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 ;

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

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 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 .

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

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 ' ' ' ) 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 .

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

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.


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

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

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 and RGCC KO NSCs differentiated by 2D cultured dual - SMAD inhibition method . Scale bar : 5μm .

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 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 .

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

(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 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 .

O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

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

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


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 .

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


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 .

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


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 .

O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_LINE B-DISEASE O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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


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 .

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

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 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 .

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

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

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

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 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 .

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

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 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 .

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

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 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 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 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 .

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

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 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 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 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 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 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 .

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

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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

(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 .

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

(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 .

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

(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 .

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

(E) 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 .

O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_LINE O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD 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 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 .

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

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 .

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

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 .

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

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 .

B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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

(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 .

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

(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 .

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

(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 B-CELL_LINE B-DISEASE O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 .

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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 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 .

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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 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 .

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

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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.


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 ) .

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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 : 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 ) .

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

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(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 ) .

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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 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 ) .

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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 ) 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 .

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(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 .

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

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

(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 .

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

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 .

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

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 .

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

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 .

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

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

(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 B-CELL_TYPE O B-GENEPROD O O O O O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-EXP_ASSAY 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 .

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

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 .

B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

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 .

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

(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 .

O B-CELL_TYPE O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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 B-CELL_TYPE O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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 .

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

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 .

O B-CELL_TYPE O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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 .

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

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 .

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

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 .

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

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 ) .

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

(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 .

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

(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 )

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE 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 ) .

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

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

(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 ) .

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

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 ) .

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

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

O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM

(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 .

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

(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.