L , PANC - 1 - transduced were subjected to a Boyden chamber migration . Migrating were quantified after 24h . n = 5 in each group .

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L, PANC-1-transduced cells were subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 5 in each group.


A - B A , Murine pancreatic tumour R211 were treated for 15 min with vehicle , α - specific A66 or pan - PI3K inhibitor BKM120 at 0 . 01 , 0 . 05 , 0 . 1 , 0 . 5 , 1 or 5 µM in presence of 10 % FBS of P - Akt ( Ser473 ) , total Akt β Actin were observed by western blot . B , P - Akt on Ser473 was quantified normalised with β Actin . n = 3 in each group .

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A-B A, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle, α-specific A66 or pan-PI3K inhibitor BKM120 at 0.01, 0.05, 0.1, 0.5, 1 or 5 µM in the presence of 10% FBS and protein levels of P-Akt (Ser473), total Akt and β Actin were observed by western blot. B, P-Akt on Ser473 was quantified and normalised with β Actin. n = 3 in each group.


R211 were treated with vehicle , α - specific inhibitor A66 or pan - PI3K inhibitor BKM120 at 1 µM D , migrating were quantified after 24h in a Boyden chamber compared to vehicle ( DMSO ) . n = 3 in each group .

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R211 cells were treated with the vehicle, α-specific inhibitor A66 or pan-PI3K inhibitor BKM120 at 1 µM D, migrating cells were quantified after 24h in a Boyden chamber assay and compared to vehicle (DMSO). n = 3 in each group.


E , Murine pancreatic tumour R211 were treated for 15 min with vehicle ( 0 . 01 % DMSO ) or α - specific A66 or pan - PI3K inhibitor BKM120 at 1µM . Phospholipids were extracted , total PIP , PIP2 PIP3 were quantified compared to vehicle ( DMSO ) . n = 3 in each group .

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E, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle (0.01% DMSO) or α-specific A66 or pan-PI3K inhibitor BKM120 at 1µM. Phospholipids were extracted, total PIP, PIP2 and PIP3 were quantified and compared to the vehicle (DMSO). n = 3 in each group.


A , Migration inhibition capacities of PI3K inhibitors at 1 µM were tested on pancreatic tumour ( R211 , PDAC8661 , PANC - 1 , 10593 , 10158 , R6344 , R6430 , R6065 , R6141 ) . mean of these values was plotted against in vitro IC50 for each class I PI3K each inhibitor . IC50 is determined on recombinant . Pearson correlation were performed its p - values were presented separately for each class I PI3K isoform .

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A, Migration inhibition capacities of PI3K inhibitors at 1 µM were tested on pancreatic tumour cells (R211, PDAC8661, PANC-1, 10593, 10158, R6344, R6430, R6065, R6141). The mean of these values was plotted against the in vitro IC50 for each class I PI3K and each inhibitor. IC50 is determined on recombinant proteins. Pearson correlation tests were performed and its p-values were presented separately for each class I PI3K isoform.


A , Representative of PI3K activation ( as shown by positive pAkt substrate ) on tumours of PDAC , with values of of cfDNA mutated KRAS allele frequency .

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A, Representative images of PI3K activation (as shown by positive pAkt substrate staining) on the primary tumours of PDAC patients, with values of quantification of cfDNA and mutated KRAS allele frequency.


C , Survival curve of KPC mice presenting different of cfDNA . n in each group is indicated . Log rank , * * * p < 0 .

C, Survival curve of KPC mice presenting different levels of cfDNA. n in each group is indicated. Log rank test, *** p<0.001


G , Survival curve of KPC mice treated with vehicle or BYL - 719 . Log rank , * p < 0 . 05 .

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G, Survival curve of KPC mice treated with the vehicle or BYL-719. Log rank test, * p<0.05.


H - J H , Representative i , of Ki67 positive in pancreatic tumours in J , metastasized liver sections after treatment with oral doses of vehicle ( 0 . 5 % methyl cellulose with 0 . 2 % Tween - 80 ) or of BYL - 719 ( 50mg / kg ) .

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H-J H, Representative images and i, quantification of Ki67 positive cells in pancreatic tumours and in J, metastasized liver sections after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg).


K - L K , Treatment regimen of tail vein injection experiment in nude mice treated with PI3Kα inhibitor or vehicle ( n is indicated ) , at two time points L , representative of luminescence via IVIS ® Spectrum in vivo imaging system .

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K-L K, Treatment regimen of the tail vein injection experiment in nude mice treated with the PI3Kα inhibitor or vehicle (n is indicated), quantification at two time points and L, representative images of luminescence via IVIS® Spectrum in vivo imaging system.


N , Tail vein injection experiment in C57 / B6 mice of murine syngeneic pancreatic cancer ( Kras mutated A338 , Kras mutated half PI3Kα inactive , A260 ; n > 8 in each group ) , of micro - macro - metastasis at final time point .

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N, Tail vein injection experiment in C57/B6 mice of murine syngeneic pancreatic cancer cells (Kras mutated A338, Kras mutated and half PI3Kα inactive, A260; n>8 in each group), quantification of micro- and macro-metastasis at final time point.


A - D Blood of A , blood , B , monocytes , C , granulocytes D , lymphocytes in KPC mice , with cohort in each group .

A-D Blood count of A, white blood cells, B, monocytes, C, granulocytes and D, lymphocytes in KPC mice, with cohort size in each group.


h - jH - J H , Representative of I , F4 / 80 positive J , CD206 positive after pharmacological inhibition of PI3Kα . Black arrowheads show positive immune .

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h-jH-J H, Representative images and quantification of I, F4/80 positive cells and J, CD206 positive cells after the pharmacological inhibition of PI3Kα. Black arrowheads show positive immune cells.


S , Murine pancreatic tumour R211 were treated with vehicle , TNF - α 20 ng / mL , BYL - 719 1 µM or both treatments subjected to a Boyden chamber migration . Migrating were quantified after 24h . n = 4 independent values .

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S, Murine pancreatic tumour cells R211 were treated with vehicle, TNF-α 20 ng/mL, BYL-719 1 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n= 4 independent values.


Expression pattern of Rgcc in embryonic day 14 . 5 ( E14 . 5 ) mouse cortex as detected by in situ hybridization . right panels showed expression patterns of NSC markers , including Pax6 , Sox2 , Ki67 , along ventricular zone ( VZ ) of E14 . 5 mouse cortex . Scale bar : 20 μm .

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Expression pattern of Rgcc in the embryonic day 14.5 (E14.5) mouse cortex as detected by in situ hybridization. The right panels showed the expression patterns of NSC markers, including Pax6, Sox2, and Ki67, along the ventricular zone (VZ) of the E14.5 mouse cortex. Scale bar: 20 μm.


Immunofluorescent of coronal sections of E14 . 5 mouse cortex 1 day post IUE . ShRNA targeting Rgcc control scramble - shRNA were nucleofected separately . Ki67 were co - stained with GFP . To quantify between NSC markers GFP positive , only in VZ SVZ are taken into account . ROIs of 800 * 1900 pixel were randomly picked for counting . Scale bars : 20 μm . of relative of Ki67 GFP double positive versus GFP positive . Immunofluorescent of coronal sections of E14 . 5 mouse cortex 1 day post IUE . Sox2 were co - stained with GFP . Scale bars : 20 μm . of relative of Sox2 GFP double positive versus GFP positive . Immunofluorescent of coronal sections of E14 . 5 mouse cortex 1 day post IUE . Pax6 were co - stained with GFP . Scale bars : 20 μm . of relative of Pax6 GFP double positive versus GFP positive .

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Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. ShRNA targeting Rgcc and control scramble-shRNA were nucleofected separately. Ki67 were co-stained with GFP. To quantify the ratio between NSC markers and GFP positive cells, only the cells in VZ and SVZ are taken into account. And ROIs of 800*1900 pixel were randomly picked for counting. Scale bars: 20 μm. Quantification of the relative ratio of Ki67 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. Sox2 were co-stained with GFP. Scale bars: 20 μm. Quantification of the relative ratio of Sox2 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. Pax6 were co-stained with GFP. Scale bars: 20 μm. Quantification of the relative ratio of Pax6 and GFP double positive cells versus GFP positive cells.


Immunofluorescent of coronal sections of E17 . 5 mouse cortex 5 days post IUE . ShRNA targeting Rgcc control scramble - shRNA were transfected separately at E12 . 5 . brains were harvest at E17 . 5 . Tbr1 were co - stained with GFP . ROIs of 300 * 300 pixel were randomly picked along Tbr1 positive region for counting . Scale bars : 100 μm . of relative of Tbr1 GFP double positive versus GFP positive . Immunofluorescent of coronal sections of E17 . 5 mouse cortex 4 days post IUE . ShRNA targeting Rgcc control scramble - shRNA were transfected separately at E13 . 5 . brains were harvest at E17 . 5 . Ctip2 were co - stained with GFP . ROIs of 300 * 300 pixel were randomly picked along Ctip2 positive region for counting . Scale bars : 100 μm . of relative of Ctip2 GFP double positive versus GFP positive . Immunofluorescent of coronal sections of E17 . 5 mouse cortex 4 days post IUE . ShRNA targeting Rgcc control scramble - shRNA were transfected separately at E13 . 5 . brains were harvest at E17 . 5 . Satb2 were co - stained with GFP . ROIs of 300 * 300 pixel were randomly picked along ventricle for counting . Scale bars : 100 μm . of relative of Satb2 GFP double positive versus GFP positive .

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Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 5 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E12.5. All the brains were harvest at E17.5. Tbr1 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along Tbr1 positive region for counting. Scale bars: 100 μm. Quantification of the relative ratio of Tbr1 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E13.5. All the brains were harvest at E17.5. Ctip2 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along Ctip2 positive region for counting. Scale bars: 100 μm. Quantification of the relative ratio of Ctip2 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E13.5. All the brains were harvest at E17.5. Satb2 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along ventricle for counting. Scale bars: 100 μm. Quantification of the relative ratio of Satb2 and GFP double positive cells versus GFP positive cells.


Immunofluorescent of coronal sections of E17 . 5 mouse cortex 4 days post IUE . Scale bars : 100 μm . of GFP positive in VZ / SVZ , IZ , CP were counted for migration .

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Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. Scale bars: 100 μm. The ratio of GFP positive cells in VZ/SVZ, IZ, and CP were counted for migration analysis.


Immunofluorescent were performed on sections of day 45 wildtype H9 RGCC - KO hCOs . Sections were stained with SOX2 . Scale bar : 200 μm of VZ - like SOX2 - positive rosette . Each plot represented an rosette . H9 , n = 19 ; # 6 , n = 24 ; # 23 , n = 19 from 2 independent experiments . Immunofluorescent were performed on sections of day 45 wildtype H9 RGCC - KO hCOs . Sections were stained with PAX6 Phospho - Histone 3 ( PH3 ) . Scale bar : 200 μm of of PH3 PAX6 double positive versus total number of PAX6 positive of each rosette . Each plot represented an organoid ( H9 , n = 6 ; # 6 , n = 8 ; # 23 , n = 8 from 2 independent experiments ) . Three to five rosettes were analyzed for each organoid . Immunofluorescent were performed on sections of day 45 wildtype H9 RGCC - KO hCOs . Sections were stained with Ki67 . Scale bar : 200 μm . of VZ - like Ki67 positive rosette . Each plot represented an rosette . H9 , n = 60 ; # 6 , n = 54 ; # 23 , n = 96 from 2 independent experiments .

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Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with SOX2. Scale bar: 200 μm Quantification of the VZ-like SOX2-positive rosette area. Each plot represented an individual rosette. H9, n=19; #6, n=24; #23, n=19 from 2 independent experiments. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with PAX6 and Phospho-Histone 3 (PH3). Scale bar: 200 μm Quantification of the ratio of PH3 and PAX6 double positive cells versus the total number of PAX6 positive cells of each rosette. Each plot represented an individual organoid (H9, n=6; #6, n=8; #23, n=8 from 2 independent experiments). Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with Ki67. Scale bar: 200 μm. Quantification of the VZ-like Ki67 positive rosette area. Each plot represented an individual rosette. H9, n=60; #6, n=54; #23, n=96 from 2 independent experiments.


Immunofluorescent were performed on sections of day 45 wildtype H9 RGCC - KO hCOs . Sections were stained with TUJ1 PAX6 . Scale bar : 200 μm . of of TUJ1 versus PAX6 . ( H9 , n = 15 ; # 6 , n = 6 ; # 23 , n = 9 from 2 independent experiment ) . Each plot represented an organoid . Three to five rosettes were analyzed for each organoid . Immunofluorescent were performed on sections of day 45 wildtype H9 RGCC - KO hCOs . Sections were stained with TBR1 PAX6 . Scale bar : 200 μm . of of TBR1 - positive around PAX6 - positive versus PAX6 - positive in each rosette . ( H9 , n = 10 ; # 6 , n = 10 ; # 23 , n = 7 from 2 independent experiment ) . Each plot represented an organoid . Three to five rosettes were analyzed for each organoid . Immunofluorescent were performed on sections of day 45 wildtype H9 RGCC - KO hCOs . Sections were stained with CTIP2 PAX6 . Scale bar : 200 μm . of of CTIP2 versus PAX6 . ( H9 , n = 8 ; # 6 , n = 8 ; # 23 , n = 8 from 2 independent experiment ) . Each plot represented an organoid . Three to five rosettes were analyzed for each organoid .

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Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with TUJ1 and PAX6. Scale bar: 200 μm. Quantification of the ratio of TUJ1 area versus PAX6 area. (H9, n=15; #6, n=6; #23, n=9 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with TBR1 and PAX6. Scale bar: 200 μm. Quantification of the ratio of TBR1-positive area around PAX6-positive area versus PAX6-positive area in each rosette. (H9, n=10; #6, n=10; #23, n=7 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with CTIP2 and PAX6. Scale bar: 200 μm. Quantification of the ratio of CTIP2 area versus PAX6 area. (H9, n=8; #6, n=8; #23, n=8 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid.


Immunofluorescent of coronal sections of E14 . 5 mouse cortex one - day post IUE . ShRNA targeting Rgcc control scramble - shRNA were nucleofected separately . PH3 were co - stained with GFP . punctuated PH3 were at G2 - phase , while PH3 with continuous pattern were at M - phase . To quantify between PH3 GFP double positive GFP , only in VZ SVZ are taken into account . Scale bar : 20 μm . n ≥ 3 mouse brains for each group . Data are presented as mean ± ; n ≥ 3 mouse brains for each group ; Statistical significance was tested by Student ' s t - . * * , p < 0 . 01 ; * * * , p < 0 . 001 ;

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Immunofluorescent staining of coronal sections of the E14.5 mouse cortex one-day post IUE. ShRNA targeting Rgcc and control scramble-shRNA were nucleofected separately. PH3 were co-stained with GFP. The punctuated PH3 cells were at G2-phase, while PH3 cells with continuous pattern were at M-phase. To quantify the ratio between PH3 and GFP double positive cells and GFP cells, only the cells in VZ and SVZ are taken into account. Scale bar: 20 μm. n≥3 mouse brains for each group. Data are presented as mean ± SEM; n≥3 mouse brains for each group; Statistical significance was tested by Student's t-test. **, p<0.01; ***, p<0.001;


Representative pictures of mitotic NSC marked by P - Vimentin PH3 revealed by DAPI at apical surface of E14 . 5 mouse cortex after IUE . A representative vertically dividing was shown in upper panel , a representative horizontally dividing was shown in lower panel . dash indicates apical surface splitting plane . Scale bar : 5 μm .

Representative pictures of mitotic NSC marked by P-Vimentin and PH3 revealed by DAPI at the apical surface of the E14.5 mouse cortex after IUE. A representative vertically dividing cell was shown in upper panel, and a representative horizontally dividing cell was shown in lower panel. White dash line indicates the apical surface and splitting plane. Scale bar: 5 μm.


RGCC was co - stained with PCNT on 3D hCO slices . RGCC was with stronger expression in NSCs during M - phase . prometaphase ( B ' ) , metaphase ( B ' ' ) anaphase ( B ' ' ' ) telophase ( B ' ' ' ' ) . RGCC was expressed around nucleus in interphase ( B ' ' ' ' ' ) . NSCs undergoing prometaphase , metaphase , anaphase telophase were pointed out by yellow arrow . Interphase were pointed out by arrow ( B ' ' ' ' ' ) . Immunostaining of 2D - differentiated NSCs in metaphase showed partial colocalization of RGCC with α - TUBULIN in spindle ( B ' ' ' ' ' ' , yellow arrow ) . Scale bar : 5 μm .

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RGCC was co-stained with PCNT on 3D hCO slices. RGCC was with stronger expression in NSCs during M-phase. prometaphase (B'), metaphase (B'') anaphase (B''') and telophase (B''''). RGCC was expressed around nucleus in interphase (B''''') . NSCs undergoing prometaphase, metaphase, anaphase and telophase were pointed out by yellow arrow. Interphase were pointed out by white arrow (B'''''). Immunostaining of 2D-differentiated NSCs in metaphase showed partial colocalization of RGCC with α-TUBULIN in the spindle (B'''''', yellow arrow). Scale bar: 5 μm.


Western blots results . were collected from WT hCOs RGCC KO hCOs # 6 at day 45 . B - ACTIN were used as references .

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The Western blots results. Proteins were collected from WT hCOs and RGCC KO hCOs #6 at day 45. B-ACTIN were used as references.


PCNT was co - stained with β - TUBULIN on WT NSCs RGCC KO NSCs differentiated by 2D cultured dual - SMAD inhibition method . Scale bar : 5μm .

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PCNT was co-stained with β -TUBULIN on WT NSCs and RGCC KO NSCs differentiated by 2D cultured dual-SMAD inhibition method. Scale bar: 5μm.


( A ) Immunoblot of phosphorylated Smad2 ( P - Smad2 ) PMEPA1 ( high low exposures displayed ) in MB sample lysates from different groups : WNT ( blue ) , SHH ( red ) , Group 3 ( yellow ) or Group 4 ( green ) . β - actin was used as a loading control . Relative of P - Smad2 signal to β - actin ( P - S2 / β - Actin ) total Smad2 ( P - S2 / Tot - S2 ) are indicated below blots .

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(A) Immunoblot analysis of phosphorylated Smad2 (P-Smad2) and PMEPA1 (high and low exposures displayed) in MB patient sample lysates from different groups: WNT (blue), SHH (red), Group 3 (yellow) or Group 4 (green). β-actin was used as a loading control. Relative quantification of P-Smad2 signal to β-actin (P-S2/β-Actin) and total Smad2 (P-S2/Tot-S2) are indicated below the blots.


( B ) Boxplots summarizing expression of INHBB , TGFB1 TGFB3 ligands of TGFβ / Activin pathway in different groups of MB ( blue WNT , red SHH , yellow Group 3 green Group 4 ) in adult cerebellum ( grey ) in dataset of " Data ref : Cavalli et al , 2017 " . Wilcoxon rank - sum were performed to determine p - values for panel B . p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(B) Boxplots summarizing the expression of INHBB, TGFB1 and TGFB3 ligands of the TGFβ/Activin pathway in the different groups of MB (blue WNT, red SHH, yellow Group 3 and green Group 4) and in fetal and adult cerebellum (grey) in the dataset of "Data ref: Cavalli et al, 2017". Wilcoxon rank-sum tests were performed to determine p-values for panel B. p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( C ) Immunoblot of phosphorylated Smad2 ( P - Smad2 ) in non - Group 3 ( blue ) Group 3 ( yellow ) MB lines on left panel . level of total Smad2 ( Smad2 ) was assessed β - actin was used as a loading control . On right panel , relative level of P - Smad2 ( P - S2 ) were quantified to total β - actin . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Bars represent 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

(C) Immunoblot analysis of phosphorylated Smad2 (P-Smad2) in non-Group 3 (blue) and Group 3 (yellow) MB cell lines on the left panel. The level of total Smad2 (Smad2) was assessed and β-actin was used as a loading control. On the right panel, relative level of P-Smad2 (P-S2) were quantified to total β-actin. The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.


( D ) RT - qPCR were performed on extracted from non - Group 3 ( blue ) Group 3 ( yellow ) MB lines to compare expression of INHBB ( left ) TGFB3 ( right ) . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Bars represent 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

(D) RT-qPCR were performed on RNA extracted from non-Group 3 (blue) and Group 3 (yellow) MB cell lines to compare expression levels of INHBB (left) and TGFB3 (right). The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.


level of phosphorylated Smad2 ( P - Smad2 ) total Smad2 ( Smad2 ) was assessed by immunoblotting β - actin was used as a loading control . Lower bar graphs show WB of P - Smad2 ( P - S2 ) normalized to β - actin . ( A ) Activation of pathway was assessed in non - Group 3 ( blue ) Group 3 ( yellow ) MB lines in response to TGFβ or ActivinB stimulation for 1h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 level of phosphorylated Smad2 (P-Smad2) and total Smad2 (Smad2) was assessed by immunoblotting and β-actin was used as a loading control. Lower bar graphs show WB quantification of P-Smad2 (P-S2) normalized to β-actin. (A) Activation of the pathway was assessed in non-Group 3 (blue) and Group 3 (yellow) MB cell lines in response to TGFβ or ActivinB stimulation for 1h.


level of phosphorylated Smad2 ( P - Smad2 ) total Smad2 ( Smad2 ) was assessed by immunoblotting β - actin was used as a loading control . Lower bar graphs show WB of P - Smad2 ( P - S2 ) normalized to β - actin . ( B ) 1603MED were treated with PBS ( vehicle ) or a blocking antibody targeting ActivinB ( Ab α - ActB ) or Follistatin . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent 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

The level of phosphorylated Smad2 (P-Smad2) and total Smad2 (Smad2) was assessed by immunoblotting and β-actin was used as a loading control. Lower bar graphs show WB quantification of P-Smad2 (P-S2) normalized to β-actin. (B) 1603MED cells were treated with PBS (vehicle) or a blocking antibody targeting ActivinB (Ab α-ActB) or Follistatin. 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.


level of phosphorylated Smad2 ( P - Smad2 ) total Smad2 ( Smad2 ) was assessed by immunoblotting β - actin was used as a loading control . Lower bar graphs show WB of P - Smad2 ( P - S2 ) normalized to β - actin . ( C ) Conditioned media experiments were performed on HDMB03 MB . Phosphorylation of Smad2 was analyzed by immunoblot upon treatment with either non conditioned media ( NCM ) , media conditioned with HDMB03 ( CM - HDMB03 ) or media conditioned with 1603MED ( CM - 1603 ) . Pre - incubation with blocking antibody against ActivinB ( Ab α - ActB ) or vehicule ( PBS ) was performed before HDMB03 - treatment as indicated . Relative level of P - Smad2 ( P - S2 ) were quantified to β - actin ( below ) . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent 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 O O O O O O O O O O O O O O O O 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 level of phosphorylated Smad2 (P-Smad2) and total Smad2 (Smad2) was assessed by immunoblotting and β-actin was used as a loading control. Lower bar graphs show WB quantification of P-Smad2 (P-S2) normalized to β-actin. (C) Conditioned media experiments were performed on the HDMB03 MB cell line. Phosphorylation of Smad2 was analyzed by immunoblot upon treatment with either non conditioned media (NCM), media conditioned with HDMB03 cells (CM-HDMB03) or media conditioned with 1603MED cells (CM-1603). Pre-incubation with blocking antibody against ActivinB (Ab α-ActB) or vehicule (PBS) was performed before HDMB03 cell-line treatment as indicated. Relative level of P-Smad2 (P-S2) were quantified to β-actin (below). 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 ) RT - qPCR were performed on total extracted from 1603MED 48 hours after transfection with siRNA targeting INHBB . Relative INHBB expression was assessed . siCTRL condition was set at 1 . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent 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

(D) RT-qPCR were performed on total RNA extracted from 1603MED cells 48 hours after transfection with siRNA targeting INHBB. Relative INHBB expression was assessed. siCTRL condition was set at 1. 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.


level of phosphorylated Smad2 ( P - Smad2 ) total Smad2 ( Smad2 ) was assessed by immunoblotting β - actin was used as a loading control . Lower bar graphs show WB of P - Smad2 ( P - S2 ) normalized to β - actin . ( E ) 1603MED were transfected with indicated control siRNA ( siCTRL , blue ) or targeting INHBB ( siINHBB , red ) . Lysates were prepared 48 hours after transfection . Lower bar graphs represent of relative level of P - Smad2 ( P - S2 ) to β - actin . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent 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

The level of phosphorylated Smad2 (P-Smad2) and total Smad2 (Smad2) was assessed by immunoblotting and β-actin was used as a loading control. Lower bar graphs show WB quantification of P-Smad2 (P-S2) normalized to β-actin. (E) 1603MED cells were transfected with the indicated control siRNA (siCTRL, blue) or targeting INHBB (siINHBB, red). Lysates were prepared 48 hours after transfection. Lower bar graphs represent the quantification of the relative level of P-Smad2 (P-S2) 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.


( F ) Growth curve of 1603MED after transfection with either siCTRL ( blue ) or siINHBB ( red ) . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth curve of 1603MED cells after transfection with either siCTRL (blue) or siINHBB (red). The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001.


D458 lines were treated with PBS ( vehicle , black ) or with ActivinB ( green ) . Immunoblot of phosphorylated Smad2 ( P - Smad2 ) , total Smad2 β - actin in response to ActivinB stimulation for 24 hours . of P - Smad2 ( P - S2 ) to β - actin are shown on right panels . Bars represent 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

D458 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin in response to ActivinB stimulation for 24 hours. Quantification of P-Smad2 (P-S2) to β-actin are shown on right panels. Bars represent the mean ± SD. Number of replicates is n≥3.


D458 lines were treated with PBS ( vehicle , black ) or with ActivinB ( green ) . Growth curve experiments showing proliferation upon ActivinB treatment . p - values were determined by unpaired t - two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

D458 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Growth curve experiments showing cell proliferation upon ActivinB treatment. The p-values were determined by unpaired t-test and two-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.


D458 lines were treated with PBS ( vehicle , black ) or with ActivinB ( green ) . cycle by FACS measuring BrdU incorporation 7AAD labelling at 48 hours upon ActivinB stimulation . of in different phases of cycle is represented ( G0 / G1 , S G2 / M phases ) . Bars represent 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

D458 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon ActivinB stimulation. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3.


D458 lines were treated with PBS ( vehicle , black ) or with ActivinB ( green ) . of apoptotic measured by FACS of caspase3 48h hours after treatment with ActivinB . Bars represent 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

D458 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48h hours after treatment with ActivinB. Bars represent the mean ± SD. Number of replicates is n≥3.


D283 lines were treated with PBS ( vehicle , black ) or with ActivinB ( green ) . Immunoblot of phosphorylated Smad2 ( P - Smad2 ) , total Smad2 β - actin in response to ActivinB stimulation for 24 hours . of P - Smad2 ( P - S2 ) to β - actin are shown on right panels . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin in response to ActivinB stimulation for 24 hours. Quantification of P-Smad2 (P-S2) to β-actin are shown on right panels. Bars represent the mean ± SD. Number of replicates is n≥3.


D283 lines were treated with PBS ( vehicle , black ) or with ActivinB ( green ) . Growth curve experiments showing proliferation upon ActivinB treatment . p - values were determined by unpaired t - two - way ANOVA for F . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Growth curve experiments showing cell proliferation upon ActivinB treatment. The p-values were determined by unpaired t-test and two-way ANOVA for F. *p < 0.05, **p < 0.01, ***p < 0.001.


D283 lines were treated with PBS ( vehicle , black ) or with ActivinB ( green ) . cycle by FACS measuring BrdU incorporation 7AAD labelling at 48 hours upon ActivinB stimulation . of in different phases of cycle is represented ( G0 / G1 , S G2 / M phases ) . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon ActivinB stimulation. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3.


D283 lines were treated with PBS ( vehicle , black ) or with ActivinB ( green ) . of apoptotic measured by FACS of caspase3 48h hours after treatment with ActivinB . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48h hours after treatment with ActivinB. Bars represent the mean ± SD. Number of replicates is n≥3.


1603MED were treated with DMSO ( vehicle , black ) , with LY364947 ( red ) or with SB431542 ( orange ) . Immunoblot of phosphorylated Smad2 ( P - Smad2 ) , total Smad2 β - actin upon inhibition of TGFβ / Activin signaling using LY364947 SB431542 inhibitors for 24 hours . Bar graphs on right panel represent of relative level of P - Smad2 ( P - S2 ) to β - actin . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

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

1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin upon inhibition of TGFβ/Activin signaling using LY364947 and SB431542 inhibitors for 24 hours. Bar graphs on the right panel represent the quantification of the relative level of P-Smad2 (P-S2) to β-actin. Bars represent the mean ± SD. Number of replicates is n≥3.


1603MED were treated with DMSO ( vehicle , black ) , with LY364947 ( red ) or with SB431542 ( orange ) . Growth curve experiments showing proliferation upon TGFβ / Activin signaling inhibition . p - values were determined by unpaired t - two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Growth curve experiments showing cell proliferation upon TGFβ/Activin signaling inhibition. The p-values were determined by unpaired t-test and two-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.


1603MED were treated with DMSO ( vehicle , black ) , with LY364947 ( red ) or with SB431542 ( orange ) . cycle by FACS measuring BrdU incorporation 7AAD labelling at 48 hours upon inhibition . of in different phases of cycle is represented ( G0 / G1 , S G2 / M phases ) . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

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

1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon inhibition. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3.


1603MED were treated with DMSO ( vehicle , black ) , with LY364947 ( red ) or with SB431542 ( orange ) . of apoptotic measured by FACS of caspase3 48 hours after TGFβ / Activin signaling inhibition . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

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

1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48 hours after TGFβ/Activin signaling inhibition. Bars represent the mean ± SD. Number of replicates is n≥3.


D283 were treated with DMSO ( vehicle , black ) , with LY364947 ( red ) or with SB431542 ( orange ) . Immunoblot of phosphorylated Smad2 ( P - Smad2 ) , total Smad2 β - actin upon inhibition of TGFβ / Activin signaling using LY364947 SB431542 inhibitors for 24 hours . Bar graphs on right panel represent of relative level of P - Smad2 ( P - S2 ) to β - actin . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin upon inhibition of TGFβ/Activin signaling using LY364947 and SB431542 inhibitors for 24 hours. Bar graphs on the right panel represent the quantification of the relative level of P-Smad2 (P-S2) to β-actin. Bars represent the mean ± SD. Number of replicates is n≥3.


D283 were treated with DMSO ( vehicle , black ) , with LY364947 ( red ) or with SB431542 ( orange ) . Growth curve experiments showing proliferation upon TGFβ / Activin signaling inhibition . p - values were determined by unpaired t - two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Growth curve experiments showing cell proliferation upon TGFβ/Activin signaling inhibition. The p-values were determined by unpaired t-test and two-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.


D283 were treated with DMSO ( vehicle , black ) , with LY364947 ( red ) or with SB431542 ( orange ) . cycle by FACS measuring BrdU incorporation 7AAD labelling at 48 hours upon inhibition . of in different phases of cycle is represented ( G0 / G1 , S G2 / M phases ) . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon inhibition. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3.


D283 were treated with DMSO ( vehicle , black ) , with LY364947 ( red ) or with SB431542 ( orange ) . of apoptotic measured by FACS of caspase3 48 hours after TGFβ / Activin signaling inhibition . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48 hours after TGFβ/Activin signaling inhibition. Bars represent the mean ± SD. Number of replicates is n≥3.


( A ) Ranking of top genes whose expression is correlated with INHBB in Group 3 MB samples . Spearman ' s rank correlation coefficient ρ p - value are indicated . p - values were determined by Spearman rank correlation * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ranking of top genes whose expression is correlated with INHBB in Group 3 MB patient samples. Spearman's rank correlation coefficient ρ and p-value are indicated. The p-values were determined by Spearman rank correlation test *p < 0.05, **p < 0.01, ***p < 0.001.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 PMEPA1 gene expression in MB groups . Colored dots represent each samples colors represent MB groups ( WNT in blue , SHH in red , Group 3 in yellow Group 4 in green ) . p - values were determined by Spearman rank correlation * 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.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 level of P - Smad2 ( x axis ) PMEPA1 ( Y axis ) normalized to β - actin in each samples . p - values were determined by Wilcoxon rank - sum * 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.


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

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

(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 of PMEPA1 in 1603MED ( G ) 48 hours after transfection with either siCTRL ( blue ) or siPMEPA1 ( red ) . Bar graphs on right represent of relative level of PMEPA1 level normalized to β - actin . p - values were determined by unpaired t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent 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

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 ) after transfection with either siCTRL ( blue ) or siPMEPA1 ( red ) . 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 of PMEPA1 in D283 ( I ) 48 hours after transfection with either siCTRL ( blue ) or siPMEPA1 ( red ) . Bar graphs on right represent of relative level of PMEPA1 level normalized to β - actin . p - values were determined by unpaired t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent 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 ) after transfection with either siCTRL ( blue ) or siPMEPA1 ( red ) . 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 of phosphorylated Smad2 ( P - Smad2 ) in Group 3 MB lines PDXs . level of total Smad2 ( Smad2 ) was assessed β - actin was used as a loading control . of P - Smad2 ( P - S2 ) to β - actin are shown on right panel . Right ( A ) panels represent relative of P - Smad2 normalized to β - actin . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

(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 lines PDXs relative to HDMB03 ( set at 1 ) by RT - qPCR . Bars represent 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 β - actin upon ActivinB or TGFβ stimulation for 1 hour . lower panels represent relative of P - Smad2 normalized to β - actin . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent 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 . Phosphorylation of Smad2 ( P - Smad2 ) was analyzed by immunoblot upon treatment with either non - conditioned media ( NCM ) , media conditioned with HDMB03 ( CM - HDMB03 ) or media conditioned on PDX4 ( CM - PDX4 ) . Pre - incubation with blocking antibody against ActivinB ( Ab α - ActB ) or with vehicle was performed before HDMB03 - treatment as indicated . lower panels represent relative of P - Smad2 normalized to β - actin . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

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(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 β - actin were performed on extracts from cultures of PDX4 , PDX3 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 cisplatin ( purple ) after orthotopic grafting of PDX4 into cerebellum . pink rectangle represents Galunisertib treatment duration , while blue dotted lines represent 3 cisplatin administrations . p - values were determined by Log - Rank ( Mantel - Cox ) on panel A * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

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

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(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 by in 3 representative tumors per group after 25 days of treatment . scale represents 500μm 100μm on left 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 expression level of INHBB , PMEPA1 , MYC NRL in 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 . samples are colored by subtypes as indicated . ( E ) Scatter plot of INHBB PMEPA1 , expression in Group 3 samples . Colored dots represent each sample colors represent group 3 MB subtypes ( α in yellow , β in brown δ in orange ) . Wilcoxon rank - sum were performed for panel D . Spearman ' s rank correlation coefficient ρ p - value are indicated on panel E . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 . Bars represent 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 , pictures of SVZ of a 4D + mouse of proportion of RFP - ( black ) RFP + ( red ) progenitors among B1 , C A 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 , pictures of SVZ of a 4D - ( left ) 4D + ( right insets magnified ) mice of proportion of BrdU + among B1 , C A ( identified as in B ) . Note that in 4D + mice was restricted to 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 ) of absolute number of B1 , C A ( identified as in B ) in SVZ of 4D - ( ) 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 : pictures of SVZ absolute number of in 4D - ( bars ) or 4D + ( black or red bars for or RFP + , 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 : pictures of SVZ absolute number of in 4D - ( bars ) or 4D + ( black or red bars for or RFP + , respectively ) mice scored positive for various markers as indicated . Insets in ( C ) are magnified ( right ) with arrowheads pointing label - retaining NSC ( ) or astrocytes ( empty ) .

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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 : pictures of SVZ or proportions ( C - E ) of in 4D - ( bars ) or 4D + ( black or red bars for or RFP + , respectively ) mice scored positive for various markers as indicated . Arrowheads in ( D ) point doublets ( among RFP + - reteining 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 : pictures of OB ( E ) absolute number or proportions of in 4D - ( bars ) or 4D + ( black or red bars for or RFP + , respectively ) mice scored positive for various markers as indicated . ( E ) GL = glomerular , EPL = external plexiform , MCL = mitral GCL = granule layers .

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 ) pictures ( left ) of immunolabeled GFP + RFP - ( 4D - ) GFP + RFP + ( 4D + ) apical dendrites of superficial granule neurons quantifications ( middle right ) of spine density total dendritic ( box whiskers ) 3D - Sholl ( graph profile ) of apical dendrites starting from soma ( drawings ) as 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 showing co - localization with pre - post - synaptic markers VIAAT ( granule neuron ) gephyrin ( mitral ) , respectively ( magnification shown in insets ) .

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(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 in superficial granule 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 + ) 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 ) 4D + ( bottom ) mice . Note in G that lag preventing spike initiation is longer at lower currents ( green ) shorter at higher ( black ) , suggesting presence of an A - type K current typical of granule 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 whiskers plots representing electrophysiological properties of neurons derived from 4D - 4D + NSC ( black red , respectively ) including from left to right : spike number , resting potential ( Vrest ) , input resistance ( Rin ) lag to spiking of recorded superficial granule ( 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 ) graph indicating proportion of correct responses ( performance ) for bins of 100 trials each during testing with binary mixtures of concentrated octanols . Discontinuous indicates similar 95 % plateau performance of 4D - ( black ) 4D + ( red ) mice . ( D ) Whiskers box plots of performance ( left ) lick frequency ( right ) during a probe of 200 trials with 1 : 10 diluted octanols after testing as in ( C ) .

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

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(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 ) - sex - matched Atx ∆ ΜΕ / ∆ ΜΕ ; Il10 - / - mice ( n = 9 ) Atx + / + ; Il10 - / - ( n = 9 ) were examined for development of spontaneous colitis . changes were monitored every other day , starting at of 37 days ending at of 6 months . Results are means ± SD . Data were compared by Two - way ANOVA ( with treatment times ) , followed by multiple - comparison Bonferroni t 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 full - mouse colon . Each 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 E ) H & E stained section of mouse colon . Insets are enlarged in 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 ) shown as mean ± .

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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 ) Atx + / + ; Il10 - / - ( n = 18 ) was analyzed by 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 small intestine in a ' Swiss - Roll ' form . Inset areas are enlarged in 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 lysates of peritoneal macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + were subjected to immunoblotting 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 mice were subjected to ESI - MS / MS method to quantify endogenous LPA . molecular species of LPA total LPA ( inset graph ) were compared . data are shown as mean ± ( n = 3 / group ) * P < 0 . 05 ( one - unpaired t - ) .

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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 D ) Thioglycolate - elicited peritoneal immune harvested from mice were immediately fixed for transmission electron microscopy to visualize appearance of macrophages ( C ) 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 . was purified concentrated with 3K cut - off filter , followed by Western blot with antibody recognizing lipid raft marker 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 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 . was purified concentrated with 3K cut - off filter , followed by Western blot with antibody recognizing lipid raft marker Flotillin - 1 .

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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 ) . plasma was examined with FV10i confocal scanning microscopy . Inset areas were magnified at right panel . Each scale bar indicates 10 μm .

(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 ) 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 ( B ) were stimulated with LPS ( 20 ng / mL , 20 min ) in presence / absence of ATX inhibitor ( 50 μM , 30 min ) or vehicle ( Veh , DMSO 0 . 1 % ) . 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.


physical interaction of TLR4 - CD14 was examined by FRET . HEK293 were transfected with TLR4 - EYFP ( yellow ) CD14 - ECFP ( blue ) ( C ) followed by ATX inhibitor ( 50 μM , 30 min ) or vehicle treatment ( Veh , DMSO 0 . 1 % ) . were washed with PBS fixed , then visualized with filter sets for CFP YFP . FRET were visualized with FRET filter set , expressed as corrected FRET efficiency displayed in quantitative pseudocolor indicating between ( arbitrary linear units of ) . Presented are representative 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.


physical interaction of TLR4 - TLR4 was examined by FRET . HEK293 were transfected with TLR4 - EYFP TLR4 - ECFP ( D ) , followed by ATX inhibitor ( 50 μM , 30 min ) or vehicle treatment ( Veh , DMSO 0 . 1 % ) . were washed with PBS fixed , then visualized with filter sets for CFP YFP . FRET were visualized with FRET filter set , expressed as corrected FRET efficiency displayed in quantitative pseudocolor indicating between ( arbitrary linear units of ) . Presented are representative 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 B ) Peritoneal macrophages from Atx + / + ( A ) 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 with TLR4 - anti rabbit FITC ( Green ) , early endosomal marker EEA1 - anti mouse Dylight405 ( Blue ) , Alexa Fluor 594 - CTXB ( Red ) . TLR4 internalization was examined with confocal microscopy . Presented are representative from 3 independent experiments in which more than 95 % of exhibited similar results . Scale bar , 10 μm .

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(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.


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

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

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

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

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.


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

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 ERK1/2, were used for a loading control. Presented are the representative image from at least three independent experiments.


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

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

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 wild type ( C ) were stimulated by LPS indicated time point lysates were subjected to immunoblotting with antibodies as indicated . RasGAP were used for a loading control . Presented are 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 were stimulated by LPS with ATX inhibitor . lysates were subjected to immunoblotting with antibodies as indicated . , β - Actin were used for a loading control . Presented are 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.


macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + were activated with TLR2 ligand Peptidoglycan ( PGN , 30 ng / mL ) ( E ) for indicated time points . lysates were subjected to immunoblotting with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are 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.


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

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 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.


macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + were activated with IL - 1β ( 100 ng / mL ) for indicated time points . : lysates were subjected to immunoblotting with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are 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.


macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + were activated with TNFα ( 25 ng / mL ) ( H ) for indicated time points . lysates were subjected to immunoblotting with antibodies as indicated . Regular ERK1 / 2 were used for a loading control . Presented are 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 Atx + / + ( A ) were stimulated with LPS ( 50 ng / mL , 8 h ) culture medium was collected for ELISA to measure level of secreted cytokines . assays were performed in triplicate , data are shown as mean ± .

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

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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 . With scatter dot plot , gate was set so that viable were selected to analyze of signal by flow cytometry . overlay plot represents 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 ) , ( 9 - 10 ) - sex - matched Atx ∆ ΜΕ / ∆ ΜΕ mice ( n = 9 ) Atx + / + ( n = 21 ) were injected with LPS ( i . p . 25 mg / kg ) . Survival was monitored for up to 16 h , analyzed by 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 ) Atx + / + ( n = 6 ) mice 10 h after LPS injection , after which serum TNFα level was measured . data are shown as mean ± .

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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 scanning of macrophages from Atx ∆ ΜΕ / ∆ ΜΕ mice Atx + / + . were incubated with IgG - opsonized latex beads in absence / presence of LPS ( 20 ng / mL , 2 h ) stained with Phalloidin ( F - actin ) 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 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

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

O O O O O O 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

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 with Phalloidin - iFluor 647 . arrow indicates phagocytic cup formed in plasma . 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 ) , 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 production was examined by immunoblotting ( 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 isolates in presence of meropenem ( MEM10 , 10 µg / ml ) . were grown overnight sub - cultured for 30 minutes at 37 ° C before being exposed to meropenem at 10 µg / mL moved to a 37 ° C stationary incubator . Samples were taken at each indicated time point spot - titered onto LB medium to quantify colony forming units ( CFU / mL ) throughout meropenem treatment . ( B ) of corresponding to time points in ( A ) were taken after labeling with stain FM4 - 64 . E . cloacae , Enterobacter cloacae ; K . aerogenes , Klebsiella aerogenes ; K . pneumoniae , Klebsiella pneumoniae ; E . coli , Escherichia coli

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 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 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

(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