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

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

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


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

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


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

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


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

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

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


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

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


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

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

(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 ) .

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

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


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

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

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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

(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 .

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


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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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

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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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 positive for NDP52. HeLa cells expressing the indicated NDP52 variants fused to YFP were infected with S. Typhimurium.


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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(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 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 ) .

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

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(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 of supernatant of choroid plexus explants from PBS - or LPS - injected mice ( n = 6 ) . Data are displayed as mean ± analyzed by Student ' s t - . Significance are indicated on graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

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

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

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


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


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

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

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(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 ( ) , nuclei with Hoechst ( blue ) , pan - cytokeratin ( panCK ; green ) was used to label choroid plexus . dotted shows ventricular border arrow heads point to EVs that crossed ependymal layer .

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

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

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

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(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 ( IL - 6 , IL - 1β 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 of CSF ( right ; in vivo ) from untreated ( black ; n = 3 ) LPS treated mice ( grey ; n = 6 ) .

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

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(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 of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 Irak1 ( G ) inflammatory genes Il - 1β , Tnf , Il - 6 , iNos iκbα ( H ) in brain tissue from LPS - injected mice icv injected with vehicle ( black ) or GW4869 ( grey ; n = 7 ) . Data are displayed as mean ± analyzed by Student ' s t - . Significance are indicated on graphs : * , 0 . 01 ≤ P < 0 . 05 .

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

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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 of Nrg3 , Nrg1β type III ( Nrg1β − III ) , Nrg2β , Nrg1β type II ( Nrg1β − II ) , Nrg2α Nrg1β type I ( Nrg1β − I ) in cortex ( Cx ) , hippocampus ( HC ) 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 was performed to assess statistical significance ( * * * * P < 0 . 0001 , * * * P < 0 . 001 , ns = not significant ) .

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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 in fractions from brain lysates : S1 , crude lysate ; S2 , cytosol membranes ; S2 ' , cytosol ; LM , membranes ; P2 ' , crude synaptosomes ; S3 ' , synaptosomal cytoplasm ; SV , synaptic vesicles ; P3 , synaptosomal membranes ; SPM , synaptic plasma membranes .

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

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

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

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

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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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C , D ) Neurons from Nrg3 - / - ; ErbB4 - / - ; vGAT - Cre mice were transduced with a lentivirus expressing either ( C ) ErbB4wt or ( D ) ErbB4kd ( kinase dead ErbB4 ) in a Cre - dependent manner , with a second at a low titer expressing Nrg3 / SypCFP . Immunocytochemical of ErbB4 SypCFP ; ( C , C ' ) ( D , D ' ) each show same image , ( C ' , D ' ) show SypCFP signal

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C, D) Neurons from Nrg3-/-;ErbB4-/-;vGAT-Cre mice were transduced with a lentivirus expressing either (C) ErbB4wt or (D) ErbB4kd (kinase dead ErbB4) in a Cre-dependent manner, and with a second virus at a low titer expressing Nrg3/SypCFP. Immunocytochemical analysis of ErbB4 and SypCFP; (C,C') and (D,D') each show the same image, (C',D') show the SypCFP signal


E - G ) of Synapse preference towards ErbB4 + neurons ( E ) , ErbB4 enrichment ( F ) SypCFP enrichment ( G ) in synapses on neurons expressing wildtype ErbB4 ( wt ) or ErbB4kd ( kd )

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E-G) Quantification of Synapse preference towards ErbB4+ neurons (E), ErbB4 enrichment (F) and SypCFP enrichment (G) in synapses on neurons expressing wildtype ErbB4 (wt) or ErbB4kd (kd)


H ) Nrg3 - / - neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 GFP immunostained with antibodies against ErbB4 , GFP GluA ; ( H ) shows triple stained image , while ( H ' ) ( H ' ' ) only show ErbB4 GluA signals , respectively

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H) Nrg3-/- neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4, GFP and GluA; (H) shows the triple stained image, while (H') and (H'') only show ErbB4 and GluA signals, respectively


I ) Co - cultures of Nrg3 - / - neurons with HEK293 expressing Nrg3 . Cultures were stained with antibodies against Nrg3 , ErbB4 GluA . Note that Nrg3 , ErbB4 GluA are enriched at contact sites between Nrg3 - expressing HEK293 ErbB4 + neurons

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I) Co-cultures of Nrg3-/- neurons with HEK293 cells expressing Nrg3. Cultures were stained with antibodies against Nrg3, ErbB4 and GluA. Note that Nrg3, ErbB4 and GluA are enriched at the contact sites between Nrg3-expressing HEK293 cells and ErbB4+ neurons


C ) Example traces of evoked excitatory postsynaptic currents ( eEPSCs ) averaged over 50 sweeps . D , E ) of peak amplitude of first eEPSC ( D ) of paired ( E )

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C) Example traces of evoked excitatory postsynaptic currents (eEPSCs) averaged over 50 sweeps. D, E) Quantification of the peak amplitude of the first eEPSC (D) and of the paired pulse ratio (E)


F - I ) sEPSC recordings from CA1 pyramidal ( F , H ) PV neurons ( G , I ) in acute slices . sEPSC frequencies ( F , G ) amplitudes ( H , I ) are

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F-I) sEPSC recordings from CA1 pyramidal (F,H) and PV neurons (G, I) in acute slices. sEPSC frequencies (F,G) and amplitudes (H,I) are shown


TRF1 immunofluorescence of lungs . Notice absence presence of TRF1 signal in carcinomas surrounding healthy tissue of Trf1Δ / Δ mice , respectively .

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TRF1 immunofluorescence of the lungs. Notice the absence and presence of TRF1 signal in the carcinomas and surrounding healthy tissue of Trf1Δ/Δ mice, respectively.


of Trf1 excision by PCR . Notice completed excision in carcinomas of Trf1lox / lox lungs .

Analysis of Trf1 excision by PCR. Notice the completed excision in carcinomas of Trf1lox/lox lungs.


Detection of β - galactosidase activity in lungs as a surrogate marker of oncogenic K - RasG12V expression .

Detection of β-galactosidase activity in the lungs as a surrogate marker of oncogenic K-RasG12V expression.


growth curve of Trf1 + / + K - Ras + / G12Vp53 + / + Trf1Δ / ΔK - Ras + / G12Vp53 + / + measured by computed tomography ( CT ) . of number of Trf1 + / + K - Ras + / G12Vp53 + / + Trf1Δ / ΔK - Ras + / G12Vp53 + / + carcinomas at point .

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Tumor growth curve of Trf1+/+K-Ras+/G12Vp53+/+ and Trf1Δ/ΔK-Ras+/G12Vp53+/+ measured by computed tomography (CT).Quantification of the number and size of Trf1+/+K-Ras+/G12Vp53+/+ and Trf1Δ/ΔK-Ras+/G12Vp53+/+carcinomas at death point.


of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed . Post - mortem of Trf1 deletion in each revealed that none of Trf1lox / lox K - Ras + / G12V p53 + / + ones had excised Trf1 .

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Percentage of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed. Post-mortem analysis of Trf1 deletion in each tumor revealed that none of the Trf1lox/lox K-Ras+/G12V p53+/+ ones had excised Trf1.


growth curve growth slope of Trf1 + / + K - Ras + / G12V p53 − / − Trf1 − / − K - Ras + / G12V p53 − / − measured by CT .

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Tumor growth curve and tumor growth slope of Trf1+/+ K-Ras+/G12V p53−/− and Trf1−/− K-Ras+/G12V p53−/− measured by CT.


maximal section of Trf1 + / + K - Ras + / G12Vp53 − / − Trf1Δ / ΔK - Ras + / G12Vp53 − / − lungs measured by histological before point by CT .

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Tumor maximal section of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs measured by histological analysis before death point by CT.


Maximum 18F - FDG - glucose uptake by Trf1 + / + K - Ras + / G12Vp53 − / − Trf1Δ / ΔK - Ras + / G12Vp53 − / − tumors 22 after infection by positron emission tomography ( PET ) . Representative PET - CT image of Trf1 + / + K - Ras + / G12Vp53 − / − Trf1Δ / ΔK - Ras + / G12Vp53 − / − lungs .

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Maximum 18F-FDG-glucose uptake by Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−tumors 22 weeks after infection by positron emission tomography (PET).Representative PET-CT image of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs.


Survival curve of Trf1 + / + K - Ras + / G12V p53 − / − Trf1 − / − K - Ras + / G12V p53 − / − mice .

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Survival curve of Trf1+/+ K-Ras+/G12V p53−/− and Trf1−/− K-Ras+/G12V p53−/− mice.


of showing γH2AX foci in carcinomas of indicated genotypes ( left panel ) . Representative of γH2AX immunohistochemistry ( right panel ) .

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Percentage of cells showing γH2AX foci in carcinomas of the indicated genotypes (left panel). Representative images of γH2AX immunohistochemistry (right panel).


of showing 3 or more γH2AX RAP1 colocalizing foci ( TIFs ) ( left panel ) . Representative of γH2AX RAP1 double immunofluorescence ( right panel ) . Yellow arrowheads : colocalization of γH2AX RAP1 .

Percentage of cells showing 3 or more γH2AX and RAP1 colocalizing foci (TIFs) (left panel). Representative images of γH2AX and RAP1 double immunofluorescence (right panel). Yellow arrowheads: colocalization of γH2AX and RAP1.


of active caspase - 3 ( AC3 ) - positive ( left panel ) . Representative of AC3 immunohistochemistry ( right panel ) .

Percentage of active caspase-3 (AC3)-positive cells (left panel). Representative images of AC3 immunohistochemistry (right panel).


of Ki67 - positive in carcinomas of indicated genotypes ( left panel ) . Representative of Ki67 immunohistochemistry ( right panel ) .

Percentage of Ki67-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of Ki67 immunohistochemistry (right panel).


of pH3 - positive in carcinomas of indicated genotypes ( left panel ) . Representative of pH3 immunohistochemistry ( right panel ) . Red arrowheads : pH3 - positive .

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Percentage of pH3-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of pH3 immunohistochemistry (right panel). Red arrowheads: pH3-positive cells.


of giant nuclei in carcinomas of indicated genotype . of anaphase bridges out of total anaphases in carcinomas of indicated genotypes . Representative of giant nuclei , multilobulated nuclei , anaphase bridges , multipolar mitoses . Red arrowheads indicate corresponding mitotic aberrations indicated in image .

Percentage of giant nuclei in the carcinomas of the indicated genotype.Percentage of anaphase bridges out of total anaphases in the carcinomas of the indicated genotypes.Representative images of giant nuclei, multilobulated nuclei, anaphase bridges, and multipolar mitoses. Red arrowheads indicate the corresponding mitotic aberrations indicated in the image.


A - C latency ( A ) , volume ( B ) , ( C ) of subcutaneous tumors generated by control Trf1 - downregulated K - RasG12V - transformed lung in athymic mice . D Representative of subcutaneous tumors .

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A-C The latency (A), volume (B), and weight (C) of subcutaneous tumors generated by control and Trf1-downregulated K-RasG12V-transformed lung cells in athymic mice.D Representative images of the subcutaneous tumors.


E Trf1 expression measured by qPCR in injected in generated subcutaneous tumors .

E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors.


F - H Number of Ki67 - positive ( F ) , number of γH2AX - positive ( G ) , number of active caspase - 3 - positive ( H ) per field in subcutaneous tumors .

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F-H Number of Ki67-positive (F), number of γH2AX-positive (G), and number of active caspase-3-positive (H) cells per field in the subcutaneous tumors.


I Representative of aberrant giant nuclei anaphase bridges in Trf1 - downregulated subcutaneous tumors compared to normal nuclei of control tumors .

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I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors.


J TRF1 immunofluorescence shows downregulation of Trf1 in lung tumors of mice intravenously injected with control Trf1 - downregulated .

J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells.


K measured in lungs of mice intravenously injected with control Trf1 - downregulated . L Representative of lungs colonized by control Trf1 - downregulated , respectively .

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K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively.


M - O Number of Ki67 - positive ( M ) , number of γH2AX - positive ( N ) , number of active caspase - 3 - positive ( O ) per field in lung tumors .

M-O Number of Ki67-positive (M), number of γH2AX-positive (N), and number of active caspase-3-positive (O) cells per field in the lung tumors.


P Trf1 expression measured by qPCR in A549 infected either with sh - scrambled or sh - Trf1 .

P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.


Trf1 expression in indicated tissues of wild - type Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 .

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Trf1 expression levels in the indicated tissues of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


Representative of TRF1 immunofluorescence of of TRF1 - positive in skin intestine sections of wild - type Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 .

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Representative images of TRF1 immunofluorescence and quantification of the percentage of TRF1-positive cells in skin and intestine sections of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


Survival curve of wild - type Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 .

Survival curve of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


of histological alterations observed in tamoxifen - treated Trf1lox / lox hUBC - CreERT2 mice 4 months after tamoxifen retrieval compared to their wild - type counterparts .

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Quantification of the histological alterations observed in tamoxifen-treated Trf1lox/lox hUBC-CreERT2 mice and 4 months after tamoxifen retrieval compared to their wild-type counterparts.


of blood populations in wild - type Trf1lox / lox hUBC - CreERT2mice subjected to a tamoxifen - containing diet for 7 after 3 4 months of tamoxifen retrieval .

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Quantification of blood cell populations in wild-type and Trf1lox/lox hUBC-CreERT2mice subjected to a tamoxifen-containing diet for 7 weeks and after 3 weeks and 4 months of tamoxifen retrieval.


Trf1 expression in blood , intestine , skin , bone marrow of Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet either for 7 or for 6 months after 3 tamoxifen retrieval compared to wild - type mice .

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Trf1 expression levels in blood, intestine, skin, and bone marrow of Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet either for 7 weeks or for 6 months and after 3 weeks tamoxifen retrieval compared to wild-type mice.


of TRF1 by immunofluorescence in lung - derived treated with DMSO , with 10 μM ETP - 47228 ( 24 h ) , with 10 μM ETP - 47037 ( 48 h ) . Representative are shown to right .

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Quantification of TRF1 levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with 10 μM ETP-47228 (24 h), and with 10 μM ETP-47037 (48 h). Representative images are shown to the right.


of γH2AX by immunofluorescence in lung - derived treated with DMSO , with ETP - 47228 ( 24 h ) , with ETP - 47037 ( 48 h ) . Representative are shown to right .

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Quantification of γH2AX levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with ETP-47228 (24 h), and with ETP-47037 (48 h). Representative images are shown to the right.


of telomere - induced foci ( TIFs ) by double immunofluorescence with anti - RAP1 anti - γH2AX antibodies . Representative are shown to right . arrowheads : colocalization of γH2AX RAP1 .

Quantification of telomere-induced foci (TIFs) by double immunofluorescence with anti-RAP1 and anti-γH2AX antibodies. Representative images are shown to the right. White arrowheads: colocalization of γH2AX and RAP1.


Effect of different ETP - 47228 ETP - 47037 concentrations during 24 h on proliferation in lung - derived relative to growth of DMSO - treated .

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Effect of different ETP-47228 and ETP-47037 concentrations during 24 h on proliferation in lung tumor-derived cell line relative to the growth of DMSO-treated cells.


growth in allograft model ETP - 47037 or with ETP - 47228 .

Tumor growth quantification in allograft model ETP-47037 or with ETP-47228.


of growth relative to initial . Representative CT are shown to right . arrowhead indicates a within a highly inflammatory region .

Quantification of tumor growth relative to initial tumor size. Representative CT images are shown to the right. The white arrowhead indicates a tumor within a highly inflammatory region.


of TRF1 by immunofluorescence in intestine lung tumors samples of mice treated with vehicle or with ETP - 47037 for 10 days . Representative are shown to right ( n = 4 ) .

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Quantification of TRF1 levels by immunofluorescence in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).


Number of showing γH2AX foci in intestine lung tumors samples of mice treated with vehicle or with ETP - 47037 for 10 days . Representative are shown to right ( n = 4 ) .

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Number of cells showing γH2AX foci in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).


A - C of number of ( A ) Ki67 - , ( B ) pan - p - H3 pattern - , ( C ) foci p - H3 pattern - positive in untreated ETP - 470037 - treated lungcarcinomas . data represent mean values obtained for three mice in each group . Error bars represent standard errors . D Representative Ki67 p - H3 .

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A-C Quantification of the number of (A) Ki67-, (B) pan-nuclear p-H3 pattern-, and (C) foci p-H3 pattern-positive cells in untreated and ETP-470037-treated lungcarcinomas. The data represent the mean values obtained for three mice in each group. Error bars represent standard errors.D Representative Ki67 and p-H3 images.


E Representative H & amp ; amp ; E of intestine samples corresponding to untreated ETP - 47037 - treated . High - magnification are shown to right indicating presence of normal mitosis , giant multinucleated aberrant mitotic figures .

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E Representative H&amp;E images of intestine samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown to the right indicating the presence of normal mitosis, giant multinucleated and aberrant mitotic figures.