( E ) Western blot ( inset ) and quantification ( bar graph ) of Mili protein abundance in lysates from cultured mouse hippocampal neurons and undifferentiated aNPCs . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by two - tailed Student ' s t - test ( in E ) .

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(E) Western blot (inset) and quantification (bar graph) of Mili protein abundance in lysates from cultured mouse hippocampal neurons and undifferentiated aNPCs. Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by two-tailed Student's t-test (in E).


( F ) Representative immunofluorescence micrograph of Mili ( white ) and Td + NSCs ( red ) in hippocampal subgranular zone ( SGZ ) ; arrows indicate Td + Mili + double - positive cells . Data information : In F : GCL , granular cell layer ; H , Hilus . The scale bars represent 10 µm .

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(F) Representative immunofluorescence micrograph of Mili (white) and Td+ NSCs (red) in hippocampal subgranular zone (SGZ); arrows indicate Td+ Mili + double-positive cells. Data information: In F: GCL, granular cell layer; H, Hilus. The scale bars represent 10 µm.


( G ) Scheme of the experiment ( top ) and Mili mRNA expression in sorted Td + and Td - cells after in vivo transduction with split - Cre viruses in the hippocampus ( bottom ) . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by one - way ANOVA with Bonferroni test G )

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(G) Scheme of the experiment (top) and Mili mRNA expression in sorted Td+ and Td- cells after in vivo transduction with split-Cre viruses in the hippocampus (bottom). Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test G)


( E ) Mili mRNA expression ( left bar graph ) ; western blot ( inset ) and quantification ( right bar graph ) of Mili protein abundance in aNPCs upon viral transduction of scrambled shRNA ( Control ) or shRNA targeting Mili ( Mili KD ) . Data information : data are expressed as mean ± SEM unless differently indicated , n = 3 biological replicates ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test .

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

(E) Mili mRNA expression (left bar graph); western blot (inset) and quantification (right bar graph) of Mili protein abundance in aNPCs upon viral transduction of scrambled shRNA (Control) or shRNA targeting Mili (Mili KD). Data information: data are expressed as mean ± SEM unless differently indicated, n = 3 biological replicates ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test.


Western blot ( G ) of the endogenous piCS after co - immunoprecipitation ( IP ) with endogenous Mili ( IP Mili ) , or control IgG ( IP IgG ) in lysates of DIF4 aNPCs .

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Western blot (G) of the endogenous piCS after co-immunoprecipitation (IP) with endogenous Mili (IP Mili), or control IgG (IP IgG) in lysates of DIF4 aNPCs.


( C ) Mili mRNA expression ( left bar graph ) ; western blot ( inset ) and quantification ( right bar graph ) of Mili protein abundance in lysates from the DG of mouse hippocampi 48 hours after the injection of scrambled ( Control ) or GapmeR1 against Mili ( Mili KD ) . Data information : data are expressed as mean ± SEM , n = 3 ( C , biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test .

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

(C) Mili mRNA expression (left bar graph); western blot (inset) and quantification (right bar graph) of Mili protein abundance in lysates from the DG of mouse hippocampi 48 hours after the injection of scrambled (Control) or GapmeR1 against Mili (Mili KD). Data information: data are expressed as mean ± SEM, n = 3 (C, biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test.


( E ) Representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP at 30 dpi of scrambled ( Control ) and GapmeR1 against Mili ( Mili KD ) ; Right panels : Fold change in GFAP fluorescence intensity level ( upper graph ) in a hippocampal region of interest ( ROI ) of 500µm2 in brain slices upon Mili KD compared to Control ; Gfap mRNA levels ( lower graph ) in the DG from mouse hippocampi 48 hours after the injection of scrambled ( Control ) or GapmeR1 ( Mili KD ) . Data information : data are expressed as mean ± SEM , n = 3 E mRNA and 5 ( E biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . SGZ , subgranular zone . The scale bars represent 100 µm ( E ,

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

(E) Representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP at 30 dpi of scrambled (Control) and GapmeR1 against Mili (Mili KD); Right panels: Fold change in GFAP fluorescence intensity level (upper graph) in a hippocampal region of interest (ROI) of 500µm2 in brain slices upon Mili KD compared to Control; Gfap mRNA levels (lower graph) in the DG from mouse hippocampi 48 hours after the injection of scrambled (Control) or GapmeR1 (Mili KD). Data information: data are expressed as mean ± SEM, n = 3 E mRNA and 5 (E biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. SGZ, subgranular zone. The scale bars represent 100 µm (E,


( F ) ( Left ) representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP ( green ) , BrdU ( red ) , NeuN ( white ) and nuclear DNA ( blue ) at 30 dpi of scrambled ( Control ) or GapmeR1 against Mili ( Mili KD ) ; ( right graphs ) percentages of NeuN + BrdU + ( white arrowheads in the images ) , or GFAP + BrdU + ( yellow arrowheads in the images ) double - positive cells over total BrdU + cells . Data information : data are expressed as mean ± SEM , n = F ) biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . GCL , granular cell layer , SGZ , subgranular zone . The scale bars represent 100 µm F ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (Left) representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP (green), BrdU (red), NeuN (white) and nuclear DNA (blue) at 30 dpi of scrambled (Control) or GapmeR1 against Mili (Mili KD); (right graphs) percentages of NeuN+BrdU+ (white arrowheads in the images), or GFAP+BrdU+ (yellow arrowheads in the images) double-positive cells over total BrdU+ cells. Data information: data are expressed as mean ± SEM, n = F) biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. GCL, granular cell layer, SGZ, subgranular zone. The scale bars represent 100 µm F).


( B ) Representative bright - field microscopy images ( left ) and quantification ( right ) of ß - galactosidase + aNPCs as percent of total cells upon Mili KD , or control 48 h after induction of spontaneous differentiation . Data information : data are expressed as mean ± SEM , n = 5 ( B biological replicates p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . The scale bars represent 50 µm ( B ,

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

(B) Representative bright-field microscopy images (left) and quantification (right) of ß-galactosidase+ aNPCs as percent of total cells upon Mili KD, or control 48 h after induction of spontaneous differentiation. Data information: data are expressed as mean ± SEM, n = 5 (B biological replicates p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. The scale bars represent 50 µm (B,


( C ) Representative fluorescence microscopy images ( left ) and quantification ( right ) of control or Mili KD neuroblasts 48h after spontaneous differentiation , immunostained with anti - BrdU ( white ) and Ki67 ( purple ) antibodies . ( Right ) Percentage of BrdU + and Ki67 - cells over BrdU + cells . Data information : data are expressed as mean ± SEM , n = C ) biological replicates p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . The scale bars represent 50 µm C )

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 B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative fluorescence microscopy images (left) and quantification (right) of control or Mili KD neuroblasts 48h after spontaneous differentiation, immunostained with anti-BrdU (white) and Ki67 (purple) antibodies. (Right) Percentage of BrdU+ and Ki67- cells over BrdU+ cells. Data information: data are expressed as mean ± SEM, n = C) biological replicates p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. The scale bars represent 50 µm C)


F , Chow fed hIGFREO had similar body mass to WT , however , hIGFREO did not gain as much weight as WT after 8 weeks of HFD ( n = 6 - 10 mice per group ) .

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F, Chow fed hIGFREO had similar body mass to WT, however, hIGFREO did not gain as much weight as WT after 8 weeks of HFD (n=6-10 mice per group).


G , Representative images of difference in fat and water distribution shown by magnetic resonance ( MR ) imaging in hIGFREO and WT . Scale bar = 1cm .

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G, Representative images of difference in fat and water distribution shown by magnetic resonance (MR) imaging in hIGFREO and WT. Scale bar = 1cm.


hIGFREO glucose intolerance measured by glucose tolerance test ( n = 5 - 7 mice per group ) .

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hIGFREO glucose intolerance measured by glucose tolerance test (n=5-7 mice per group).


G , H , Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve ( n = 10 - 12 mice per group ) .

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G, H, Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve (n=10-12 mice per group).


hIGFREO , food consumption using indirect calorimeter assessment after HFD compared to wildtype littermates ( WT ) after HFD . ( n = 4 per genotype ) .

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hIGFREO , food consumption using indirect calorimeter assessment after HFD compared to wildtype littermates (WT) after HFD. (n=4 per genotype).


D , Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT ( n = 6 per genotype ) .

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D, Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT (n= 6 per genotype).


Faith ' s phylogenetic diversity ( PD ) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF - 1R endothelial overexpressing mice ( hIGFREO ) mice and wildtype littermates ( WT ) mice after high fat diet feeding ( n = 4 - 5 mice per group ) . Chao - 1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT ( n = 4 - 5 mice per group ) .

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Faith's phylogenetic diversity (PD) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF-1R endothelial overexpressing mice (hIGFREO) mice and wildtype littermates (WT) mice after high fat diet feeding (n=4-5 mice per group). Chao-1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT (n=4-5 mice per group).


B , Bi , human IGF - 1R endothelial overexpressing mice ( hIGFREO ) had comparable weight gain as wildtype littermates ( WT ) after 8 weeks of HFD + antibiotics ( ABs ) when compared to WT . Bii , both hIGFREO and WT gained significant weight compared to chow fed mice ( n = 7 = 9 mice per group ) .

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B, Bi, human IGF-1R endothelial overexpressing mice (hIGFREO) had comparable weight gain as wildtype littermates (WT) after 8 weeks of HFD + antibiotics (ABs) when compared to WT. Bii, both hIGFREO and WT gained significant weight compared to chow fed mice (n=7=9 mice per group).


D , E , There was no difference in hIGFREO and WT glucose tolerance ( as measured by glucose tolerance test and area under the curve ( AUC ) ) ( n = 7 - 9 mice per group ) .

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D, E, There was no difference in hIGFREO and WT glucose tolerance (as measured by glucose tolerance test and area under the curve (AUC)) (n=7-9 mice per group).


Characteristic micrographs for both data sets , respectively ( left and right ) .

Characteristic micrographs for both data sets, respectively (left and right).


g - h ) Representative β - catenin ChIP - qPCR of indicated target genes in wt and TCF1 / LEF1 long isoform double KO ( DLKO ) clones ( g ) and Kaiso KO clones ( h ) in RPMI8402 cells . Enrichment of each ChIP is normalized to its respective negative IgG control , indicated by a red - dashed line . DLKO1 , DLKO2 , KKO1 and KKO2 designate independent single - cell derived clones . Graph represents the mean and sd of 3 technical replicates .

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

g-h) Representative β-catenin ChIP-qPCR of indicated target genes in wt and TCF1/LEF1 long isoform double KO (DLKO) clones (g) and Kaiso KO clones (h) in RPMI8402 cells. Enrichment of each ChIP is normalized to its respective negative IgG control, indicated by a red-dashed line. DLKO1, DLKO2, KKO1 and KKO2 designate independent single-cell derived clones. Graph represents the mean and sd of 3 technical replicates.


e ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell line ( right panel ) after knockdown of β - catenin ( left panel ) compared to control . Graph represents the mean and sd of 6 independent experiments . f ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell line after treatment with the β - catenin inhibitor ICG - 001 ( 10µM , overnight ) . Graph represents the mean and sd of 3 independent experiments . g ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell lines ( right panel ) after knockdown of ZBTB33 / Kaiso ( left panel ) compared to control . Graph represents the mean and sd of 3 independent experiments . Data information : Statistical significance was determined by two - sided Student ' s t test .

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

e) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell line (right panel) after knockdown of β-catenin (left panel) compared to control. Graph represents the mean and sd of 6 independent experiments. f) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell line after treatment with the β-catenin inhibitor ICG-001 (10µM, overnight). Graph represents the mean and sd of 3 independent experiments. g) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell lines (right panel) after knockdown of ZBTB33/Kaiso (left panel) compared to control. Graph represents the mean and sd of 3 independent experiments. Data information: Statistical significance was determined by two-sided Student's t test.


c ) Kaplan - Meier curves representing disease free ( DSF , top panel ) and overall survival ( OS , bottom panel ) probability for groups of patients P1 ( N = 7 ) , P2 ( N = 5 ) , P3 ( N = 4 ) , P4 ( N = 9 ) and P5 ( N = 15 ) obtained in a ) . Time is represented in years . Statistical significance among groups was determined by log - rank test .

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c) Kaplan-Meier curves representing disease free (DSF, top panel) and overall survival (OS, bottom panel) probability for groups of patients P1 (N = 7), P2 (N = 5), P3 (N = 4), P4 (N = 9) and P5 (N = 15) obtained in a). Time is represented in years. Statistical significance among groups was determined by log-rank test.


a ) Effect of Vincristine ( VCR ) treatment on cell viability of RPMI8402 cells transduced with sh β - catenin or sh - control . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * p ≤ 0 . 001 of IC50 comparison with respect to sh control Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for a ( at day 0 )

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of Vincristine (VCR) treatment on cell viability of RPMI8402 cells transduced with sh β-catenin or sh-control. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. *** p ≤ 0.001 of IC50 comparison with respect to sh control Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for a (at day 0)


Effect of VCR ( b ) treatment in the presence or absence of increasing doses of the β - catenin inhibitor ICG - 001 . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 . 05 of IC50 comparison with respect VCR - treated cells . Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for , b , ( at day 0 )

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

Effect of VCR (b) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 comparison with respect VCR-treated cells. Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for , b, (at day 0)


Methotrexate ( MTX ) ( c left ) , L - Asparaginase ( L - ASP ) ( c central ) or Cytarabine ( Ara - C ) ( c right ) treatment in the presence or absence of increasing doses of the β - catenin inhibitor ICG - 001 . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 . 05 of IC50 Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for c , ( at day 0 )

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

Methotrexate (MTX) (c left), L-Asparaginase (L-ASP) (c central) or Cytarabine (Ara-C) (c right) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for c, (at day 0)


c ) Percentage of RPMI8402 cells PB , BM , spleen and liver at sacrifice . RPMI8402 cells identified as human CD45 + HLA - ABC + CD5 + by flow cytometry and gated on DAPI negative cells . Control : animals left untreated ( n = 2 ) ; Chemo + DMSO : animals receiving chemotherapy and vehicle ( n = 4 ) ; Chemo + ICG - 001 : animals receiving chemotherapy and the β - catenin inhibitor ICG - 001 ( n = 4 ) Data information : For all applicable figure panels , data from Chemo + DMSO and Chemo + ICG - 001 groups are mean ± SD and dots represent individual values per animal . Control group data is only represented by individual values per animal . Statistical significance between Chemo + DMSO and Chemo + ICG - 001 groups was determined by 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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

c) Percentage of RPMI8402 cells PB, BM, spleen and liver at sacrifice. RPMI8402 cells identified as human CD45+HLA-ABC+CD5+ by flow cytometry and gated on DAPI negative cells. Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.


g ) H & E staining of representative images per each mouse treated as indicated . h ) Percentage of liver area covered by cellular colonies per 10X micrograph . 4 different images were quantified per animal and mean values per animal are represented by dots . Control : animals left untreated ( n = 2 ) ; Chemo + DMSO : animals receiving chemotherapy and vehicle ( n = 4 ) ; Chemo + ICG - 001 : animals receiving chemotherapy and the β - catenin inhibitor ICG - 001 ( n = 4 ) Data information : For all applicable figure panels , data from Chemo + DMSO and Chemo + ICG - 001 groups are mean ± SD and dots represent individual values per animal . Control group data is only represented by individual values per animal . Statistical significance between Chemo + DMSO and Chemo + ICG - 001 groups was determined by 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 O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

g) H&E staining of representative images per each mouse treated as indicated. h) Percentage of liver area covered by cellular colonies per 10X micrograph. 4 different images were quantified per animal and mean values per animal are represented by dots. Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.


Representative micrographs of a time - lapse microscopy of K and KM non - regressed tumour cells ( H2B - GFP green ) . Top : Mitotic cell with a chromatin bridge ( yellow arrow ) . Bottom : Mitotic cell with misalignment and cytokinesis failure resulting in binucleation ( yellow arrow ) .

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Representative micrographs of a time-lapse microscopy of K and KM non-regressed tumour cells (H2B-GFP green). Top: Mitotic cell with a chromatin bridge (yellow arrow). Bottom: Mitotic cell with misalignment and cytokinesis failure resulting in binucleation (yellow arrow).


Percentage of mitotic errors in K and KM primary tumours and in K and KM non - regressed tumours . Percentage of cells in K and KM primary tumours and in K and KM non - regressed tumours with the indicated mitotic errors . Scale bar 20 µm .

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Percentage of mitotic errors in K and KM primary tumours and in K and KM non-regressed tumours. Percentage of cells in K and KM primary tumours and in K and KM non-regressed tumours with the indicated mitotic errors. Scale bar 20 µm.


Somatic copy number alterations in each individual Kras and Kras / Mad2 non - regressed tumours . 7 K non - regressed and 16 KM non - regressed tumours were sequenced . Focal deletion ( DEL , green ) , Focal amplification ( AMP , light green ) , whole chromosome gain ( WCG , dark grey ) and loss ( WCL , light grey ) , partial chromosome gain ( PCG , dark blue ) and loss ( PCL , light blue ) and gross chromosomal rearrangement ( GCR , red ) .

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Somatic copy number alterations in each individual Kras and Kras/Mad2 non-regressed tumours. 7 K non-regressed and 16 KM non-regressed tumours were sequenced. Focal deletion (DEL, green), Focal amplification (AMP, light green), whole chromosome gain (WCG, dark grey) and loss (WCL, light grey), partial chromosome gain (PCG, dark blue) and loss (PCL, light blue) and gross chromosomal rearrangement (GCR, red).


Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene . Colour of the block corresponds to the segment mean ( the degree to which a genome segment is lost ( blue ) or gained ( red ) .

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Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene. Colour of the block corresponds to the segment mean (the degree to which a genome segment is lost (blue) or gained (red).


Immunostaining of phospho - cMet in non - regressed tumours carrying or not the amplification in chromosome 6 . Yellow numbers indicate the total number of cMet positive cells / total number of cells counted . Scale bar 100 µm .

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Immunostaining of phospho-cMet in non-regressed tumours carrying or not the amplification in chromosome 6. Yellow numbers indicate the total number of cMet positive cells/total number of cells counted. Scale bar 100 µm.


Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification . 0 indicates when doxycycline was removed . Each colour represents one tumour . Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth .

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Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification. 0 indicates when doxycycline was removed. Each colour represents one tumour. Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth.


Genome - wide log2 - ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non - regressed tumour showing no amplification in the primary tumour ( upper panels ) and a small amplification in the non - regressed tumours ( bottom panels , yellow arrow ) .

Genome-wide log2-ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non-regressed tumour showing no amplification in the primary tumour (upper panels) and a small amplification in the non-regressed tumours (bottom panels, yellow arrow).


Immunostaining of phospho - cMet in 3 biopsied primary tumours ( PT ) and their corresponding non - regressed tumours ( KM5 , KM7 and KM8 , which are also shown in Fig 2C ) . Scale bar 100 µm .

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Immunostaining of phospho-cMet in 3 biopsied primary tumours (PT) and their corresponding non-regressed tumours (KM5, KM7 and KM8, which are also shown in Fig 2C). Scale bar 100 µm.


Representative two - dimensional scatter plots constructed with overlaid dPCR data of the reference ( VIC ) and cMet ( FAM ) from one tumour without cMet amplification and one with cMet amplified . Dots represent results of independent PCR reactions in the wells of a digital PCR chip . Reactions in the bottom left corner ( yellow ) are negative for both targets , while the ones in the top right corner ( green ) are double positives . Reactions in the top left ( blue ) and bottom right ( red ) corners are positive for cMet and the reference targets , respectively .

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Representative two-dimensional scatter plots constructed with overlaid dPCR data of the reference (VIC) and cMet (FAM) from one tumour without cMet amplification and one with cMet amplified. Dots represent results of independent PCR reactions in the wells of a digital PCR chip. Reactions in the bottom left corner (yellow) are negative for both targets, while the ones in the top right corner (green) are double positives. Reactions in the top left (blue) and bottom right (red) corners are positive for cMet and the reference targets, respectively.


Representative photographs of FISH staining with a probe for Met ( red signal ) and a probe for a reference gene EML4 ( green signals ) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots ( white arrows ) . The lower panel shows an example of a tumour with cMet amplification ( several red dots ) and 2 green dots ( yellow arrow ) . Scale bar 10 µm .

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Representative photographs of FISH staining with a probe for Met (red signal) and a probe for a reference gene EML4 (green signals) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots (white arrows). The lower panel shows an example of a tumour with cMet amplification (several red dots) and 2 green dots (yellow arrow). Scale bar 10 µm.


Relative volume of tumours grown in 12 Rag2 - / - animals after treatment with Tepotinib or vehicle control ( 3K and 3KM tumours for each condition ) . No statistical significance was found by One - way ANOVA . Mean + / - SEM .

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Relative volume of tumours grown in 12 Rag2-/- animals after treatment with Tepotinib or vehicle control (3K and 3KM tumours for each condition). No statistical significance was found by One-way ANOVA. Mean +/- SEM.


Relative volume of tumours grown in Rag2 - / - animals after treatment with Tepotinib or vehicle control . One - way ANOVA , Tukey ' s multiple comparison . Ns : not significant ; * * p < 0 . 024 . Mean + / - SEM . A total of 19 Rag2 - / - mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls . For tumours with no cMet , 13 Rag2 - / - mice were treated with Tepotinib and 9 were used as control .

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Relative volume of tumours grown in Rag2-/- animals after treatment with Tepotinib or vehicle control. One-way ANOVA, Tukey's multiple comparison. Ns: not significant; ** p<0.024. Mean +/- SEM. A total of 19 Rag2-/- mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls. For tumours with no cMet, 13 Rag2-/- mice were treated with Tepotinib and 9 were used as control.


Immunostaining of phospho - cMet in the same tumours grown in Rag2 - / - mice after treatment with Tepotinib or vehicle control . Scale bar 200 µm .

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Immunostaining of phospho-cMet in the same tumours grown in Rag2-/- mice after treatment with Tepotinib or vehicle control. Scale bar 200 µm.


Quantification of pH3 in tumours grown in Rag2 - / - animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control .

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Quantification of pH3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.


Quantification of caspase 3 in tumours grown in Rag2 - / - animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control .

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Quantification of caspase 3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.


A , B Representative size - exclusion chromatography profiles of the MICU1 - MICU2 complex and chimaera in 2 mM EGTA ( A ) and 2 mM CaCl2 ( B ) conditions .

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A, B Representative size-exclusion chromatography profiles of the MICU1-MICU2 complex and chimaera in 2 mM EGTA (A) and 2 mM CaCl2 (B) conditions.


A The binding of fluorescently ( GFP ) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA . B The binding of fluorescently ( GFP ) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2 + .

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A The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA. B The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2+.


C Comparison of binding of MICU1 and EMRE peptide in both conditions .

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C Comparison of binding of MICU1 and EMRE peptide in both conditions.


D MICU1 - MICU2 heterodimer interacts with the EMRE peptide at a 1 : 1 ratio .

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D MICU1-MICU2 heterodimer interacts with the EMRE peptide at a 1:1 ratio.


E Carbachol ( CCh ) - induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt - EMRE and scramble peptides . Solid lines , mean ; shaded regions , ± SEM , n = 3 .

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E Carbachol (CCh)-induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt-EMRE and scramble peptides. Solid lines, mean; shaded regions, ± SEM, n = 3.


F Resting mitochondrial Ca2 + levels in mt - EMRE and scramble peptides treated cells , evaluated via ratiometric imaging of the mitochondrial targeted mt - riG6m . Mean ± s . d . , student t - test , * * p < 0 . 01 , n = 3 . Data information : Error bars for some points are too small to be visible .

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F Resting mitochondrial Ca2+ levels in mt-EMRE and scramble peptides treated cells, evaluated via ratiometric imaging of the mitochondrial targeted mt-riG6m. Mean ± s.d., student t-test, **p < 0.01, n = 3. Data information: Error bars for some points are too small to be visible.


G Mean mitochondrial Ca2 + peaks response to 100 μM CCh . Mean ± s . d . , student t - test , * * * p ≤ 0 . 001 , n = 3 . Data information : Error bars for some points are too small to be visible .

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G Mean mitochondrial Ca2+ peaks response to 100 μM CCh. Mean ± s.d., student t-test, ***p ≤ 0.001, n = 3. Data information: Error bars for some points are too small to be visible.


H Representative [ Ca2 + ] c clearance traces of U2OS cells treated with PBS and mt - EMRE peptide .

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H Representative [Ca2+]c clearance traces of U2OS cells treated with PBS and mt-EMRE peptide.


I Quantification of mitochondria Ca2 + uptake rate in response to 100 μM histamine . Mean ± s . d . , student t - test , n . s . , not significant , n = 4 . Data information : Error bars for some points are too small to be visible .

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I Quantification of mitochondria Ca2+ uptake rate in response to 100 μM histamine. Mean ± s.d., student t-test, n.s., not significant, n = 4. Data information: Error bars for some points are too small to be visible.


5C or mES medium was used during reprogramming . On day 15 , Oct4GFP + colonies were counted ( A ) .

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5C or mES medium was used during reprogramming. On day 15, Oct4GFP+ colonies were counted (A).


5C or mES medium was used during reprogramming . The percentages of Oct4GFP + cells were also determined with FACS ( B ) .

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5C or mES medium was used during reprogramming. The percentages of Oct4GFP+ cells were also determined with FACS (B).


( C ) The expression of several core pluripotency markers was determined with qPCR at different time points during reprogramming and normalized against those in MEFs .

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(C) The expression of several core pluripotency markers was determined with qPCR at different time points during reprogramming and normalized against those in MEFs.


( D - E ) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument . Increase in extracellular acidification rate ( ECAR ) after adding glucose was considered as glycolysis ability of the cells ( D ) , while the decrease in oxygen consumption rate ( OCR ) was considered as the ATP production ability of the cells ( E ) .

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(D-E) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument. Increase in extracellular acidification rate (ECAR) after adding glucose was considered as glycolysis ability of the cells (D), while the decrease in oxygen consumption rate (OCR) was considered as the ATP production ability of the cells (E).


( F - G ) Expression of Hif1α was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming ( F ) . The numbers of Oct4GFP + colonies were determined on day 15 ( G ) .

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(F-G) Expression of Hif1α was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming (F). The numbers of Oct4GFP+ colonies were determined on day 15 (G).


( H - I ) Expression of Pdk1 / 2 was modulated with a retrovirus system , and oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) were used during reprogramming ( H ) . The numbers of Oct4GFP + colonies were determined on day 15 ( I ) .

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(H-I) Expression of Pdk1/2 was modulated with a retrovirus system, and oligomycin (1 µM) and 2-DG (5 mM) were used during reprogramming (H). The numbers of Oct4GFP+ colonies were determined on day 15 (I).


( A - B ) 5C and mES medium were used during reprogramming . Representative alkaline phosphatase ( AP ) staining on day 15 was provided in ( A ) . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 ( B ) .

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(A-B) 5C and mES medium were used during reprogramming. Representative alkaline phosphatase (AP) staining on day 15 was provided in (A). AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (B).


( C ) Thirty - two epigenetic factors were selected because they had higher expression in 5C - Oct4GFP - than in mES - Oct4GFP - . Their expression in ESCs , iPSCs , and pre - iPSCs in two previous reported assay ( GSE14012 and GSE10871 ) was also listed .

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(C) Thirty-two epigenetic factors were selected because they had higher expression in 5C-Oct4GFP- than in mES-Oct4GFP-. Their expression in ESCs, iPSCs, and pre-iPSCs in two previous reported assay (GSE14012 and GSE10871) was also listed.


( D ) The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , or Wdr5 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 .

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(D) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15.


During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The expression of pluripotency markers was determined with qPCR on day 6 during reprogramming ( E ) .

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During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The expression of pluripotency markers was determined with qPCR on day 6 during reprogramming (E).


During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The numbers of Oct4GFP + colonies were determined on day 15 ( F ) .

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

During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The numbers of Oct4GFP+ colonies were determined on day 15 (F).


During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The histone methylation on core pluripotency loci was determined on day 6 with ChIP - qPCR ( G ) .

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

During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The histone methylation on core pluripotency loci was determined on day 6 with ChIP-qPCR (G).


( A ) The enrichment of HIF1α , SNAI2 , TWIST1 / 2 , and ZEB1 binding sites on the promoters of Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 was determined by Pscan Zambelli et al , 2009 ( ) . The resulted Z - scores were listed . " Max . M " suggested the maximum Z - score generated with SNAI2 , TWIST1 / 2 , and ZEB1 binding sites on one indicated promoter . " Average " suggested the average Z - score of all transcriptional factors tested in the Pscan software on one indicated promoter .

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

(A) The enrichment of HIF1α, SNAI2, TWIST1/2, and ZEB1 binding sites on the promoters of Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 was determined by Pscan Zambelli et al, 2009(). The resulted Z-scores were listed. "Max. M" suggested the maximum Z-score generated with SNAI2, TWIST1/2, and ZEB1 binding sites on one indicated promoter. "Average" suggested the average Z-score of all transcriptional factors tested in the Pscan software on one indicated promoter.


The expression of H1f1α , Snai2 , Twist1 / 2 , and Zeb1 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming with mES ( B ) The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs . The comparisons were performed between all groups and corresponding Flag groups .

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

The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with mES (B) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups.


The expression of H1f1α , Snai2 , Twist1 / 2 , and Zeb1 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming with 5C medium ( C ) . The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs . The comparisons were performed between all groups and corresponding Flag groups .

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

The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with 5C medium (C). The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups.


pre - iPSCs were isolated during reprogramming with mES - Vc , mES or 5C medium . These pre - iPSCs were further cultured with a different medium ( mES - Vc and mES medium ) for seven days . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . The numbers of Oct4GFP + colonies were determined on day 7 ( E ) .

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

pre-iPSCs were isolated during reprogramming with mES-Vc, mES or 5C medium. These pre-iPSCs were further cultured with a different medium (mES-Vc and mES medium) for seven days. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The numbers of Oct4GFP+ colonies were determined on day 7 (E).


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 ( G ) .

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

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (G).


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . In addition , energy metabolism was determined on day 6 with Seahorse

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

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. In addition, energy metabolism was determined on day 6 with Seahorse instrument


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . Cell migration was determined by measuring the migration rate with live - cell imaging on day 6 ( J ) .

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

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. Cell migration was determined by measuring the migration rate with live-cell imaging on day 6 (J).


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , or Wdr5 was determined on day 6 with qPCR ( K ) .

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

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was determined on day 6 with qPCR (K).


Expression of Hif1α was modulated via a retrovirus system or small - molecule compounds , oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) , during reprogramming . The expression of mesenchymal markers ( A )

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

Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers (A)


Expression of Hif1α was modulated via a retrovirus system or small - molecule compounds , oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) , during reprogramming . The expression of mesenchymal markers cell migration ( B ) was determined with qPCR and live - cell imaging , respectively , on day 6 .

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

Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers cell migration (B) was determined with qPCR and live-cell imaging, respectively, on day 6.


Repsox ( 1 μM ) was used on day 2 - 7 to inhibit early EMT during reprogramming with 5C medium . Except for OK + M + S group , the four Yamanaka factors were delivered simultaneously . Energy metabolism was determined on day 6 with Seahorse instrument ( E - F ) .

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

Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. Except for OK+M+S group, the four Yamanaka factors were delivered simultaneously. Energy metabolism was determined on day 6 with Seahorse instrument (E-F).


Repsox ( 1 μM ) was used on day 2 - 7 to inhibit early EMT during reprogramming with 5C medium . The expression of glycolysis markers was determined on day 6 with qPCR ( G ) .

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

Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. The expression of glycolysis markers was determined on day 6 with qPCR (G).


On day 15 during reprogramming , the expression of E - cadherin ( E - CAD ) and N - cadherin ( N - CAD ) , was determined with immunofluorescence together with the morphology in Oct4GFP + colonies ( A , Scale bar , 25 μM ) .

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

On day 15 during reprogramming, the expression of E-cadherin (E-CAD) and N-cadherin (N-CAD), was determined with immunofluorescence together with the morphology in Oct4GFP+ colonies (A, Scale bar, 25 μM).


After sorting Oct4GFP + cells out ( 5C - Oct4GFP + and mES - Oct4GFP + cells ) , the expression of markers of different pluripotent states ( 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

After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of different pluripotent states (B)


After sorting Oct4GFP + cells out ( 5C - Oct4GFP + and mES - Oct4GFP + cells ) , the expression of markers of Xist ( C )

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

After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of Xist (C)


After sorting Oct4GFP + cells out ( 5C - Oct4GFP + and mES - Oct4GFP + cells ) their abilities to form chimeras ( D ) were determined .

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

After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells) their abilities to form chimeras (D) were determined.


( F ) The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days , and the morphology changed towards R1 ESCs ( naïve PSCs ) and R1 ESCs - primed ( primed PSCs ) , respectively . The obtained cells were maintained for additional 20 passages . The 5C - Oct4GFP + cells cultured in primed medium lost GFP fluorescence . R1 ESCs - primed were primed PSCs generated from R1 ESCs . Scale bar , 100 μM .

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

(F) The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. The obtained cells were maintained for additional 20 passages. The 5C-Oct4GFP+ cells cultured in primed medium lost GFP fluorescence. R1 ESCs-primed were primed PSCs generated from R1 ESCs. Scale bar, 100 μM.


The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days , and the morphology changed towards R1 ESCs ( naïve PSCs ) and R1 ESCs - primed ( primed PSCs ) , respectively . ( G ) PSCs mentioned in ( F ) were used for RNA - Seq analysis . Similarity among gene expression profiles was determined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. (G) PSCs mentioned in (F) were used for RNA-Seq analysis. Similarity among gene expression profiles was determined.


The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days to generate 5C - Oct4GFP + ( naïve ) and 5C - Oct4GFP + ( primed ) cells . The expression of Xist ( H )

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

The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. The expression of Xist (H)


The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days to generate 5C - Oct4GFP + ( naïve ) and 5C - Oct4GFP + ( primed ) cells . the abilities to form chimeras ( I - J ) were determined .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. the abilities to form chimeras (I-J) were determined.


The expression Oct4GFP and E - cadherin was determined with FACS on day 15 during reprogramming ( A ) .

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

The expression Oct4GFP and E-cadherin was determined with FACS on day 15 during reprogramming (A).


Based on the FACS results , the expression of Oct4GFP and E - cadherin was summarized in Oct4GFP + E - cadherin + cells ( B ) and in Oct4GFP - E - cadherin - cells ( C ) .

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

Based on the FACS results, the expression of Oct4GFP and E-cadherin was summarized in Oct4GFP+E-cadherin+ cells (B) and in Oct4GFP-E-cadherin- cells (C).


The percentage of E - cadherin + cells in Oct4GFP + cells was listed in ( D ) , while the percentage of Oct4GFP + cells in E - cadherin + cells was listed in ( E ) .

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

The percentage of E-cadherin+ cells in Oct4GFP+ cells was listed in (D), while the percentage of Oct4GFP+ cells in E-cadherin+ cells was listed in (E).


( F - G ) Energy metabolism was analyzed in 5C - Oct4GFP + and mES - Oct4GFP + cells with Seahorse instrument .

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-G) Energy metabolism was analyzed in 5C-Oct4GFP+ and mES-Oct4GFP+ cells with Seahorse instrument.


The numbers of three types of Oct4GFP + colonies ( J )

The numbers of three types of Oct4GFP+ colonies (J)


the expression of four epithelial markers in Oct4GFP + cells ( K ) were determined on day 15 . " Average " in ( K ) suggested the average expression of four epithelia markers .

O O O O O O O O O O O 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 four epithelial markers in Oct4GFP+ cells (K) were determined on day 15. "Average" in (K) suggested the average expression of four epithelia markers.


( A - B ) Energy metabolism was analyzed with 5C - Oct4GFP + , R1 ESCs ( naïve PSCs ) , and R1 ESCs - primed ( primed PSCs ) with Seahorse instrument .

(A-B) Energy metabolism was analyzed with 5C-Oct4GFP+, R1 ESCs (naïve PSCs), and R1 ESCs-primed (primed PSCs) with Seahorse instrument.


( C ) Expression of epithelial , mesenchymal , glycolysis and oxidative phosphorylation ( OXPHOS ) markers ( listed in Dataset EV4 ) was summarized in R1 ESCs , 5C - Oct4GFP + ( naïve ) , 5C - Oct4GFP + , 5C - Oct4GFP + ( primed ) , and R1 ESCs - primed , based on the current RNA - Seq . For each marker , Log2 values of FKPM were normalized by subtracting the average of five groups . Log2 values of FKPM of each group of markers were then averaged and plotted . 5C - Oct4GFP + ( naïve ) and 5C - Oct4GFP + ( primed ) were 5C - Oct4GFP + cultured in naïve and primed medium , respectively , for three days .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of epithelial, mesenchymal, glycolysis and oxidative phosphorylation (OXPHOS) markers (listed in Dataset EV4) was summarized in R1 ESCs, 5C-Oct4GFP+ (naïve), 5C-Oct4GFP+, 5C-Oct4GFP+ (primed), and R1 ESCs-primed, based on the current RNA-Seq. For each marker, Log2 values of FKPM were normalized by subtracting the average of five groups. Log2 values of FKPM of each group of markers were then averaged and plotted. 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) were 5C-Oct4GFP+ cultured in naïve and primed medium, respectively, for three days.


( D ) Genes with higher expression at different developmental stages were selected out from previously reported RNA - seq ( Data ref : Bertone , 2015 ) . " Mor " , " ICM " , " pre - EPI " and " post - EPI " represented genes that have high expression only in morula , inner cell mass , pre - implantation epiblast and post - implantation epiblast , respectively . " Mor + ICM " , " ICM + pre " , and " EPI " indicated genes that have high expression in both morula and inner cell mass , in both inner cell mass and pre - implantation epiblast , and in both pre - implantation and post - implantation epiblast , respectively . The results of previously reported RNA - seq were summarized in the left panel , while the expression of these genes in current RNA - seq was summarized in the right panel . Log2 values of FKPM were presented similarly as in ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Genes with higher expression at different developmental stages were selected out from previously reported RNA-seq (Data ref: Bertone, 2015). "Mor", "ICM", "pre-EPI" and "post-EPI" represented genes that have high expression only in morula, inner cell mass, pre-implantation epiblast and post-implantation epiblast, respectively. "Mor+ICM", "ICM+pre", and "EPI" indicated genes that have high expression in both morula and inner cell mass, in both inner cell mass and pre-implantation epiblast, and in both pre-implantation and post-implantation epiblast, respectively. The results of previously reported RNA-seq were summarized in the left panel, while the expression of these genes in current RNA-seq was summarized in the right panel. Log2 values of FKPM were presented similarly as in (C).


( F ) Markers of 5C , naïve and primed states ( Dataset EV4 ) were used to analyze expression profiles of epiblast during the early development of mouse embryos ( Data ref : Mohammed et al , 2017a ) ( F ) . Single - cell was plotted based on the average expression of each group of markers .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Markers of 5C, naïve and primed states (Dataset EV4) were used to analyze expression profiles of epiblast during the early development of mouse embryos (Data ref: Mohammed et al, 2017a) (F). Single-cell was plotted based on the average expression of each group of markers.


( G ) The average expression of both naïve and primed markers was calculated based on GSE100597 . The top - 2 cells from E4 . 5 epiblast and top - 5 cells from E5 . 5 epiblast were selected . The expression of several naïve markers ( Dppa3 , Esrrb , Klf4 , Klf5 , Tbx3 , and Zfp42 ) and several mesenchymal markers ( Fgf5 , Lef1 , Nodal , and Pou3f1 ) in these selected cells were listed ( upper part ) . The average expression of these genes in E3 . 5 , 4 . 5 , 5 . 5 and 6 . 5 epiblast were also listed ( lower part ) . These results were all normalized against the average expression of these genes in all tested cells .

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

(G) The average expression of both naïve and primed markers was calculated based on GSE100597. The top-2 cells from E4.5 epiblast and top-5 cells from E5.5 epiblast were selected. The expression of several naïve markers (Dppa3, Esrrb, Klf4, Klf5, Tbx3, and Zfp42) and several mesenchymal markers (Fgf5, Lef1, Nodal, and Pou3f1) in these selected cells were listed (upper part). The average expression of these genes in E3.5, 4.5, 5.5 and 6.5 epiblast were also listed (lower part). These results were all normalized against the average expression of these genes in all tested cells.


( H ) The expression of representative markers of 5C , naïve and primed states was summarized in E3 . 5 , 4 . 5 , 5 . 5 and 6 . 5 epiblast .

(H) The expression of representative markers of 5C, naïve and primed states was summarized in E3.5, 4.5, 5.5 and 6.5 epiblast.


Cells from E5 . 5 epiblast were separated into 8 groups ( I - VIII ) basing on the average expression of epithelial , mesenchymal , and glycolysis markers ( H ) . The average expression of three groups of markers were summarized in ( J ) .

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

Cells from E5.5 epiblast were separated into 8 groups (I-VIII) basing on the average expression of epithelial, mesenchymal, and glycolysis markers (H). The average expression of three groups of markers were summarized in (J).


The expression of six important markers of 5C state was summarized in these eight groups of cells ( K )

The expression of six important markers of 5C state was summarized in these eight groups of cells (K)


( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography Aberrant Replication Products ( ARPs ) are also indicated , as are the different species of catenated dimers : nicked - nicked catenanes , ' n - nCats ' ; nicked - supercoiled catenanes , ' n - scCats ' ; supercoiled - supercoiled catenanes ' sc - scCats ' . ( C ) Quantification of total DNA synthesis from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . Total lane signal ( AU ) was normalized to the maximum recorded signal within each individual experiment . ( D ) Quantification of total DNA synthesis from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . Total lane signal ( AU ) was normalized to the maximum recorded signal within each individual experiment . ( E ) Quantification of supercoiled circular monomers ( ' scCMs ' ) from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . ( F ) Quantification of scCMs from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . ( G ) Quantification of linears ( ' Lins ' ) from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . ( H ) Quantification of Lins from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . Data information : For ( E ) - ( H ) , abundance of the indicated species was calculated as a percentage of total lane signal ( % of total ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Samples from (A) were separated on a native agarose gel and visualized by autoradiography Aberrant Replication Products (ARPs) are also indicated, as are the different species of catenated dimers: nicked-nicked catenanes, 'n-nCats'; nicked-supercoiled catenanes, 'n-scCats'; supercoiled-supercoiled catenanes 'sc-scCats'. (C) Quantification of total DNA synthesis from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Total lane signal (AU) was normalized to the maximum recorded signal within each individual experiment. (D) Quantification of total DNA synthesis from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. Total lane signal (AU) was normalized to the maximum recorded signal within each individual experiment. (E) Quantification of supercoiled circular monomers ('scCMs') from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. (F) Quantification of scCMs from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. (G) Quantification of linears ('Lins') from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. (H) Quantification of Lins from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. Data information: For (E)-(H), abundance of the indicated species was calculated as a percentage of total lane signal (% of total).


( A ) Plasmid DNA was replicated in the presence of [ α - 32P ] dATP and vehicle control , etoposide , or doxorubicin . Purified DNA intermediates were digested with XmnI , which cuts the plasmid template once , to allow for identification of DNA breaks . ( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography . ( C ) Quantification of DNA breaks from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of breaks was calculated as a percentage of total lane signal ( % of total ) . ( D ) Quantification of linears ( ' Lins ' ) from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of linears ( AU ) was normalized to the maximum recorded signal within each individual experiment .

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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plasmid DNA was replicated in the presence of [α-32P]dATP and vehicle control, etoposide, or doxorubicin. Purified DNA intermediates were digested with XmnI, which cuts the plasmid template once, to allow for identification of DNA breaks. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. (C) Quantification of DNA breaks from (B). Mean ± SD, n=3 independent experiments. Abundance of breaks was calculated as a percentage of total lane signal (% of total). (D) Quantification of linears ('Lins') from (B). Mean ± SD, n=3 independent experiments. Abundance of linears (AU) was normalized to the maximum recorded signal within each individual experiment.


( F ) Samples from ( E ) were separated on native agarose gels and visualized by autoradiography . ( G ) Quantification of DPC - containing molecules from ( F ) . Mean ± SD , n = 3 independent experiments . Abundance of the DPC - containing molecules was calculated as a percentage of total lane signal ( % of total ) . ( H ) Quantification of the amount of supercoiled circular monomers ( ' scCMs ' ) from ' + ProK ' samples from ( F ) . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers ( AU ) was normalized to the maximum recorded signal within each individual experiment .

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

(F) Samples from (E) were separated on native agarose gels and visualized by autoradiography. (G) Quantification of DPC-containing molecules from (F). Mean ± SD, n=3 independent experiments. Abundance of the DPC-containing molecules was calculated as a percentage of total lane signal (% of total). (H) Quantification of the amount of supercoiled circular monomers ('scCMs') from '+ProK' samples from (F). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers (AU) was normalized to the maximum recorded signal within each individual experiment.


( A ) Plasmid DNA was replicated in Mock - or TOP2α - immunodepleted extracts in the presence or absence of vehicle control , etoposide , or doxorubicin . In mock - depleted extracts ( ΔMock ) , the final replication product are supercoiled circular monomers ( ' scCMs ' ) . In TOP2α - depleted extracts ( ΔTOP2α ) , the final replication product are catenanes ( ' Cats ' ) because TOP2α is required for DNA unlinking . ( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography . Linear molecules , arising from DNA breaks , are indicated with a red arrow migrate at a similar molecular weight to a subset of catenanes . ( C ) Quantification of scCMs from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a percentage of total lane signal ( % of total ) . ( D ) Quantification of Cats from ( B ) lanes 16 - 30 . Mean ± SD , n = 3 independent experiments . Abundance of catenanes was calculated as a percentage of total lane signal ( % of total ) .

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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plasmid DNA was replicated in Mock- or TOP2α-immunodepleted extracts in the presence or absence of vehicle control, etoposide, or doxorubicin. In mock-depleted extracts (ΔMock), the final replication product are supercoiled circular monomers ('scCMs'). In TOP2α-depleted extracts (ΔTOP2α), the final replication product are catenanes ('Cats') because TOP2α is required for DNA unlinking. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. Linear molecules, arising from DNA breaks, are indicated with a red arrow migrate at a similar molecular weight to a subset of catenanes. (C) Quantification of scCMs from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total). (D) Quantification of Cats from (B) lanes 16-30. Mean ± SD, n=3 independent experiments. Abundance of catenanes was calculated as a percentage of total lane signal (% of total).


( F ) Samples from ( E ) were separated on native agarose gels and visualized by autoradiography . Linear molecules are indicated with a red arrow . ( G ) Quantification of scCMs from + ProK gel in ( F ) lanes 6 - 20 . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a percentage of total lane signal ( % of total ) . ( H ) Quantification of DPC Containing Molecules from ( F ) lanes 6 - 20 . Mean ± SD , n = 3 independent experiments . Abundance of DPC - containing molecules was calculated as a percentage of total lane signal ( % of total ) .

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

(F) Samples from (E) were separated on native agarose gels and visualized by autoradiography. Linear molecules are indicated with a red arrow. (G) Quantification of scCMs from + ProK gel in (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total). (H) Quantification of DPC Containing Molecules from (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of DPC-containing molecules was calculated as a percentage of total lane signal (% of total).


( A ) Plasmid DNA was replicated in the presence or absence of etoposide and p97 - i , which inhibits removal of replisomes ( green ) from DNA . At different timepoints chromatin - bound proteins were recovered via plasmid pulldowns . ( B ) Proteins from ( A ) were recovered and analyzed via Western blotting . Input corresponds to 1 % of total reaction . Modified TOP2α species are also indicated . ( C ) Quantification of TOP2α ( both modified and unmodified species ) from ( B ) lanes 1 - 8 . ( D ) Quantification of TOP2α ( both modified and unmodified species ) from ( B ) lanes 9 - 16 . ( E ) Quantification of CDC45 from ( B ) lanes 1 - 8 . ( F ) Quantification of CDC45 from ( B ) lanes 9 - 16 . Data information : ( C - F Mean ± SD , n = 3 independent experiments . Protein abundance ( AU ) was normalized to the maximum recorded signal within each individual experiment .

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

(A) Plasmid DNA was replicated in the presence or absence of etoposide and p97-i, which inhibits removal of replisomes (green) from DNA. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns. (B) Proteins from (A) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction. Modified TOP2α species are also indicated. (C) Quantification of TOP2α (both modified and unmodified species) from (B) lanes 1-8. (D) Quantification of TOP2α (both modified and unmodified species) from (B) lanes 9-16. (E) Quantification of CDC45 from (B) lanes 1-8. (F) Quantification of CDC45 from (B) lanes 9-16. Data information: (C-F Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment.


( G ) Plasmid DNA was replicated in the presence or absence of etoposide and aphidicolin ( Aph ) , which prevents DNA synthesis but still permits unwinding by replisomes . At different timepoints chromatin - bound proteins were recovered via plasmid pulldowns . ( H ) Proteins from ( G ) were recovered and analyzed via Western blotting . Input corresponds to 1 % of total reaction . ( I ) Quantification of RPA30 from ( H ) lanes 9 - 16 . ( J ) Quantification of RPA30 from ( H ) lanes 1 - 8 . ( K ) Quantification of TOP2α ( both modified and unmodified species ) from ( H ) lanes 1 - 4 and 9 - 12 . ( L ) Quantification of TOP2α ( both modified and unmodified species ) from ( H ) lanes 5 - 8 and 13 - 16 . Data information : I - L ) Mean ± SD , n = 3 independent experiments . Protein abundance ( AU ) was normalized to the maximum recorded signal within each individual experiment .

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

(G) Plasmid DNA was replicated in the presence or absence of etoposide and aphidicolin (Aph), which prevents DNA synthesis but still permits unwinding by replisomes. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns. (H) Proteins from (G) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction. (I) Quantification of RPA30 from (H) lanes 9-16. (J) Quantification of RPA30 from (H) lanes 1-8. (K) Quantification of TOP2α (both modified and unmodified species) from (H) lanes 1-4 and 9-12. (L) Quantification of TOP2α (both modified and unmodified species) from (H) lanes 5-8 and 13-16. Data information: I-L) Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment.


( A ) Plasmid DNA was replicated in the presence of [ α - 32P ] dATP and vehicle control , aclarubicin , or doxorubicin . ( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography . ( C ) Quantification of supercoiled circular monomers from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a percentage of total lane signal ( % of total ) .

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 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

(A) Plasmid DNA was replicated in the presence of [α-32P]dATP and vehicle control, aclarubicin, or doxorubicin. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. (C) Quantification of supercoiled circular monomers from (B). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total).


( E ) Samples from ( D ) were separated on a native agarose gel and visualized by autoradiography . ( F ) Quantification of fork merger from ( E ) lanes 1 - 8 . Mean ± SD , n = 3 independent experiments . ( G ) Quantification of fork merger from ( E ) lanes 9 - 17 . Mean ± SD , n = 3 independent experiments . Fork merger was calculated as a percentage of total lane signal ( % of total ) for ( F ) - ( G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Samples from (D) were separated on a native agarose gel and visualized by autoradiography. (F) Quantification of fork merger from (E) lanes 1-8. Mean ± SD, n=3 independent experiments. (G) Quantification of fork merger from (E) lanes 9-17. Mean ± SD, n=3 independent experiments. Fork merger was calculated as a percentage of total lane signal (% of total) for (F)-(G).


B . DCC and netrin - 1 expression ( FACS analysis ) in B - cells lymphocytes isolated from DCC + / + and DCCmut / mut mice spleen and lymph nodes . Results are presented as expression ratios between means expression levels in mutant animals ( n = 3 ) and control ones ( n = 3 ) . No significant difference was observed between wild - type and mutant animals .

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

B. DCC and netrin-1 expression (FACS analysis) in B-cells lymphocytes isolated from DCC+/+ and DCCmut/mut mice spleen and lymph nodes. Results are presented as expression ratios between means expression levels in mutant animals (n=3) and control ones (n=3). No significant difference was observed between wild-type and mutant animals.


C . DCCmut / mut mouse with splenomegaly and systemic lymphadenopathy . Left panel : black and white arrows indicate respectively the inguinal and mesenteric lymph nodes . Right panel : enlarged spleen in a lymphoma - bearing mutant mouse ( left ) as compared to control organ ( right ) .

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

C. DCCmut/mut mouse with splenomegaly and systemic lymphadenopathy. Left panel: black and white arrows indicate respectively the inguinal and mesenteric lymph nodes. Right panel: enlarged spleen in a lymphoma-bearing mutant mouse (left) as compared to control organ (right).


D . Incidence of lymphoid proliferations and lymphoma in DCC + / + ( n = 19 ) and DCCmut / mut ( n = 29 ) mice . Tumors classification was performed blinded to genotype according to anatomopathologists consensus ( 29 ) . * : p = 0 . 036 ; one - tailed Fisher ' s exact test .

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

D. Incidence of lymphoid proliferations and lymphoma in DCC+/+ (n=19) and DCCmut/mut (n=29) mice. Tumors classification was performed blinded to genotype according to anatomopathologists consensus (29). *: p=0.036; one-tailed Fisher's exact test.


E . Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice . Sections from control spleen , low - grade FL and high - grade DLBCL were stained with hematoxylin - eosin - safran or with antibodies to B220 or Ki67 . w : white pulp , r : red pulp , cr : compressed red pulp .

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

E. Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice. Sections from control spleen, low-grade FL and high-grade DLBCL were stained with hematoxylin-eosin-safran or with antibodies to B220 or Ki67. w: white pulp, r: red pulp, cr: compressed red pulp.


A - B . Analysis of DCC ( A ) and netrin - 1 ( B ) expression in a cohort of normal tonsils ( n = 9 ) , Germinal - Center - B - Cell - like - DLBCL ( n = 13 ) , Activated - B - Cell - like - DLBCL ( n = 18 ) and MCL ( n = 36 ) . Quantification of genes expression was performed by Q - RT - PCR , relatively to HPRT housekeeping gene . * * : p < 0 . 005 ; * * * * : p < 0 . 0001 ; two - sided Mann - Whitney U - test .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of DCC (A) and netrin-1 (B) expression in a cohort of normal tonsils (n=9), Germinal-Center-B-Cell-like-DLBCL (n=13), Activated-B-Cell-like-DLBCL (n=18) and MCL (n=36). Quantification of genes expression was performed by Q-RT-PCR, relatively to HPRT housekeeping gene. **: p<0.005; ****: p<0.0001; two-sided Mann-Whitney U-test.


C - D . Analysis of netrin - 1 expression in DLBCL ( C ) and MCL ( D ) by immunohistochemistry . C . 21 DLBCL patients biopsies were used to build a TMA . Normal tissues ( normal tonsils , reactive lymph nodes , muscle , liver ) are included ( the left - most spot , upper panel , corresponds to liver ) . Enlarged representative image of netrin - 1 expression in DLBCL is shown ( lower panel ) . D . Representative images of netrin - 1 expression in paraffin - embedded biopsies of MCL ( n = 9 ) . Five tumors were positive for netrin - 1 ( left panel ) and four were negative ( right panel ) .

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

C-D. Analysis of netrin-1 expression in DLBCL (C) and MCL (D) by immunohistochemistry. C. 21 DLBCL patients biopsies were used to build a TMA. Normal tissues (normal tonsils, reactive lymph nodes, muscle, liver) are included (the left-most spot, upper panel, corresponds to liver). Enlarged representative image of netrin-1 expression in DLBCL is shown (lower panel). D. Representative images of netrin-1 expression in paraffin-embedded biopsies of MCL (n=9). Five tumors were positive for netrin-1 (left panel) and four were negative (right panel).