positive Paneth cells


F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 , P = 0 . 024 ) . Arrow indicate Paneth cell . Scale bars = 10 μm

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

F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6-Ogt KO mice (n = 5, P = 0.024). Arrow indicate Paneth cell. Scale bars = 10 μm


G Expression of AMP genes in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 Pan Defesin P = 0 . 0291 , Defa3 P = 0 . 0462 )

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G Expression of AMP genes in the ileum of wildtype and Defa6-Ogt KO mice (n = 5 Pan Defesin P = 0.0291, Defa3 P = 0.0462)


H Representative images of cultured organoid on Day 3


I Expression of inflammatory markers in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 )

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I Expression of inflammatory markers in the ileum of wildtype and Defa6-Ogt KO mice (n = 5)


J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis . Daily changes in body weight ( J ) are shown ( n = 4 - 5 ) . ( L : P = 0 .

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J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes in body weight (J) are shown (n = 4-5). (L: P = 0.049


J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis . Daily changes i colitis scores ( K are shown ( n = 4 - 5 ) . ( L : P = 0 .

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 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

J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes i colitis scores (K are shown (n = 4-5). (L: P = 0.049


J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis RT - qPCR of inflammatory genes in the ileum ( L ) are shown ( n = 4 - 5 ) . ( L : P = 0 .

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J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis RT-qPCR of inflammatory genes in the ileum (L) are shown (n = 4-5). (L: P = 0.049


A Expression of phosphorylated and total STAT3 and NF - κB p65 in the colon of wildtype and Vil - Ogt KO mice

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

A Expression of phosphorylated and total STAT3 and NF-κB p65 in the colon of wildtype and Vil-Ogt KO mice


B Ileum and colon IECs from 10 - week - old wildtype and Vil - Ogt KO mice were isolated for RNA - sequencing analyses . The heatmaps of the sample - to - sample distances are shown

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

B Ileum and colon IECs from 10-week-old wildtype and Vil-Ogt KO mice were isolated for RNA-sequencing analyses. The heatmaps of the sample-to-sample distances are shown


C Volcano plots of gene expression changes . The x - axis specifies the fold - changes ( FC ) and the y - axis specifies the negative logarithm to the base 10 of the p - values adjusted for multiple testing with the Benjamini - Hochberg procedure . FC > 2 and p < 0 . 05 genes are shown in solid dots

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Volcano plots of gene expression changes. The x-axis specifies the fold-changes (FC) and the y-axis specifies the negative logarithm to the base 10 of the p-values adjusted for multiple testing with the Benjamini-Hochberg procedure. FC > 2 and p < 0.05 genes are shown in solid dots


F Levels of STAT1 protein in IECs determined by immunoblotting and O - GlcNAc modification of STAT1 determined by immunoprecipitation using the O - GlcNAc antibody followed by Immunoblotting

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F Levels of STAT1 protein in IECs determined by immunoblotting and O-GlcNAc modification of STAT1 determined by immunoprecipitation using the O-GlcNAc antibody followed by Immunoblotting


G , H Caco - 2 cells were treated with 100 uM Ac45S - GlcNAc for 24 hours , levels of STAT1 and global O - GlcNAcylatio were

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

G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 and global O-GlcNAcylatio were determined


G , H Caco - 2 cells were treated with 100 uM Ac45S - GlcNAc for 24 hours , levels of STAT1 an STAT1 O - GlcNAcylation ( H ) were

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

G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 an STAT1 O-GlcNAcylation (H) were determined


I siRNAs against scrambled sequence or human OGT were transfected in 293T cells and STAT1 levels were determined 48 hours later

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J The pGAS - luc Cis - Reporter plasmid was co - transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 ( n = 4 , P = 0 . 0003 )

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

J The pGAS-luc Cis-Reporter plasmid was co-transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 (n = 4, P=0.0003)


A , B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS . Daily changes in body weight ( A ) and colitis score ( B ) ( n = 5 ) . ( B : day6 P = 0 . 0171 , day7 P = 0 . 0445

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

A, B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS. Daily changes in body weight (A) and colitis score (B) (n= 5). (B: day6 P = 0.0171, day7 P= 0.0445


C Intestinal barrier functional assays measuring serum FITC - dextran before DSS treatment ( n = 5 , P = 0 . 0117 )

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

C Intestinal barrier functional assays measuring serum FITC-dextran before DSS treatment (n = 5, P = 0.0117)


D - G Albumin levels in feces ( D on day 4 of recovery ( n = 5 )

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

D-G Albumin levels in feces (D on day 4 of recovery (n = 5)


colon length ( E ) on day 4 of recovery ( n = 5 )

colon length (E) on day 4 of recovery (n = 5)


H & E staining of colon tissues ( F on day 4 of recovery ( n = 5 ) . Scale bars = 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

H&E staining of colon tissues (F on day 4 of recovery (n = 5). Scale bars = 100 μm


RT - qPCR of inflammatory genes in the colon ( G ) on day 4 of recovery ( n = 5 ) . Scale bars = 100 μm . ( E : P = 0 . 0474 ; G : Tnfa P = 0 . 0190 , Il1b P = 0 .

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

RT-qPCR of inflammatory genes in the colon (G) on day 4 of recovery (n = 5). Scale bars = 100 μm. (E: P = 0.0474; G: Tnfa P = 0.0190, Il1b P = 0.0301


( 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

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 O O O O O O O O O O O

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 ) .

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

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 ) .

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

(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 ) .

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(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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 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 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 .

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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 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 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 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 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

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 .

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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 .

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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 .

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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 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

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 .

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 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 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 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 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

(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 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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 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 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O 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 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 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 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 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 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 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 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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 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 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 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 ) .

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

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 ) .

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

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 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

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 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 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

(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

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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 ) .

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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 ) .

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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 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 .

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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 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 .

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

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

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


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


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 .

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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 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 .

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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 . The 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 and 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 ) and MLH1 ( red ) .

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


Frequencies of autosomal chromosomes with 0 , 1 , and 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 the 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 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 .

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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.


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

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


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

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


The mean number of MSH4 foci per nucleus in control and M1apKI / KI spermatocytes at the 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 staining with antibodies against SYCP3 ( red ) and RAD51 ( green ) on spermatocyte spreads .

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


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

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


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

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


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

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


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 .

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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 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 .

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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 and M1apKI / KI mice . Follicle counts and total oocyte counts 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 and M1ap - / - mice , followed by western blot analyses of M1AP , SHOC1 , and 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 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 .

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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 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 .

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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 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 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 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 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 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 .

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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 the cells .

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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 . 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 .

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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 . 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

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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 . 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 .

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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 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 .

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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 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

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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 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

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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 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

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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 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 .

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

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.