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

O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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 expressing GFP ‐ tagged wild ‐ type Atg13 , either wild ‐ type Atg1 or Atg1 ‐ VE mutant ( left panel ) wild ‐ type containing Atg13 ‐ GFP 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 Supplementary data .

O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY 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 of OG immunohistochemistry on paraffin - embedded colon sections from control , UC CD ( n = 4 ) . Intensities of were scored on 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 B-GENEPROD B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O 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 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 o O - GlcNAc ( B ) immunohistochemistry on paraffin - embedded colon sections from control , UC CD ( n = 4 ) . Intensities of were scored on 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 of intestinal epithelial OGT O - GlcNAcylation in a second cohort of control , UC , CD . ( 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 B-TISSUE O B-CELL_TYPE O B-GENEPROD B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O 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 - scores of OGT O - GlcNAcylation against histological scores in UC ( D ) 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 B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 of wildtype Vil - Ogt KO mice at 6 of ( 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 wildtype Vil - Ogt KO mice ( WT n = 17 , KO n = 9 , P < 0 . 0001 )

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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 of wildtype Vil - Ogt KO mice

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


D of wildtype Vil - Ogt KO mice at 9 of ( 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 wildtype Vil - Ogt KO mice ( WT n = 9 , heterozygous KO n = 8 , homozygous KO n = 6 , P = 0 . 0439 )

O O O B-TISSUE B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-ORGANISM 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 of duodenum , jejunum , ileum , colon , rectum of 10 - week - old wildtype 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 in ileum of wildtype Vil - Ogt KO mice ( n = 6 , P = 0 . 0048 )

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


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

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


C Expression of AMP genes in ileum tissues of wildtype 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 B-EXP_ASSAY O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD 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 isolated from WT Vil - Ogt KO mice

O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O

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


E Representative of O - GlcNAc in Paneth of wildtype Defa6 - Ogt KO mice . Stars indicate lysozyme - positive Paneth

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

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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 ileum of wildtype 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 of cultured organoid on Day 3

H Representative images of cultured organoid on Day 3


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

O O B-EXP_ASSAY O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O O O

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


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

O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-TISSUE O B-ORGANISM O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE O O 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 in body weight (J) are shown (n = 4-5). (L: P = 0.049


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

O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O B-TISSUE O B-ORGANISM O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE O O O 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 Defa6 - Ogt KO mice were transplanted with fecal microbiota from Vil - Ogt KO mice then induced with DSS for colitis RT - qPCR of inflammatory genes in ileum ( L ) are shown ( n = 4 - 5 ) . ( L : P = 0 .

O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O B-TISSUE O B-ORGANISM O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE 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 RT-qPCR of inflammatory genes in the ileum (L) are shown (n = 4-5). (L: P = 0.049


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

O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O B-TISSUE O O B-GENEPROD O B-GENEPROD O B-ORGANISM

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


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

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

O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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 of STAT1 in IECs determined by immunoblotting O - GlcNAc modification of STAT1 determined by immunoprecipitation using O - GlcNAc antibody followed by Immunoblotting

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 were treated with 100 uM Ac45S - GlcNAc for 24 hours , of STAT1 global O - GlcNAcylatio were

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

O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 or human OGT were transfected in 293T STAT1 were determined 48 hours later

O O O O O O B-ORGANISM O B-GENEPROD O O O B-CELL_LINE B-GENEPROD O O O O O O

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


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

O O O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE O B-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD 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 before induction of colitis by DSS . Daily changes in ( A ) colitis score ( B ) ( n = 5 ) . ( B : day6 P = 0 . 0171 , day7 P = 0 . 0445

O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O 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 B-TISSUE O O O O B-TISSUE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 in feces ( D on day 4 of recovery ( n = 5 )

O O O O B-GENEPROD O B-TISSUE 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 ( E ) on day 4 of recovery ( n = 5 )

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


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

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O O O O 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-GENEPROD O O O O O O B-GENEPROD 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 of M . tb 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 mean ± SD ( n = 3 wells per group ) data shown are representative of three independent experiments ( biological replicates ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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 for IFN - β mRNA ( B ) in wild type BMMs 5 24 hr post - treatment with EVs respectively ( 1 μg / ml equals approximately 3 x 108 vesicles / ml ) . 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (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 ( C ) in wild type BMMs 5 24 hr post - treatment with EVs respectively ( 1 μg / ml equals approximately 3 x 108 vesicles / ml ) . 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of 10μg / ml for times indicated . 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 B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 for IFN - β mRNA ( E ) in either WT , MAVS - / 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 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ( 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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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 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 B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O

(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 .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O O

(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-GENEPROD B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_TYPE 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 .

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


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( 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 B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-SUBCELLULAR O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-SUBCELLULAR O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

(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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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 .

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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 ) 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 - .

B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

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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 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 - .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O O B-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-SUBCELLULAR O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O O B-CELL_TYPE O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SUBCELLULAR O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O 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 B-SUBCELLULAR O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-SUBCELLULAR O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O O O O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-SUBCELLULAR O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-SUBCELLULAR O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O O O O O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

B-GENEPROD O O O B-CELL_TYPE O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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(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

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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 ) .

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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

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 homozygous M1AP splicing mutation in heterozygous mutation in their fertile brother . arrowhead indicates position of 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 of M1AP expression in various tissues from adult wild - type ( WT ) mice . GAPDH served as 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 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 .

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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 .

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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 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-TISSUE B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O

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 .

O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O 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 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

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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

O O O O B-EXP_ASSAY B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_LINE O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O B-EXP_ASSAY B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_LINE O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE 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.