Figure 1 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.


Figure 2The 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2The 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.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.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.


Figure 3D458 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 . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3D458 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.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.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.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.


Figure 41603MED 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 . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 41603MED 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 < 0.001.1603MED 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.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.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.


Figure 5 ( 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 . ( 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 . ( 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 . 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 . 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.(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.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.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.


Figure 6 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.(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.(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.


Figure 7 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.(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.


Figure 1B Representative adhesion frequency ( Pa ) vs . contact duration ( tc ) curves forNKG2D expressing NK cells ( n ≥ 3 ) in contact with RBCs ( n ≥ 3 ) coated with a ligand ( MICA in red , MICB in orange , ULBP1 in green , or ULBP3 in blue ) at different contact durations , fitted by non - linear in - situ binding kinetics model Site densities of NKG2D ( mr ) and its ligands ( ml ) are indicated . Data information : Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired t - test ) . In - situ force - free affinities ( C ) , on - rates ( D ) of NKG2D binding with indicated ligands from mammalian cells . The in - situ force - free kinetics were obtained from fittings with in - situ binding kinetics modeling in ( B ) . Data information : Every dot represents one independent binding experiment . Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired t - test ) . In - situ force - free off - rates ( E ) of NKG2D binding with indicated ligands from mammalian cells . The in - situ force - free kinetics were obtained from fittings with in - situ binding kinetics modeling in ( B ) . Data information : Every dot represents one independent binding experiment . Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired t - test ) . F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in - situ and in - solution assay . Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1 . Data information : In - solution affinities of proteins purified from E . coli In - solution affinities of proteins purified from mammalian cells were measured by

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B Representative adhesion frequency (Pa) vs. contact duration (tc) curves forNKG2D expressing NK cells (n ≥ 3) in contact with RBCs (n ≥ 3) coated with a ligand (MICA in red, MICB in orange, ULBP1 in green, or ULBP3 in blue) at different contact durations, fitted by non-linear in-situ binding kinetics model Site densities of NKG2D (mr) and its ligands (ml) are indicated. Data information: Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).In-situ force-free affinities (C), on-rates (D) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).In-situ force-free off-rates (E) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in-situ and in-solution assay. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1. Data information: In-solution affinities of proteins purified from E. coli In-solution affinities of proteins purified from mammalian cells were measured by BLI


Figure 2IFN - γ release assay and IFN - γ production ( B ) by human peripheral NK cells stimulated with plate - coated NKG2D ligands ( MICA in red , MICB in green and ULBP3 in blue ) assessed by Cytometric Bead Array , of which are their 50 % effective concentration ( EC50 ) ( C ) . Data information : The IFN - γ release assay in ( B ) was one representative experiment of three total independent experiments . Data points in ( B ) and bars in ( C ) represent mean values . Error bars in ( B ) and ( C ) represent mean ± SEM . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) . D - F Plots of reciprocals of EC50 vs . the effective in - situ affinities ( AcKa ) ( D ) , the effective in - situ on - rate ( AcKon ) ( E ) and in - situ off - rate ( koff ) ( F ) .

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Figure 2IFN-γ release assay and IFN-γ production (B) by human peripheral NK cells stimulated with plate-coated NKG2D ligands (MICA in red, MICB in green and ULBP3 in blue) assessed by Cytometric Bead Array, of which are their 50% effective concentration (EC50) (C). Data information: The IFN-γ release assay in (B) was one representative experiment of three total independent experiments. Data points in (B) and bars in (C) represent mean values. Error bars in (B) and (C) represent mean ± SEM. ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).D-F Plots of reciprocals of EC50 vs. the effective in-situ affinities (AcKa) (D), the effective in-situ on-rate (AcKon) (E) and in-situ off-rate (koff) (F).


Figure 3C Verification of the binding specificity of NKG2D with different ligands . Data information : Every dot represents the adhesion frequency of one cell - bead pair and the experimental data came from at least two to three repeated trials . Horizontal lines represent mean values . Error bars represent mean ± SEM . E , F Force - dependent bond lifetimes of NKG2D and various ligands at 5 pN , 10 pN and 15 pN ( E ) and full force spectrum ( F ) . Data information : Bond lifetimes in ( E ) and ( F ) ( in which n = 1505 for MICA , n = 1852 for MICB , n = 531 for ULBP1 , n = 1241 for ULBP3 ) of all ligands with NKG2D came from at least 19 NK cell - bead pairs of at least four independent biological experiments . bars in ( E ) , and data points in ( F ) represent mean values . Error bars represent mean ± SEM .

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Figure 3C Verification of the binding specificity of NKG2D with different ligands. Data information: Every dot represents the adhesion frequency of one cell-bead pair and the experimental data came from at least two to three repeated trials. Horizontal lines represent mean values. Error bars represent mean ± SEM.E, F Force-dependent bond lifetimes of NKG2D and various ligands at 5 pN, 10 pN and 15 pN (E) and full force spectrum (F). Data information: Bond lifetimes in (E) and (F) (in which n = 1505 for MICA, n = 1852 for MICB, n = 531 for ULBP1, n = 1241 for ULBP3) of all ligands with NKG2D came from at least 19 NK cell-bead pairs of at least four independent biological experiments. bars in (E), and data points in (F) represent mean values. Error bars represent mean ± SEM.


Figure 4C The force vs . extension trajectories for the simulations are shown in ( A ) and ( B ) . Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in ( A ) and ( B ) are marked . F Distance vs . time curves for force - induced H - bond formation between indicated residues within NKG2D - MICA binding interfaces . The dashed red lines represent H - bonds whose distances are less than 3 . 5 Å .

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Figure 4C The force vs. extension trajectories for the simulations are shown in (A) and (B). Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in (A) and (B) are marked.F Distance vs. time curves for force-induced H-bond formation between indicated residues within NKG2D-MICA binding interfaces. The dashed red lines represent H-bonds whose distances are less than 3.5 Å.


Figure 5A , B Abolishments of force - induced binding residues by 3A mutations ( MICA 3A , n = 526 ) and TAT mutations ( MICA TAT , n = 580 ) impairs NKG2D ' s catch bond with MICA WT ( n = 1505 ) ( A ) and IFN - γ release ( B ) . Data information : Bond lifetimes in ( A ) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments . IFN - γ release of NK cells in ( B ) was one representative experiment of three total independent experiments . Data points represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) . 50 % effective concentration ( EC50 ) of NK cells stimulated by indicated MICA mutants Data information : horizontal lines represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) . IFN - γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM ( D ) and 22nM ( E ) . Data information : bars in ( E ) , and data point in ( F ) represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) .

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Figure 5A, B Abolishments of force-induced binding residues by 3A mutations (MICA 3A, n = 526) and TAT mutations (MICA TAT, n= 580) impairs NKG2D's catch bond with MICA WT (n = 1505) (A) and IFN-γ release (B). Data information: Bond lifetimes in (A) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments. IFN-γ release of NK cells in (B) was one representative experiment of three total independent experiments. Data points represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).50% effective concentration (EC50) of NK cells stimulated by indicated MICA mutants Data information: horizontal lines represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).IFN-γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM (D) and 22nM (E). Data information: bars in (E), and data point in (F) represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


Figure 6A - C Plots and Pearson correlation analysis of reciprocals of EC50 vs . the force - dependent in - situ koff at 5 pN ( A ) , 10 pN ( B ) 15pN ( C ) . I Detection range comparison in force - dependent affinity of NKG2D and indicated ligands under difference force . Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1 .

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Figure 6A-C Plots and Pearson correlation analysis of reciprocals of EC50 vs. the force-dependent in-situ koff at 5 pN (A), 10 pN (B) 15pN (C).I Detection range comparison in force-dependent affinity of NKG2D and indicated ligands under difference force. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1.


Figure 7D Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on - rates and force - dependent off - rates .

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Figure 7D Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on-rates and force-dependent off-rates.


Figure 2A : Snapshots of LimE - GFP , GFP - myo , and corresponding kymographs for type 1 cells . Corresponding speeds are 10 . 8µm / min and 9 . 4µm / min . White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint , as extracted from the corresponding stress kymographs . C : Snapshots of LimE - GFP , GFP - myo , and corresponding kymographs for type 2 cells . Corresponding speeds are 5 . 6µm / min and 8 . 7µm / min . White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint . B : Stress maps quantifying the magnitude of the force per area using a color scale with blue / red corresponding to small / large stresses and the direction of forces using vectors . Fan - shaped cells were rotated so that the vertical ( x ) axis is the direction of motion ( see Methods ) . Shown are the overall stress T , the stress in the direction of motion Tx and the stress perpendicular to motion Ty for the type 1 cell with the LimE marker , and the corresponding Tx and Ty kymographs along the cells outlines . D : Stress maps of T , Tx and Ty for the type 2 cell with the LimE marker , and corresponding Tx and Ty kymographs along the cells outlines .

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Figure 2A: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 1 cells. Corresponding speeds are 10.8µm/min and 9.4µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint, as extracted from the corresponding stress kymographs. C: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 2 cells. Corresponding speeds are 5.6µm/min and 8.7µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint.B: Stress maps quantifying the magnitude of the force per area using a color scale with blue/red corresponding to small/large stresses and the direction of forces using vectors. Fan-shaped cells were rotated so that the vertical (x) axis is the direction of motion (see Methods). Shown are the overall stress T, the stress in the direction of motion Tx and the stress perpendicular to motion Ty for the type 1 cell with the LimE marker, and the corresponding Tx and Ty kymographs along the cells outlines. D: Stress maps of T, Tx and Ty for the type 2 cell with the LimE marker, and corresponding Tx and Ty kymographs along the cells outlines.


Figure 3A : Sequence of snapshots of the LimE - GFP distribution and stress T for an oscillatory cell together with snapshots of the GFP - myo distribution for a separate oscillatory cell . Corresponding speeds are 7 . 0µm / min and 4 . 6µm / min . The sequences show an entire cycle of oscillation ( spreading and contraction ) . White arrows represent the direction of motion before the cell spreads and contracts during which no net motion exists .

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Figure 3A: Sequence of snapshots of the LimE-GFP distribution and stress T for an oscillatory cell together with snapshots of the GFP-myo distribution for a separate oscillatory cell. Corresponding speeds are 7.0µm/min and 4.6µm/min. The sequences show an entire cycle of oscillation (spreading and contraction). White arrows represent the direction of motion before the cell spreads and contracts during which no net motion exists.


Figure 4A : Sequence of snapshots of the LimE - GFP distribution and stress T for an amoeboid cell and the GFP - myo distribution for a separate amoeboid cell . Corresponding speeds are 7 . 4µm / min and 4 . 5µm / min . The sequences show both the phases of protrusion and retraction . White arrows represent the direction of motion .

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Figure 4A: Sequence of snapshots of the LimE-GFP distribution and stress T for an amoeboid cell and the GFP-myo distribution for a separate amoeboid cell. Corresponding speeds are 7.4µm/min and 4.5µm/min. The sequences show both the phases of protrusion and retraction. White arrows represent the direction of motion.


Figure 5A : Protrusion and retraction speed for amoeboid , oscillatory and fan - shaped cells , defined as the average of the pixels with the 20 % lowest and highest membrane speed . C : Average edge velocity in regions of low and high LimE - GFP and GFP - myo fluorescence . High fluorescence was defined as the 20 % brightest LimE - GFP and GFP - myo pixels in the kymographs while low fluorescence consisted of the remaining 80 % pixels .

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Figure 5A: Protrusion and retraction speed for amoeboid, oscillatory and fan-shaped cells, defined as the average of the pixels with the 20% lowest and highest membrane speed.C: Average edge velocity in regions of low and high LimE-GFP and GFP-myo fluorescence. High fluorescence was defined as the 20% brightest LimE-GFP and GFP-myo pixels in the kymographs while low fluorescence consisted of the remaining 80% pixels.


Figure 1 ( a ) Lipid phosphatase activity of Paladin , wild - type and phosphatase - dead C / S mutant , towards PI ( 4 , 5 ) P2 and PI ( 3 , 4 , 5 ) P3 substrates . Positive control ; wild - type phosphatase and tensin homolog ( PTEN ) ; negative control ; lipid phosphatase - dead C124S PTEN . Mean ± SEM , Paired t - test , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( b ) Representative confocal image of HDMEC stained for Paladin ( green ) , Vascular Endothelial Cadherin ( VEC , red ) and nuclei ( DAPI , blue ) , scale bar : 10 μm . White boxes in the upper image are showed at higher magnification below , scale bar : 3 µm . ( c ) HDMEC stained for VEGFR2 ( green ) , Paladin ( red ) and nuclei ( DAPI , blue ) and with or without stimulation of 50 ng / ml VEGF - A for 0 , 2 or 10 min . Scale bar : 10 µm . ( d ) Quantification of VEGFR2 - Paladin co - staining before and after VEGF - A stimulation as shown in ( C ) . Mean ± SEM , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( e ) HDMEC analysed using Proximity Ligand Assay ( PLA ) for Paladin and VEGFR2 . Green dots indicate complex formation , VE - cadherin ( red ) and nuclei ( DAPI , blue ) . Cells stimulated with 50 ng / ml VEGF - A for 0 , 2 or 10 min . Scale bar : 10 µm . ( f ) Quantification of ( e ) , the number of PLA Paldin - VEGFR2 complexes per cell at 0 , 2 and 10 minutes after VEGF - A stimulation ( top ) . Quantification of ( e ) , average distance for the PLA complexes to the nearest VE - cadherin positive junction at 0 , 2 and 10 minutes after VEGF - A stimulation ( bottom ) . Mean ± SEM , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( g ) HDMEC stained for Paladin ( green ) and EEA1 ( red ) , colocalization in yellow , and nuclei ( DAPI , blue ) after VEGF - A stimulation for 0 , 2 or 10 minutes , a representative image from a single confocal plane is shown , scale bar : 10 µm . White boxes in the upper image are showed at higher magnification below , scale bar 3 µm . ( h ) Quantification of ( g ) , EEA1 / Paladin double positive particles per field of view at 0 , 2 and 10 minutes after VEGF - A stimulation . Mean ± SEM , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(a) Lipid phosphatase activity of Paladin, wild-type and phosphatase-dead C/S mutant, towards PI(4,5)P2 and PI(3,4,5)P3 substrates. Positive control; wild-type phosphatase and tensin homolog (PTEN); negative control; lipid phosphatase-dead C124S PTEN. Mean±SEM, Paired t-test, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(b) Representative confocal image of HDMEC stained for Paladin (green), Vascular Endothelial Cadherin (VEC, red) and nuclei (DAPI, blue), scale bar: 10 μm. White boxes in the upper image are showed at higher magnification below, scale bar: 3 µm.(c) HDMEC stained for VEGFR2 (green), Paladin (red) and nuclei (DAPI, blue) and with or without stimulation of 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (d) Quantification of VEGFR2-Paladin co-staining before and after VEGF-A stimulation as shown in (C). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(e) HDMEC analysed using Proximity Ligand Assay (PLA) for Paladin and VEGFR2. Green dots indicate complex formation, VE-cadherin (red) and nuclei (DAPI, blue). Cells stimulated with 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (f) Quantification of (e), the number of PLA Paldin-VEGFR2 complexes per cell at 0, 2 and 10 minutes after VEGF-A stimulation (top). Quantification of (e), average distance for the PLA complexes to the nearest VE-cadherin positive junction at 0, 2 and 10 minutes after VEGF-A stimulation (bottom). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(g) HDMEC stained for Paladin (green) and EEA1 (red), colocalization in yellow, and nuclei (DAPI, blue) after VEGF-A stimulation for 0, 2 or 10 minutes, a representative image from a single confocal plane is shown, scale bar: 10 µm. White boxes in the upper image are showed at higher magnification below, scale bar 3 µm. (h) Quantification of (g), EEA1/Paladin double positive particles per field of view at 0, 2 and 10 minutes after VEGF-A stimulation. Mean±SEM, One-way ANOVA. n= 3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


Figure 2 ( a ) Cell surface VEGFR2 levels detected by cell surface biotinylation , using thiol - cleavable sulfo - NHS - SS - biotin , of HDMEC transfected with PALD1 siRNA ( # 1 and # 2 ) or non - targeting control ( " c " ) siRNA , followed by VEGF - A stimulation ( 50 ng / ml ) for indicated time periods . Total lysates ( input ) and streptavidin ( SA ) pull down , immunoblotted for VEGFR2 , Paladin , and actin . ' No biotin ctrl ' , cells not treated with sulfo - NHS - SS - biotin . ( b ) Quantification of data in ( a ) . VEGFR2 surface levels ( data pooled for the indicated time points ) normalized to total VEGFR2 levels and compared between control and siRNA treated HDMEC . n = 4 for each time point , biological replicates , Mean ± SEM . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( c ) Internalized pool of VEGFR2 after VEGF - A treatment ( 50 ng / ml ) of non - transfected HDMEC ( " NT " ) or HDMEC transfected with PALD1 siRNA ( # 1 and # 2 ) or non - targeting control siRNA ( " c " ) . Cell surface biotinylation was performed prior to VEGF - A stimulation and at indicated time points , remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down . Immunoblotting of the total lysate ( input ) and SA pull down fraction for VEGFR2 , Paladin and actin . ( d ) Quantification of data in ( c ) . Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples . Mean ± SEM , unpaired t - test for indicated time points , normalized to cntrl siRNA sample . n = 3 for each time point , biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( e ) Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown . Representative images of VEGFR2 ( green ) / EEA1 ( red ) double - positive ( yellow ) vesicles in negative control siRNA , and PALD1 KD # 2 siRNA - silenced HDMEC at 0 , 2 and 10 min of VEGF - A stimulation ( 50 ng / ml ) . DAPI in blue , scale bar : 10 µm . Inset shows only EEA1 channel , scale bar : 10 µm . ( f ) Quantification of ( e ) , number of EEA1 positive ( top ) or VEGFR2 - EEA1 double - positive vesicles ( bottom ) per field of view . Mean ± SEM , Two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( g ) HDMEC stained for PI ( 4 , 5 ) P2 ( cyan ) , VE - cadherin ( red ) and Paladin ( yellow in inset ) following treatment using negative control or PALD1 ( KD # 2 ) siRNA . VEGF - A stimulation for 0 , 2 or 10 minutes ( 50 ng / ml ) . DAPI in blue . Scale bar : 10 µm ( h ) Quantification of ( g ) , Total PI ( 4 , 5 ) P2 signal ( top ) or intracellular PI ( 4 , 5 ) P2 not overlapping with VE - cadherin ( bottom ) . Mean ± SEM , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(a) Cell surface VEGFR2 levels detected by cell surface biotinylation, using thiol-cleavable sulfo-NHS-SS-biotin, of HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control ("c") siRNA, followed by VEGF-A stimulation (50 ng/ml) for indicated time periods. Total lysates (input) and streptavidin (SA) pull down, immunoblotted for VEGFR2, Paladin, and actin. 'No biotin ctrl', cells not treated with sulfo-NHS-SS-biotin. (b) Quantification of data in (a). VEGFR2 surface levels (data pooled for the indicated time points) normalized to total VEGFR2 levels and compared between control and siRNA treated HDMEC. n=4 for each time point, biological replicates, Mean±SEM. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(c) Internalized pool of VEGFR2 after VEGF-A treatment (50 ng/ml) of non-transfected HDMEC ("NT") or HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control siRNA ("c"). Cell surface biotinylation was performed prior to VEGF-A stimulation and at indicated time points, remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down. Immunoblotting of the total lysate (input) and SA pull down fraction for VEGFR2, Paladin and actin. (d) Quantification of data in (c). Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples. Mean±SEM, unpaired t-test for indicated time points, normalized to cntrl siRNA sample. n=3 for each time point, biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(e) Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown. Representative images of VEGFR2 (green)/EEA1 (red) double-positive (yellow) vesicles in negative control siRNA, and PALD1 KD#2 siRNA-silenced HDMEC at 0, 2 and 10 min of VEGF-A stimulation (50 ng/ml). DAPI in blue, scale bar: 10 µm. Inset shows only EEA1 channel, scale bar: 10 µm. (f) Quantification of (e), number of EEA1 positive (top) or VEGFR2-EEA1 double-positive vesicles (bottom) per field of view. Mean±SEM, Two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(g) HDMEC stained for PI(4,5)P2 (cyan), VE-cadherin (red) and Paladin (yellow in inset) following treatment using negative control or PALD1 (KD#2) siRNA. VEGF-A stimulation for 0, 2 or 10 minutes (50 ng/ml). DAPI in blue. Scale bar: 10 µm (h) Quantification of (g), Total PI(4,5)P2 signal (top) or intracellular PI(4,5)P2 not overlapping with VE-cadherin (bottom). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


Figure 3Signalling downstream of VEGF - A / VEGFR2 assessed in HDMEC , untreated ( NT , non - transfected ) , transfected with non - targeting siRNA ( c / cntrl ) , or with two different PALD1 - targeting siRNAs ( KD # 1 and KD # 2 ) , and treated with VEGF - A for 0 - 10 and 0 - 20 min , respectively . Immunoblotting of cell lysates for phosphorylated ( p ) VEGFR2 ( pY1175 ) , total VEGFR2 Actin served as loading control ( a PALD1 knockdown was verified by blotting for Paladin . Quantification of pVEGFR2 ( normalized to total VEGFR2 ) ( b ) Mean ± SEM , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 . Signalling downstream of VEGF - A / VEGFR2 assessed in HDMEC , untreated ( NT , non - transfected ) , transfected with non - targeting siRNA ( c / cntrl ) , or with two different PALD1 - targeting siRNAs ( KD # 1 and KD # 2 ) , and treated with VEGF - A for 0 - 10 and 0 - 20 min , respectively . Immunoblotting of cell lysates for phosphorylated Erk1 / 2 ( pT202 and pY204 ) , and total Erk . Actin served as loading control c ) . PALD1 knockdown was verified by blotting for Paladin . Quantification of pErk1 / 2 ( normalized to total Erk1 / 2 ) ( d ) . Mean ± SEM , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 . ( e - f ) Immunoblotting on total heart lysates from adult Pald1 + / + and Pald1 - / - mice , tail - vein injected with VEGF - A ( 0 . 25µg / g body weight ) or PBS for the indicated time points , for Paladin , phosphorylated and total levels of VEGFR2 , Erk1 / 2 and β2 - microglobulin ( β 2 - MG , loading control ) . ( f ) Quantification of pVEGFR pY1173 normalized to total VEGFR2 ( n = 3 ) , total VEGFR2 levels normalized to total loading control ( n = 4 ) , pT202 / pY204 Erk1 / 2 normalized to Erk1 / 2 ( n = 5 ) . Mean ± SEM , multiple t - test with Holm - Sidak correction ( total VEGFR2 ) , two - way ANOVA ( others ) . Data information : * p < 0 . 05 , * * p < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated (p) VEGFR2 (pY1175), total VEGFR2 Actin served as loading control (a PALD1 knockdown was verified by blotting for Paladin. Quantification of pVEGFR2 (normalized to total VEGFR2) (b) Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01.Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated Erk1/2 (pT202 and pY204), and total Erk. Actin served as loading control c). PALD1 knockdown was verified by blotting for Paladin. Quantification of pErk1/2 (normalized to total Erk1/2) (d). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01.(e-f) Immunoblotting on total heart lysates from adult Pald1+/+ and Pald1-/- mice, tail-vein injected with VEGF-A (0.25µg/g body weight) or PBS for the indicated time points, for Paladin, phosphorylated and total levels of VEGFR2, Erk1/2 and β2-microglobulin (β 2-MG, loading control). (f) Quantification of pVEGFR pY1173 normalized to total VEGFR2 (n=3), total VEGFR2 levels normalized to total loading control (n=4), pT202/pY204 Erk1/2 normalized to Erk1/2 (n=5). Mean±SEM, multiple t-test with Holm-Sidak correction (total VEGFR2), two-way ANOVA (others). Data information: *p < 0.05, **p < 0.01.


Figure 4 ( a , b ) Delayed vascular outgrowth and hyperdense vascular front in isolectinB4 - stained P5 retina from Pald1 - / - mouse compared with Pald1 + / + ( a ) . Orange arrow indicates radial expansion of the vascular plexus in the Pald1 + / + retina . Scale bar : 1 mm . Quantification of radial expansion ( b ) as shown in ( a ) normalized to wild type litter mates . Mean ± SEM , unpaired t - test . n = 14 litters , 18 wild type and 23 knock - out pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( c , d ) Increased filopodia number in Pald1 - / - mouse retina at vascular front , visualized by isolectinB4 staining ( c ) . Scale bar : 50 μm . Quantification of filopodia per 100 µm at the vascular front ( d ) . Mean ± SEM , unpaired t - test . n = 3 litters , 7 wild type and 5 knock - out pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( e , f ) P5 retina vascular front ( isolectinB4 , green ) and endothelial nuclei ( Erg , red ) ( e ) . Scale bar : 100 μm . Magenta stippled square area quantified in ( f ) . Vascular density was determined in the capillary bed ( left ) ( 11 wild type , 12 knock - out ) and in area around veins ( right ) ( 10 wild type , 9 knock - out , biological replicates ) . Mean ± SEM , unpaired t - test . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( g , h ) pT202 / pY204 Erk1 / 2 immunostaining ( black ) in Pald1 + / + and Pald1 - / - P5 pups . IsolectinB4 ( white ) visualizes the entire vasculature . Scale bar : 100 μm . Quantification of pT202 / pY204 Erk1 / 2 area ( h ) as in the black stippled square in ( g ) ( 400 µm from the retina rim ) , normalized to isolectinB4 area . Mean ± SEM , unpaired t - test . n = 5 litters , 14 wild type and 10 knock - out retinas per genotype . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( i ) Quantitative real - time PCR analysis of P4 - P5 retinas from Pald1 + / + and Pald1 - / - pups . Ccnd1 transcript levels , normalized to Gapdh . Mean ± SEM , unpaired t - test . n = 10 Pald1 + / + and 14 Pald1 - / - pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( j ) Cyclin D1 ( black ) and isolectinB4 ( white ) staining of P5 retinas from Pald1 + / + and Pald1 - / - pups . Scale bar : 100 µm . ( k ) Quantification of number of Cyclin D1 positive nuclei normalized for isolectinB4 area in retinas from Pald1 + / + and Pald1 - / P5 pups . Mean ± SEM , unpaired t - test . n = 7 Pald1 + / + and 6 Pald1 - / - pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( l ) Quantification of relative radial expansion in vehicle ( n = 2 litters , 5 Pald1 + / + and 4 Pald1 - / - pups ) and MEK inhibitor ( U0126 ) - treated pups ( n = 4 litters , 7 Pald1 + / + and 5 Pald1 - / - pups ) . MEK inhibitor / vehicle was administered twice at 12h interval at P4 and eyes collected at P5 . Each dot is one mouse . Mean ± SEM , one - way ANOVA , n = 4 - 7 . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( m ) Quantification of the tip cell number in vehicle - ( n = 3 litters , 5 Pald1 + / + and 5 Pald1 - / - pups ) and MEK inhibitor ( U0126 ) - treated pups ( n = 3 litters , 5 Pald1 + / + and 4 Pald1 - / - pups ) . MEK inhibitor / vehicle administered twice at P5 at 2 - h intervals , and eyes collected 2 h after the second injection . Each dot is one mouse . Mean ± SEM , one - way ANOVA . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(a, b) Delayed vascular outgrowth and hyperdense vascular front in isolectinB4-stained P5 retina from Pald1-/- mouse compared with Pald1+/+ (a). Orange arrow indicates radial expansion of the vascular plexus in the Pald1+/+ retina. Scale bar: 1 mm. Quantification of radial expansion (b) as shown in (a) normalized to wild type litter mates. Mean±SEM, unpaired t-test. n=14 litters, 18 wild type and 23 knock-out pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.(c, d) Increased filopodia number in Pald1-/- mouse retina at vascular front, visualized by isolectinB4 staining (c). Scale bar: 50 μm. Quantification of filopodia per 100 µm at the vascular front (d). Mean±SEM, unpaired t-test. n=3 litters, 7 wild type and 5 knock-out pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.(e, f) P5 retina vascular front (isolectinB4, green) and endothelial nuclei (Erg, red) (e). Scale bar: 100 μm. Magenta stippled square area quantified in (f). Vascular density was determined in the capillary bed (left) (11 wild type, 12 knock-out) and in area around veins (right) (10 wild type, 9 knock-out, biological replicates). Mean±SEM, unpaired t-test. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.(g, h) pT202/pY204 Erk1/2 immunostaining (black) in Pald1+/+ and Pald1-/- P5 pups. IsolectinB4 (white) visualizes the entire vasculature. Scale bar: 100 μm. Quantification of pT202/pY204 Erk1/2 area (h) as in the black stippled square in (g) (400 µm from the retina rim), normalized to isolectinB4 area. Mean±SEM, unpaired t-test. n=5 litters, 14 wild type and 10 knock-out retinas per genotype. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.(i) Quantitative real-time PCR analysis of P4-P5 retinas from Pald1+/+ and Pald1-/- pups. Ccnd1 transcript levels, normalized to Gapdh. Mean±SEM, unpaired t-test. n=10 Pald1+/+ and 14 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.(j) Cyclin D1 (black) and isolectinB4 (white) staining of P5 retinas from Pald1+/+ and Pald1-/-pups. Scale bar: 100 µm.(k) Quantification of number of Cyclin D1 positive nuclei normalized for isolectinB4 area in retinas from Pald1+/+ and Pald1-/ P5 pups. Mean±SEM, unpaired t-test. n=7 Pald1+/+ and 6 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.(l) Quantification of relative radial expansion in vehicle (n=2 litters, 5 Pald1+/+ and 4 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=4 litters, 7 Pald1+/+ and 5 Pald1-/- pups). MEK inhibitor/vehicle was administered twice at 12h interval at P4 and eyes collected at P5. Each dot is one mouse. Mean±SEM, one-way ANOVA, n=4-7. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.(m) Quantification of the tip cell number in vehicle- (n=3 litters, 5 Pald1+/+ and 5 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=3 litters, 5 Pald1+/+ and 4 Pald1-/- pups). MEK inhibitor/vehicle administered twice at P5 at 2-h intervals, and eyes collected 2 h after the second injection. Each dot is one mouse. Mean±SEM, one-way ANOVA. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


Figure 5 ( a ) Eyes from Pald1 + / LacZ mice collected at P17 from untreated animals or animals with oxygen - induced retinopathy ( OIR ) . Pald1 - promoter driven LacZ expression and X - gal staining generated signals in capillaries , veins and arteries in the OIR retina at P17 compared to the normoxia control with predominantly arterial LacZ expression . Scale bar : 1 mm . ( b ) Paladin levels in primary human umbilical vein endothelial cells ( HUVEC ) untreated or treated for 24 h with VEGF - A ( 50 ng / ml ) , FGF2 ( 50 ng / ml ) , or SDF1α ( 30 ng / ml ) , quantified by immunoblotting for Paladin and β - actin ( loading control ) . Mean ± SEM , one way - ANOVA , n = 5 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 . ( c ) Eyes from adult Pald1LacZ / + mice collected at 72 h after single - bolus intravitreal injection of 1 μg VEGF - A , and PBS in the contralateral eye , followed by X - gal staining . Arrow indicates Pald1 promoter activity in veins ( outlined by dashed line ) specifically after VEGF - A treatment . A , artery ; V , vein . Scale bar : 100 μm . ( d - f ) Representative images of isolectinB4 - stained P17 retinas from OIR - challenged Pald1 + / + and Pald1 - / - mice ( detailed view in the insets ) ( d ) . Scale bar : 1 mm ( inset 250 µm ) . Quantification of neovascular tuft area ( e ) and avascular area ( f ) . Each dot represents the mean of both retinas per mouse . Mean ± SEM , unpaired t - test , n = 3 litters , 4 Pald1 + / + and 3 Pald1 - / - pups . Data information : * p < 0 . 05 , * * p < 0 . 01 . g ) Representative images of retina vasculature immunostained for isolectinB4 and pT202 / pY204 Erk1 / 2 ( pErk1 / 2 ) . pErk1 / 2 staining within the vessels is also visualized using a 16 - color heatmap to display staining intensity . Scale bar : 30 µm ( inset 10µm ) . ( h ) Quantification of pErk1 / 2 immunostaining as shown in ( g ) within isolectinB4 - positive vessels , as fold - change of pErk1 / 2 stained area . Each dot represents the mean of both retinas per mouse . Mean ± SEM , unpaired t - test , n = 3 litters , 5 Pald1 + / + and 5 Pald1 - / - pups . Data information : * p < 0 . 05 , * * p < 0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(a) Eyes from Pald1+/LacZ mice collected at P17 from untreated animals or animals with oxygen-induced retinopathy (OIR). Pald1-promoter driven LacZ expression and X-gal staining generated signals in capillaries, veins and arteries in the OIR retina at P17 compared to the normoxia control with predominantly arterial LacZ expression. Scale bar: 1 mm.(b) Paladin levels in primary human umbilical vein endothelial cells (HUVEC) untreated or treated for 24 h with VEGF-A (50 ng/ml), FGF2 (50 ng/ml), or SDF1α (30 ng/ml), quantified by immunoblotting for Paladin and β-actin (loading control). Mean±SEM, one way-ANOVA, n=5 biological replicates. Data information: *p < 0.05, **p < 0.01.(c) Eyes from adult Pald1LacZ/+ mice collected at 72 h after single-bolus intravitreal injection of 1 μg VEGF-A, and PBS in the contralateral eye, followed by X-gal staining. Arrow indicates Pald1 promoter activity in veins (outlined by dashed line) specifically after VEGF-A treatment. A, artery; V, vein. Scale bar: 100 μm.(d-f) Representative images of isolectinB4-stained P17 retinas from OIR-challenged Pald1+/+ and Pald1-/- mice (detailed view in the insets) (d). Scale bar: 1 mm (inset 250 µm). Quantification of neovascular tuft area (e) and avascular area (f). Each dot represents the mean of both retinas per mouse. Mean±SEM, unpaired t-test, n=3 litters, 4 Pald1+/+ and 3 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01.g) Representative images of retina vasculature immunostained for isolectinB4 and pT202/pY204 Erk1/2 (pErk1/2). pErk1/2 staining within the vessels is also visualized using a 16-color heatmap to display staining intensity. Scale bar: 30 µm (inset 10µm). (h) Quantification of pErk1/2 immunostaining as shown in (g) within isolectinB4-positive vessels, as fold-change of pErk1/2 stained area. Each dot represents the mean of both retinas per mouse. Mean±SEM, unpaired t-test, n=3 litters, 5 Pald1+/+ and 5 Pald1-/- pups. Data information: *p < 0.05, **p < 0.01.


Figure 1A NHF , Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation , then immunostained with LAMP1 antibody . Dashed lines in images outline cell boundaries . Scale bar , 10 μm . C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A . The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods . n = 3 per group in which 30 cells were counted per condition per experiment . Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test . B Wildtype ( Ocrl + / y ) and Ocrl - / - MEF cells were grown in complete media or 5 h serum starvation , then immunostained with indicated antibodies . Dashed lines in images outline cell boundaries . Scale bar , 10 μm . D , E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B . The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods . n = 3 per group in which 30 cells were counted per condition per experiment . Data information : , data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test . I Snap images of WT ( Ocrl + / y ) , OCRL knockout ( Ocrl - / - ) and IOB MEFs transfected with lysosome - GFP . After transfection , WT , OCRL knockout and IOB MEFs were left in serum starvation for 8 h . Scale bar , 5 μm . J Quantitative analyses of tubulated lysosomes in the experiments shown in H . n = 3 per group in which 10 cells were counted per condition per experiment . D Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A NHF, Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation, then immunostained with LAMP1 antibody. Dashed lines in images outline cell boundaries. Scale bar, 10 μm. C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A. The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.B Wildtype (Ocrl+/y) and Ocrl-/- MEF cells were grown in complete media or 5 h serum starvation, then immunostained with indicated antibodies. Dashed lines in images outline cell boundaries. Scale bar, 10 μm.D, E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B. The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: , data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.I Snap images of WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs transfected with lysosome-GFP. After transfection, WT, OCRL knockout and IOB MEFs were left in serum starvation for 8 h. Scale bar, 5 μm. J Quantitative analyses of tubulated lysosomes in the experiments shown in H. n = 3 per group in which 10 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.