( A ) Immunoblot of lysates from wild - type , PERK - KO GCN2 - KO MEF incubated in presence of DMSO ( Control ) , Tunicamycin ( 0 . 1 µg / ml ) , or starved in EBSS for 16 h . Data are representative of three independent experiments .

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(A) Immunoblot of protein lysates from wild-type, PERK-KO and GCN2-KO MEF cells incubated in the presence of DMSO (Control), Tunicamycin (0.1 µg/ml), or starved in EBSS for 16 h. Data are representative of three independent experiments.


( B - D ) Wild - type ( B ) , GCN2 - KO ( C ) , PERK - KO ( D ) MEF incubated in presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) as indicated in ( A ) . were fixed , permeabilized stained with antibodies against TFE3 ( green ) CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments . ( E - F ) of of MEF with TFE3 upon DMSO ( Control ) , Tunicamycin treatments for 16 h ( E ) , or starvation for 2 h ( F ) ( mean ± SD of three independent experiments , one - way ANOVA versus DMSO treated , ns = not significant , * * * p < 0 . 001 ; n > 400 per condition ) .

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(B-D) Wild-type (B), GCN2-KO (C), and PERK-KO (D) MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) as indicated in (A). Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(E-F) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control), Tunicamycin treatments for 16 h (E), or starvation for 2 h (F) (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated cells, ns= not significant, ***p < 0.001; n>400 cells per condition).


( G ) of phospho - 4EBP1 in wild - type GCN2 - KO MEF upon starvation for 2 h ( mean ± SD of fold increase of phospho - 4EBP1 to total 4EBP1 from three independent experiments , one - way ANOVA versus starvation treated wild - type , * * * p < 0 . 001 ) .

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(G) Quantification of phospho-4EBP1 protein levels in wild-type and GCN2-KO MEF cells upon starvation for 2 h (the mean ± SD of the fold increase of the phospho-4EBP1 to total 4EBP1 ratio from three independent experiments, one-way ANOVA analysis versus starvation treated wild-type cells, ***p < 0.001).


( H ) of of MEF with TFE3 upon DMSO ( Control ) or Torin - 1 ( 250 nM ) treatments for 1 h ( mean ± SD of three independent experiments , one - way ANOVA versus DMSO treated wild - type , * * * p < 0 . 001 ; n > 400 per condition ) .

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(H) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or Torin-1 (250 nM) treatments for 1 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated wild-type cells, ***p < 0.001; n>400 cells per condition).


( A ) ARPE - 19 infected with either adenovirus expressing TFE3 - WT - Myc or TFE3 - S321A - Myc for 16h . were fixed , permeabilized stained with antibodies against Myc . Scale bar , 10 µm . of of ARPE - 19 with TFE3 - Myc ( means ± SD of 3 independent experiments , students t - , * * * * p < 0 . 0001 ; n > 400 per condition ) .

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(A) ARPE-19 cells infected with either adenovirus expressing TFE3-WT-Myc or TFE3-S321A-Myc for 16h. Cells were fixed, permeabilized and stained with antibodies against Myc. Scale bar, 10 µm. Quantification of the percentage of ARPE-19 cells with nuclear TFE3-Myc (means ± SD of 3 independent experiments, students t-test, ****p < 0.0001; n>400 cells per condition).


( B ) Immunoblots of lysates from ARPE - 19 infected as indicated in ( A ) . Data are representative of three independent experiments .

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(B) Immunoblots of protein lysates from ARPE-19 cells infected as indicated in (A). Data are representative of three independent experiments.


( C ) Immunoblots of TFE3 - Ser321 phosphorylation state in fractions of ARPE - 19 incubated with DMSO ( Ctrl . ) , Tunicamycin ( Tun . ) , or Torin - 1 . Data are representative of three independent experiments .

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(C) Immunoblots of TFE3-Ser321 phosphorylation state in nuclear fractions of ARPE-19 cells incubated with DMSO (Ctrl.), Tunicamycin (Tun.), or Torin-1. Data are representative of three independent experiments.


( D ) Immunoblots of of a GST pull - down from fractions of MEF treated with DMSO or Tunicamycin ( 5 µg / ml ) ( Tun . ) for 16 h . Data are representative of three independent experiments .

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(D) Immunoblots of protein of a GST pull-down from nuclear fractions of MEF cells treated with DMSO or Tunicamycin (5 µg/ml) (Tun.) for 16 h. Data are representative of three independent experiments.


( E ) Immunoblots of lysates from ARPE - 19 incubated in presence of DMSO ( Ctrl . ) , Tunicamycin ( 5 µg / ml ) ( Tun . ) , or starved in EBSS ( Strv . ) for 16 h . Data are representative of three independent experiments .

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(E) Immunoblots of protein lysates from ARPE-19 cells incubated in the presence of DMSO (Ctrl.), Tunicamycin (5 µg/ml) (Tun.), or starved in EBSS (Strv.) for 16 h. Data are representative of three independent experiments.


( F ) of of ARPE - 19 with TFE3 upon Tunicamycin ( Tun . ) or Tunicamycin + FK506 treatments for 16 h ( mean ± SD of three independent experiments , one - way ANOVA versus Tunicamycin treated , * * * p < 0 . 001 ; n > 400 per condition ) .

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(F) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Tunicamycin + FK506 treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus Tunicamycin treated cells, ***p < 0.001; n>400 cells per condition).


( G ) of of calcineurin depleted ARPE - 19 with TFE3 upon Tunicamycin ( Tun . ) or Starvation ( Strv . ) treatments for 16 h ( mean ± SD of three independent experiments , one - way ANOVA versus siNon - target treated within indicated treatment , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; n > 400 per condition ) .

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(G) Quantification of the percentage of calcineurin depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Starvation (Strv.) treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment, ns= not significant, *p < 0.05, **p < 0.01, ***p < 0.001; n>400 cells per condition).


( H ) Immunoblots of lysates from ARPE - 19 depleted of Calcineurin catalytic subunits PPP3CA PPP3CB . Data are representative of three independent experiments .

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(H) Immunoblots of protein lysates from ARPE-19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB. Data are representative of three independent experiments.


( I ) Relative Quantitative Real - Time PCR of PPP3CA PPP3CB mRNA transcript in ARPE - 19 depleted of Calcineurin catalytic subunits PPP3CA PPP3CB ( mean ± SD of fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analsysis versus siNon - Target ( siNT ) treated , * * * p < 0 . 001 ) .

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(I) Relative Quantitative Real-Time PCR analysis of PPP3CA and PPP3CB mRNA transcript levels in ARPE-19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analsysis versus the siNon-Target (siNT) treated cells, ***p < 0.001).


( J ) of of ATF4 depleted ARPE - 19 with TFE3 upon Tunicamycin treatment for 16 h ( mean ± SD of two independent experiments , one - way ANOVA versus siNon - target treated within indicated treatment * * * p < 0 . 001 ; n > 400 per condition ) .

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(J) Quantification of the percentage of ATF4 depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin treatment for 16 h (mean ± SD of two independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment***p < 0.001; n>400 cells per condition).


( A ) Relative Quantitative Real - Time PCR of TFEB TFE3 mRNA transcript in ARPE - 19 depleted of TFEB TFE3 ( mean ± SD of fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , student ' s t - versus siNon - Target ( siNT ) treated , * * * p < 0 . 001 ) .

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(A) Relative Quantitative Real-Time PCR analysis of TFEB and TFE3 mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, student's t-test versus siNon-Target (siNT) treated cells, ***p < 0.001).


( B ) Relative Quantitative Real - Time PCR of MCOLN1 , ATP6V1C1 , HEXA UVRAG mRNA transcript in ARPE - 19 depleted of TFEB TFE3 upon incubation with DMSO ( Control ) or Tunicamycin ( 5 µg / ml ) for 16 h ( mean ± SD of fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA versus same treatment condition in siNon - Target ( siNT ) treated or versus DMSO condition in siNon - Target ( siNT ) treated , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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(B) Relative Quantitative Real-Time PCR analysis of MCOLN1, ATP6V1C1, HEXA and UVRAG mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 upon incubation with DMSO (Control) or Tunicamycin (5 µg/ml) for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the same treatment condition in the siNon-Target (siNT) treated cells or versus the DMSO condition in siNon-Target (siNT) treated cells, *p < 0.05, **p < 0.01, ***p < 0.001).


( A ) Immunoblots of lysates from ARPE - 19 infected with either adenovirus expressing Null , TFEB - S211A , or TFE3 upon incubation with DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) or starved in EBSS for 16 h . Data are representative of five independent experiments . ( B ) of ATF4 , CHOP GADD34 in ARPE - 19 treated as indicated in ( A ) ( mean ± SD of fold change of indicated to actin from three independent experiments , one - way ANOVA versus corresponding treatment condition in adenovirus - Null infected , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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(A) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A, or TFE3 upon incubation with DMSO (Control), Tunicamycin (5 µg/ml) or starved in EBSS for 16 h. Data are representative of five independent experiments.(B) Quantification of ATF4, CHOP and GADD34 protein levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, *p < 0.05, **p < 0.01, ***p < 0.001).


( C ) Relative Quantitative Real - Time PCR of ATF4 , CHOP , GADD34 MCOLN1 mRNA transcript in ARPE - 19 treated as indicated in ( A ) ( mean ± SD of fold change of indicated genes normalized to actin mRNA from three independent experiments , one - way ANOVA versus corresponding treatment condition in adenovirus - Null infected , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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(C) Relative Quantitative Real-Time PCR analysis of ATF4, CHOP, GADD34 and MCOLN1 mRNA transcript levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the RNA fold change of indicated genes normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, **p < 0.01, ***p < 0.001).


( A - D ) Relative Quantitative Real - Time PCR of ATF4 ( A ) , ASNS ( B ) , CAT1 ( C ) xCT ( D ) mRNA transcript in ATF4 - depleted ARPE - 19 infected with either adenovirus expressing Null or TFEB - S211A upon incubation with DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) , or starved in EBSS for 16 h ( mean ± SD of fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA versus corresponding treatment condition in adenovirus - Null infected siRNA Non - Target ( siNT ) treated , * p < 0 . 05 , * * * p < 0 . 001 ) .

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(A-D) Relative Quantitative Real-Time PCR analysis of ATF4 (A), ASNS (B), CAT1 (C) and xCT (D) mRNA transcript levels in ATF4-depleted ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A upon incubation with DMSO (Control), Tunicamycin (5 µg/ml), or starved in EBSS for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected and siRNA Non-Target (siNT) treated cells, *p < 0.05, ***p < 0.001).


( E ) ChIP - qPCR of ATF4 promoter 2000bp upstream of region of interest ( ATF4 - Control ) from MEF that were untreated ( control ) , starved for 2 h , or treated with Tunicamycin ( 0 . 1 µg / ml ) for 16 h . Amplification regions are indicated by arrows . was immunoprecipitated with antibodies for anti - TFE3 . Bar graphs show amount of immunoprecipitated detected by real - time PCR . Values were normalized to input plotted as relative enrichment compared to untreated conditions ( means ± SD of three independent experiments , students t - , * p < 0 . 05 , * * p < 0 . 01 ) .

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(E) ChIP-qPCR analysis of the ATF4 promoter and 2000bp upstream of the region of interest (ATF4-Control) from MEF cells that were untreated (control), starved for 2 h, or treated with Tunicamycin (0.1 µg/ml) for 16 h. Amplification regions are indicated by arrows. Chromatin DNA was immunoprecipitated with antibodies for anti-TFE3. Bar graphs show the amount of immunoprecipitated DNA detected by the real-time PCR assay. Values were normalized to the input and plotted as relative enrichment compared to untreated conditions (means ± SD of three independent experiments, students t-test, *p < 0.05, **p < 0.01).


( F ) Relative Quantitative Real - Time PCR of SUMF1 , DERL1 , ATF6 , APEX1 GPX1 mRNA transcript in ARPE - 19 infected with either adenovirus expressing Null or TFEB - S211A ( mean ± SD of fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , students t - , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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(F) Relative Quantitative Real-Time PCR analysis of SUMF1, DERL1, ATF6, APEX1 and GPX1 mRNA transcript levels in ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, students t-test, **p < 0.01, ***p < 0.001).


( G ) Immunoblots of lysates from ARPE - 19 infected with either adenovirus expressing Null or TFEB - S211A . Data are representative of three independent experiments . ( H ) of IRE1α APEX1 in ARPE - 19 infected as indicated in ( G ) ( mean ± SD of fold increase of indicated to actin from three independent experiments , students t - , * * * p < 0 . 001 ) .

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(G) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A. Data are representative of three independent experiments.(H) Quantification of IRE1α and APEX1 protein levels in ARPE-19 cells infected as indicated in (G) (mean ± SD of the fold increase of the indicated protein to actin ratio from three independent experiments, students t-test, ***p < 0.001).


( A ) Immunoblots of lysates from Null - or TFEB / 3 knockout - MEF incubated in presence of DMSO ( Control ) or Tunicamycin ( Tun . ) ( 0 . 1 µg / ml ) for indicated times . Data are representative of three independent experiments . ( B ) of ATF4 in Null - or TFEB / 3 knockout - MEF treated as indicated in ( A ) ( mean ± SD of ATF4 to actin from three independent experiments , student ' s t - versus corresponding time point in Null - MEF , ns = not significant , * * p < 0 . 01 ) .

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(A) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times. Data are representative of three independent experiments.(B) Quantification of ATF4 protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (A) (mean ± SD of the ATF4 to actin protein ratio from three independent experiments, student's t-test versus the corresponding time point in Null-MEF cells, ns= not significant, **p < 0.01).


( C ) viability was determined in Null - or TFEB / 3 knockout - MEF incubated in presence of DMSO ( Control ) or Tunicamycin ( Tun . ) ( 0 . 1 µg / ml ) for indicated times ( mean ± SD of of viable compared to control from three independent experiments , one - way ANOVA versus corresponding time point in Null - MEF * * * p < 0 . 001 ) .

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(C) Cell viability was determined in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times (mean ± SD of the percentage of viable cells compared to control from three independent experiments, one-way ANOVA analysis versus the corresponding time point in Null-MEF cells ***p < 0.001).


( D ) condensation , an indication of undergoing apoptosis , was assessed in Null - or TFEB / 3 knockout - MEF incubated in presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) for indicated times . Confocal microscopy were obtained from fixed stained with Hoechst 33342 . Scale bar , 20 µm . Data are representative of three independent experiments .

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(D) Chromatin condensation, an indication of cells undergoing apoptosis, was assessed in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Confocal microscopy images were obtained from fixed cells stained with Hoechst 33342. Scale bar, 20 µm. Data are representative of three independent experiments.


( E ) Immunoblots of lysates from Null - or TFEB / 3 knockout - MEF treated as described in ( A ) . Data are representative of three independent experiments . ( F - H ) of CHOP ( F ) , PUMA ( G ) NOXA ( H ) in Null - or TFEB / 3 knockout - MEF treated as indicated in ( E ) ( mean ± SD of indicated to actin from three independent experiments , student ' s t - versus corresponding time point in Null - MEF , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ) .

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(E) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells treated as described in (A). Data are representative of three independent experiments.(F-H) Quantification of CHOP (F), PUMA (G) and NOXA (H) protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (E) (mean ± SD of the indicated protein to actin ratio from three independent experiments, student's t-test analysis versus the corresponding time point in Null-MEF cells, ns= not significant, *p < 0.05, **p < 0.01).


( I ) adenovirus of lysates from ARPE - 19 infected with either adenovirus expressing Null , TFEB - S211A or TFE3 . Data are representative of three independent experiments . ( J ) of PUMA in ARPE - 19 infected as indicated in ( I ) ( mean ± SD of fold change of indicated to actin from three independent experiments , one - way ANOVA versus adenovirus - Null infected , * p < 0 . 05 , * * * p < 0 . 001 ) .

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(I) adenovirus of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A or TFE3. Data are representative of three independent experiments.(J) Quantification of PUMA protein levels in ARPE-19 cells infected as indicated in (I) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the adenovirus-Null infected cells, *p < 0.05, ***p < 0.001).


( A ) Serum IgG ELISA reactivity with MTB ( H37Rv ) whole lysate , purified fraction secreted culture filtrate ( CFP ) for TB healthy donors ( HD ) . ELISA graphs ( left ; TB : black lines ; n = 25 ; HD : red dotted lines ; n = 2 ) under curve ( AUC ) values for tested samples ( right ; TB : n = 25 ; HD : n = 17 ) are shown . Median is shown .

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(A) Serum IgG ELISA reactivity with MTB (H37Rv) whole cell lysate, purified cell membrane fraction and secreted culture filtrate proteins (CFP) for TB patients and healthy donors (HD). ELISA graphs (left; TB: black lines; n=25; HD: red dotted lines; n=2) and area under the curve (AUC) values for all tested samples (right; TB: n=25; HD: n=17) are shown. Median is shown.


( B ) AUC values for IgA serum ELISA response against MTB whole lysate , purified fraction secreted culture filtrate ( CFP ) of TB ( n = 25 ) healthy donors ( HD ; n = 9 ) . Median is shown .

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(B) AUC values for the IgA serum ELISA response against MTB whole cell lysate, purified cell membrane fraction and secreted culture filtrate proteins (CFP) of TB patients (n=25) and healthy donors (HD; n=9). Median is shown.


( C ) Flow cytometry gating strategy frequency of circulating plasmablasts ( CD19 + CD27 + + CD38 + ) for one representative TB ( TB24 ) HD ( left ) . Frequency of circulating CD19 + CD27 + + CD38 + plasmablasts of CD19 + B in peripheral blood of TB ( n = 24 ) HD ( n = 8 ) . Dashed indicates threshold for detectable plasmablast populations ( right ) . Median are shown .

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(C) Flow cytometry gating strategy and frequency of circulating plasmablasts (CD19+CD27++CD38+) for one representative TB patient (TB24) and HD (left). Frequency of circulating CD19+CD27++CD38+plasmablasts of all CD19+ B cells in peripheral blood of TB patients (n=24) and HD (n=8). Dashed line indicates threshold for detectable plasmablast populations (right). Median and SEM are shown.


( D ) Expression of CD19 activation markers ( CD86 , CD84 CD24 ) in plasmablasts memory B as measured by flow cytometry .

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(D) Expression of CD19 and activation markers (CD86, CD84 and CD24) in plasmablasts and memory B cells as measured by flow cytometry.


( E ) Plasmablast response of TB7 ( top ) IgG serum response against MTB lysate of TB7 , TB19 TB39 ( bottom ) one HD at indicated time points before after treatment onset .

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(E) Plasmablast response of patient TB7 (top) and IgG serum response against MTB cell lysate of patient TB7, TB19 and TB39 (bottom) and one HD at the indicated time points before and after treatment onset.


( F ) Dots indicate AUC values for anti - MTB lysate IgG serum ELISA response ( y - axis ) versus frequency of circulating plasmablasts ( x - axis ) for TB .

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(F) Dots indicate AUC values for the anti-MTB cell lysate IgG serum ELISA response (y-axis) versus frequency of circulating plasmablasts (x-axis) for individual TB patients.


( A ) Gating strategy , phenotype frequency of circulating plasmablasts ( CD19 + CD27 + + CD38 + ) isolated by flow cytometric sorting from 3 TB ( TB7 , TB24 , TB33 ) in comparison to one representative HD . Boxes indicate sort gates . plasmablast frequency is indicated .

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(A) Gating strategy, phenotype and frequency of circulating plasmablasts (CD19+CD27++CD38+) isolated by flow cytometric cell sorting from 3 TB patients (TB7, TB24, TB33) in comparison to one representative HD. Boxes indicate sort gates. The plasmablast frequency is indicated.


( B ) Absolute number of somatic hypermutations ( SHM ) in IGHV , IGKV IGLV segments of IgA IgG plasmablast antibody genes sequenced from TB7 , TB24 TB33 . absolute number of sequences analyzed is indicated below graph . Geometric means with are indicated in grey . SHM means of historic data from sorted CD27 + IgA + or CD27 + IgG + from peripheral blood of HD are indicated in red for comparison ( Berkowska et al , 2015a ; Tiller et al , 2007 ) .

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(B) Absolute number of somatic hypermutations (SHM) in the IGHV, IGKV and IGLV segments of IgA and IgG plasmablast antibody genes sequenced from TB7, TB24 and TB33. The absolute number of sequences analyzed is indicated below the graph. Geometric means with SEM are indicated in grey. SHM means of historic data from sorted CD27+IgA+ or CD27+IgG+ cells from peripheral blood of HD are indicated in red for comparison (Berkowska et al, 2015a; Tiller et al, 2007).


( C ) Isotype distribution of plasmablast memory B from TB7 , TB24 TB33 . PB , plasmablasts ( CD19 + CD27 + + CD38 + ) ; M , memory B ( CD19 + CD27 + ) .

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(C) Isotype distribution of plasmablast and memory B cells from TB7, TB24 and TB33. PB, plasmablasts (CD19+CD27++CD38+); M, memory B cells (CD19+CD27+).


( D ) Gating strategy , phenotype frequency of HBHA - reactive memory B ( CD19 + CD27 + HBHA + ) in peripheral blood of one representative TB ( TB29 ) , health - care worker ( HCW ) 2 HD , respectively .

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(D) Gating strategy, phenotype and frequency of HBHA-reactive memory B cells (CD19+CD27+HBHA+) in peripheral blood of one representative TB patient (TB29), health-care worker (HCW) 2 and HD, respectively.


( E ) Dots indicate frequency of HBHA - reactive memory B out of CD27 + memory B in TB ( n = 23 ) HCWs ( n = 7 ) . Mean are indicated . P - value was determined using Wilcoxon - Mann - Whitney ; * * * * p < 0 . 0001 .

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(E) Dots indicate frequency of HBHA-reactive memory B cells out of all CD27+ memory B cells in individual TB patients (n=23) and HCWs (n=7). Mean and SEM are indicated. P-value was determined using Wilcoxon-Mann-Whitney test; ****p<0.0001.


( F ) Dots indicate frequency of resting memory B ( CD19 + CD27 + CD10 - ) out of B in peripheral blood of TB compared to HD .

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(F) Dots indicate the frequency of resting memory B cells (CD19+CD27+CD10-) out of all B cells in peripheral blood of individual TB patients compared to HD.


( G ) Anti - HBHA serum IgG ELISA reactivity for TB HCW ( black lines ) compared to two representative HD ( red lines ) . Dashed indicates threshold OD405nm for positive reactivity ( left ) . Asterisks indicate serum responses of donors selected for single sorting of HBHA - reactive memory B . Data are representative of two independent experiments .

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(G) Anti-HBHA serum IgG ELISA reactivity for TB patients and HCW (black lines) compared to two representative HD (red lines). Dashed line indicates the threshold OD405nm for positive reactivity (left). Asterisks indicate serum responses of donors selected for single sorting of HBHA-reactive memory B cells. Data are representative of two independent experiments.


( H ) Absolute number of somatic hypermutations ( SHM ) in IGHV ( IGHA , IGHG IGHM ) , IGKV or IGLV segments of sorted anti - HBHAmemory from TB HCWs . Geometric means with are indicated in grey . For comparison , red lines indicate historic SHM means for randomly sorted CD27 + IgA + , CD27 + IgG + or CD27 + IgM + from peripheral blood of HDs ( Berkowska et al , 2015a ; Tiller et al , 2007 ; Tsuiji et al , 2006 ) .

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(H) Absolute number of somatic hypermutations (SHM) in the IGHV (IGHA, IGHG and IGHM), IGKV or IGLV segments of sorted anti-HBHAmemory cells from TB patients and HCWs. Geometric means with SEM are indicated in grey. For comparison, red lines indicate the historic SHM means for randomly sorted CD27+IgA+, CD27+IgG+ or CD27+IgM+ cells from peripheral blood of HDs (Berkowska et al, 2015a; Tiller et al, 2007; Tsuiji et al, 2006).


( I ) Number of clonally expanded B clusters among HBHA - reactive memory B from two TB ( TB35 , TB29 ) HCWs ( HCW1 , HCW 2 ) . in clusters are indicated in grey , single are indicated in . No B clusters were shared between donors .

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(I) Number and size of clonally expanded B cell clusters among HBHA-reactive memory B cells from two TB patients (TB35, TB29) and HCWs (HCW1, HCW 2). Cells in clusters are indicated in grey, single cells are indicated in white. No B cell clusters were shared between donors.


( J ) IgA , IgG IgM isotype distribution of single - - sorted HBHA - reactive memory . Mean are indicated .

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(J) IgA, IgG and IgM isotype distribution of single-cell-sorted HBHA-reactive memory cells. Mean and SEM are indicated.


( A , B ) Representative ELISA graphs show reactivity of antibodies from TB7 to ( A ) MTB whole lysate or ( B ) MTB ( left ) . Dashed red indicates threshold OD405nm for positive reactivity . Green indicates negative control antibody ( mGO53 ; ( Wardemann et al , 2003 ) ) . AUC ( under curve ) values indicate reactivity to ( A ) MTB lysate or ( B ) whole MTB for antibodies from TB7 , TB24 , TB33 . Total numbers of analyzed antibodies are indicated ( right ) .

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(A, B) Representative ELISA graphs show reactivity of antibodies from patient TB7 to (A) MTB whole cell lysate or (B) MTB bacteria (left). Dashed red line indicates the threshold OD405nm for positive reactivity. Green line indicates negative control antibody (mGO53; (Wardemann et al, 2003)). AUC (area under curve) values indicate the reactivity to (A) MTB cell lysate or (B) whole MTB bacteria for antibodies from TB7, TB24, TB33. Total numbers of analyzed antibodies are indicated (right).


( D ) ELISA graph shows reactivity to MTB fraction for MTB - reactive ( n = 26 ) non - reactive ( n = 14 ) antibodies .

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(D) ELISA graph shows reactivity to the MTB cell membrane fraction for MTB-reactive (n=26) and non-reactive (n=14) antibodies.


( E ) Reactivity to whole BCG as determined by flow cytometry for three representative MTB lysate - reactive antibodies one non - reactive antibody ( isotype control , mGO53 ) .

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(E) Reactivity to whole BCG bacteria as determined by flow cytometry for three representative MTB cell lysate-reactive antibodies and one non-reactive antibody (isotype control, mGO53).


( A ) Binding of antibodies TB24PB037 TB33PB123 to LAM purified from MTB as determined by Western Blot . negative isotype control ( mGO53 ) a commercially available anti - LAM antibody ( positive control ) are shown for comparison .

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(A) Binding of antibodies TB24PB037 and TB33PB123 to LAM purified from MTB as determined by Western Blot. The negative isotype control (mGO53) and a commercially available anti-LAM antibody (positive control) are shown for comparison.


( B ) Anti - BCG specificity of LAM - reactive antibody TB24PB037 ( black ) compared to E . coli as determined by ELISA with whole ( left ) flow cytometry ( right ) .

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(B) Anti-BCG specificity of the LAM-reactive antibody TB24PB037 (black) compared to E.coli as determined by ELISA with whole bacteria (left) and flow cytometry (right).


( C ) Anti - LAMELISA for TB24PB037 or negative control antibody at indicated concentrations . mean ± SD of absorbance was calculated .

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(C) Anti-LAMELISA for TB24PB037 or negative control antibody at the indicated concentrations. The mean ±SD of the absorbance was calculated.


( D ) Antibody TB24PB037 binds to MTB - LAM but not to LAM from M . smegmatis . ELISA performed as described in ( C ) .

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(D) Antibody TB24PB037 binds to MTB-LAM but not to LAM from M. smegmatis. ELISA performed as described in (C).


( E ) Representative HBHA ELISA for antibodies cloned from HBHA - reactive memory B of TB35 HCW1 . Dashed red indicates threshold OD405nm for positive reactivity . Green indicates negative control antibody ( mGO53 ; ( Wardemann et al , 2003 ) ) .

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(E) Representative HBHA ELISA for antibodies cloned from HBHA-reactive memory B cells of patient TB35 and HCW1. Dashed red line indicates the threshold OD405nm for positive reactivity. Green line indicates negative control antibody (mGO53; (Wardemann et al, 2003)).


( F ) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity .

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(F) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity.


( G ) microscopy shows BCG - MTB - reactivity of representative anti - HBHA antibodies compared to a non - reactive isotype control antibody . Scale bar 10 μm ( top / bottom ) .

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(G) Fluorescence microscopy shows BCG- and MTB-reactivity of representative anti-HBHA antibodies compared to a non-reactive isotype control antibody. Scale bar 10 μm (top/bottom).


Relative ( CFU ) compared to medium control ( 100 % ) in human lung epithelial A549 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies non - reactive isotype control as indicated . ( A ) Representative results for MTB - reactive plasmablast antibodies . Mean are indicated .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (A) Representative results for individual MTB-reactive plasmablast antibodies. Mean and SEM are indicated.


Relative ( CFU ) compared to medium control ( 100 % ) in human lung epithelial A549 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies non - reactive isotype control as indicated . ( B ) Direct comparison of tested MTB - reactive IgA ( n = 12 ) IgG ( n = 26 ) antibodies . Mean are indicated .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (B) Direct comparison of all tested MTB-reactive IgA (n=12) and IgG (n=26) antibodies. Mean and SEM are indicated.


Relative ( CFU ) compared to medium control ( 100 % ) in human lung epithelial A549 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies non - reactive isotype control as indicated . ( C ) Representative results for HBHA - reactive memory B antibodies from TB HCW .

Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (C) Representative results for individual HBHA-reactive memory B cell antibodies from TB patients and HCW.


Relative ( CFU ) compared to medium control ( 100 % ) in human lung epithelial A549 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies non - reactive isotype control as indicated . ( D ) Same as in ( B ) but with HBHA - reactive antibodies as shown in ( C ) . ( D ) Direct comparison tested HBHA - reactive IgA ( n = 6 ) IgG ( n = 7 ) antibodies .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (D) Same as in (B) but with HBHA-reactive antibodies as shown in (C). (D) Direct comparison all tested HBHA-reactive IgA (n=6) and IgG (n=7) antibodies.


Relative ( CFU ) compared to medium control ( 100 % ) in human lung epithelial A549 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies non - reactive isotype control as indicated . ( E ) Direct comparison of indicated MTB - reactive antibodies expressed as IgA IgG . Mean are indicated . P - values were determined using non - parameric t - , * p < 0 . 1 ; * * p < 0 . 01 , ns : p > 0 . 5 .

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Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (E) Direct comparison of the indicated MTB-reactive antibodies expressed as IgA and IgG. Mean and SEM are indicated. P-values were determined using non-parameric t-test, * p<0.1; **p<0.01, ns: p>0.5.


( A ) Human lung epithelial A549 bronchial epithelial ( hAECB ) lack FcγR ( CD16 , CD64 CD32 ) FcαR ( CD89 ) surface expression as analyzed by flow cytometry . Human THP1 serve as positive control .

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(A) Human lung epithelial A549 cells and primary bronchial epithelial cells (hAECB) lack FcγR (CD16, CD64 and CD32) and FcαR (CD89) cell surface expression as analyzed by flow cytometry. Human THP1 cells serve as positive control.


( B ) neonatal FcR ( FcRn ) is expressed in both A549 human THP1 as determined by Western Blotting . Data are representative of at least two independent experiments .

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(B) The neonatal FcR (FcRn) is expressed in both A549 cells and human THP1 cells as determined by Western Blotting. Data are representative of at least two independent experiments.


( C ) Absolute ( CFUs ) in human THP1 macrophages 1 h after infection with MTB pre - incubated with LAM - reactive IgA1 ( left ) or IgG1 ( right ) antibody TB24PB037 or respective isotype control antibody as indicated . Mean are indicated .

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(C) Absolute bacterial counts (CFUs) in human THP1 macrophages 1 h after infection with MTB pre-incubated with the LAM-reactive IgA1 (left) or IgG1 (right) antibody TB24PB037 or the respective isotype control antibody as indicated. Mean and SEM are indicated.


( A ) Absolute ( CFUs ) in human lung epithelial A549 1 h after infection with MTB pre - incubated with purified serum IgA ( left ) or IgG ( right ) antibodies from TB ( TB ) compared to HD as indicated . Mean are indicated . Data are representative of three independent experiments with three technical replicates each .

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(A) Absolute bacterial counts (CFUs) in human lung epithelial A549 cells 1 h after infection with MTB pre-incubated with purified serum IgA (left) or IgG (right) antibodies from TB patients (TB) compared to HD as indicated. Mean and SEM are indicated. Data are representative of three independent experiments with three technical replicates each.


( B ) Direct comparison of purified serum IgA IgG antibodies on epithelial invasion as measured in ( A ) . P - value was determined using Wilcoxon - Mann - Whitney ; * * * * p < 0 . 0001 .

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(B) Direct comparison of purified serum IgA and IgG antibodies on epithelial cell invasion as measured in (A). P-value was determined using Wilcoxon-Mann-Whitney test; ****p<0.0001.


Ten - fold serial dilutions of 1 OD600 unit / mL of indicated strains were spotted on SD plates containing 0 ( C - ) or 1 mM choline ( C + ) 0 ( I - ) or 75 μM inositol ( I + ) incubated at 30oC for 3 d . A representative experiment is shown ( from n = 5 ) .

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Ten-fold serial dilutions of 1 OD600 unit/mL of the indicated strains were spotted on SD plates containing 0 (C-) or 1 mM choline (C+) and 0 (I-) or 75 μM inositol (I+) and incubated at 30oC for 3 d. A representative experiment is shown (from n=5).


A - L Wild type cultured in C - ( A ) , cho2opi3 cultured in C + ( B ) , coS # 4 ( C - D ) , coS # 3 ( E - G ) , coS # 2 ( I - K ) cultured in C - were analyzed by electron microscopy . In addition , coS # 3 ( H ) coS # 2 ( L ) are shown after culture in C + for 3 generations . arrow heads ( F , I , J ) point to protrusions in envelope . CW , wall ; ER , endoplasmic reticulum ; PM , plasma ; M , mitochondria ; N , nucleus ; V , vacuole ; * , lipid droplet . Scale bars correspond to 200 nm ( A , B , D , F , G ) or 500 nm ( C , E , H , I , J , K , L ) .

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A-L Wild type cultured in C- (A), cho2opi3 cultured in C+ (B ), coS#4 (C-D), coS#3 (E-G), and coS#2 (I-K) cultured in C- were analyzed by electron microscopy. In addition, coS#3 (H) and coS#2 (L) are shown after culture in C+ for 3 generations. The arrow heads (F, I, J) point to protrusions in the nuclear envelope. CW, cell wall; ER, endoplasmic reticulum; PM, plasma membrane; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 200 nm (A, B, D, F, G) or 500 nm (C, E, H, I, J, K, L).


Serial dilutions of strains indicated were spotted on SD C + / - I + / - incubated at 30 oC for 4 d .

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Serial dilutions of the strains indicated were spotted on SD C+/- I+/- and incubated at 30 oC for 4 d.


lipid TAG content , ergosterolester content ( EE , inset ) per OD600 unit of yeast strains indicated after culture to mid - log phase in SD with or without 1 mM choline ( C ) ; * p < 0 . 05 , * * p < 0 . 01 , unpaired two - t - of indicated bar compared to C + condition . Data information : data were obtained by mass spectrometry are presented as mean ± SD ( n = 3 biological replicates ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , unpaired two - t - of indicated bar compared to cho2opi3 parent unless indicated otherwise .

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

Membrane lipid and TAG content, and ergosterolester content (EE, inset) per OD600 unit of the yeast strains indicated after culture to mid-log phase in SD with or without 1 mM choline (C); * p < 0.05, ** p < 0.01, unpaired two-tailed t-test of the indicated bar compared to the C+ condition. Data information: All data were obtained by mass spectrometry and are presented as mean ±SD (n=3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise.


Generation of suppressors of choline auxotrophy of cho2opi3 , cho2opi3lro1 , cho2opi3lro1 pLRO1 on choline - free medium as indicated at 30 oC for 7 d .

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Generation of suppressors of choline auxotrophy of cho2opi3, cho2opi3lro1, and cho2opi3lro1 pLRO1 on choline-free medium as indicated at 30 oC for 7 d.


Growth ( 30 oC for 6 d ) phospholipid composition of co acc1N / H lro1 pLRO1 cultured in SD C - ura - , containing glucose / galactose mixtures ( 2 % , w / v ) as carbon source with of galactose indicated . Phospholipid composition analyzed by TLC is presented as mean of total phospholipid of 3 biological replicates with values indicated .

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

Growth (30 oC for 6 d) and phospholipid composition of co acc1N/H lro1 pLRO1 cultured in SD C- ura-, containing glucose/galactose mixtures (2%, w/v) as carbon source with the percentage of galactose indicated. Phospholipid composition analyzed by TLC is presented as mean percentage of total phospholipid of 3 biological replicates with the individual values indicated.


ACC1 transcript after switching strains indicated from SD C + I + to medium indicated at OD600 0 . 02 or 0 . 2 ( co diploid in C - ) , culture for 24 h at 30 oC . Data were normalized to ACT1 expressed as means of 3 biological replicates relative to corresponding cultured in C + I + , with values indicated .

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

ACC1 transcript levels after switching the strains indicated from SD C+I+ to the medium indicated at OD600 0.02 or 0.2 (co diploid in C-), and culture for 24 h at 30 oC. Data were normalized to ACT1 and expressed as means of 3 biological replicates relative to the corresponding strain cultured in C+I+, with the individual values indicated.


( F - G ) EM of cho2opi3 that after preculture in SD C + , were transferred to OD600 0 . 05 subsequently cultured to mid - log phase in SD C + ( F ) or SD C - ( G ) both containing 0 . 05 μg / mL SorA . CW , wall ; ER , endoplasmic reticulum ; M , mitochondria ; N , nucleus ; V , vacuole ; * , lipid droplet . Scale bars correspond to 500 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) EM analysis of cho2opi3 cells that after preculture in SD C+, were transferred to OD600 0.05 and subsequently cultured to mid-log phase in SD C+ (F) or SD C- (G) both containing 0.05 μg/mL SorA. CW, cell wall; ER, endoplasmic reticulum; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 500 nm.


POX1 transcript after switching strains indicated from SD C + I + to medium indicated at OD600 0 . 02 or 0 . 2 ( co diploid in C - ) , culture for 24 h at 30 oC . Data were normalized to ACT1 expressed as means of 3 biological replicates relative to corresponding cultured in C + I + , with values indicated .

POX1 transcript levels after switching the strains indicated from SD C+I+ to the medium indicated at OD600 0.02 or 0.2 (co diploid in C-), and culture for 24 h at 30 oC. Data were normalized to ACT1 and expressed as means of 3 biological replicates relative to the corresponding strain cultured in C+I+, with the individual values indicated.


Growth of co S ( 2n - 1 ) in absence of choline is lost when GPT2 or DGA1 is overexpressed , restored by SorA . Plasmids pHEYg - 1 - DGA1 , pHEYg - 1 - GPT2 empty vector control ( pEV ) were transformed into strains indicated . After preculture in SD C + , ten - fold serial dilutions were spotted on SD C + / - with or without 0 . 1 μg / mL SorA incubated at 30 oC for 3 d .

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

Growth of co S(2n-1) in the absence of choline is lost when GPT2 or DGA1 is overexpressed, and restored by SorA. Plasmids pHEYg-1-DGA1, pHEYg-1-GPT2 and the empty vector control (pEV) were transformed into the strains indicated. After preculture in SD C+, ten-fold serial dilutions were spotted on SD C+/- with or without 0.1 μg/mL SorA and incubated at 30 oC for 3 d.


encoded GP - of indicated C - Laurdan - stained yeast strains cultured with or without choline , accompanied by corresponding weighted GP - histogram fitted to a single Gaussian function ( blue ) , transmission image . arrows point to plasma membranes exhibiting higher lipid packing than internal , organellar membranes ; blue horizontal arrows indicate highly packed lipid material in co S ( 2n - 1 ) . To deplete cho2opi3 parent of PC , were transferred to SD C - at OD600 0 . 1 , cultured into mid - log phase . scale bars correspond to 10 µm ; red scale bars in transmission correspond to 5 µm .

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

Intensity encoded GP-images of the indicated C-Laurdan-stained yeast strains cultured with or without choline, accompanied by the corresponding intensity weighted GP-histogram fitted to a single Gaussian function (light blue line), and transmission image. White arrows point to plasma membranes exhibiting higher lipid packing than internal, organellar membranes; blue horizontal arrows indicate highly packed lipid material in co S(2n-1). To deplete the cho2opi3 parent of PC, cells were transferred to SD C- at OD600 0.1, and cultured into mid-log phase. White scale bars correspond to 10 µm; red scale bars in transmission images correspond to 5 µm.


( A ) Mettl14 cKO leads to perinatal lethality . Newborn cKO pups are smaller with tight shiny skin .

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

(A) Mettl14 cKO leads to perinatal lethality. Newborn cKO pups are smaller with tight and shiny skin.


( B ) Immunohistochemistry confirms loss of Mettl14 expression in skin in cKO .

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

(B) Immunohistochemistry confirms loss of Mettl14 expression in skin in cKO animals.


( C ) H / E of newborn skin sections from WT Mettl14 cKO mice .

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

(C) H/E staining of newborn skin sections from WT and Mettl14 cKO mice.


( D ) Expression of p63 in skin epidermis was examined by immunofluorescence in WT cKO mice . ( E ) Number of p63 - positive in WT cKO skin was quantified shown as box whisker plots . plot indicates mean ( solid diamond within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - ) .

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

(D) Expression of p63 in skin epidermis was examined by immunofluorescence staining in WT and cKO mice. (E) Number of p63-positive cells in WT and cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test).


( F ) Skin stratification in WT cKO skin was determined by immunofluorescence with different antibodies as indicated . Krt14 : Keratin 14 ; Krt10 : Keratin 10 ; β4 : β4 - integrin ; DAPI for nucleus . dashed denotes basement that separates dermis epidermis ( Epi ) .

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

(F) Skin stratification in WT and cKO skin was determined by immunofluorescence staining with different antibodies as indicated. Krt14: Keratin 14; Krt10: Keratin 10; β4: β4-integrin; DAPI for nucleus staining. The dashed line denotes the basement membrane that separates dermis and epidermis (Epi).


( G ) Wound healing as monitored by wound 8 days post injury . n = 3 ; P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D . ( standard deviation ) .

(G) Wound healing as monitored by wound size 8 days post injury. n=3; P<0.01 (Student's t-test). Error bar represents S.D. (standard deviation).


H ) Histological of skin sections at wound edges . Halves of wound sections are shown . Note significant reduction of HPE ( hyperproliferative epidermis ) in cKO skin . Es : eschar . Dotted lines denote epidermal boundaries . ( I ) of Ki67 - positive present in wound HPE . plot indicates mean ( open circles within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 19 , P < 0 . 01 ( Student ' s t - ) .

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

H) Histological staining of skin sections at the wound edges. Halves of wound sections are shown. Note significant reduction of HPE (hyperproliferative epidermis) in cKO skin. Es: eschar. Dotted lines denote epidermal boundaries. (I) Quantification of Ki67-positive cells present in wound HPE. The plot indicates the mean (open circles within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=19, P<0.01 (Student's t-test).


( A ) EdU of WT or Mettl14 cKO skin after - chase labeling . Skin samples were counterstained with antibody against β4 - integrin . Note reduced EdU label - retaining in cKO skin epidermis . Arrows indicate Edu - positive . ( B ) Label - retaining in WT or Mettl14 cKO skin were quantified shown as box plots . plot indicates mean ( solid diamond within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - ) . n = 19 , P < 0 . 01 ( Student ' s t - ) .

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

(A) EdU staining of WT or Mettl14 cKO skin after pulse-chase labeling. Skin samples were counterstained with antibody against β4-integrin. Note reduced EdU label-retaining cells in cKO skin epidermis. Arrows indicate Edu-positive cells. (B) Label-retaining cells in WT or Mettl14 cKO skin were quantified and shown as box plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=19, P<0.01 (Student's t-test).


( C ) Number of holoclones derived from WT Mettl14 cKO skin was quantified shown as box whisker plots . plot indicates mean ( solid diamond within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - ) . n = 24 , P < 0 . 01 ( Student ' s t - ) .

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

(C) Number of holoclones derived from WT and Mettl14 cKO skin was quantified and shown as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=24, P<0.01 (Student's t-test).


( D ) of keratinocytes isolated from WT or Mettl14 cKO skin .

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(D) Morphology of primary keratinocytes isolated from WT or Mettl14 cKO skin.


( E ) Expression of Mettl14 p63 in WT KO was determined by immunoblots with respective antibodies . Immunoblot for GAPDH was used as loading control .

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

(E) Expression of Mettl14 and p63 in WT and KO cells was determined by immunoblots with respective antibodies. Immunoblot for GAPDH was used as loading control.


( F ) Proliferation of WT Mettl14 null in vitro was quantified shown as dot plots . n = 3 , P < 0 . 01 ( Student ' s t - ) for Day 6 , 9 , 12 , P < 0 . 05 ( Student ' s t - ) for Day 3 . Error bar represents S . D .

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

(F) Proliferation of WT and Mettl14 null cells in vitro was quantified and shown as dot plots. n=3, P<0.01 (Student's t-test) for Day 6, 9, and 12, and P<0.05 (Student's t-test) for Day 3. Error bar represents S.D.


( G - H ) CFE ( colony formation efficiency ) of WT Mettl14 null was determined in vitro . Results were quantified shown as bar graph ( F ) . n = 19 , P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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

(G-H) CFE (colony formation efficiency) of WT and Mettl14 null cells was determined in vitro. Results were quantified and shown as bar graph (F). n=19, P<0.01 (Student's t-test). Error bar represents S.D.


( I ) microscopy demonstrates different survival capability of WT Mettl14 inducible KO with or without Tamoxifen ( TAM ) treatment . ( J ) of WT Mettl14 inducible KO in co - culture model was quantified shown as dot plots . n = 8 , P < 0 . 01 ( Student ' s t - ) for KO with TAM treatment compared with WT or KO without TAM stimulation at both Day 7 14 . Error bar represents S . D .

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

(I) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 inducible KO cells with or without Tamoxifen (TAM) treatment. (J) Ratio of WT and Mettl14 inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with TAM treatment compared with WT cells or KO cells without TAM stimulation at both Day 7 and 14. Error bar represents S.D.


B ) Mutations in predicted m6A modification sites can significantly reduce Pvt1 methylation . n = 3 . Error bar represents S . D .

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

B) Mutations in the predicted m6A modification sites can significantly reduce Pvt1 methylation.n=3. Error bar represents S.D.


C ) Deletion of endogenous Pvt1 by an inducible Cas9 ( iCas9 ) system can significantly reduce Pvt1 level ( from RT - PCR , left panel ) MYC level ( from immunoblot , right panel ) expression . n = 3 , P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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

C) Deletion of endogenous Pvt1 by an inducible Cas9 (iCas9) system can significantly reduce Pvt1 RNA level (quantification from RT-PCR, left panel) and MYC protein level (quantification from immunoblot, right panel) expression. n=3, P<0.01 (Student's t-test). Error bar represents S.D.


D ) CFE of WT , Pvt1 inducible KO , Pvt1 inducible KO rescued with WT or mutant Pvt1 was quantified presented as bar graph . n = 3 , * * : P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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

D) CFE of WT, Pvt1 inducible KO, and Pvt1 inducible KO cells rescued with WT or mutant Pvt1 was quantified and presented as bar graph. n=3, **: P<0.01 (Student's t-test). Error bar represents S.D.


E ) Skin organoids derived from WT or Pvt1 inducible KO were grafted to nude mice . regenerated skin was analyzed by H / E .

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

E) Skin organoids derived from WT or Pvt1 inducible KO cells were grafted to nude mice. The regenerated skin was analyzed by H/E staining.


F ) Epidermal thickness of WT Pvt1 KO skin grafts was quantified presented as box whisker plots . plot indicates mean ( solid diamond within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - ) . n = 18 , P < 0 . 01 ( Student ' s t - ) .

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

F) Epidermal thickness of WT and Pvt1 KO skin grafts was quantified and presented as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=18, P<0.01 (Student's t-test).


G ) Number of p63 - positive in WT cKO skin was quantified shown as bar graph . n = 5 , P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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

G) Number of p63-positive cells in WT and cKO skin was quantified and shown as bar graph. n=5, P<0.01 (Student's t-test). Error bar represents S.D.


H ) microscopy demonstrates different survival capability of WT Pvt1 inducible KO with or without Doxycycline ( Dox ) treatment .

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

H) Fluorescence microscopy demonstrates different survival capability of WT and Pvt1 inducible KO cells with or without Doxycycline (Dox) treatment.


I ) of WT Pvt1 inducible KO in co - culture model was quantified shown as dot plots . n = 8 , P < 0 . 01 ( Student ' s t - ) for KO with Dox treatment compared with WT or KO without Dox stimulation at both Day 7 14 . Error bar represents S . D .

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

I) Ratio of WT and Pvt1 inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with Dox treatment compared with WT cells or KO cells without Dox stimulation at both Day 7 and 14. Error bar represents S.D.


A ) Interaction between Pvt1 MYC was determined by immunoprecipitation followed with RT - PCR . Note significant decrease in Pvt1 MYC interaction upon FTO treatment ( left panel ) , calcium shift - induced differentiation ( middle panel , Hi : high calcium ) , Pvt1 methylation site mutations ( right panel ) . n = 3 , P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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

A) Interaction between Pvt1 and MYC was determined by immunoprecipitation followed with RT-PCR. Note significant decrease in Pvt1 and MYC interaction upon FTO treatment (left panel), calcium shift-induced differentiation (middle panel, Hi: high calcium), and Pvt1 methylation site mutations (right panel). n=3, P<0.01 (Student's t-test). Error bar represents S.D.


B ) Immunoblots show decreased MYC level in differentiated keratinocytes Mettl14 KO .

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

B) Immunoblots show decreased MYC protein level in differentiated keratinocytes and Mettl14 KO cells.


Undifferentiated WT Mettl14 KO ( C ) were treated with cycloheximide . lysates were collected at 0 , 30 , 60 , 90 , 120 , 180 , 300 minutes post treatment subjected to immunoblotting with different antibodies as indicated .

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

Undifferentiated WT and Mettl14 KO cells (C) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.


undifferentiated WT Pvt1 KO ( D ) were treated with cycloheximide . lysates were collected at 0 , 30 , 60 , 90 , 120 , 180 , 300 minutes post treatment subjected to immunoblotting with different antibodies as indicated .

undifferentiated WT and Pvt1 KO cells (D) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.


WT before after calcium shift - induced differentiation ( E ) were treated with cycloheximide . lysates were collected at 0 , 30 , 60 , 90 , 120 , 180 , 300 minutes post treatment subjected to immunoblotting with different antibodies as indicated .

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

WT cells before and after calcium shift-induced differentiation (E) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.


( F ) Skin organoids derived from Mettl14 KO or KO rescued with exogenous expression of MYC were grafted to nude mice . regenerated skin was analyzed by H / E .

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

(F) Skin organoids derived from Mettl14 KO cells or KO cells rescued with exogenous expression of MYC were grafted to nude mice. The regenerated skin was analyzed by H/E staining.


( G ) Epidermal thickness of KO KO skin rescued with MYC expression was quantified presented as box whisker plots . plot indicates mean ( solid diamond within box ) , 25th percentile ( bottom of box ) , median ( middle of box ) , 75th percentile ( top of box ) , 5th 95th percentile ( whiskers ) , 1st 99th percentile ( solid triangles ) minimum maximum measurements ( solid squares ) . n = 6 ( biological repeats ) , P < 0 . 05 ( Student ' s t - ) . n = 18 , P < 0 . 01 ( Student ' s t - ) .

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(G) Epidermal thickness of KO and KO skin rescued with MYC expression was quantified and presented as box and whisker plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=18, P<0.01 (Student's t-test).


( H ) Number of p63 - positive in KO KO skin rescued with MYC expression was quantified shown as bar graph . n = 5 , P < 0 . 01 ( Student ' s t - ) . Error bar represents S . D .

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(H) Number of p63-positive cells in KO and KO skin rescued with MYC expression was quantified and shown as bar graph. n=5, P<0.01 (Student's t-test). Error bar represents S.D.


( I ) microscopy demonstrates different survival capability of WT Mettl14 KO with inducible expression of MYC .

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(I) Fluorescence microscopy demonstrates different survival capability of WT and Mettl14 KO cells with inducible expression of MYC.


( J ) of WT Mettl14 KO with inducible expression of MYC in co - culture model was quantified shown as dot plots . n = 8 , P < 0 . 01 ( Student ' s t - ) for KO without Dox treatment ( no MYC expression ) compared with WT or KO with Dox stimulation ( exogenous MYC expression ) at both Day 7 14 . Error bar represents S . D .

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(J) Ratio of WT and Mettl14 KO cells with inducible expression of MYC in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells without Dox treatment (no MYC expression) compared with WT cells or KO cells with Dox stimulation (exogenous MYC expression) at both Day 7 and 14. Error bar represents S.D.


a , b , of HeLa stably expressing YFP fused to indicated galectins infected with S . Typhimurium for 1 h . a , of coated by indicated galectins . YFP - positive were counted by microscopy . Mean standard deviation ( s . d . ) of triplicate HeLa cultures , n > 100 per coverslip . b , Confocal microscopy . Arrowheads , shown in insets . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole .

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a, b, Analysis of HeLa cells stably expressing YFP fused to the indicated galectins and infected with S. Typhimurium for 1 h. a, Percentage of bacteria coated by the indicated galectins. YFP-positive bacteria were counted by microscopy. Mean and standard deviation (s.d.) of triplicate HeLa cultures, n > 100 bacteria per coverslip. b, Confocal microscopy. Arrowheads, bacteria shown in insets. DAPI, 4′,6-diamidino-2-phenylindole.


c , Kinetics of fold replication for S . Typhimurium in HeLa transfected with indicated siRNAs . were counted on basis of their ability to form colonies on agar plates . Mean s . d . of triplicate HeLa cultures duplicate colony . siRNAs are further characterized in Supplementary Fig . 2a - c . * * P 0 . 01 , Student ' s t - . Scale bar , 10 µm .

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c, Kinetics of fold replication for S. Typhimurium in HeLa cells transfected with the indicated siRNAs. Bacteria were counted on the basis of their ability to form colonies on agar plates. Mean and s.d. of triplicate HeLa cultures and duplicate colony counts. siRNAs are further characterized in Supplementary Fig. 2a-c. **P  0.01, Student's t-test. Scale bar, 10 µm.


a , LUMIER binding : normalized between luciferase activity bound to beads present in lysates . Lysates of 293ET expressing NDP52 , p62 or optineurin each fused to luciferase , indicated Flag - tagged galectins were incubated with anti - Flag beads . Flag - tagged are further characterized in Supplementary Fig .

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a, LUMIER binding assay: normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells expressing NDP52, p62 or optineurin each fused to luciferase, and the indicated Flag-tagged galectins were incubated with anti-Flag beads. Flag-tagged proteins are further characterized in Supplementary Fig. 4a


b , Lysates of 293ET , expressing Flag - tagged as indicated , were immunoprecipitated with anti - Flag beads . Lysates immunoprecipitates ( IP ) were blotted for presence of Flag - tagged endogenous NDP52 .

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b, Lysates of 293ET cells, expressing Flag-tagged proteins as indicated, were immunoprecipitated with anti-Flag beads. Lysates and immunoprecipitates (IP) were blotted for the presence of Flag-tagged proteins and endogenous NDP52.


c , Confocal of HeLa infected with S . Typhimurium for 1 h stained with antisera against NDP52 galectin 8 . Arrowheads , shown in insets .

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c, Confocal images of HeLa cells infected with S. Typhimurium for 1 h and stained with antisera against NDP52 and galectin 8. Arrowheads, bacteria shown in insets.


d , Co - localization of NDP52 with galectin - positive in HeLa stably expressing YFP fused to indicated galectins , infected with S . Typhimurium stained with NDP52 antiserum 1 h after infection . Mean s . d . of duplicate HeLa cultures , n > 100 per coverslip , representative of two independent experiments . Scale bar , 10 µm .

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d, Co-localization of NDP52 with galectin-positive bacteria in HeLa cells stably expressing YFP fused to the indicated galectins, infected with S. Typhimurium and stained with NDP52 antiserum 1 h after infection. Mean and s.d. of duplicate HeLa cultures, n > 100 bacteria per coverslip, representative of two independent experiments. Scale bar, 10 µm.


a , of S . Typhimurium coated by indicated galectins . HeLa stably expressing YFP - tagged galectins were treated with indicated siRNAs . YFP - positive were counted by microscopy at 1 h p . i . siRNAs are further characterized in Supplementary Fig . 2 .

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a, Percentage of S. Typhimurium coated by the indicated galectins. HeLa cells stably expressing YFP-tagged galectins were treated with the indicated siRNAs. YFP-positive bacteria were counted by microscopy at 1 h p.i. siRNAs are further characterized in Supplementary Fig. 2.


b , of HeLa treated with indicated siRNAs stained with NDP52 antiserum . NDP52 - positive were counted by microscopy 1 h after infection with S . Typhimurium . siRNAs are further characterized in Supplementary Fig . 2 .

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b, Analysis of HeLa cells treated with the indicated siRNAs and stained with NDP52 antiserum. NDP52-positive bacteria were counted by microscopy 1 h after infection with S. Typhimurium. siRNAs are further characterized in Supplementary Fig. 2.


c , of coated by indicated galectin 8 alleles . HeLa stably expressing indicated galectin 8 alleles fused to YFP were infected with S . Typhimurium . YFP - positive S . Typhimurium were counted by microscopy at 75 min p . i . WT , wild type .

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c, Percentage of bacteria coated by the indicated galectin 8 alleles. HeLa cells stably expressing the indicated galectin 8 alleles fused to YFP were infected with S. Typhimurium. YFP-positive S. Typhimurium were counted by microscopy at 75 min p.i. WT, wild type.


d , Binding of galectin 8 to HeLa . indicated HeLa were incubated with His - GST - ubiquitin ( Ub ) , His - GST - galectin 8 or buffer as indicated , followed by murine anti - His antibody PE - labelled anti - mouse serum .

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d, Binding of galectin 8 to bacteria and HeLa cells. The indicated bacteria and HeLa cells were incubated with His-GST-ubiquitin (Ub), His-GST-galectin 8 or buffer as indicated, followed by murine anti-His antibody and PE-labelled anti-mouse serum.


e , of S . Typhimurium coated by indicated galectins . Wild - type CHO mutant Lec3 . 2 . 8 . 1 stably expressing YFP - tagged galectins were infected with S . Typhimurium . YFP - positive were counted by microscopy at 1 h p . i .

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e, Percentage of S. Typhimurium coated by the indicated galectins. Wild-type CHO cells and mutant Lec3.2.8.1 cells stably expressing YFP-tagged galectins were infected with S. Typhimurium. YFP-positive bacteria were counted by microscopy at 1 h p.i.


f , Confocal of HeLa expressing indicated YFP - tagged galectins . were left untreated or were exposed to hypertonic conditions , with or without ( w / o ) PEG as indicated , followed by hypotonic shock .

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f, Confocal images of HeLa cells expressing the indicated YFP-tagged galectins. Cells were left untreated or were exposed to hypertonic conditions, with or without (w/o) PEG as indicated, followed by hypotonic shock.


g , Fold replication of S . Typhimurium in HeLa expressing indicated galectin 3 variants transfected with indicated siRNAs . At 2 h 6 h after infection , were lysed counted on basis of their ability to form colonies on agar plates . Galectin 3 are further characterized in Supplementary Fig . 4b . Mean s . d . of duplicate coverslips > 100 counted per coverslip . Data are representative of at least two repeats . * P < 0 . 05 , Student ' s t - . Scale bar , 10 µm .

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g, Fold replication of S. Typhimurium in HeLa cells expressing the indicated galectin 3 variants and transfected with the indicated siRNAs. At 2 h and 6 h after infection, cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates. Galectin 3 proteins are further characterized in Supplementary Fig. 4b. Mean and s.d. of duplicate coverslips>100 bacteria counted per coverslip. Data are representative of at least two repeats. *P< 0.05, Student's t-test. Scale bar, 10 µm.


a , Confocal micrograph of HeLa expressing GFP - LC3 mCherry - galectin - 8 , stained for NDP52 1 h after infection with S . Typhimurium . lower right panel contains a scan along yellow in merge inset . Arrowheads , shown in insets .

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a, Confocal micrograph of HeLa cells expressing GFP-LC3 and mCherry-galectin-8, stained for NDP52 1 h after infection with S. Typhimurium. The lower right panel contains a fluorescence line scan along the yellow line in the merge inset. Arrowheads, bacteria shown in insets.


b , of GFP - LC3 - positive S . Typhimurium at 1 h p . i . in HeLa treated with indicated siRNAs .

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b, Percentage of GFP-LC3-positive S. Typhimurium at 1 h p.i. in HeLa cells treated with the indicated siRNAs.


c , of positive for NDP52 . HeLa expressing indicated NDP52 variants fused to YFP were infected with S . Typhimurium .

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c, Percentage of bacteria positive for NDP52. HeLa cells expressing the indicated NDP52 variants fused to YFP were infected with S. Typhimurium.


d , of positive for indicated markers . HeLa , either wild type or expressing YFP - galectin - 8 as indicated , were infected with S . Typhimurium . Ubiquitin was detected by antibody .

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d, Percentage of bacteria positive for the indicated markers. HeLa cells, either wild type or expressing YFP-galectin-8 as indicated, were infected with S. Typhimurium. Ubiquitin was detected by antibody staining.


e , HeLa treated with indicated siRNAs were infected with S . Typhimurium stained for NDP52 at indicated time points . were counted by microscopy at indicated time points . Graphs , representing at least two independent repeats , show mean s . d . of duplicate coverslips for which > 200 were counted . * P 0 . 05 , Student ' s t - . Scale bar , 10 µm .

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e, HeLa cells treated with the indicated siRNAs were infected with S. Typhimurium and stained for NDP52 at the indicated time points. Fluorescent bacteria were counted by microscopy at the indicated time points. Graphs, representing at least two independent repeats, show mean and s.d. of duplicate coverslips for which >200 bacteria were counted. *P  0.05, Student's t-test. Scale bar, 10 µm.


( A ) Representative transmission electron microscope ( ) image showing presence of EVs in CSF in two independent experiments .

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(A) Representative transmission electron microscope (TEM) image showing the presence of EVs in the CSF in two independent experiments.


( B ) NanoSightquantification of amount of particles in CSF 0 , 1 , 2 , 4 6 hours after i . p . LPS injection ( n = 3 - 5 ) .

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(B) NanoSightquantification of the amount of particles in the CSF 0, 1, 2, 4 and 6 hours after i.p. LPS injection (n=3-5).


( C ) distribution of EVsin vivo in CSF before ( black ; n = 5 ) 6 h after ( grey ; n = 3 ) LPS treatment determined by NanoSight .

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(C) Size distribution of the EVsin vivo in the CSF before (black; n=5) and 6 h after (grey; n=3) LPS treatment determined by NanoSight analysis.


( D - G ) Quantitative real - time polymerase chain reaction of miR - 1a ( D ) , miR - 9 ( E ) , miR - 146a ( F ) , miR - 155 ( G ) ( n = 4 ) . was isolated from pooled CSF ( 50 µl ) from different mice ( n = 3 ) . Data are displayed as mean ± analyzed by Student ' s t - . Significance are indicated on graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

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(D-G) Quantitative real-time polymerase chain reaction analysis of miR-1a (D), miR-9 (E), miR-146a (F), and miR-155 (G) (n=4). RNA was isolated from pooled CSF (50 µl) from different mice (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A - B ) In vitro ( A ) distribution ( B ) of EVs isolated from conditioned medium of CPE grown in transwell system after 12 h in absence ( black ) or presence ( grey ) of LPS ( n = 5 ) determined by NanoSight .

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(A-B) In vitro quantification (A) and size distribution (B) of EVs isolated from conditioned medium of primary CPE cells grown in transwell system after 12 h in the absence (black) or presence (grey) of LPS (n=5) determined by NanoSight analysis.


( C - E ) TaqMan qPCR for of miR - 9 ( C ) , miR - 146a ( D ) miR - 155 ( E ) on exosomal pellet isolated from conditioned medium of CPE grown in a transwell system stimulated for 12 h with LPS ( n = 3 ) .

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(C-E) TaqMan qPCR assay for the quantification of miR-9 (C), miR-146a (D) and miR-155 (E) on the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS (n=3).


( F - I ) of miRNAs miR - 1a ( F ) , miR - 9 ( G ) , miR - 146a ( H ) miR - 155 ( I ) by TaqMan qPCR from CPE grown in a transwell system without or with LPS stimulation ( n = 3 ) . Significance are indicated on graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

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(F-I) Quantification of the miRNAs miR-1a (F), miR-9 (G), miR-146a (H) and miR-155 (I) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation (n=3). Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A ) In vitro of EVs isolated from conditioned medium of LPS - stimulated CPE grown in a transwell system in absence or presence of exosome inhibitor GW4869 ( n = 3 ) .

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(A) In vitro quantification of EVs isolated from conditioned medium of LPS-stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 (n=3).


( B - D ) TaqMan of miRNAs miR - 9 ( B ) , miR - 146a ( C ) miR - 155 ( D ) in supernatant of LPS - stimulated CPE grown in a transwell system either left untreated or pretreated with GW4869 to inhibit exosome secretion ( n = 3 ) . miR - 1a were below detection limit .

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(B-D) TaqMan assay quantification of the miRNAs miR-9 (B), miR-146a (C) and miR-155 (D) in supernatant of LPS-stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion (n=3). miR-1a levels were below detection limit.


( E - H ) TaqMan of miRNAs miR - 1a ( E ) , miR - 9 ( F ) , miR - 146a ( G ) miR - 155 ( H ) in lysate of LPS - stimulated CPE grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion ( n = 3 ) . Data are displayed as mean ± analyzed by Student ' s t - . Significance are indicated on graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

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(E-H) TaqMan assay quantification of the miRNAs miR-1a (E), miR-9 (F), miR-146a (G) and miR-155 (H) in cell lysate of LPS-stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A ) Representative confocal of CD63 , RAB5 ANXA2 ( red ) in choroid plexus ( CP ) 0 , 4 8 h after LPS treatment . Hoechst ( blue ) was used to stain nucleus . dotted indicates ependymal that ventricle square boxes indicate zoomed insert displayed at right corner of each image . Scalebar = 100 µm .

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(A) Representative confocal images of CD63, RAB5 and ANXA2 (red) in the choroid plexus (CP) 0, 4 and 8 h after LPS treatment. Hoechst (blue) was used to stain the nucleus. The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image. Scalebar = 100 µm.


( B - C ) Representative showing presence of MVBs in CPE before ( B ) 6 hours after ( C ) LPS administration in vivo . arrow heads point to exosomes present in MVBs ( n = 3 ) . Scale bar = 9 µm .

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(B-C) Representative TEM images showing the presence of MVBs in the CPE cells before (B) and 6 hours after (C) LPS administration in vivo. White arrow heads point to exosomes present in MVBs (n=3). Scale bar = 9 µm.


( D - F ) of amount of MVBs per section ( D ) , amount of exosomes per MVB ( E ) , amount of exosomes per section ( F ) , on .

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(D-F) Quantification of amount of MVBs per cell section (D), amount of exosomes per MVB (E), and amount of exosomes per cell section (F), based on TEM analysis.