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

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

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


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

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

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


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

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

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


level of phosphorylated Smad2 ( P - Smad2 ) total Smad2 ( Smad2 ) was assessed by immunoblotting β - actin was used as a loading control . Lower bar graphs show WB of P - Smad2 ( P - S2 ) normalized to β - actin . ( B ) 1603MED were treated with PBS ( vehicle ) or a blocking antibody targeting ActivinB ( Ab α - ActB ) or Follistatin . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

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

The level of phosphorylated Smad2 (P-Smad2) and total Smad2 (Smad2) was assessed by immunoblotting and β-actin was used as a loading control. Lower bar graphs show WB quantification of P-Smad2 (P-S2) normalized to β-actin. (B) 1603MED cells were treated with PBS (vehicle) or a blocking antibody targeting ActivinB (Ab α-ActB) or Follistatin. The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3.


level of phosphorylated Smad2 ( P - Smad2 ) total Smad2 ( Smad2 ) was assessed by immunoblotting β - actin was used as a loading control . Lower bar graphs show WB of P - Smad2 ( P - S2 ) normalized to β - actin . ( C ) Conditioned media experiments were performed on HDMB03 MB . Phosphorylation of Smad2 was analyzed by immunoblot upon treatment with either non conditioned media ( NCM ) , media conditioned with HDMB03 ( CM - HDMB03 ) or media conditioned with 1603MED ( CM - 1603 ) . Pre - incubation with blocking antibody against ActivinB ( Ab α - ActB ) or vehicule ( PBS ) was performed before HDMB03 - treatment as indicated . Relative level of P - Smad2 ( P - S2 ) were quantified to β - actin ( below ) . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

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

The level of phosphorylated Smad2 (P-Smad2) and total Smad2 (Smad2) was assessed by immunoblotting and β-actin was used as a loading control. Lower bar graphs show WB quantification of P-Smad2 (P-S2) normalized to β-actin. (C) Conditioned media experiments were performed on the HDMB03 MB cell line. Phosphorylation of Smad2 was analyzed by immunoblot upon treatment with either non conditioned media (NCM), media conditioned with HDMB03 cells (CM-HDMB03) or media conditioned with 1603MED cells (CM-1603). Pre-incubation with blocking antibody against ActivinB (Ab α-ActB) or vehicule (PBS) was performed before HDMB03 cell-line treatment as indicated. Relative level of P-Smad2 (P-S2) were quantified to β-actin (below). The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3.


( D ) RT - qPCR were performed on total extracted from 1603MED 48 hours after transfection with siRNA targeting INHBB . Relative INHBB expression was assessed . siCTRL condition was set at 1 . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

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

(D) RT-qPCR were performed on total RNA extracted from 1603MED cells 48 hours after transfection with siRNA targeting INHBB. Relative INHBB expression was assessed. siCTRL condition was set at 1. The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3.


level of phosphorylated Smad2 ( P - Smad2 ) total Smad2 ( Smad2 ) was assessed by immunoblotting β - actin was used as a loading control . Lower bar graphs show WB of P - Smad2 ( P - S2 ) normalized to β - actin . ( E ) 1603MED were transfected with indicated control siRNA ( siCTRL , blue ) or targeting INHBB ( siINHBB , red ) . Lysates were prepared 48 hours after transfection . Lower bar graphs represent of relative level of P - Smad2 ( P - S2 ) to β - actin . p - values were determined by unpaired t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bars represent mean ± SD . Number of replicates is n ≥ 3 .

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

The level of phosphorylated Smad2 (P-Smad2) and total Smad2 (Smad2) was assessed by immunoblotting and β-actin was used as a loading control. Lower bar graphs show WB quantification of P-Smad2 (P-S2) normalized to β-actin. (E) 1603MED cells were transfected with the indicated control siRNA (siCTRL, blue) or targeting INHBB (siINHBB, red). Lysates were prepared 48 hours after transfection. Lower bar graphs represent the quantification of the relative level of P-Smad2 (P-S2) to β-actin. The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3.


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of PMEPA1 level normalized to β - actin across groups . p - values were determined by unpaired t - for D * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

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


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

O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 β - actin were performed on extracts from 1603MED or D283 or D458 treated with either DMSO ( vehicle ) , LY364947 , SB431542 , blocking antibody against ActivinB ( Ab α - ActB ) , Follistatin , PBS or ActivinB ( ActB ) for 24 hours .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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 + ) is magnified ( bottom ) .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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 mice were subjected to ESI - MS / MS method to quantify endogenous LPA . molecular species of LPA total LPA ( inset graph ) were compared . data are shown as mean ± ( n = 3 / group ) * P < 0 . 05 ( one - unpaired t - ) .

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O 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 production was examined by immunoblotting ( E ) .

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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