( B ) Boxplots representing PMEPA1 expression levels in the different MB groups ( WNT in blue , SHH in red , Group 3 in yellow and Group 4 in green ) and in fetal and adult cerebellum ( grey ) in the dataset " Data ref : Cavalli et al , 2017 " . Only p - values corresponding to comparisons between Group 3 and the other groups are indicated . The p - values were determined by Wilcoxon rank - sum test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(B) Boxplots representing PMEPA1 expression levels in the different MB groups (WNT in blue, SHH in red, Group 3 in yellow and Group 4 in green) and in fetal and adult cerebellum (grey) in the dataset " Data ref: Cavalli et al, 2017". Only p-values corresponding to comparisons between Group 3 and the other groups are indicated. The p-values were determined by Wilcoxon rank-sum test *p < 0.05, **p < 0.01, ***p < 0.001.


( C ) Scatter plot of INHBB and PMEPA1 gene expression levels in all MB groups . Colored dots represent each patient samples and colors represent the MB groups ( WNT in blue , SHH in red , Group 3 in yellow and Group 4 in green ) . The p - values were determined by Spearman rank correlation test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(C) Scatter plot of INHBB and PMEPA1 gene expression levels in all MB groups. Colored dots represent each patient samples and colors represent the MB groups (WNT in blue, SHH in red, Group 3 in yellow and Group 4 in green). The p-values were determined by Spearman rank correlation test *p < 0.05, **p < 0.01, ***p < 0.001.


( D ) Boxplot represents the quantification of PMEPA1 protein level normalized to β - actin levels across groups . The p - values were determined by unpaired t - test for D * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(D) Boxplot represents the quantification of PMEPA1 protein level normalized to β-actin levels across groups. The p-values were determined by unpaired t-test for D *p < 0.05, **p < 0.01, ***p < 0.001.


( E ) Scatter plot represents Log2 relative protein level of P - Smad2 ( x axis ) and PMEPA1 ( Y axis ) normalized to β - actin in each individual samples . The p - values were determined by Wilcoxon rank - sum test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(E) Scatter plot represents Log2 relative protein level of P-Smad2 (x axis) and PMEPA1 (Y axis) normalized to β-actin in each individual samples. The p-values were determined by Wilcoxon rank-sum test *p < 0.05, **p < 0.01, ***p < 0.001.


( F ) Immunoblots of phosphorylated Smad2 ( P - Smad2 ) , total Smad2 , MYC , PMEPA1 , OTX2 and β - actin were performed on extracts from 1603MED or D283 or D458 cells treated with either DMSO ( vehicle ) , LY364947 , SB431542 , blocking antibody against ActivinB ( Ab α - ActB ) , Follistatin , PBS or ActivinB ( ActB ) for 24 hours .

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(F) Immunoblots of phosphorylated Smad2 (P-Smad2), total Smad2, MYC, PMEPA1, OTX2 and β-actin were performed on extracts from 1603MED or D283 or D458 cells treated with either DMSO (vehicle), LY364947, SB431542, blocking antibody against ActivinB (Ab α-ActB), Follistatin, PBS or ActivinB (ActB) for 24 hours.


Immunoblot analysis of PMEPA1 levels in 1603MED ( G ) cells 48 hours after transfection with either siCTRL ( blue ) or siPMEPA1 ( red ) . Bar graphs on the right represent the quantification of the relative level of PMEPA1 protein level normalized to β - actin . The p - values were determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent the mean ± SD . Number of replicates is n ≥ 3 .

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Immunoblot analysis of PMEPA1 levels in 1603MED (G) cells 48 hours after transfection with either siCTRL (blue) or siPMEPA1 (red). Bar graphs on the right represent the quantification of the relative level of PMEPA1 protein level normalized to β-actin. The p-values were determined by unpaired t-test *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3.


Growth curves of 1603MED ( H ) cells after transfection with either siCTRL ( blue ) or siPMEPA1 ( red ) . The p - values were determined by two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

Growth curves of 1603MED (H) cells after transfection with either siCTRL (blue) or siPMEPA1 (red). The p-values were determined by two-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.


Immunoblot analysis of PMEPA1 levels in D283 ( I ) cells 48 hours after transfection with either siCTRL ( blue ) or siPMEPA1 ( red ) . Bar graphs on the right represent the quantification of the relative level of PMEPA1 protein level normalized to β - actin . The p - values were determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent the mean ± SD . Number of replicates is n ≥ 3 .

Immunoblot analysis of PMEPA1 levels in D283 (I) cells 48 hours after transfection with either siCTRL (blue) or siPMEPA1 (red). Bar graphs on the right represent the quantification of the relative level of PMEPA1 protein level normalized to β-actin. The p-values were determined by unpaired t-test *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3.


Growth curves of D283 ( J ) cells after transfection with either siCTRL ( blue ) or siPMEPA1 ( red ) . The p - values were determined by two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

Growth curves of D283 (J) cells after transfection with either siCTRL (blue) or siPMEPA1 (red). The p-values were determined by two-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.


( A ) Immunoblot analysis of phosphorylated Smad2 ( P - Smad2 ) in Group 3 MB cell lines and PDXs . The level of total Smad2 ( Smad2 ) was assessed and β - actin was used as a loading control . Quantification of P - Smad2 ( P - S2 ) to β - actin are shown on right panel . Right ( A ) panels represent the relative quantification of P - Smad2 normalized to β - actin . Bars represent the mean ± SD . Number of replicates is n ≥ 3 .

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(A) Immunoblot analysis of phosphorylated Smad2 (P-Smad2) in Group 3 MB cell lines and PDXs. The level of total Smad2 (Smad2) was assessed and β-actin was used as a loading control. Quantification of P-Smad2 (P-S2) to β-actin are shown on right panel. Right (A) panels represent the relative quantification of P-Smad2 normalized to β-actin. Bars represent the mean ± SD. Number of replicates is n≥3.


( B ) Expression of INHBB in Group 3 MB cell lines and PDXs relative to HDMB03 ( set at 1 ) by RT - qPCR . Bars represent the mean ± SD . Number of replicates is n ≥ 3 .

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(B) Expression of INHBB in Group 3 MB cell lines and PDXs relative to HDMB03 (set at 1) by RT-qPCR. Bars represent the mean ± SD. Number of replicates is n≥3.


( C ) Immunoblot of phosphorylated Smad2 ( P - Smad2 ) , total Smad2 and β - actin upon ActivinB or TGFβ stimulation for 1 hour . lower panels represent the relative quantification of P - Smad2 normalized to β - actin . The p - values were determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent the mean ± SD . Number of replicates is n ≥ 3 .

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(C) Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin upon ActivinB or TGFβ stimulation for 1 hour. lower panels represent the relative quantification of P-Smad2 normalized to β-actin. The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3.


( D ) Conditioned media experiments were performed on HDMB03 MB cell line . Phosphorylation of Smad2 ( P - Smad2 ) was analyzed by immunoblot upon treatment with either non - conditioned media ( NCM ) , media conditioned with HDMB03 cells ( CM - HDMB03 ) or media conditioned on PDX4 cells ( CM - PDX4 ) . Pre - incubation with blocking antibody against ActivinB ( Ab α - ActB ) or with vehicle was performed before HDMB03 cell - line treatment as indicated . lower panels represent the relative quantification of P - Smad2 normalized to β - actin . The p - values were determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent the mean ± SD . Number of replicates is n ≥ 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Conditioned media experiments were performed on HDMB03 MB cell line. Phosphorylation of Smad2 (P-Smad2) was analyzed by immunoblot upon treatment with either non-conditioned media (NCM), media conditioned with HDMB03 cells (CM-HDMB03) or media conditioned on PDX4 cells (CM-PDX4). Pre-incubation with blocking antibody against ActivinB (Ab α-ActB) or with vehicle was performed before HDMB03 cell-line treatment as indicated. lower panels represent the relative quantification of P-Smad2 normalized to β-actin. The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3.


( E ) Immunoblots of phosphorylated Smad2 ( P - Smad2 ) , total Smad2 , MYC , PMEPA1 , OTX2 and β - actin were performed on extracts from cell cultures of PDX4 , PDX3 and PDX7 treated by either DMSO ( vehicle ) , LY364947 , SB431542 , blocking antibody against ActivinB ( Ab α - ActB ) , Follistatin , PBS or ActivinB for 24 hours .

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(E) Immunoblots of phosphorylated Smad2 (P-Smad2), total Smad2, MYC, PMEPA1, OTX2 and β-actin were performed on extracts from cell cultures of PDX4, PDX3 and PDX7 treated by either DMSO (vehicle), LY364947, SB431542, blocking antibody against ActivinB (Ab α-ActB), Follistatin, PBS or ActivinB for 24 hours.


( A ) Kaplan - Meier representing survival of mice treated with either vehicle ( black ) or Galunisertib ( LY2157299 , red ) or Cisplatin ( blue ) or a combination of Galunisertib and cisplatin ( purple ) after orthotopic grafting of PDX4 cells into the cerebellum . The pink rectangle represents Galunisertib treatment duration , while the blue dotted lines represent the 3 cisplatin administrations . The p - values were determined by Log - Rank ( Mantel - Cox ) test on panel A * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 . Bars represent the mean ± SD . Number of replicates is n ≥ 3 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O 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) Kaplan-Meier representing survival of mice treated with either vehicle (black) or Galunisertib (LY2157299, red) or Cisplatin (blue) or a combination of Galunisertib and cisplatin (purple) after orthotopic grafting of PDX4 cells into the cerebellum. The pink rectangle represents Galunisertib treatment duration, while the blue dotted lines represent the 3 cisplatin administrations. The p-values were determined by Log-Rank (Mantel-Cox) test on panel A *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.


( B ) Boxplot of tumor area after 25 days of treatment . On the right , boxplots represent quantification of P - Smad2 staining on tumor ( IHC ) . The p - values were determined by unpaired t - test on panel B . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 . Bars represent the mean ± SD . Number of replicates is n ≥ 3 .

(B) Boxplot of tumor area after 25 days of treatment. On the right, boxplots represent quantification of P-Smad2 staining on tumor (IHC). The p-values were determined by unpaired t-test on panel B. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.


( C ) P - Smad2 staining by IHC in 3 representative tumors per group after 25 days of treatment . The scale represents 500μm and 100μm on the left and right panels , respectively .

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(C) P-Smad2 staining by IHC in 3 representative tumors per group after 25 days of treatment. The scale represents 500μm and 100μm on the left and right panels, respectively.


( D ) Boxplots representing the expression level of INHBB , PMEPA1 , MYC and NRL in the different MB subtypes , as defined in Cavalli et al . ( Cavalli et al . , 2017 ) . Only p - values corresponding to comparisons between Group 3 subtypes are indicated . Patient samples are colored by subtypes as indicated . ( E ) Scatter plot of INHBB and PMEPA1 , expression levels in Group 3 patient samples . Colored dots represent each patient sample and colors represent the group 3 MB subtypes ( α in yellow , β in brown and δ in orange ) . Wilcoxon rank - sum tests were performed for panel D . Spearman ' s rank correlation coefficient ρ and p - value are indicated on panel E . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 . Bars represent the mean ± SD . Number of replicates is n ≥ 3 .

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(D) Boxplots representing the expression level of INHBB, PMEPA1, MYC and NRL in the different MB subtypes, as defined in Cavalli et al. (Cavalli et al., 2017). Only p-values corresponding to comparisons between Group 3 subtypes are indicated. Patient samples are colored by subtypes as indicated. (E) Scatter plot of INHBB and PMEPA1, expression levels in Group 3 patient samples. Colored dots represent each patient sample and colors represent the group 3 MB subtypes (α in yellow, β in brown and δ in orange). Wilcoxon rank-sum tests were performed for panel D. Spearman's rank correlation coefficient ρ and p-value are indicated on panel E. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.


( B ) From top to bottom : experimental design of 4D induction , fluorescence pictures of the SVZ of a 4D + mouse and quantification of the proportion of RFP - ( black ) and RFP + ( red ) progenitors among B1 , C and A cells identified with markers as indicated .

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(B) From top to bottom: experimental design of 4D induction, fluorescence pictures of the SVZ of a 4D+ mouse and quantification of the proportion of RFP- (black) and RFP+ (red) progenitors among B1, C and A cells identified with markers as indicated.


( C ) From top to bottom : experimental design , fluorescence pictures of the SVZ of a 4D - ( left ) and 4D + ( right and insets magnified ) mice and quantification of the proportion of BrdU + among B1 , C and A cells ( identified as in B ) . Note that in 4D + mice quantification was restricted to the RFP + subpopulation ( red bars ) .

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(C) From top to bottom: experimental design, fluorescence pictures of the SVZ of a 4D- (left) and 4D+ (right and insets magnified) mice and quantification of the proportion of BrdU+ among B1, C and A cells (identified as in B). Note that in 4D+ mice quantification was restricted to the RFP+ subpopulation (red bars).


( D ) Quantification of the absolute number of B1 , C and A cells ( identified as in B ) in the SVZ of 4D - ( white ) and 4D + ( black ) mice regardless of RFP expression ( RFP + / - ) .

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(D) Quantification of the absolute number of B1, C and A cells (identified as in B) in the SVZ of 4D- (white) and 4D+ (black) mice regardless of RFP expression (RFP+/-).


From top to bottom : fluorescence pictures of the SVZ and absolute number of cells in 4D - ( white bars ) or 4D + ( black or red bars for all or RFP + cells , respectively ) mice scored positive for various markers as indicated .

From top to bottom: fluorescence pictures of the SVZ and absolute number of cells in 4D- (white bars) or 4D+ (black or red bars for all or RFP+ cells, respectively) mice scored positive for various markers as indicated.


From top to bottom : fluorescence pictures of the SVZ and absolute number of cells in 4D - ( white bars ) or 4D + ( black or red bars for all or RFP + cells , respectively ) mice scored positive for various markers as indicated . Insets in ( C ) are magnified ( right ) with arrowheads pointing label - retaining NSC ( white ) or astrocytes ( empty ) .

From top to bottom: fluorescence pictures of the SVZ and absolute number of cells in 4D- (white bars) or 4D+ (black or red bars for all or RFP+ cells, respectively) mice scored positive for various markers as indicated. Insets in (C) are magnified (right) with arrowheads pointing label-retaining NSC (white) or astrocytes (empty).


From top to bottom : fluorescence pictures of the SVZ or proportions ( C - E ) of cells in 4D - ( white bars ) or 4D + ( black or red bars for all or RFP + cells , respectively ) mice scored positive for various markers as indicated . Arrowheads in ( D ) point cell doublets ( among RFP + protein - reteining cells in 4D + ) .

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From top to bottom: fluorescence pictures of the SVZ or proportions (C-E) of cells in 4D- (white bars) or 4D+ (black or red bars for all or RFP+ cells, respectively) mice scored positive for various markers as indicated. Arrowheads in (D) point cell doublets (among RFP+ protein-reteining cells in 4D+).


From top to bottom : fluorescence pictures of OB ( E ) and absolute number or proportions of cells in 4D - ( white bars ) or 4D + ( black or red bars for all or RFP + cells , respectively ) mice scored positive for various markers as indicated . ( E ) GL = glomerular , EPL = external plexiform , MCL = mitral cell and GCL = granule cell layers .

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From top to bottom: fluorescence pictures of OB (E) and absolute number or proportions of cells in 4D- (white bars) or 4D+ (black or red bars for all or RFP+ cells, respectively) mice scored positive for various markers as indicated. (E) GL=glomerular, EPL=external plexiform, MCL=mitral cell and GCL=granule cell layers.


( B ) Fluorescence pictures ( left ) of immunolabeled GFP + RFP - ( 4D - ) and GFP + RFP + ( 4D + ) apical dendrites of superficial granule neurons and quantifications ( middle and right ) of spine density and total dendritic length ( box and whiskers ) and 3D - Sholl ( line graph profile ) of apical dendrites starting from the soma ( drawings ) as the mean number of intersections at 10 μm intervals .

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(B) Fluorescence pictures (left) of immunolabeled GFP+RFP- (4D-) and GFP+RFP+ (4D+) apical dendrites of superficial granule neurons and quantifications (middle and right) of spine density and total dendritic length (box and whiskers) and 3D-Sholl (line graph profile) of apical dendrites starting from the soma (drawings) as the mean number of intersections at 10 μm intervals.


( C ) 3D reconstruction of multi - channel confocal stacks acquired from a RFP + apical dendrite of a granule cell showing co - localization with the pre - and post - synaptic markers VIAAT ( granule neuron ) and gephyrin ( mitral cell ) , respectively ( magnification shown in insets ) .

(C) 3D reconstruction of multi-channel confocal stacks acquired from a RFP+ apical dendrite of a granule cell showing co-localization with the pre- and post-synaptic markers VIAAT (granule neuron) and gephyrin (mitral cell), respectively (magnification shown in insets).


( D ) Anti - RFP immunogold labeling of a cell in the superficial granule cell layer of a 4D + mouse . Inset shows a representative RFP + synapse ( out of > 10 analyzed from 4D + mice ) .

(D) Anti-RFP immunogold labeling of a cell in the superficial granule cell layer of a 4D+ mouse. Inset shows a representative RFP+ synapse (out of >10 analyzed from 4D+ mice).


Current clamp recordings showing examples of spontaneous barrages of synaptic potentials ( E ) , Inset in E ( 4D + cell ) is magnified ( bottom ) .

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Current clamp recordings showing examples of spontaneous barrages of synaptic potentials (E), Inset in E (4D+ cell) is magnified (bottom).


Current clamp recordings showing examples of repetitive spiking in response to depolarizing current steps ( F - G ) of 4D - ( top ) and 4D + ( bottom ) mice . Note in G that the lag preventing spike initiation is longer at lower currents ( green ) and shorter at higher ( black ) , suggesting the presence of an A - type K current typical of granule cells in 4D - vs . 4D + .

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Current clamp recordings showing examples of repetitive spiking in response to depolarizing current steps (F-G) of 4D- (top) and 4D+ (bottom) mice. Note in G that the lag preventing spike initiation is longer at lower currents (green) and shorter at higher (black), suggesting the presence of an A-type K current typical of granule cells in 4D- vs. 4D+.


( H ) Box and whiskers plots representing electrophysiological properties of neurons derived from 4D - and 4D + NSC ( black and red , respectively ) including from left to right : spike number , resting membrane potential ( Vrest ) , input resistance ( Rin ) and lag to spiking of the recorded superficial granule cells ( see Fig . EV3 for additional parameters ) .

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(H) Box and whiskers plots representing electrophysiological properties of neurons derived from 4D- and 4D+ NSC (black and red, respectively) including from left to right: spike number, resting membrane potential (Vrest), input resistance (Rin) and lag to spiking of the recorded superficial granule cells (see Fig. EV3 for additional parameters).


( C ) Line graph indicating the proportion of correct responses ( performance ) for bins of 100 trials each during testing with binary mixtures of concentrated octanols . Discontinuous line indicates the similar 95 % plateau performance of 4D - ( black ) and 4D + ( red ) mice . ( D ) Whiskers box plots of performance ( left ) and lick frequency ( right ) during a probe of 200 trials with 1 : 10 diluted octanols after testing as in ( C ) .

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

(C) Line graph indicating the proportion of correct responses (performance) for bins of 100 trials each during testing with binary mixtures of concentrated octanols. Discontinuous line indicates the similar 95% plateau performance of 4D- (black) and 4D+ (red) mice. (D) Whiskers box plots of performance (left) and lick frequency (right) during a probe of 200 trials with 1:10 diluted octanols after testing as in (C).


( A ) ATX serum protein levels were measured in blood serum samples from IBD patients and healthy controls using ELISA : active UC ( n = 26 ) , active CD ( n = 34 ) , control ( Con ) ( n = 26 ) .

(A) ATX serum protein levels were measured in blood serum samples from IBD patients and healthy controls using ELISA: active UC (n=26), active CD (n=34), control (Con) (n=26).


( B ) Age - and sex - matched Atx ∆ ΜΕ / ∆ ΜΕ ; Il10 - / - mice ( n = 9 ) and Atx + / + ; Il10 - / - littermates ( n = 9 ) were examined for the development of spontaneous colitis . Body weight changes were monitored every other day , starting at the age of 37 days and ending at the age of 6 months . Results are means ± SD . Data were compared by Two - way ANOVA ( with treatment and times ) , followed by the multiple - comparison Bonferroni t test to assess differences between groups .

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(B) Age- and sex-matched Atx∆ΜΕ/∆ΜΕ;Il10-/- mice (n=9) and Atx+/+;Il10-/- littermates (n=9) were examined for the development of spontaneous colitis. Body weight changes were monitored every other day, starting at the age of 37 days and ending at the age of 6 months. Results are means ± SD. Data were compared by Two-way ANOVA (with treatment and times), followed by the multiple-comparison Bonferroni t test to assess differences between groups.


( C ) Gross appearance of the full - length mouse colon . Each line on a ruler represents a millimeter ( mm ) .

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(C) Gross appearance of the full-length mouse colon. Each line on a ruler represents a millimeter (mm).


( D and E ) H & E stained section of the mouse colon . Insets are enlarged in the lower panel . Colonic mucosa thickness was measured using ZEISS Axio Imager Z1 microscope ( n = 14 / group ) ( E ) .

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(D and E) H&E stained section of the mouse colon. Insets are enlarged in the lower panel. Colonic mucosa thickness was measured using ZEISS Axio Imager Z1 microscope (n=14/group) (E).


( F ) Histological parameters were quantified with H & E - colon sections ( n = 10 / group ) ( D ) and shown as mean ± SEM .

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(F) Histological parameters were quantified with H&E-colon sections (n=10/group) (D) and shown as mean ± SEM.


( G ) Survival of Atx ∆ ΜΕ / ∆ ΜΕ ; Il10 - / - ( n = 17 ) and Atx + / + ; Il10 - / - littermates ( n = 18 ) was analyzed by the Kaplan - Meier method ( Log - rank P = 0 . 0046 ) .

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(G) Survival of Atx∆ΜΕ/∆ΜΕ;Il10-/- (n=17) and Atx+/+;Il10-/- littermates (n=18) was analyzed by the Kaplan-Meier method (Log-rank P=0.0046).


( H ) Presented are H & E - stained sections of the small intestine in a ' Swiss - Roll ' form . Inset areas are enlarged in the right panel .

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(H) Presented are H&E-stained sections of the small intestine in a 'Swiss-Roll' form. Inset areas are enlarged in the right panel.


( A ) Total cell lysates of peritoneal macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were subjected to immunoblotting analysis to confirm Atx deletion .

(A) Total cell lysates of peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were subjected to immunoblotting analysis to confirm Atx deletion.


( B ) Peritoneal macrophages from the mice were subjected to the ESI - MS / MS method to quantify endogenous LPA . Individual molecular species of LPA and total LPA ( inset graph ) were compared . The data are shown as mean ± SEM ( n = 3 / group ) * P < 0 . 05 ( one - tailed unpaired t - test ) .

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(B) Peritoneal macrophages from the mice were subjected to the ESI-MS/MS method to quantify endogenous LPA. Individual molecular species of LPA and total LPA (inset graph) were compared. The data are shown as mean ± SEM (n=3/group) *P < 0.05 (one-tailed unpaired t-test).


( C and D ) Thioglycolate - elicited peritoneal immune cells harvested from the mice were immediately fixed for transmission electron microscopy to visualize the appearance of macrophages ( C ) and DCs ( D ) . Scale bar indicates 1 μm .

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(C and D) Thioglycolate-elicited peritoneal immune cells harvested from the mice were immediately fixed for transmission electron microscopy to visualize the appearance of macrophages (C) and DCs (D). Scale bar indicates 1 μm.


Lipid rafts from peritoneal macrophages ( E , F ) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100 , 000g for 17 h . Protein was purified and concentrated with the 3K cut - off filter , followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Caveolin or Flotillin - 1 .

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Lipid rafts from peritoneal macrophages (E, F) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Caveolin or Flotillin-1.


Lipid rafts from mouse macrophage Raw264 . 7 cells treated with Atx inhibitor ( Atx - i ) PF8380 ( 50 μM , 30 min ) or vehicle ( Veh . , DMSO 0 . 1 % ) ( G ) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100 , 000g for 17 h . Protein was purified and concentrated with the 3K cut - off filter , followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Flotillin - 1 .

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

Lipid rafts from mouse macrophage Raw264.7 cells treated with Atx inhibitor (Atx-i) PF8380 (50 μM, 30 min) or vehicle (Veh., DMSO 0.1%) (G) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Flotillin-1.


( H ) Peritoneal macrophages were stained with Alexa Fluor 594 - cholera toxin subunit B ( Red ) . The plasma membrane was examined with FV10i confocal scanning microscopy . Inset areas were magnified at the right panel . Each scale bar indicates 10 μm .

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(H) Peritoneal macrophages were stained with Alexa Fluor 594-cholera toxin subunit B (Red). The plasma membrane was examined with FV10i confocal scanning microscopy. Inset areas were magnified at the right panel. Each scale bar indicates 10 μm.


Peritoneal macrophages ( A ) were stimulated with LPS ( 20 ng / mL , 20 min ) Cell lysates were subjected to co - immunoprecipitation ( IP ) with CD14 antibody , followed by immunoblotting ( IB ) with TLR4 antibody .

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Peritoneal macrophages (A) were stimulated with LPS (20 ng/mL, 20 min) Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.


Raw264 . 7 cells ( B ) were stimulated with LPS ( 20 ng / mL , 20 min ) in the presence / absence of ATX inhibitor ( 50 μM , 30 min ) or vehicle ( Veh , DMSO 0 . 1 % ) . Cell lysates were subjected to co - immunoprecipitation ( IP ) with CD14 antibody , followed by immunoblotting ( IB ) with TLR4 antibody .

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Raw264.7 cells (B) were stimulated with LPS (20 ng/mL, 20 min) in the presence/absence of ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%). Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.


The physical interaction of TLR4 - CD14 was examined by FRET analysis . HEK293 cells were transfected with TLR4 - EYFP ( yellow ) and CD14 - ECFP ( blue ) ( C ) followed by ATX inhibitor ( 50 μM , 30 min ) or vehicle treatment ( Veh , DMSO 0 . 1 % ) . Cells were washed with PBS and fixed , then visualized with filter sets for CFP and YFP . FRET images were visualized with the FRET filter set , expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins ( arbitrary linear units of fluorescence intensity ) . Presented are the representative images from at least three independent experiments . Scale bar indicates 5 μm .

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The physical interaction of TLR4-CD14 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP (yellow) and CD14-ECFP (blue) (C) followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.


The physical interaction of TLR4 - TLR4 was examined by FRET analysis . HEK293 cells were transfected with TLR4 - EYFP and TLR4 - ECFP ( D ) , followed by ATX inhibitor ( 50 μM , 30 min ) or vehicle treatment ( Veh , DMSO 0 . 1 % ) . Cells were washed with PBS and fixed , then visualized with filter sets for CFP and YFP . FRET images were visualized with the FRET filter set , expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins ( arbitrary linear units of fluorescence intensity ) . Presented are the representative images from at least three independent experiments . Scale bar indicates 5 μm .

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The physical interaction of TLR4-TLR4 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP and TLR4-ECFP (D), followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.


( A and B ) Peritoneal macrophages from Atx + / + ( A ) and Atx ∆ ΜΕ / ∆ ΜΕ ( B ) mice were stimulated with LPS ( 20 ng / mL , 20 min ) , followed by fixation in 4 % paraformaldehyde for 15 min at room temperature and staining with TLR4 - anti rabbit FITC ( Green ) , early endosomal marker EEA1 - anti mouse Dylight405 ( Blue ) , and Alexa Fluor 594 - CTXB ( Red ) . TLR4 internalization was examined with confocal microscopy . Presented are the representative from 3 independent experiments in which more than 95 % of the cells exhibited similar results . 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 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

(A and B) Peritoneal macrophages from Atx+/+ (A) and Atx∆ΜΕ/∆ΜΕ (B) mice were stimulated with LPS (20 ng/mL, 20 min), followed by fixation in 4% paraformaldehyde for 15 min at room temperature and staining with TLR4-anti rabbit FITC (Green), early endosomal marker EEA1-anti mouse Dylight405 (Blue), and Alexa Fluor 594-CTXB (Red). TLR4 internalization was examined with confocal microscopy. Presented are the representative from 3 independent experiments in which more than 95% of the cells exhibited similar results. Scale bar, 10 μm.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated with LPS ( 20 ng / mL , 20 min ) The cell lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) analysis with the antibodies indicated . Presented is the representative from three independent experiments .

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

The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.


Raw264 . 7 cells were stimulated by LPS with the ATX inhibitor ( 50 μM , 30 min ) or vehicle ( Veh , DMSO 0 . 1 % ) The cell lysates were subjected to immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) analysis with the antibodies indicated . Presented is the representative from three independent experiments .

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

Raw264.7 cells were stimulated by LPS with the ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated by LPS [ 20 ng / mL , 20 min : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 , were used for a loading control . Presented are the representative image from at least three independent experiments .

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

The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated by LPS [20 ng/mL, 20 min : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2, were used for a loading control. Presented are the representative image from at least three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ and Atx + / + mice were stimulated by LPS [ 20 ng / mL , 20 min The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . regular Akt were used for a loading control . Presented are the representative image from at least three independent experiments .

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

The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated by LPS [20 ng/mL, 20 min The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. regular Akt were used for a loading control. Presented are the representative image from at least three independent experiments.


Atx - heterozygous ( Atx - Het ) mice and wild type littermates ( C ) were stimulated by LPS indicated time point The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . RasGAP levels were used for a loading control . Presented are the representative image from at least three independent experiments .

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Atx-heterozygous (Atx-Het) mice and wild type littermates (C) were stimulated by LPS indicated time point The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. RasGAP levels were used for a loading control. Presented are the representative image from at least three independent experiments.


( D ) Raw264 . 7 cells were stimulated by LPS with the ATX inhibitor . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . , β - Actin were used for a loading control . Presented are the representative image from at least three independent experiments .

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(D) Raw264.7 cells were stimulated by LPS with the ATX inhibitor. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. , β-Actin were used for a loading control. Presented are the representative image from at least three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with TLR2 ligand Peptidoglycan ( PGN , 30 ng / mL ) ( E ) for the indicated time points . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments .

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The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with TLR2 ligand Peptidoglycan (PGN, 30 ng/mL) (E) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with Pam3CSK4 ( 1 μg / mL ) ( F ) for the indicated time points . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments .

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

The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with Pam3CSK4 (1 μg/mL) (F) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with IL - 1β ( 100 ng / mL ) for the indicated time points . : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments .

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The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with IL-1β (100 ng/mL) for the indicated time points. : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


The macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates were activated with TNFα ( 25 ng / mL ) ( H ) for the indicated time points . The cell lysates were subjected to immunoblotting analysis with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are the representative image from at least three independent experiments .

The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with TNFα (25 ng/mL) (H) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.


Peritoneal macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates ( A ) were stimulated with LPS ( 50 ng / mL , 8 h ) The culture medium was collected for ELISA to measure the level of secreted cytokines . All assays were performed in triplicate , and data are shown as mean ± SEM .

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Peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates (A) were stimulated with LPS (50 ng/mL, 8 h) The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.


Raw264 . 7 cells ( B ) were stimulated with LPS ( 50 ng / mL , 8 h ) in the presence / absence of the ATX inhibitor ( 50 μM , 30 min ) . The culture medium was collected for ELISA to measure the level of secreted cytokines . All assays were performed in triplicate , and data are shown as mean ± SEM .

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Raw264.7 cells (B) were stimulated with LPS (50 ng/mL, 8 h) in the presence/absence of the ATX inhibitor (50 μM, 30 min). The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.


Peritoneal macrophages were stimulated with LPS or vehicle ( endotoxin free water ) , after which intracellular cytokine production was measured through FACS analysis . With the scatter dot plot , the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry . The overlay plot represents the cytometry data .

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Peritoneal macrophages were stimulated with LPS or vehicle (endotoxin free water), after which intracellular cytokine production was measured through FACS analysis. With the scatter dot plot, the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry. The overlay plot represents the cytometry data.


( F ) To induce sepsis via LPS injection ( Shirey et al . , 2013 ; Voss et al . , 2016 ) , age ( 9 - 10 weeks ) - and sex - matched Atx ∆ ΜΕ / ∆ ΜΕ mice ( n = 9 ) and Atx + / + littermates ( n = 21 ) were injected with LPS ( i . p . 25 mg / kg ) . Survival was monitored for up to 16 h , and analyzed by the Kaplan - Meier method ( Log - rank P = 0 . 0004 ) .

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(F) To induce sepsis via LPS injection (Shirey et al., 2013; Voss et al., 2016), age (9-10 weeks)- and sex-matched Atx∆ΜΕ/∆ΜΕ mice (n = 9) and Atx+/+ littermates (n = 21) were injected with LPS (i.p. 25 mg/kg). Survival was monitored for up to 16 h, and analyzed by the Kaplan-Meier method (Log-rank P=0.0004).


( G ) Blood samples were collected from Atx ∆ ΜΕ / ∆ ΜΕ ( n = 7 ) and Atx + / + ( n = 6 ) mice 10 h after LPS injection , after which the serum TNFα protein level was measured . The data are shown as mean ± SEM .

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(G) Blood samples were collected from Atx∆ΜΕ/∆ΜΕ (n = 7) and Atx+/+ (n = 6) mice 10 h after LPS injection, after which the serum TNFα protein level was measured. The data are shown as mean ± SEM.


Confocal laser scanning micrographs of the macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates . Cells were incubated with IgG - opsonized latex beads in the absence / presence of LPS ( 20 ng / mL , 2 h ) and stained with Phalloidin ( F - actin ) and DAPI . Internalized beads were examined under a higher ( A )

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

Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a higher(A)


Confocal laser scanning micrographs of the macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice and Atx + / + littermates . Cells were incubated with IgG - opsonized latex beads in the absence / presence of LPS ( 20 ng / mL , 2 h ) and stained with Phalloidin ( F - actin ) and DAPI . Internalized beads were examined under a lower magnification ( B ) to quantify % phagocytosis by dividing latex bead - positive cell numbers by the total number of DAPI - positive cells ( n = 11 - 16 per group ) . The data are analyzed with results accumulated from three independent experiments and shown as mean ± SEM ( B ) . * * P < 0 . 01 ( Mann - Whitney U test ) .

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

Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a lower magnification (B) to quantify % phagocytosis by dividing latex bead-positive cell numbers by the total number of DAPI-positive cells (n=11 - 16 per group). The data are analyzed with results accumulated from three independent experiments and shown as mean ± SEM (B). **P < 0.01 (Mann-Whitney U test).


( C ) LPS - stimulated macrophages were co - incubated with IgG - opsonized latex beads for 40 min . Phagocytic cups were visualized by F - actin staining with Phalloidin - iFluor 647 . The arrow indicates the phagocytic cup formed in the plasma membrane . Scale bar is 10 μm .

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(C) LPS-stimulated macrophages were co-incubated with IgG-opsonized latex beads for 40 min. Phagocytic cups were visualized by F-actin staining with Phalloidin-iFluor 647. The arrow indicates the phagocytic cup formed in the plasma membrane. Scale bar is 10 μm.


After LPS ( 20 ng / mL , 4 h ) treatment , LPS - induced iNos , Arginase - 1 ( Arg - 1 ) , and Ym - 1 mRNA expression were evaluated by semi - quantitative PCR ( D ) .

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After LPS (20 ng/mL, 4 h) treatment, LPS-induced iNos, Arginase-1 (Arg-1), and Ym-1 mRNA expression were evaluated by semi-quantitative PCR (D).


With LPS stimulation ( 20 ng / mL , 8 h ) , iNOS protein production was examined by immunoblotting analysis ( E ) .

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With LPS stimulation (20 ng/mL, 8 h), iNOS protein production was examined by immunoblotting analysis (E).


( A ) Time - dependent killing of a panel of bacterial isolates in the presence of meropenem ( MEM10 , 10 µg / ml ) . Cells were grown overnight and sub - cultured for 30 minutes at 37 ° C before being exposed to meropenem at 10 µg / mL and moved to a 37 ° C stationary incubator . Samples were taken at each indicated time point and spot - titered onto LB medium to quantify colony forming units ( CFU / mL ) throughout meropenem treatment . ( B ) images of cells corresponding to the time points in ( A ) were taken after labeling with membrane stain FM4 - 64 . E . cloacae , Enterobacter cloacae ; K . aerogenes , Klebsiella aerogenes ; K . pneumoniae , Klebsiella pneumoniae ; E . coli , Escherichia coli

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(A) Time-dependent killing of a panel of bacterial isolates in the presence of meropenem (MEM10, 10 µg/ml). Cells were grown overnight and sub-cultured for 30 minutes at 37°C before being exposed to meropenem at 10 µg/mL and moved to a 37°C stationary incubator. Samples were taken at each indicated time point and spot-titered onto LB medium to quantify colony forming units (CFU/mL) throughout meropenem treatment. (B) images of cells corresponding to the time points in (A) were taken after labeling with membrane stain FM4-64. E. cloacae, Enterobacter cloacae; K. aerogenes, Klebsiella aerogenes; K. pneumoniae, Klebsiella pneumoniae; E. coli, Escherichia coli


( D ) Regulatory modes of representative VxrB - controlled genes in response to PenG . Upper plot shows VxrB binding patterns ( assessed via ChIP - Seq ) , lower panel shows transcriptional response upon overexpression of VxrB D78E ( assessed via RNA - Seq ) . RNA expression was confirmed by S1 nuclease mapping assay in wild type and ΔvxrAB mutant strains after exposure to PenG ( 100 µg / ml for 3 hours ) . White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes . Source data is available as supplemental material .

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(D) Regulatory modes of representative VxrB-controlled genes in response to PenG. Upper plot shows VxrB binding patterns (assessed via ChIP-Seq), lower panel shows transcriptional response upon overexpression of VxrB D78E (assessed via RNA-Seq). RNA expression was confirmed by S1 nuclease mapping assay in wild type and ΔvxrAB mutant strains after exposure to PenG (100 µg/ml for 3 hours). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.


Reduction of cell wall synthesis gene expression causes a tolerance defect . ( C ) Survival of a ∆ pbp1b ∆ pbp1a Piptg : pbp1a strain after 6 hours of PenG exposure . Pup indicates conditions of high IPTG concentrations ( 100 µM ) , Pdown low IPTG concentration ( 2 µM ) . Survival fraction is cfu / ml of PenG - treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration .

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Reduction of cell wall synthesis gene expression causes a tolerance defect. (C) Survival of a ∆pbp1b ∆pbp1a Piptg:pbp1a strain after 6 hours of PenG exposure. Pup indicates conditions of high IPTG concentrations (100 µM), Pdown low IPTG concentration (2 µM). Survival fraction is cfu/ml of PenG-treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration.


( D ) Time - dependent killing experiment in the presence of PenG ( 100 µg / ml , 10 x MIC ) . All data are means ( + / - standard error ) of 3 independent biological replicates .

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(D) Time-dependent killing experiment in the presence of PenG (100 µg/ml, 10 x MIC). All data are means (+/- standard error) of 3 independent biological replicates.


( C ) Time - course of iron accumulation in PenG - treated WT and ∆ vxrAB cells as measured by ICP - MS . Data are average of 3 independent biological replicates , error bars represent standard deviation . ∗ ∗ P = 0 . 001 ; ∗ ∗ ∗ P < 0 . 0003 ( paired t - test ) .

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(C) Time-course of iron accumulation in PenG-treated WT and ∆vxrAB cells as measured by ICP-MS. Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗P=0.001; ∗∗∗P<0.0003 (paired t-test).


( B ) . To test PenG - mediated induction of Fur - regulated genes , S1 nuclease mapping assays were performed on the small RNA ryhB , the RyhB target sodB , hutA / B , coding for heme transport systems and vc2212 , which encodes a component of a ferric citrate uptake system ) . Cells were grown to mid - exponential phase followed by exposure to H2O2 or PenG for the indicated duration . Numbers represent expression levels normalized to untreated control ( averages from three independent replicates ) . White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes . Source data is available as supplemental material .

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(B). To test PenG-mediated induction of Fur-regulated genes, S1 nuclease mapping assays were performed on the small RNA ryhB, the RyhB target sodB, hutA/B, coding for heme transport systems and vc2212, which encodes a component of a ferric citrate uptake system). Cells were grown to mid-exponential phase followed by exposure to H2O2 or PenG for the indicated duration. Numbers represent expression levels normalized to untreated control (averages from three independent replicates). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.


( B ) and ( C ) Iron uptake mutants restore ∆ vxrAB growth on 10 % sucrose . The indicated strains were grown to exponential phase ( OD600 ~ 0 . 5 ) in LB medium and spot - plated on 10 % Sucrose plates with or without additional iron sulfate . An example is shown in ( B ) , quantification of cfu is shown in ( C ) . Data information : Data are average of 3 independent biological replicates , error bars represent standard deviation . ∗ ∗ ∗ P < 0 . 0003 ; ∗ ∗ ∗ ∗ P < 0 . 0001 ( paired t - test ) .

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(B) and (C) Iron uptake mutants restore ∆vxrAB growth on 10% sucrose. The indicated strains were grown to exponential phase (OD600~0.5) in LB medium and spot-plated on 10% Sucrose plates with or without additional iron sulfate. An example is shown in (B), quantification of cfu is shown in (C). Data information: Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗∗ P<0.0003; ∗∗∗∗ P<0.0001 (paired t-test).


( B ) Excretion of V . cholerae . Fecal samples were collected every two hours and assessed for their viable cell content ( Colony Forming Units , CFU ) to monitor the level of V . cholerae excretion . Each symbol represents fecal samples from an individual mouse ( n = 4 ) . The number of bacterial CFU was normalized per milligram of feces ( CFU / mg ) . Error bars represent standard deviation . ∗ ∗ P < 0 . 005 ; ∗ ∗ ∗ P < 0 . 001 ( two - way ANOVA ) .

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(B) Excretion of V. cholerae. Fecal samples were collected every two hours and assessed for their viable cell content (Colony Forming Units, CFU) to monitor the level of V. cholerae excretion. Each symbol represents fecal samples from an individual mouse (n=4). The number of bacterial CFU was normalized per milligram of feces (CFU/mg). Error bars represent standard deviation. ∗∗ P<0.005; ∗∗∗ P<0.001 (two-way ANOVA).


( E ) Time - dependent killing of respiratory chain mutants ( ∆ nqrA , encoding NADH dehydrogenase and ∆ ubiA , encoding an early step of ubiquinone synthesis ) in the presence of PenG ( 100 µg / ml , 10xMIC ) . All strains were grown in LB 0 . 2 % glucose . Data information : Data are mean of three independent biological replicates and error bars represent standard deviation .

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(E) Time-dependent killing of respiratory chain mutants (∆nqrA, encoding NADH dehydrogenase and ∆ubiA, encoding an early step of ubiquinone synthesis) in the presence of PenG (100 µg/ml, 10xMIC). All strains were grown in LB 0.2 % glucose. Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.


( F ) Growth - phase dependent killing assay of ROS detoxification systems ( ∆ fur , ∆ sodB , and ∆ oxyR1 ∆ katG ∆ katB ) . At designated time - points , a 5 ml aliquot was withdrawn and exposed to 100 µg / ml PenG for 3 h . CFU / mL for each time point was assessed before ( solid lines ) and after addition of PenG ( dotted lines ) . Data information : Data are mean of three independent biological replicates and error bars represent standard deviation .

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(F) Growth-phase dependent killing assay of ROS detoxification systems (∆fur, ∆sodB, and ∆oxyR1∆katG∆katB). At designated time-points, a 5 ml aliquot was withdrawn and exposed to 100 µg/ml PenG for 3 h. CFU/mL for each time point was assessed before (solid lines) and after addition of PenG (dotted lines). Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.


IIA1 . 6 B lymphoma cells were stimulated with BCR - ligand - ( IgM ) or BCR - ligand + ( IgG ) beads for 60 min , fixed and stained for F - actin ( top ) and α - tubulin ( bottom ) . Scale bar : 3 µm .

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IIA1.6 B lymphoma cells were stimulated with BCR-ligand- (IgM) or BCR-ligand+ (IgG) beads for 60 min, fixed and stained for F-actin (top) and α-tubulin (bottom). Scale bar: 3 µm.


Histograms show the quantifications of the polymerized tubulin and filamentous F - actin at the centrosome ( dashed outline on the image , values correspond to the fraction of fluorescence in a 2 - micron - wide area around the centrosome relative to the total fluorescence in the cell ) and the total amount of polymerized fluorescent tubulin ( bottom right , values were normalized with respect to the mean of control condition ) . Measurements were pooled from 3 independent experiments ; IgM ( BCR - ligand - ) : n = 88 ; IgG ( BCR - ligand + ) : n = 93 . Error bars correspond to standard deviations . P values were calculated with Mann - Whitney test . Scale bar : 2 µm .

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Histograms show the quantifications of the polymerized tubulin and filamentous F-actin at the centrosome (dashed outline on the image, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell) and the total amount of polymerized fluorescent tubulin (bottom right, values were normalized with respect to the mean of control condition). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test. Scale bar: 2 µm.


Percentage differences of centrosomal F - actin and centrosomal microtubule fluorescence intensities in cells stimulated with BCR - ligand + beads with respect to cells stimulated with BCR - ligand - beads . The data set is identical to panel ( B ) . Measurements were pooled from 3 independent experiments ; IgM ( BCR - ligand - ) : n = 88 ; IgG ( BCR - ligand + ) : n = 93 . Error bars correspond to standard deviations . P values were calculated with one - sample t - test ( i . e . comparison to a theoretical mean of " 0 " ) .

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Percentage differences of centrosomal F-actin and centrosomal microtubule fluorescence intensities in cells stimulated with BCR-ligand+ beads with respect to cells stimulated with BCR-ligand- beads. The data set is identical to panel (B). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome in an XY representation of individual measurements . The two lines correspond to linear regressions of the two sets of data relative to cells stimulated with BCR - ligand + ( activated cells ) or BCR - ligand - ( resting cells ) beads .

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The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome in an XY representation of individual measurements. The two lines correspond to linear regressions of the two sets of data relative to cells stimulated with BCR-ligand+ (activated cells) or BCR-ligand- (resting cells) beads.


IIA1 . 6 B lymphoma cells were fixed and immuno - stained for F - actin ( red ) and microtubules ( green ) . Images show the projection of maximal intensity of three confocal slices spaced by 0 . 5 µm apart from the centrosome . Scale bars : 2 µm ( 0 . 5 µm in the zoomed insets ) .

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IIA1.6 B lymphoma cells were fixed and immuno-stained for F-actin (red) and microtubules (green). Images show the projection of maximal intensity of three confocal slices spaced by 0.5 µm apart from the centrosome. Scale bars: 2 µm (0.5 µm in the zoomed insets).


IIA1 . 6 B lymphoma cells were treated 45 min with indicated inhibitors ( CK666 at 25 µM , SMIFH2 at 25µM ) or DMSO as control prior to being fixed and stained for α - tubulin ( left column ) and F - actin ( right column ) . Scale bar : 3 µm .

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IIA1.6 B lymphoma cells were treated 45 min with indicated inhibitors (CK666 at 25 µM, SMIFH2 at 25µM) or DMSO as control prior to being fixed and stained for α-tubulin (left column) and F-actin (right column). Scale bar: 3 µm.


Histograms show the quantifications of the amount of polymerized fluorescent tubulin ( right , values were normalized with respect to the mean of control condition ) and filamentous actin at the centrosome ( left , values correspond to the fraction of fluorescence in a 2 - micron - wide area around the centrosome relative to the total fluorescence in the cell ) . Measurements were pooled from 3 independent experiments ; DMSO : n = 91 , CK666 : n = 82 , SMIFH2 : n = 74 , latrunculinA : n = 96 . Error bars correspond to standard deviations . P values were calculated with Mann - Whitney test .

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Histograms show the quantifications of the amount of polymerized fluorescent tubulin (right, values were normalized with respect to the mean of control condition) and filamentous actin at the centrosome (left, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell). Measurements were pooled from 3 independent experiments; DMSO: n= 91, CK666: n= 82, SMIFH2: n= 74, latrunculinA: n= 96. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test.


Percentage differences of centrosomal F - actin and microtubule fluorescence intensities in cells treated with cytoskeleton inhibitors in comparison with the respective densities in cells treated with DMSO . Errors bars represent standard deviations . P values were calculated with one - sample t - test ( i . e . comparison to a theoretical mean of " 0 " ) .

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Percentage differences of centrosomal F-actin and microtubule fluorescence intensities in cells treated with cytoskeleton inhibitors in comparison with the respective densities in cells treated with DMSO. Errors bars represent standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


The graph shows the same measurements as in panel ( B ) in an XY representation of individual measurements . The three lines correspond to linear regressions of the three sets of data relative to cells stimulated with each actin drug .

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The graph shows the same measurements as in panel (B) in an XY representation of individual measurements. The three lines correspond to linear regressions of the three sets of data relative to cells stimulated with each actin drug.


IIA1 . 6 B lymphoma cells were transfected to transiently express centrin1 - GFP ( red ) and EB3 - mCherry ( green ) and video - recorded at the contact site with the glass coverslip ( left ) and at the centrosome ( right ) . Scale bar : 3 µm .

IIA1.6 B lymphoma cells were transfected to transiently express centrin1-GFP (red) and EB3-mCherry (green) and video-recorded at the contact site with the glass coverslip (left) and at the centrosome (right). Scale bar: 3 µm.


The duration of EB3 - positive comets presence in the bottom plane was measured in DMSO and CK666 treated cells ( left ) . Error bars correspond to standard deviations . The number of EB3 - positive comets exiting the a 2 - µm wide centrosomal area was also compared between the two conditions ( right ) . In both cases P values were calculated with Mann - Whitney test .

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The duration of EB3-positive comets presence in the bottom plane was measured in DMSO and CK666 treated cells (left). Error bars correspond to standard deviations. The number of EB3-positive comets exiting the a 2-µm wide centrosomal area was also compared between the two conditions (right). In both cases P values were calculated with Mann-Whitney test.


IIA1 . 6 B lymphoma cells were transfected to transiently express centrin1 - VCA - GFP ( bottom ) or centrin1 - GFP ( top ) as control prior to be fixed and stained for α - tubulin ( left column ) and F - actin ( middle column ) . The GFP signal of centrin1 or centrin1 - VCA is shown in the right column to illustrate the proper centrosome targeting . Scale bar : 3 µm .

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IIA1.6 B lymphoma cells were transfected to transiently express centrin1-VCA-GFP (bottom) or centrin1-GFP (top) as control prior to be fixed and stained for α-tubulin (left column) and F-actin (middle column). The GFP signal of centrin1 or centrin1-VCA is shown in the right column to illustrate the proper centrosome targeting. Scale bar: 3 µm.


Histograms show the quantifications of the amount of polymerized fluorescent tubulin ( right ) and filamentous actin at the centrosome ( left ) . Values correspond to the fraction of fluorescence in a 2 - micron - wide area around the centrosome relative to the total fluorescence in the cell . Measurements were pooled from 3 independent experiments ; centrin1 - GFP : n = 88 , centrin1 - VCA - GFP : n = 87 . Errors bars represent standard deviations . P values were calculated with Mann - Whitney test .

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Histograms show the quantifications of the amount of polymerized fluorescent tubulin (right) and filamentous actin at the centrosome (left). Values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell. Measurements were pooled from 3 independent experiments; centrin1-GFP: n= 88, centrin1-VCA-GFP: n= 87. Errors bars represent standard deviations. P values were calculated with Mann-Whitney test.


Percentage differences of F - actin and microtubule fluorescence intensities at the centrosome were compared in cells transfected either with centrin1 - VCA - GFP or with centrin1 - GFP . Errors bars represent standard deviations . P values were calculated with one - sample t - test ( i . e . comparison to a theoretical mean of " 0 " ) .

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Percentage differences of F-actin and microtubule fluorescence intensities at the centrosome were compared in cells transfected either with centrin1-VCA-GFP or with centrin1-GFP. Errors bars represent standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome . The two lines correspond to linear regressions of the two sets of data relative to cells transfected with centrin1 - VCA - GFP or centrin1 - GFP .

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The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome. The two lines correspond to linear regressions of the two sets of data relative to cells transfected with centrin1-VCA-GFP or centrin1-GFP.


Two sets of representative images showing fluorescent microtubules and actin filaments assembled from isolated centrosomes . Centrosomes were isolated from Jurkat cells expressing centrin1 - GFP . Upper and lower lines show actin filaments and microtubules radiating from two distinct centrosomes with low ( top ) and high ( bottom ) densities of actin filaments . Scale bars : 10 μm .

Two sets of representative images showing fluorescent microtubules and actin filaments assembled from isolated centrosomes. Centrosomes were isolated from Jurkat cells expressing centrin1-GFP. Upper and lower lines show actin filaments and microtubules radiating from two distinct centrosomes with low (top) and high (bottom) densities of actin filaments. Scale bars: 10 μm.


The graph shows the number of microtubules per centrosome relative to the density of actin filaments . Inset shows actin filaments at the centrosome with a FIRE look - up table and a 20 µm - wide circle in which actin fluorescence intensity is measured . Measurements were pooled from 5 independent experiments ; n = 50 .

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The graph shows the number of microtubules per centrosome relative to the density of actin filaments. Inset shows actin filaments at the centrosome with a FIRE look-up table and a 20 µm-wide circle in which actin fluorescence intensity is measured. Measurements were pooled from 5 independent experiments; n= 50.


Microtubules ( top line ) and actin filaments ( bottom line ) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin ( from left to right ) . Scale bar : 20 μm .

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Microtubules (top line) and actin filaments (bottom line) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin (from left to right). Scale bar: 20 μm.


The graph shows the number of microtubules per centrosome in response to increasing concentrations of monomeric actin . Data were pooled from 2 independent experiments ; 0 µM : n = 21 ; 0 . 3 µM : n = 17 ; 0 . 5 µM : n = 17 ; 1 . 0 µM : n = 17 . * * * * : P < 0 . 001 Mann - Whitney test .

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The graph shows the number of microtubules per centrosome in response to increasing concentrations of monomeric actin. Data were pooled from 2 independent experiments; 0 µM: n=21; 0.3 µM: n=17; 0.5 µM: n=17; 1.0 µM: n=17. ****:P<0.001 Mann-Whitney test.


The image shows the density of actin filaments ( in the presence of 1µM of actin monomers ) at the centrosome color - coded with the FIRE look - up table and the definition of central , peripheral and distal regions corresponding to decreasing concentrations of actin filaments . Scale bar : 20 μm . The graph shows the measurements of microtubule growth rate in each region . Data were pooled from 3 independent experiments : central zone : n = 58 , peripheral : n = 104 , outside : n = 61 ; n . s . means no statistical difference between the data set according to Mann - Whitney test .

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The image shows the density of actin filaments (in the presence of 1µM of actin monomers) at the centrosome color-coded with the FIRE look-up table and the definition of central, peripheral and distal regions corresponding to decreasing concentrations of actin filaments. Scale bar: 20 μm. The graph shows the measurements of microtubule growth rate in each region. Data were pooled from 3 independent experiments: central zone: n=58, peripheral: n=104, outside: n=61; n. s. means no statistical difference between the data set according to Mann-Whitney test.


The graphs show the various intensities of centrosome immuno - staining with antibodies against gamma - tubulin on the same coverslip depending on the presence / absence of actin filaments ( left ) or on the amount of actin filaments ( right ) . Data were pooled from 3 independent experiments . Left graph : without actin n = 69 , with actin n = 26 , right graph n = 26 . N . s . means no statistical difference between the data set according to Mann - Whitney test .

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The graphs show the various intensities of centrosome immuno-staining with antibodies against gamma-tubulin on the same coverslip depending on the presence/absence of actin filaments (left) or on the amount of actin filaments (right). Data were pooled from 3 independent experiments. Left graph: without actin n=69, with actin n=26, right graph n=26. N.s. means no statistical difference between the data set according to Mann-Whitney test.


Schematic illustration of the first dynamic assay : sequential addition of tubulin followed by tubulin and actin on isolated centrosomes . Representative images showing microtubules ( top line ) and the merged images of actin and microtubules ( bottom line ) for the two steps of the assay ; in the presence of tubulin only ( left column ) and in the presence of tubulin and actin ( right column ) . Scale bar : 10 µm . Quantification of the differences in the number of microtubules per centrosome between the two stages of the experiment described above on centrosomes capable ( first condition ) , or not ( second condition ) , to grow actin filaments . Data were collected from a single experiment ; asters without actin filaments : n = 29 ; asters with actin filaments : n = 13 . Data were analyzed using Mann - Whitney test .

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Schematic illustration of the first dynamic assay: sequential addition of tubulin followed by tubulin and actin on isolated centrosomes. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) for the two steps of the assay; in the presence of tubulin only (left column) and in the presence of tubulin and actin (right column). Scale bar: 10 µm. Quantification of the differences in the number of microtubules per centrosome between the two stages of the experiment described above on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were collected from a single experiment; asters without actin filaments: n= 29; asters with actin filaments: n= 13. Data were analyzed using Mann-Whitney test.


Schematic illustration of the second dynamic assay : tubulin is added to measure centrosome nucleation capacity and washed out . Then actin is added followed by actin and tubulin . Representative images showing microtubules ( top line ) and the merged images of actin and microtubule ( bottom line ) during the three steps of the assay ; in the presence of tubulin only ( left column ) , in the absence of tubulin and presence of actin ( middle column ) and in the presence of tubulin and actin ( right column ) . Scale bar : 10 µm . Arrowheads indicate microtubules unable to re - grow after assembly of actin filaments . Quantification of the differences in the number of microtubules per centrosome between the first and last steps of the experiment described above ( panels D and E ) on centrosomes capable ( first condition ) , or not ( second condition ) , to grow actin filaments . Data were pooled from 2 independent experiments ; asters without actin filaments : n = 24 ; asters with actin filaments : n = 13 . * * * * : P < 0 . 001 Student t - test .

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Schematic illustration of the second dynamic assay: tubulin is added to measure centrosome nucleation capacity and washed out. Then actin is added followed by actin and tubulin. Representative images showing microtubules (top line) and the merged images of actin and microtubule (bottom line) during the three steps of the assay; in the presence of tubulin only (left column), in the absence of tubulin and presence of actin (middle column) and in the presence of tubulin and actin (right column). Scale bar:10 µm. Arrowheads indicate microtubules unable to re-grow after assembly of actin filaments. Quantification of the differences in the number of microtubules per centrosome between the first and last steps of the experiment described above (panels D and E) on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were pooled from 2 independent experiments; asters without actin filaments: n= 24; asters with actin filaments: n= 13. ****:P<0.001 Student t-test.


Representative images of microtubules ( top ) and actin filaments ( bottom ) growth from micropatterns . Scale bars : 20 μm .

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Representative images of microtubules (top) and actin filaments (bottom) growth from micropatterns. Scale bars: 20 μm.


Schematic illustration of the assay on micropatterned substrate . Tubulin was first added alone to measure the nucleation capacity of each micropattern , and then washed out . Later on , actin was added followed by actin and tubulin . Finally , actin was rinsed out and gelsolin was added to fully disassemble actin filaments . Representative images showing microtubules ( top line ) and the merged images of actin and microtubules ( bottom line ) during the four steps of the assay ; in the presence of tubulin only , in the absence of tubulin and presence of actin , in the presence of tubulin and actin , and finally in the presence of tubulin and gelsolin but in the absence of actin filaments ( from left to right ) . Scale bar : 10 μm .

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Schematic illustration of the assay on micropatterned substrate. Tubulin was first added alone to measure the nucleation capacity of each micropattern, and then washed out. Later on, actin was added followed by actin and tubulin. Finally, actin was rinsed out and gelsolin was added to fully disassemble actin filaments. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) during the four steps of the assay; in the presence of tubulin only, in the absence of tubulin and presence of actin, in the presence of tubulin and actin, and finally in the presence of tubulin and gelsolin but in the absence of actin filaments (from left to right). Scale bar: 10 μm.


Quantification of the number of microtubules per micropattern in the presence of tubulin only ( left ) , actin and tubulin ( middle ) and tubulin only after actin filaments disassembly ( right ) . Data were pooled from 2 independent experiments ; n = 133 . * * * * : P < 0 . 001 Student t - test . The graph shows the same measurements as in panel ( E ) in an XY representation of individual measurements . It illustrates the differences in the number of microtubules per micropattern between the first to the second step ( tubulin only versus actin and tubulin together ) with respect to the density of actin filaments per micropattern .

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Quantification of the number of microtubules per micropattern in the presence of tubulin only (left), actin and tubulin (middle) and tubulin only after actin filaments disassembly (right). Data were pooled from 2 independent experiments; n= 133. ****:P<0.001 Student t-test. The graph shows the same measurements as in panel (E) in an XY representation of individual measurements. It illustrates the differences in the number of microtubules per micropattern between the first to the second step (tubulin only versus actin and tubulin together) with respect to the density of actin filaments per micropattern.


RPE1 cells stably expressing centrin1 - GFP were plated for 3 h on coverslips coated with different ratios ( 100 : 0 ; 50 : 50 or 1 : 99 ) of fibronectin and PLL - PEG prior to fixation and staining for F - actin ( top line and magnified views around centrosome below . Scale bars : 10 µm and 2 µm , respectively ) and α - tubulin ( bottom line . Scale bar : 10 µm ) .

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RPE1 cells stably expressing centrin1-GFP were plated for 3 h on coverslips coated with different ratios (100:0; 50:50 or 1:99) of fibronectin and PLL-PEG prior to fixation and staining for F-actin (top line and magnified views around centrosome below. Scale bars: 10 µm and 2 µm, respectively) and α-tubulin (bottom line. Scale bar: 10 µm).


IIA1 . 6 B lymphoma cells were plated for 60 min on poly - L - lysine , fibronectin or ICAM - 1 coated cover slides prior to be fixed and stained for F - actin ( top line ) and α - tubulin ( bottom line ) . Scale bar : 3 µm .

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IIA1.6 B lymphoma cells were plated for 60 min on poly-L-lysine, fibronectin or ICAM-1 coated cover slides prior to be fixed and stained for F-actin (top line) and α-tubulin (bottom line). Scale bar: 3 µm.


Quantification of the area occupied by RPE1 cells on the substrate ( top left ) , F - actin content at the centrosome ( top right ) polymerized tubulin at the centrosome ( bottom left ) and in the entire cell ( bottom right ) for the three conditions of cell adhesion described in ( B ) . Measurements came from 3 independent experiments with more than 60 analyzed cells in each . Errors bars represent standard deviations . F - actin and microtubule contents were compared using Mann - Whitney test , and variations of the cell area were compared using unpaired t - test .

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Quantification of the area occupied by RPE1 cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (B). Measurements came from 3 independent experiments with more than 60 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.


Quantification of the area occupied by B lymphoma cells on the substrate ( top left ) , F - actin content at the centrosome ( top right ) polymerized tubulin at the centrosome ( bottom left ) and in the entire cell ( bottom right ) for the three conditions of cell adhesion described in ( D ) . Measurements came from 3 independent experiments with more than 80 analyzed cells in each . Errors bars represent standard deviations . F - actin and microtubule contents were compared using Mann - Whitney test , and variations of the cell area were compared using unpaired t - test .

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Quantification of the area occupied by B lymphoma cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (D). Measurements came from 3 independent experiments with more than 80 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.


( G ) GTT or ITT in 17 - week - old wild - type or JFKTG mice fed on HFD . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

(G) GTT or ITT in 17-week-old wild-type or JFKTG mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


( H ) H & E and Oil Red O staining of liver sections from wild - type or JFKTG mice fed on ND or HFD . Scale bar , 100 μm .

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(H) H&E and Oil Red O staining of liver sections from wild-type or JFKTG mice fed on ND or HFD. Scale bar, 100 μm.


( D ) The body weight of male wild - type or JfkKO mice fed on HFD . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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(D) The body weight of male wild-type or JfkKO mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


( G ) H & E and Oil Red O staining of liver sections from wild - type or JfkKO mice fed on HFD . Scale bar , 100 μm .

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(G) H&E and Oil Red O staining of liver sections from wild-type or JfkKO mice fed on HFD. Scale bar, 100 μm.


( H ) Wild - type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally - delivered vector or JFK via tail vein . Seven days after injection , total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , one - way ANOVA with Tukey ' s HSD test ) . H & E and Oil Red O staining of liver sections are shown . Scale bar , 100 μm .

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(H) Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector or JFK via tail vein. Seven days after injection, total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content. Error bars represent mean ± SEM (n = 6, *p < 0.05, one-way ANOVA with Tukey's HSD test). H&E and Oil Red O staining of liver sections are shown. Scale bar, 100 μm.


( A ) HepG2 cells were stably transfected with vector or FLAG - JFK . Cellular extracts were immunopurified with anti - FLAG immunoafﬁnity resin and eluted with FLAG peptides . The eluates were resolved on SDS - PAGE and silver - stained . The proteins bands were retrieved and analyzed by mass spectrometry .

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(A) HepG2 cells were stably transfected with vector or FLAG-JFK. Cellular extracts were immunopurified with anti-FLAG immunoafﬁnity resin and eluted with FLAG peptides. The eluates were resolved on SDS-PAGE and silver-stained. The proteins bands were retrieved and analyzed by mass spectrometry.