( b ) Representative electron micrographs of ypt7Δ , atg19Δ yeast cells grown under cytoplasm - to - vacuole targetting ( Cvt ) conditions expressing the indicated proteins labelled with an anti - Myc antibody . The white arrowheads indicate the isolation membrane . The dashed line indicates the circumference of the prApe1 oligomer . Gold particles , 10 nm . Scale bars , 200 nm . See Supplementary Fig . 5 for full images .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative electron micrographs of ypt7Δ, atg19Δ yeast cells grown under cytoplasm-to-vacuole targetting (Cvt) conditions expressing the indicated proteins labelled with an anti-Myc antibody. The white arrowheads indicate the isolation membrane. The dashed line indicates the circumference of the prApe1 oligomer. Gold particles, 10 nm. Scale bars, 200 nm. See Supplementary Fig. 5 for full images.


( c ) Quantification of electron micrographs of yeast cells expressing either Myc - Atg19 or Myc - Atg19 - Atg34 co - labelled with anti - Myc ( 10 nm gold ) and anti - prApe1 ( 5 nm gold ) . See also Supplementary Fig . 5 . Three independent experiments ; Myc - Atg19 , N = 48 , Myc - Atg19 - 34 , N = 24 .

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

(c) Quantification of electron micrographs of yeast cells expressing either Myc-Atg19 or Myc-Atg19-Atg34 co-labelled with anti-Myc (10 nm gold) and anti-prApe1 (5 nm gold). See also Supplementary Fig. 5. Three independent experiments; Myc-Atg19, N = 48, Myc-Atg19-34, N = 24.


( d ) Anti - Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs . The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole . Supplementary Fig . 6 shows the expression of the Myc - tagged proteins and a quantification of the assay .

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

(d) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. Supplementary Fig. 6 shows the expression of the Myc-tagged proteins and a quantification of the assay.


( e ) Y2H assay testing for the interaction of Atg11 with Atg19 and the indicated Atg19 mutants .

(e) Y2H assay testing for the interaction of Atg11 with Atg19 and the indicated Atg19 mutants.


( f ) Atg19Δ yeast cells expressing the indicated Atg19 proteins and prApe1 - RFP ( red fluorescent protein ) were labelled with the vacuolar membrane dye MDY - 64 . Scale bars : 2 μM . The experiments in a - d , f have been conducted three times , the experiment in e twice . Images of uncropped western blots and gels can be found in Supplementary Fig . 7 .

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

(f) Atg19Δ yeast cells expressing the indicated Atg19 proteins and prApe1-RFP (red fluorescent protein) were labelled with the vacuolar membrane dye MDY-64. Scale bars: 2 μM. The experiments in a-d,f have been conducted three times, the experiment in e twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.


The expression of the 20 genes encoding TFs was measured 24 h after mannitol treatment during the proliferating ( n = 192 plants ) , expanding ( n = 16 plants ) and mature ( n = 16 ) leaf developmental stage . Expression levels in wild - type plants transferred to mannitol - induced stress were compared to those transferred to control conditions at the same developmental stage . Data information : Data are presented as mean ± SEM , n = 4 independent experiments . FC = Fold change . * = FDR < 0 . 05 , Unpaired two - sided Student ' s t - test .

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

The expression of the 20 genes encoding TFs was measured 24 h after mannitol treatment during the proliferating (n = 192 plants), expanding (n = 16 plants) and mature (n = 16) leaf developmental stage. Expression levels in wild-type plants transferred to mannitol-induced stress were compared to those transferred to control conditions at the same developmental stage. Data information: Data are presented as mean ± SEM, n = 4 independent experiments. FC = Fold change. * = FDR < 0.05, Unpaired two-sided Student's t-test.


A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . The first group contains TFs that reached the log2 ( FC ) threshold 40 min after mannitol treatment ( A ) The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 .

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

A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. The first group contains TFs that reached the log2(FC) threshold 40 min after mannitol treatment (A) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1.


A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . the second group reached the threshold after 1 h ( B ) The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 .

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

A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the second group reached the threshold after 1 h (B) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1.


A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . the third group after 2 h ( C ) The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 .

A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the third group after 2 h (C) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1.


A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . the fourth group after 4 h ( D ) . The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 .

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

A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the fourth group after 4 h (D). The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1.


C Heatmap of the significant regulations upon induction of overexpression of the five members of the core network , the activators ( green ) ERF6 , ERF59 , ERF98 and the repressors ( red ) ERF8 and ERF9 . Color code represents FDR - corrected p - values with thresholds at FDR = 0 . 01 , 0 . 05 and 0 . 1 . Data information : In A , data are extrapolated from estimated averages , n = 3 independent experiments , FDR - corrected p - values < 0 . 1 ( mixed model analysis , user - defined Wald tests ) . The thickness of the arrows represents the FDR value . In B , data are presented as averages , n = 3 independent experiments . The intensity of the color of the arrows represents the strength of the regulation according to the TEA values and the thickness the FDR value of the nCounter Nanostring experiment . In C , data are represented as FDR - corrected p - values , n = 3 independent experiments .

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

C Heatmap of the significant regulations upon induction of overexpression of the five members of the core network, the activators (green) ERF6, ERF59, ERF98 and the repressors (red) ERF8 and ERF9. Color code represents FDR-corrected p-values with thresholds at FDR=0.01, 0.05 and 0.1. Data information: In A, data are extrapolated from estimated averages, n = 3 independent experiments, FDR-corrected p-values < 0.1 (mixed model analysis, user-defined Wald tests). The thickness of the arrows represents the FDR value. In B, data are presented as averages, n = 3 independent experiments. The intensity of the color of the arrows represents the strength of the regulation according to the TEA values and the thickness the FDR value of the nCounter Nanostring experiment. In C, data are represented as FDR-corrected p-values, n = 3 independent experiments.


A At 22 days after stratification ( DAS ) , leaf series of the LOF lines were made and the rosette area was calculated as the sum of the area of all individual leaves ( n = 10 plants ) . The rosette area is presented relative to the corresponding wild type .

A At 22 days after stratification (DAS), leaf series of the LOF lines were made and the rosette area was calculated as the sum of the area of all individual leaves (n = 10 plants). The rosette area is presented relative to the corresponding wild type.


B Average area of the individual leaves of rap2 . 6L and myb51 , two knock - out lines with a smaller average rosette area .

O O 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 Average area of the individual leaves of rap2.6L and myb51, two knock-out lines with a smaller average rosette area.


C Rosette area of two or three independent GOF lines per TF ( calculated as the projected area or the sum of the leaves , n = 10 plants ) germinated and grown on DEX - containing medium . Measurements were performed at 22 DAS relative to the control line . " Independent line 1 " is the line with the highest overexpression level .

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

C Rosette area of two or three independent GOF lines per TF (calculated as the projected area or the sum of the leaves, n = 10 plants) germinated and grown on DEX-containing medium. Measurements were performed at 22 DAS relative to the control line. "Independent line 1" is the line with the highest overexpression level.


D A representative picture of the rosette of GOF lines with significant growth phenotypes in both independent lines at 22 DAS . Scale bar is 1 cm .

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

D A representative picture of the rosette of GOF lines with significant growth phenotypes in both independent lines at 22 DAS. Scale bar is 1 cm.


E , F Pavement cell number and area of the third leaf at 22 DAS of LOF ( E ) that showed a significant rosette area phenotype . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s test ) . In B , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( mixed model , partial F - tests ) . In E and F , data are presented as mean ± SEM , n = 3 independent experiments , . = P < 0 . 1 , * = P < 0 . 05 , * * = P < 0 . 01 , * * * = P < 0 . 001 ( Tukey ' s test ) .

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

E, F Pavement cell number and area of the third leaf at 22 DAS of LOF (E) that showed a significant rosette area phenotype. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test). In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests). In E and F, data are presented as mean ± SEM, n = 3 independent experiments, . = P<0.1, * = P<0.05, ** = P<0.01, *** = P<0.001 (Tukey's test).


E , F Pavement cell number and area of the third leaf at 22 DAS of GOF lines ( F ) that showed a significant rosette area phenotype . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s test ) . In B , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( mixed model , partial F - tests ) . In E and F , data are presented as mean ± SEM , n = 3 independent experiments , . = P < 0 . 1 , * = P < 0 . 05 , * * = P < 0 . 01 , * * * = P < 0 . 001 ( Tukey ' s test ) .

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

E, F Pavement cell number and area of the third leaf at 22 DAS of GOF lines (F) that showed a significant rosette area phenotype. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test). In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests). In E and F, data are presented as mean ± SEM, n = 3 independent experiments, . = P<0.1, * = P<0.05, ** = P<0.01, *** = P<0.001 (Tukey's test).


A The expression level of two GA - degradation and two GA - biosynthesis enzymes in expanding leaf tissue ( third leaf at 15 DAS ) of wild - type plants 20 min , 40 min , 1 h , 2 h , 4 h , 8 h , 12 h , 16 h , and 24 h after mannitol treatment . The fold changes ( FC ) were calculated relative to control conditions . Data information : In A , data are presented as mean ± SEM , n = 4 independent experiments , * = FDR < 0 . 05 ( unpaired two - sided Student ' s t - test )

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

A The expression level of two GA-degradation and two GA-biosynthesis enzymes in expanding leaf tissue (third leaf at 15 DAS) of wild-type plants 20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h after mannitol treatment. The fold changes (FC) were calculated relative to control conditions.Data information: In A, data are presented as mean ± SEM, n = 4 independent experiments, *= FDR<0.05 (unpaired two-sided Student's t-test)


B The expression of GA2 - OX6 and GA20 - OX1 in expanding leaf tissue ( third leaf - 15 DAS ) , 8 h after transfer to DEX - containing medium to induce overexpression . The FC was calculated relative to control conditions . Data information : In B , data are presented as mean ± SEM , n = 3 independent experiments , * = FDR < 0 . 1 ( mixed model analysis , user - defined Wald tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The expression of GA2-OX6 and GA20-OX1 in expanding leaf tissue (third leaf - 15 DAS), 8 h after transfer to DEX-containing medium to induce overexpression. The FC was calculated relative to control conditions.Data information: In B, data are presented as mean ± SEM, n = 3 independent experiments, * = FDR < 0.1 (mixed model analysis, user-defined Wald tests).


C The effect of ERF6 , ERF9 , ERF98 and WRKY15 on the GA2 - OX6 promoter determined with transient expression assays . The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Data information : In C , data are presented as mean ± SEM , n = 3 independent experiments .

C The effect of ERF6, ERF9, ERF98 and WRKY15 on the GA2-OX6 promoter determined with transient expression assays. The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Data information: In C, data are presented as mean ± SEM, n = 3 independent experiments.


A At 22 DAS , leaf series were made of the LOF lines ( n = 10 plants ) grown under control conditions or mild osmotic stress conditions , and the reduction under mild osmotic stress was calculated . The rosette area reduction is presented relative to the corresponding wild type . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s test ) .

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

A At 22 DAS, leaf series were made of the LOF lines (n = 10 plants) grown under control conditions or mild osmotic stress conditions, and the reduction under mild osmotic stress was calculated. The rosette area reduction is presented relative to the corresponding wild type.Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test).


B Leaf series of stz and erf11 mutants grown on control and mild osmotic stress conditions . Data information : In B , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( mixed model , partial F - tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Leaf series of stz and erf11 mutants grown on control and mild osmotic stress conditions.Data information: In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests).


C The rosette area of two or three independent GOF lines ( n = 10 plants ) germinated on DEX - containing control or mild osmotic stress medium was measured at 22 DAS and the reduction by mild osmotic stress was calculated relative to the control line . " Independent line 1 " is the line with the highest overexpression level . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s test ) .

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

C The rosette area of two or three independent GOF lines (n = 10 plants) germinated on DEX-containing control or mild osmotic stress medium was measured at 22 DAS and the reduction by mild osmotic stress was calculated relative to the control line. "Independent line 1" is the line with the highest overexpression level. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test).


A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes ERF11 ( A ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

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

A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes ERF11 (A) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes ERF5 ( B ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes ERF5 (B) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes MYB51 ( C ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes MYB51 (C) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes , RAP2 . 6L ( D ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes, RAP2.6L (D) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes ERF6 ( E ) . The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

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

A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes ERF6 (E). The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


The gain - of - function lines of all members of the core network were crossed either with each other , referred to as double cross , or with the control line ( GFP - GR ) , referred to as single cross . The projected rosette area of the F1 double and single cross and the GFP - GR , germinated and grown on DEX , was measured at 22 DAS . A representative picture of the single and double crosses is depicted . The values represent the relative increase or decrease in rosette size of the double crosses compared to their parental single crosses . The outcome of every double cross is classified into three groups : additive ( green connections ) , negative ( blue connection ) and synergistic ( orange connections ) phenotype . The precise measurements can be found in Fig EV2 .

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

The gain-of-function lines of all members of the core network were crossed either with each other, referred to as double cross, or with the control line (GFP-GR), referred to as single cross. The projected rosette area of the F1 double and single cross and the GFP-GR, germinated and grown on DEX, was measured at 22 DAS. A representative picture of the single and double crosses is depicted. The values represent the relative increase or decrease in rosette size of the double crosses compared to their parental single crosses. The outcome of every double cross is classified into three groups: additive (green connections), negative (blue connection) and synergistic (orange connections) phenotype. The precise measurements can be found in Fig EV2.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to wound - healing ( A ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar in ( A ) : 200 μm .

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

A549 cells expressing control shRNA, WDR63 shRNA#1, WDR63 shRNA#2, PCDH control, or PCDH-Flag-WDR63 were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar in (A): 200 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to transwell migration ( B ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar in ( B ) : 100 μm .

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

A549 cells expressing control shRNA, WDR63 shRNA#1, WDR63 shRNA#2, PCDH control, or PCDH-Flag-WDR63 were subjected to transwell migration (B) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar in (B): 100 μm.


( C ) Effect of WDR63 knockdown on the migration of A549 cells was analyzed by single cell tracking with time - lapse microscopy at 10 min intervals for 4 h . Scale bar : 200 μm . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA .

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

(C) Effect of WDR63 knockdown on the migration of A549 cells was analyzed by single cell tracking with time-lapse microscopy at 10 min intervals for 4 h. Scale bar: 200 μm. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA.


( D ) A549 cells expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to transwell invasion assay . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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

(D) A549 cells expressing control shRNA, WDR63 shRNA#1, WDR63 shRNA#2, PCDH control, or PCDH-Flag-WDR63 were subjected to transwell invasion assay. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to transwell migration ( E ) assays . The shown images are representative of three independent experiments . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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

A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#1 plus shRNA-resistant Flag-WDR63 were subjected to transwell migration (E) assays. The shown images are representative of three independent experiments. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar : 100 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to single cell tracking ( F ) assays . The shown images are representative of three independent experiments . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar 200 μm .

A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#1 plus shRNA-resistant Flag-WDR63 were subjected to single cell tracking (F) assays. The shown images are representative of three independent experiments. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar 200 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to transwell invasion ( G ) assays . The shown images are representative of three independent experiments . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#1 plus shRNA-resistant Flag-WDR63 were subjected to transwell invasion (G) assays. The shown images are representative of three independent experiments. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar : 100 μm.


( H ) A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 2 ( each also expressing luciferase ) were injected via tail vein into nude mice ( n = 6 for each group ) . Three weeks after injection , tumor formation was monitored by bioluminescence imaging . Data shown are mean ± SD ; * , P ˂ 0 . 05 ; One - way ANOVA .

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

(H) A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#2 (each also expressing luciferase) were injected via tail vein into nude mice (n=6 for each group). Three weeks after injection, tumor formation was monitored by bioluminescence imaging. Data shown are mean ± SD; *, P ˂ 0.05; One-way ANOVA.


( A ) WDR63 mRNA and protein levels in A549 , H292 , and HCT116 cells transduced with empty vector ( PCDH ) or PCDH - p53 . Data shown are mean ± SD ; n = 3 independent experiments . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(A) WDR63 mRNA and protein levels in A549, H292, and HCT116 cells transduced with empty vector (PCDH) or PCDH-p53. Data shown are mean ± SD; n = 3 independent experiments. **, P ˂ 0.01; ***, P ˂ 0.001; two-tailed Student's t-test.


( B ) WDR63 mRNA and protein levels in A549 , H292 , and HCT116 cells transduced with control ( shctrl ) or p53 ( shp53 ) shRNA . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WDR63 mRNA and protein levels in A549, H292, and HCT116 cells transduced with control (shctrl) or p53 (shp53) shRNA. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.


( C ) A549 cells expressing control or p53 shRNA were treated with doxorubicin ( Dox , 1μg / ml ) for the indicated periods of time . WDR63 mRNA and protein levels were then examined . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(C) A549 cells expressing control or p53 shRNA were treated with doxorubicin (Dox, 1μg/ml) for the indicated periods of time. WDR63 mRNA and protein levels were then examined. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.


( D ) WDR63 mRNA levels in H1299 cells transfected with empty vector ( - ) , wild - type p53 , or the indicated p53 mutants . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(D) WDR63 mRNA levels in H1299 cells transfected with empty vector (-), wild-type p53, or the indicated p53 mutants. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.


( F ) Lysates from A549 cells were subjected to ChIP assay using control IgG or anti - p53 antibody . ChIP products were amplified by real - time PCR . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(F) Lysates from A549 cells were subjected to ChIP assay using control IgG or anti-p53 antibody. ChIP products were amplified by real-time PCR. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.


( G ) A549 cells were transfected with empty vector ( ctrl ) or Flag - p53 plus the indicated reporter constructs . Twenty - four hours later , reporter activity was measured . Data shown are mean ± SD ; n = 3 independent experiments . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(G) A549 cells were transfected with empty vector (ctrl) or Flag-p53 plus the indicated reporter constructs. Twenty-four hours later, reporter activity was measured. Data shown are mean ± SD; n = 3 independent experiments. **, P ˂ 0.01; ***, P ˂ 0.001; two-tailed Student's t-test.


( H ) A549 cells expressing control or p53 shRNA were transfected with pGL3 - BS1 and Renilla luciferase plasmids . Twenty - four hours later , cells were treated with or without Nutlin ( 10 μM ) for another 12 h . Reporter activity was then measured . Data shown are mean ± SD ; n = 3 independent experiments . * * , P ˂ 0 . 01 ; two - tailed Student ' s t - test .

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

(H) A549 cells expressing control or p53 shRNA were transfected with pGL3-BS1 and Renilla luciferase plasmids. Twenty-four hours later, cells were treated with or without Nutlin (10 μM) for another 12 h. Reporter activity was then measured. Data shown are mean ± SD; n = 3 independent experiments. **, P ˂ 0.01; two-tailed Student's t-test.


( A ) A ~ 44 kD protein band specifically pulled down by GST - WDR63 from A549 cell lysates was identified as Arp2 by mass spectrometry analysis . The Arp2 peptide sequences obtained by MS are shown .

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

(A) A ~44 kD protein band specifically pulled down by GST-WDR63 from A549 cell lysates was identified as Arp2 by mass spectrometry analysis. The Arp2 peptide sequences obtained by MS are shown.


( B ) Lysates from HEK293T cells expressing HA - Arp2 alone or together with Flag - WDR63 were immunoprecipitated by anti - Flag antibody , followed by western blot analysis . Data shown represent three independent experiments .

(B) Lysates from HEK293T cells expressing HA-Arp2 alone or together with Flag-WDR63 were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments.


( C ) Lysates from HEK293T cells expressing GFP - WDR63 alone or together with Flag - Arp2 were immunoprecipitated by anti - Flag antibody , followed by western blot analysis . Data shown represent three independent experiments .

(C) Lysates from HEK293T cells expressing GFP-WDR63 alone or together with Flag-Arp2 were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments.


( D ) Lysates from A549 cells were immunoprecipitated with control IgG or anti - WDR63 antibody , followed by western blot analysis . Data shown represent three independent experiments .

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

(D) Lysates from A549 cells were immunoprecipitated with control IgG or anti-WDR63 antibody, followed by western blot analysis. Data shown represent three independent experiments.


( E ) Purified GST or GST - Arp2 proteins immobilized on glutathione beads were incubated with purified Flag - WDR63 . Input and bead - bound proteins were analyzed by western blot . Data shown represent three independent experiments .

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

(E) Purified GST or GST-Arp2 proteins immobilized on glutathione beads were incubated with purified Flag-WDR63. Input and bead-bound proteins were analyzed by western blot. Data shown represent three independent experiments.


( F ) Purified GST or GST - WDR63 proteins immobilized on glutathione beads were incubated with purified Arp2 . Input and bead - bound proteins were analyzed by western blot . Data shown represent three independent experiments .

(F) Purified GST or GST-WDR63 proteins immobilized on glutathione beads were incubated with purified Arp2. Input and bead-bound proteins were analyzed by western blot. Data shown represent three independent experiments.


( G ) Lysates from HEK293T cells expressing GFP - WDR63 alone or together with the indicated Flag - tagged each single subunit of the Arp2 / 3 complex were immunoprecipitated with anti - Flag antibody , followed by western blot analysis . Data shown represent three independent experiments .

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

(G) Lysates from HEK293T cells expressing GFP-WDR63 alone or together with the indicated Flag-tagged each single subunit of the Arp2/3 complex were immunoprecipitated with anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments.


( H ) Purified Flag - tagged full - length or the indicated truncated WDR63 proteins immobilized on anti - Flag M2 beads were incubated with the purified Arp2 / 3 complex ( Cytoskeleton , Inc . ) in vitro . Input and beads - bound proteins were analyzed by western blot . Data shown represent three independent experiments .

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

(H) Purified Flag-tagged full-length or the indicated truncated WDR63 proteins immobilized on anti-Flag M2 beads were incubated with the purified Arp2/3 complex (Cytoskeleton, Inc.) in vitro. Input and beads-bound proteins were analyzed by western blot. Data shown represent three independent experiments.


( A ) Immunolocalization of Arp2 in a wound - healing assay . A549 cells expressing control or WDR63 were serum - starved for 6 h before they were scratched . Cells were then treated with or without 10 % FBS for 30 min , followed by immunofluorescence assay . The branched actin networks were shown by immunostaining with anti - Cortactin antibody . Data shown represent three independent experiments . Scale bar : 20 μm . The lamellipodia formation was quantified by measuring the length of the outer margin of lamellipodia in individual cell ( n = 20 for each condition ) , and expressed as the proportion of the total length of the cell perimeter . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; One - way ANOVA .

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

(A) Immunolocalization of Arp2 in a wound-healing assay. A549 cells expressing control or WDR63 were serum-starved for 6 h before they were scratched. Cells were then treated with or without 10% FBS for 30 min, followed by immunofluorescence assay. The branched actin networks were shown by immunostaining with anti-Cortactin antibody. Data shown represent three independent experiments. Scale bar: 20 μm. The lamellipodia formation was quantified by measuring the length of the outer margin of lamellipodia in individual cell (n=20 for each condition), and expressed as the proportion of the total length of the cell perimeter. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; One-way ANOVA.


( B and C ) Effects of full - length WDR63 ( B ) and WDR63 ( 1 - 200 ) ( C ) on Arp2 / 3 - mediated actin polymerization were examined by pyrene actin polymerization assays . Various concentrations of WDR63 ( B ) or WDR63 ( 1 - 200 ) ( C ) were incubated with 2 μM actin ( 50 % pyrene - labeled ) , 10 nM Arp2 / 3 and 400 nM GST - VCA as indicated . Data shown represent three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C) Effects of full-length WDR63 (B) and WDR63 (1-200) (C) on Arp2/3-mediated actin polymerization were examined by pyrene actin polymerization assays. Various concentrations of WDR63 (B) or WDR63 (1-200) (C) were incubated with 2 μM actin (50% pyrene-labeled), 10 nM Arp2/3 and 400 nM GST-VCA as indicated. Data shown represent three independent experiments.


( D ) Effect of WDR63 on Arp2 / 3 - mediated branched actin polymerization was analyzed by electron microscopy . 0 . 6 μM WDR63 was incubated with 2 μM actin , 10 nM Arp2 / 3 and 400 nM GST - VCA as indicated . Actin structures were then stabilized with addition of 2μM phalloidin , followed by electron microscopy analysis . The arrows indicate actin branch junctions . The percentage of branched actin filaments was expressed as the ratio of the number of actin filaments with branch junctions to the total numbers of actin filaments . For each condition , over 150 actin filaments were counted . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test . Scale bar : 200 nm .

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

(D) Effect of WDR63 on Arp2/3-mediated branched actin polymerization was analyzed by electron microscopy. 0.6 μM WDR63 was incubated with 2 μM actin, 10 nM Arp2/3 and 400 nM GST-VCA as indicated. Actin structures were then stabilized with addition of 2μM phalloidin, followed by electron microscopy analysis. The arrows indicate actin branch junctions. The percentage of branched actin filaments was expressed as the ratio of the number of actin filaments with branch junctions to the total numbers of actin filaments. For each condition, over 150 actin filaments were counted. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test. Scale bar: 200 nm.


( E and F ) Increasing amounts of purified Flag - WDR63 ( 0 . 1 μM , 0 . 2 μM , and 0 . 4 μM ) ( E ) or Flag - WDR63 ( 1 - 200 ) ( 0 . 25 μM , 0 . 5 μM , and 1 μM ) ( F ) was incubated with the Arp2 / 3 complex ( 10 nM ) and GST - VCA as indicated in vitro , followed by GST pull - down assay . Data shown represent three independent experiments .

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

(E and F) Increasing amounts of purified Flag-WDR63 (0.1 μM, 0.2 μM, and 0.4 μM) (E) or Flag-WDR63 (1-200) (0.25 μM, 0.5 μM, and 1 μM) (F) was incubated with the Arp2/3 complex (10 nM) and GST-VCA as indicated in vitro, followed by GST pull-down assay. Data shown represent three independent experiments.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA were subjected to wound - healing ( A ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no significance ; One - way ANOVA . Scale bar in ( A ) : 200 μm .

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

A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bar in (A): 200 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA Lysates from these cells were also analyzed by western blot ( B ) . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no

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

A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA Lysates from these cells were also analyzed by western blot (B). Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance


A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA were subjected to transwell migration ( C ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no significance ; One - way ANOVA . Scale bars in ( C : 100 μm .

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

A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to transwell migration (C) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bars in (C : 100 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA were subjected to transwell invasion ( D ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no significance ; One - way ANOVA . Scale bars 100 μm .

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

A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to transwell invasion (D) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bars 100 μm.


A549 cells expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell migration ( E ) The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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

A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell migration (E) The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


A549 cells expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Lysates from these cells were also analyzed by western blot ( F ) . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA .

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

A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Lysates from these cells were also analyzed by western blot (F). Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA.


A549 cells expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell invasion ( G ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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

A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell invasion (G) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to wound - healing ( A ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar in ( A ) : 200 μm .

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

A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar in (A): 200 μm.


A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 Lysates from these cells were also analyzed by western blot ( B ) .

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

A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 Lysates from these cells were also analyzed by western blot (B).


A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to transwell migration ( C ) The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bars 100 μm .

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

A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to transwell migration (C) The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bars 100 μm.


A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to transwell invasion ( D ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bars 100 μm .

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

A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to transwell invasion (D) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bars 100 μm.


A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell migration ( E ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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

A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell migration (E) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell invasion ( F ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell invasion (F) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


( G ) A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 ( each also expressing luciferase ) were injected via tail vein into nude mice ( n = 6 for each group ) . Three weeks after injection , tumor formation was monitored by bioluminescence imaging . Data shown are mean ± SD ; * , P ˂ 0 . 05 ; One - way ANOVA .

(G) A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 (each also expressing luciferase) were injected via tail vein into nude mice (n=6 for each group). Three weeks after injection, tumor formation was monitored by bioluminescence imaging. Data shown are mean ± SD; *, P ˂ 0.05; One-way ANOVA.


( 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 .

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


D458 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 .

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

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


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

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 .

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

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


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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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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). 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). 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). 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 .

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


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

(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 .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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