( 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 ) .

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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 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 .

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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 and IgM isotype distribution of single - cell - sorted HBHA - reactive memory cells . Mean and SEM 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 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 ) .

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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 the MTB cell membrane fraction for MTB - reactive ( n = 26 ) and 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 bacteria as determined by flow cytometry for three representative MTB cell lysate - reactive antibodies and 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 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 .

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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 the LAM - reactive antibody TB24PB037 ( black ) compared to E . coli as determined by ELISA with whole bacteria ( left ) and 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 the indicated concentrations . The mean ± SD of the 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 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 ) ) .

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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 ) 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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. (C) Representative results for individual HBHA-reactive memory B cell antibodies from TB patients and 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 . ( 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 and IgG antibodies on epithelial cell invasion as measured in ( A ) . P - value was determined using Wilcoxon - Mann - Whitney test ; * * * * 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 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 ) .

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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 ) , 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 ) .

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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 the strains indicated were spotted on SD C + / - I + / - and 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.


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 .

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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 , and 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 ) 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 .

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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 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 .

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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 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 .

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(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 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 .

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 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 .

B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD 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 O O O O O B-SMALL_MOLECULE O 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.


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 .

O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O 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 B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD 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 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 and shiny skin .

O O O B-GENEPROD O O O B-DISEASE I-DISEASE O O O B-ORGANISM O O O O O O B-TISSUE 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 animals .

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

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


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

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

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

O O O B-TISSUE O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SUBCELLULAR B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE O B-TISSUE 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 size 8 days post injury . n = 3 ; P < 0 . 01 ( Student ' s t - test ) . Error bar represents S . D . ( standard deviation ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE O O O O O O O O O O O O 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) 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 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O B-DISEASE O O O O B-DISEASE O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O B-TISSUE O O O O O O O B-GENEPROD O O O O O B-DISEASE 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 O O O O O O O O O O O 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 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 ) .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-GENEPROD O B-TISSUE O O O O O O B-TISSUE O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) Morphology of primary keratinocytes isolated from WT or Mettl14 cKO skin .

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

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


( 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 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD 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 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 .

O O O B-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 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 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 .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 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 ) 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 .

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

(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 the predicted m6A modification sites can significantly reduce Pvt1 methylation . n = 3 . Error bar represents S . D .

O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY 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 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 .

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

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 , 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 .

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

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 cells were grafted to nude mice . The regenerated skin was analyzed by H / E staining .

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

O O B-TISSUE O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

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

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


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 .

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 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

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 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 .

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

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 protein level in differentiated keratinocytes and Mettl14 KO cells .

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

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


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 .

O O O B-GENEPROD 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 B-EXP_ASSAY 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 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 .

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 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 .

O O O O O B-SMALL_MOLECULE 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 B-EXP_ASSAY 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 cells or KO cells rescued with exogenous expression of MYC were grafted to nude mice . The regenerated skin was analyzed by H / E staining .

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

O O O B-TISSUE O O O O O B-TISSUE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

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

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

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


( 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 .

O O O O O O O B-GENEPROD 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 B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O

(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 , 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 .

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

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 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 .

O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O

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 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 .

O O O O B-CELL_LINE I-CELL_LINE O O B-GENEPROD 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 B-GENEPROD O O O O O B-GENEPROD O

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 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 .

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

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 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 .

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

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 , 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 .

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

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 , 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 .

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

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 , 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 .

O O O O O O O O O B-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O 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

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 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 .

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

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 , 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 .

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

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 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 .

O O O O O B-CELL_LINE I-CELL_LINE 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 B-SMALL_MOLECULE O O O O O O O O

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 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 .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_LINE I-CELL_LINE O O O B-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 B-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

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 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 .

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

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

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

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


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

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

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


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 .

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

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 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 .

O O B-CELL_LINE I-CELL_LINE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM 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 O O O O O O O O O O O O O O O O O O O O O O O O O O

e, HeLa cells 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 ( TEM ) image showing the presence of EVs in the CSF in two independent experiments .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O

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


( 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 ) .

O O O B-EXP_ASSAY O O O O O O O B-TISSUE 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) 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 ) 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 .

O O O O O O O B-SUBCELLULAR O O O B-TISSUE 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 B-EXP_ASSAY O O

(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 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 .

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

O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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 B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O

(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 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 ) .

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

(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 ) 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 .

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

(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 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 ) .

O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-CELL_TYPE 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-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O

(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 ) 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 .

O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O O

(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.


( G - J ) Quantitative real - time polymerase chain reaction ( qPCR ) analysis of miR - 1a ( G ) , miR - 9 ( H ) , miR - 146a ( I ) , and miR - 155 ( J ) . Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay ( 0 h , n = 4 ; 1 h , n = 5 ; 6 h , n = 5 ; 24 h , n = 3 ) .

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

(G-J) Quantitative real-time polymerase chain reaction (qPCR) analysis of miR-1a (G), miR-9 (H), miR-146a (I), and miR-155 (J). Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay (0 h, n=4; 1 h, n=5; 6 h, n=5; 24 h, n=3).


( K ) NanoSight analysis of CSF isolated from LPS - injected mice followed by icv injection of vehicle or GW4869 , a neutral sphingomyelinase inhibitor that inhibits exosome secretion ( n = 8 ) .

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

(K) NanoSight analysis of CSF isolated from LPS-injected mice followed by icv injection of vehicle or GW4869, a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 8).


( L ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle ( black ) or GW4869 ( grey ) , a neutral sphingomyelinase inhibitor that inhibits exosome secretion ( n = 4 ) .

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

(L) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle (black) or GW4869 (grey), a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 4).


( M ) NanoSight analysis of supernatant of choroid plexus explants from PBS - or LPS - injected mice ( n = 6 ) . 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 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O 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 O O O O O O O O O O O O O O O O O O O O O O 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) NanoSight analysis of supernatant of choroid plexus explants from PBS- or LPS-injected mice (n = 6). 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; ***, 0.0001 ≤ P < 0.001.


( A - B ) Representative confocal images of choroid plexus ( CP ) on brain sections from naive mice and four hours after LPS injection ( n = 3 ) . Brain sections were stained for TLR4 ( A ) and TNFR1 ( B ) ( red ) , pan - cytokeratine ( panCK ; green ) and the nuclei were stained with Hoechst ( blue ) . The ependymal cells aligning the ventricles are marked with a dotted line . Scalebar = 100 µm .

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

(A-B) Representative confocal images of choroid plexus (CP) on brain sections from naive mice and four hours after LPS injection (n=3). Brain sections were stained for TLR4 (A) and TNFR1 (B) (red), pan-cytokeratine (panCK; green) and the nuclei were stained with Hoechst (blue). The ependymal cells aligning the ventricles are marked with a dotted line. Scalebar = 100 µm.


( C ) NanoSight analysis of EVs in the CSF in sham - operated ( black ) relative to CLP - treated ( grey ) mice 10 hours after surgery .

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O O O O O B-ORGANISM O O O O O

(C) NanoSight analysis of EVs in the CSF in sham-operated (black) relative to CLP-treated (grey) mice 10 hours after surgery.


( D ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of sham - operated mice ( black ) and mice subjected to CLP ( grey ) 10 hours after surgery . ( E ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the CSF of sham - operated mice ( black ) and mice subjected to CLP ( grey ) 10 hours after surgery .

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

(D) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery. (E) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery.


( F ) NanoSight analysis of EVs in CSF of control ( black ) and TNF - injected ( 25 µg / 20 g ; grey ) mice 6 hours after TNF challenge .

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

(F) NanoSight analysis of EVs in CSF of control (black) and TNF-injected (25 µg/20 g; grey) mice 6 hours after TNF challenge.


( G ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of control mice ( black ) and in mice injected with TNF ( grey ) 6 hours after injection . ( H ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the CSF of control mice ( black ) and on mice 6 hours after TNF injection ( grey ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Scalebar 100 µm . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O B-ORGANISM O O O O O B-ORGANISM 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 O O O O O O O O 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) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of control mice (black) and in mice injected with TNF (grey) 6 hours after injection. (H) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of control mice (black) and on mice 6 hours after TNF injection (grey). Data are displayed as mean ± SEM and analyzed by Student's t-test. Scalebar 100 µm. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.


( A ) Representative confocal image of brain parenchyma , four hours after intracerebroventricular injection of PKH26 labelled EVs ( red ) . Astrocytes are stained with GFAP ( white ) , nuclei with Hoechst ( blue ) , and pan - cytokeratin ( panCK ; green ) was used to label the choroid plexus . The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer .

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

(A) Representative confocal image of brain parenchyma, four hours after intracerebroventricular injection of PKH26 labelled EVs (red). Astrocytes are stained with GFAP (white), nuclei with Hoechst (blue), and pan-cytokeratin (panCK; green) was used to label the choroid plexus. The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer.


( B - C ) Representative confocal images showing the uptake of double - labelled EVs ( Ribogreen ; green , PKH26 , red ) by astrocytes stained with GFAP ( white ) ( B ) and microglia cells stained with IBA1 ( white ) ( C ) incubated on mixed cortical cultures . Boxed areas are shown as zoomed images on right . Cell nuclei are stained with Hoechst ( blue ) . The in vivo and in vitro uptake experiments were performed 3 and 2 times ( n = 3 ) , respectively . Scalebar 100 = µm .

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

(B-C) Representative confocal images showing the uptake of double-labelled EVs (Ribogreen; green, PKH26, red) by astrocytes stained with GFAP (white) (B) and microglia cells stained with IBA1 (white) (C) incubated on mixed cortical cultures. Boxed areas are shown as zoomed images on right. Cell nuclei are stained with Hoechst (blue). The in vivo and in vitro uptake experiments were performed 3 and 2 times (n=3), respectively. Scalebar 100 = µm.


( A - B ) qPCR gene expression analysis of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated ( black ) or LPS treated ( grey ) mice ( A ) and in brain tissue ( B ) before ( black ) and 8 h after LPS injection ( grey ) ( n = 3 ) .

O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE 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

(A-B) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice (A) and in brain tissue (B) before (black) and 8 h after LPS injection (grey) (n=3).


( C ) qPCR gene expression analysis of inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα by qPCR in mixed cortical cultures ( left ; in vitro ) incubated with EVs isolated from CSF from untreated ( black ) or LPS treated ( grey ) mice and in brain tissue ( right ; in vivo ) before ( black ) and 8 h after LPS injection ( grey ) ( n = 3 ) .

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

(C) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα by qPCR in mixed cortical cultures (left; in vitro) incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice and in brain tissue (right; in vivo) before (black) and 8 h after LPS injection (grey) (n=3).


( D ) Cytokine analysis ( IL - 6 , IL - 1β and TNF ) of supernatant of mixed cortical cultures ( left ; in vitro ) incubated with EVs isolated from untreated ( black ; n = 3 ) or LPS treated ( grey ; n = 3 ) mice and of CSF ( right ; in vivo ) from untreated ( black ; n = 3 ) and LPS treated mice ( grey ; n = 6 ) .

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

(D) Cytokine analysis (IL-6, IL-1β and TNF) of supernatant of mixed cortical cultures (left; in vitro) incubated with EVs isolated from untreated (black; n=3) or LPS treated (grey; n=3) mice and of CSF (right; in vivo) from untreated (black; n=3) and LPS treated mice (grey; n=6).


( E - F ) qPCR gene expression analysis of inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα ( E ) and miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 ( F ) in mixed cortical cultures incubated with CD63 - depleted EVs isolated from untreated mice ( black ) and mice treated with LPS for 6 h ( grey ) ( n = 3 ) .

O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-ORGANISM O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O

(E-F) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (E) and miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (F) in mixed cortical cultures incubated with CD63-depleted EVs isolated from untreated mice (black) and mice treated with LPS for 6 h (grey) (n=3).


( G - H ) qPCR gene expression analysis of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 ( G ) and inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα ( H ) in brain tissue from LPS - injected mice icv injected with vehicle ( black ) or GW4869 ( grey ; n = 7 ) . 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 .

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

(G-H) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (G) and inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (H) in brain tissue from LPS-injected mice icv injected with vehicle (black) or GW4869 (grey; n=7). 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.


A ) Double fluorescence in situ hybridization using Nrg3 ( green ) and Gad1 ( red ) specific probes ; DAPI was used as a counter stain . Nrg3 is present in Gad1 - positive ( filled arrowheads ) and - negative neurons . ( A ' ) and ( A ' ' ) show higher magnifications of boxed areas indicated in ( A ) . Nrg3 - negative neurons are indicated by open arrowheads ; note that many but not all neurons express Nrg3 .

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

A) Double fluorescence in situ hybridization using Nrg3 (green) and Gad1 (red) specific probes; DAPI was used as a counter stain. Nrg3 is present in Gad1-positive (filled arrowheads) and -negative neurons. (A') and (A'') show higher magnifications of boxed areas indicated in (A). Nrg3-negative neurons are indicated by open arrowheads; note that many but not all neurons express Nrg3.


B ) Comparison of mRNA levels of Nrg3 , Nrg1β type III ( Nrg1β − III ) , Nrg2β , Nrg1β type II ( Nrg1β − II ) , Nrg2α and Nrg1β type I ( Nrg1β − I ) in the cortex ( Cx ) , hippocampus ( HC ) and striatum ( Str ) of juvenile mice ( P30 - 60 ) by quantitative RT - PCR . Data represent means ± SD of 4 biological replicates . 2 - way - ANOVA with Bonferroni ' s multiple comparisons test was performed to assess statistical significance ( * * * * P < 0 . 0001 , * * * P < 0 . 001 , ns = not significant ) .

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

B) Comparison of mRNA levels of Nrg3, Nrg1β type III (Nrg1β−III), Nrg2β, Nrg1β type II (Nrg1β−II), Nrg2α and Nrg1β type I (Nrg1β−I) in the cortex (Cx), hippocampus (HC) and striatum (Str) of juvenile mice (P30-60) by quantitative RT-PCR. Data represent means ± SD of 4 biological replicates. 2-way-ANOVA with Bonferroni's multiple comparisons test was performed to assess statistical significance (****P<0.0001, ***P<0.001, ns=not significant).


D ) Detection of Nrg3 by western blot analysis in fractions from brain lysates : S1 , crude lysate ; S2 , cytosol and light membranes ; S2 ' , cytosol ; LM , light membranes ; P2 ' , crude synaptosomes ; S3 ' , synaptosomal cytoplasm ; SV , synaptic vesicles ; P3 , synaptosomal membranes ; SPM , synaptic plasma membranes .

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

D) Detection of Nrg3 by western blot analysis in fractions from brain lysates: S1, crude lysate; S2, cytosol and light membranes; S2', cytosol; LM, light membranes; P2', crude synaptosomes; S3', synaptosomal cytoplasm; SV, synaptic vesicles; P3, synaptosomal membranes; SPM, synaptic plasma membranes.


E , F ) Immunohistochemical analysis in the hippocampus of wildtype ( E , E ' , E ' ' ) and ErbB4 mutant mice ( F ) at 2 month of age using Nrg3 - , ErbB4 - and parvalbumin ( Parv ) - specific antibodies . The white box in ( E ) is displayed magnified in ( E ' , E ' ' ) . In control mice , Nrg3 is enriched on dendrites of ErbB4 + PV interneurons , compared to the surrounding neuropil ( E ' , E ' ' ) . In ErbB4 mutants , the Nrg3 enrichment on PV interneurons is not apparent ( F ) .

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

E, F) Immunohistochemical analysis in the hippocampus of wildtype (E,E',E'') and ErbB4 mutant mice (F) at 2 month of age using Nrg3-, ErbB4- and parvalbumin (Parv)-specific antibodies. The white box in (E) is displayed magnified in (E',E''). In control mice, Nrg3 is enriched on dendrites of ErbB4+ PV interneurons, compared to the surrounding neuropil (E',E''). In ErbB4 mutants, the Nrg3 enrichment on PV interneurons is not apparent (F).


G ) Nrg3 , ErbB4 and GluA4 were identified by immunohistology and co - localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1 . Note that ( G ) displays Nrg3 / ErbB4 , ( G ' ) Nrg3 / GluA4 , and ( G ' ' ) ErbB4 / GluA4 signals of the same triple - stained image ; false colors were assigned for better signal visualization .

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

G) Nrg3, ErbB4 and GluA4 were identified by immunohistology and co-localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1. Note that (G) displays Nrg3/ErbB4, (G') Nrg3/GluA4, and (G'') ErbB4/GluA4 signals of the same triple-stained image; false colors were assigned for better signal visualization.


H ) Quantification of GluA4 puncta present on ErbB4 + dendrites in the CA1 stratum radiatum of adult wildtype ( wt ) and Nrg3 - / - mice ( age P90 - 120 ) . Data are presented as box plots with Tukey ' s whiskers and outliers ; means are indicated by a Plus symbols . n = 65 ( wt ) and n = 62 ( Nrg3 - / - ) from 5 animals each . Unpaired t - test ( 2 - tailed ) with Welch ' s correction was performed to assess statistical significance ( * * * * P < 0 . 0001 ) .

O O O O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O 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 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 O O

H) Quantification of GluA4 puncta present on ErbB4+ dendrites in the CA1 stratum radiatum of adult wildtype (wt) and Nrg3-/- mice (age P90-120). Data are presented as box plots with Tukey's whiskers and outliers; means are indicated by a Plus symbols. n=65 (wt) and n=62 (Nrg3-/-) from 5 animals each. Unpaired t-test (2-tailed) with Welch's correction was performed to assess statistical significance (****P<0.0001).


A ) His6 - tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations . Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho - tyrosine ( pY ) and ErbB4 , and of whole lysates using antibodies against pErk1 / 2 and pAkt

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

A) His6-tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations. Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho-tyrosine (pY) and ErbB4, and of whole lysates using antibodies against pErk1/2 and pAkt


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