Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two weeks post tumor cell injection , Representative microscopy images in F show STAT3 phosphorylation in spleen - colonizing primary cells exposed to either tocilizumab or isotype control antibody ( in brown , counterstained in blue with hematoxylin ) , and an H & E - stained section from the same mouse . TCZ , tocilizumab . Scale bars , 100 μm .

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, Representative microscopy images in F show STAT3 phosphorylation in spleen-colonizing primary cells exposed to either tocilizumab or isotype control antibody (in brown, counterstained in blue with hematoxylin), and an H&E-stained section from the same mouse. TCZ, tocilizumab. Scale bars, 100 μm.


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two weeks post tumor cell injection on the bone marrow lymphoma burden of OCI - Ly3 - transplanted MISTRG mice ( G ) assessed at around four weeks post tumor cell injection .

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the bone marrow lymphoma burden of OCI-Ly3-transplanted MISTRG mice (G) assessed at around four weeks post tumor cell injection.


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two weeks post tumor cell injection on the splenic lymphoma burden of RC - K8 - transplanted MISTRG6 mice ( H ) , all assessed at around four weeks post tumor cell injection .

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the splenic lymphoma burden of RC-K8-transplanted MISTRG6 mice (H), all assessed at around four weeks post tumor cell injection.


( A ) Drosophila egg chambers are composed of a large oocyte surrounded by a columnar follicle cell epithelium that activates a Hpo kinase bimolecular fluorescence complementation ( BiFC ) dimerization sensor ( HpoKD - Venus ; green ) apically in small punctae . Note that the Hpo sensor is activated in columnar cells but not in mechanically stretched cells . ( B ) Nutrient restriction ( starvation ) of adult females for 24h causes formation of large micron - scale Hpo sensor punctae .

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(A) Drosophila egg chambers are composed of a large oocyte surrounded by a columnar follicle cell epithelium that activates a Hpo kinase bimolecular fluorescence complementation (BiFC) dimerization sensor (HpoKD-Venus; green) apically in small punctae. Note that the Hpo sensor is activated in columnar cells but not in mechanically stretched cells. (B) Nutrient restriction (starvation) of adult females for 24h causes formation of large micron-scale Hpo sensor punctae.


( D ) Inhibition of PI3K by overexpression of a dominant - negative version ( the p60 subunit alone ) is sufficient to induce formation of large micron - scale Hpo sensor punctae , while activation of Akt by overexpression of a myristylated form of the protein is sufficient to reduce formation of the small apical Hpo sensor punctae that are normally observed in control animals .

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(D) Inhibition of PI3K by overexpression of a dominant-negative version (the p60 subunit alone) is sufficient to induce formation of large micron-scale Hpo sensor punctae, while activation of Akt by overexpression of a myristylated form of the protein is sufficient to reduce formation of the small apical Hpo sensor punctae that are normally observed in control animals.


( A ) Drosophila egg chambers at stages 9 / 10 of oogenesis expressing a Hpo kinase dimerization sensor that forms small punctae at the apical domain of the follicle cell epithelium that surrounds the oocyte . ( B ) Treatment of egg chambers with 1 , 6 - hexanediol ( 10 % ) for 5 min abolishes formation of Hpo sensor punctae . ( C ) Treatment of egg chambers with 1M Sorbitol to osmotically withdraw water , and thereby induce macromolecular crowding , is sufficient to induce formation of large micron - scale Hpo sensor punctae . ( D ) Nutrient restriction ( starvation ) of adult females for 24h induces large micron - scale Hpo sensor punctae that are similar in size to those in ( C ) . ( E ) Treatment of egg chambers from adult females starved for 24h with 1 , 6 - hexanediol ( 10 % ) for 5 min abolishes formation of Hpo sensor punctae .

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

(A) Drosophila egg chambers at stages 9/10 of oogenesis expressing a Hpo kinase dimerization sensor that forms small punctae at the apical domain of the follicle cell epithelium that surrounds the oocyte. (B) Treatment of egg chambers with 1,6-hexanediol (10%) for 5 min abolishes formation of Hpo sensor punctae. (C) Treatment of egg chambers with 1M Sorbitol to osmotically withdraw water, and thereby induce macromolecular crowding, is sufficient to induce formation of large micron-scale Hpo sensor punctae. (D) Nutrient restriction (starvation) of adult females for 24h induces large micron-scale Hpo sensor punctae that are similar in size to those in (C). (E) Treatment of egg chambers from adult females starved for 24h with 1,6-hexanediol (10%) for 5 min abolishes formation of Hpo sensor punctae.


( I ) Quantification of FRAP recovery as a fraction of the initial fluorescence intensity . The rapid initial slope of recovery suggests dynamic exchange between the condensate and the cytoplasmic pool of the proteins . Temporal averages are plotted as individual data points ( n > 3 biological replicates ) , error bars represent one standard deviation from the mean .

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

(I) Quantification of FRAP recovery as a fraction of the initial fluorescence intensity. The rapid initial slope of recovery suggests dynamic exchange between the condensate and the cytoplasmic pool of the proteins. Temporal averages are plotted as individual data points (n>3 biological replicates), error bars represent one standard deviation from the mean.


( A ) Apical cross - sections of Drosophila ovarian follicle cell epithelia at high magnification , showing accumulation of Hpo - YFP punctae at the apical domain , which is strongly enhanced upon 24h starvation to reduce Insulin / IGF - 1 signalling . Hpo puncta are lost following a short treatment with 1 , 6 - hexanediol . Samples were fixed before imaging . ( B ) Quantification of Hpo - YFP puncta number and size in ( A ) . Biological replicates are plotted as individual data points ( n > 13 for all samples ) , error bars represent one standard deviation from the mean , statistical significance was determined using a t - test * * * * P < 0 . 0001 * * * P < 0 . 001 * * P < 0 . 01 * P < 0 . 05 .

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

(A) Apical cross-sections of Drosophila ovarian follicle cell epithelia at high magnification, showing accumulation of Hpo-YFP punctae at the apical domain, which is strongly enhanced upon 24h starvation to reduce Insulin/IGF-1 signalling. Hpo puncta are lost following a short treatment with 1,6-hexanediol. Samples were fixed before imaging. (B) Quantification of Hpo-YFP puncta number and size in (A). Biological replicates are plotted as individual data points (n>13 for all samples), error bars represent one standard deviation from the mean, statistical significance was determined using a t-test ****P<0.0001 ***P<0.001 **P<0.01 *P<0.05.


( C ) Apical cross - sections of Drosophila ovarian follicle cell epithelia at high magnification , showing loss of Hpo - YFP punctae upon mechanical strain in ' stretch cells ' . The Drosophila follicle cell epithelium at stage 10 is comprised of anterior stretch cells ( flat ) and columnar cells . Endogenously - tagged Hpo punctae are strongly enriched at the apical domain of columnar cells but not in flattened stretch cells . White asterisks demarcate nuclei of individual stretch cells . ( D ) Quantification of Hpo - YFP total intensity , and size in ( C ) . Biological replicates are plotted as individual data points ( n > 10 for all samples ) , error bars represent one standard deviation from the mean , statistical significance was determined using a t - test * * * * P < 0 . 0001 * * * P < 0 . 001 * * P < 0 . 01 * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Apical cross-sections of Drosophila ovarian follicle cell epithelia at high magnification, showing loss of Hpo-YFP punctae upon mechanical strain in 'stretch cells'. The Drosophila follicle cell epithelium at stage 10 is comprised of anterior stretch cells (flat) and columnar cells. Endogenously-tagged Hpo punctae are strongly enriched at the apical domain of columnar cells but not in flattened stretch cells. White asterisks demarcate nuclei of individual stretch cells. (D) Quantification of Hpo-YFP total intensity, and size in (C). Biological replicates are plotted as individual data points (n>10 for all samples), error bars represent one standard deviation from the mean, statistical significance was determined using a t-test ****P<0.0001 ***P<0.001 **P<0.01 *P<0.05.


( A ) Expanded ( Ex ; endogenously tagged with YFP ) localises to the apical domain of columnar follicle cells at stages 9 / 10 of oogenesis . ( B ) The Hpo dimerization sensor localises in a similar pattern to Ex .

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(A) Expanded (Ex; endogenously tagged with YFP) localises to the apical domain of columnar follicle cells at stages 9/10 of oogenesis. (B) The Hpo dimerization sensor localises in a similar pattern to Ex.


( E ) Condensates induced by high levels of Ex in the cytoplasm co - localise with the Hpo sensor but do not co - localise with Crb , which remains apical . ( F ) Condensates induced by high levels of Ex in the cytoplasm immunostain positively for phosphorylated Wts .

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(E) Condensates induced by high levels of Ex in the cytoplasm co-localise with the Hpo sensor but do not co-localise with Crb, which remains apical. (F) Condensates induced by high levels of Ex in the cytoplasm immunostain positively for phosphorylated Wts.


( G ) Moderate overexpression of untagged Kib is sufficient to induce formation of large micron - scale HpoKD - Venus condensates that primarily localise apically . DAPI marks nuclei ( blue ) . ( H ) Overexpression of Kib - GFP with the Gal4 / UAS system is sufficient to induce formation of large micron - scale cytoplasmic condensates .

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(G) Moderate overexpression of untagged Kib is sufficient to induce formation of large micron-scale HpoKD-Venus condensates that primarily localise apically. DAPI marks nuclei (blue). (H) Overexpression of Kib-GFP with the Gal4/UAS system is sufficient to induce formation of large micron-scale cytoplasmic condensates.


( A ) Human U2OS cells in culture immunostained for endogenous Hippo pathway components reveals punctate subcellular localisation within the cytoplasm .

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(A) Human U2OS cells in culture immunostained for endogenous Hippo pathway components reveals punctate subcellular localisation within the cytoplasm.


( B ) Ectopic expression of Flag - tagged human Kibra ( KIB - Flag ) generates large spherical punctae in the cytoplasm of human Caco2 epithelial cells ( top ) . Co - localisation of KIB - GFP with active phosphorylated LATS ( pLATS ) in the cytoplasm of human Caco2 epithelial cells ( middle ) . Note that cells expressing KIB - GFP have increased Hippo signalling as indicated by reduced levels of nuclear YAP localisation ( bottom ) . Dashed lines demarcate transfected cells .

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(B) Ectopic expression of Flag-tagged human Kibra (KIB-Flag) generates large spherical punctae in the cytoplasm of human Caco2 epithelial cells (top). Co-localisation of KIB-GFP with active phosphorylated LATS (pLATS) in the cytoplasm of human Caco2 epithelial cells (middle). Note that cells expressing KIB-GFP have increased Hippo signalling as indicated by reduced levels of nuclear YAP localisation (bottom). Dashed lines demarcate transfected cells.


( A ) Expression of KIB - GFP , KIB ∆ C - GFP or KIB ∆ M + C - GFP in human HEK293T cells and co - staining for pLATS . DAPI marks nuclei in blue .

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(A) Expression of KIB-GFP, KIB∆C-GFP or KIB∆M+C-GFP in human HEK293T cells and co-staining for pLATS. DAPI marks nuclei in blue.


( A ) Human Caco2 intestinal epithelial cells are relatively flat when cultured as cell lines in 2D ( Caco - 2 cells , top , middle ) and cuboidal when cultured in 3D ( spheroid , bottom ) . Punctae of pLATS ( green ) are strongly enriched at the apical domain of cuboidal cells grown in 3D culture but not in flattened cells grown in 2D culture . Note the increased apical enrichment in medium - density culture in 2D , where cells become more cuboidal than at low - density . Phalloidin staining marks the actin cytoskeleton in red . DAPI marks nuclei in blue .

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(A) Human Caco2 intestinal epithelial cells are relatively flat when cultured as cell lines in 2D (Caco-2 cells, top, middle) and cuboidal when cultured in 3D (spheroid, bottom). Punctae of pLATS (green) are strongly enriched at the apical domain of cuboidal cells grown in 3D culture but not in flattened cells grown in 2D culture. Note the increased apical enrichment in medium-density culture in 2D, where cells become more cuboidal than at low-density. Phalloidin staining marks the actin cytoskeleton in red. DAPI marks nuclei in blue.


( C ) Mouse small intestinal organoids can become stretched upon growth as inflated spheres , which leads to mechanical strain in epithelial cells and reduced pLATS localisation apically , which correlates with reduced Hippo signalling ( leading to increased YAP nuclear localisation ) . White arrows highlight the apical domain of epithelial cells .

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(C) Mouse small intestinal organoids can become stretched upon growth as inflated spheres, which leads to mechanical strain in epithelial cells and reduced pLATS localisation apically, which correlates with reduced Hippo signalling (leading to increased YAP nuclear localisation). White arrows highlight the apical domain of epithelial cells.


( B ) High resolution imaging of intestinal organoids cultured in control medium or medium supplemented with the NRG1 growth factor for 1 h which strongly stimulates pAkt , reduces pLATS staining , and drives YAP to the nucleus .

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(B) High resolution imaging of intestinal organoids cultured in control medium or medium supplemented with the NRG1 growth factor for 1 h which strongly stimulates pAkt, reduces pLATS staining, and drives YAP to the nucleus.


B , C . Cryosections of samples from patients with prostate carcinoma and SCC of the lung . Samples were processed as described in the methods section . Ve = vessel , Tu = tumor . Scale 50µm . See also Fig 1D for comparative semi - quantitative analysis of a panel of samples from patients with prostate carcinoma , lung SCC and non - lung SCC .

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B, C. Cryosections of samples from patients with prostate carcinoma and SCC of the lung. Samples were processed as described in the methods section. Ve=vessel, Tu=tumor. Scale 50µm. See also Fig 1D for comparative semi-quantitative analysis of a panel of samples from patients with prostate carcinoma, lung SCC and non-lung SCC.


D . Semi quantitative analysis of binding of the PSMA antibodies 10B3 and J591 to cryosections from different tumor entities . For definition of the H - score reflecting binding intensity , refer to the methods section . Statistical analysis was performed using the paired , non - parametric Wilcoxon test . Sample size ( n ) and the corresponding P - values are indicated in the graphs .

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D. Semi quantitative analysis of binding of the PSMA antibodies 10B3 and J591 to cryosections from different tumor entities. For definition of the H-score reflecting binding intensity, refer to the methods section. Statistical analysis was performed using the paired, non-parametric Wilcoxon test. Sample size (n) and the corresponding P-values are indicated in the graphs.


E , F . Binding of the Fabsc - and IgGsc - molecules to PSMA - expressing LNCaP ( E ) or 22Rv1low ( F ) cells was assessed by flow cytometry , as described in the methods section . The EC50 values were calculated using GraphPad Prism software and are presented below the figure .

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E, F. Binding of the Fabsc- and IgGsc-molecules to PSMA-expressing LNCaP (E) or 22Rv1low (F) cells was assessed by flow cytometry, as described in the methods section. The EC50 values were calculated using GraphPad Prism software and are presented below the figure.


G - I . Binding of the Fabsc - and IgGsc - molecules to CD3 - expressing Jurkat - cells ( G ) and to CD4 T cells ( H ) and CD8 T cells ( I ) in PBMC preparations was assessed by flow cytometry . The results represent the average of three independent experiments performed with different PBMC donors . EC50 values are presented in the table below the figure .

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G-I. Binding of the Fabsc- and IgGsc-molecules to CD3-expressing Jurkat-cells (G) and to CD4 T cells (H) and CD8 T cells (I) in PBMC preparations was assessed by flow cytometry. The results represent the average of three independent experiments performed with different PBMC donors. EC50 values are presented in the table below the figure.


BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with LNCaP - expressing high and low amounts of PSMA , respectively . After three days , cytokine release were determined by flow cytometry as described in the methods section . Mean values and standard deviations of triplicate measurements are indicated .

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BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with LNCaP- expressing high and low amounts of PSMA, respectively. After three days, cytokine release were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated.


BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with 22Rv1low - cells expressing high and low amounts of PSMA , respectively . After three days , cytokine release were determined by flow cytometry as described in the methods section . Mean values and standard deviations of triplicate measurements are indicated .

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BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with 22Rv1low-cells expressing high and low amounts of PSMA, respectively. After three days, cytokine release were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated.


B PBMC of a healthy donor were incubated with the indicated concentrations of bsAbs , and after three days cytokine release ( B ) were measured using a 3H - thymidine uptake - and a Legendplex assay , respectively . Mean values and standard deviations from triplicate samples .

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B PBMC of a healthy donor were incubated with the indicated concentrations of bsAbs, and after three days cytokine release (B) were measured using a 3H-thymidine uptake- and a Legendplex assay, respectively. Mean values and standard deviations from triplicate samples.


C - F . 64Cu labeled bsAbs were injected into NSG mice carrying 22Rv1high tumors ( 5 animals per group ) , and uptake at the indicated locations was determined by PET quantification over time ( C , D ) and at termination of the experiment after 48 hours ( E , F ) as described in the methods section . Error bars represent standard deviation of the mean value .

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C-F. 64Cu labeled bsAbs were injected into NSG mice carrying 22Rv1high tumors (5 animals per group), and uptake at the indicated locations was determined by PET quantification over time (C, D) and at termination of the experiment after 48 hours (E, F) as described in the methods section. Error bars represent standard deviation of the mean value.


B , C . 106 LNCaP cells were injected s . c . into the right flank of NSG mice ( 5 per group ) and treatment with PBMC and bsAbs was started when tumors had reached a diameter of 5mm . BsAbs ( 2µg per dose ) were applied at day 1 , 8 and 15 ( IgGsc ) and daily at day 1 - 3 , day 8 - 10 and day 15 - 17 ( Fabsc ) , PBMC were applied at day 1 , 8 and 15 . Mean values and standard deviations of five mice per group are indicated .

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B, C. 106 LNCaP cells were injected s.c. into the right flank of NSG mice (5 per group) and treatment with PBMC and bsAbs was started when tumors had reached a diameter of 5mm. BsAbs (2µg per dose) were applied at day 1, 8 and 15 (IgGsc) and daily at day 1-3, day 8-10 and day 15-17 (Fabsc), PBMC were applied at day 1,8 and 15. Mean values and standard deviations of five mice per group are indicated.


The IgGsc - molecule was applied in escalating doses , with a starting dose of 20µg ( patient 1 ) or 40µg ( patients 2 and 3 ) to a maximum of approx . 2 . 6mg , as indicated . PSA values were monitored and T - cell activation was assessed by flow cytometry .

The IgGsc-molecule was applied in escalating doses, with a starting dose of 20µg (patient 1) or 40µg (patients 2 and 3) to a maximum of approx. 2.6mg, as indicated. PSA values were monitored and T-cell activation was assessed by flow cytometry.


B . β - catenin - mediated reporter activity in HEK293T cells expressing the indicated RNF43 cancer mutants . Cells were treated with control medium ( no Wnt3a ) or Wnt3a - conditioned medium ( Wnt3a ) overnight . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

B. β-catenin-mediated reporter activity in HEK293T cells expressing the indicated RNF43 cancer mutants. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


C . Western blot analysis showing the effect of RNF43 cancer mutants on V5 - FZD5 expression in HEK293T cells . Open and closed arrows indicate mature ( post - Golgi ) and immature ( ER - associated ) FZD5 , respectively .

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C. Western blot analysis showing the effect of RNF43 cancer mutants on V5-FZD5 expression in HEK293T cells. Open and closed arrows indicate mature (post-Golgi) and immature (ER-associated) FZD5, respectively.


D . Confocal microscopy of surface labeled SNAP - FZD5 in HEK293T cells upon expression of RNF43 WT or R519X . Cells were chased for 30 min . Scale bars represent 10 μm .

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D. Confocal microscopy of surface labeled SNAP-FZD5 in HEK293T cells upon expression of RNF43 WT or R519X. Cells were chased for 30 min. Scale bars represent 10 μm.


E . Confocal microscopy of β - catenin localization in HEK293T cells expressing the indicated RNF43 cancer mutants . Scale bars represent 10 μm .

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E. Confocal microscopy of β-catenin localization in HEK293T cells expressing the indicated RNF43 cancer mutants. Scale bars represent 10 μm.


F . Western blot analysis of RNF43 WT and R519X for cytoplasmic β - catenin levels .

F. Western blot analysis of RNF43 WT and R519X for cytoplasmic β-catenin levels.


G . β - catenin - mediated reporter activity in HEK293T cells expressing Wnt3a and WT RNF43 , oncogenic RNF43 ( R519X ) or a LOF RNF43 variant ( I48T ) after o / n treatment with DMSO or the PORCN inhibitor C59 ( 1 μM ) .

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G. β-catenin-mediated reporter activity in HEK293T cells expressing Wnt3a and WT RNF43, oncogenic RNF43 (R519X) or a LOF RNF43 variant (I48T) after o/n treatment with DMSO or the PORCN inhibitor C59 (1 μM).


A . β - catenin - mediated reporter activity in HEK293T cells expressing WT RNF43 and oncogenic RNF43 ( R519X ) harboring mutations C290S / H292S in the RING domain ( M1 ) . Cells were treated with control medium ( no Wnt3a ) or Wnt3a - conditioned medium ( Wnt3a ) overnight . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

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A. β-catenin-mediated reporter activity in HEK293T cells expressing WT RNF43 and oncogenic RNF43 (R519X) harboring mutations C290S/H292S in the RING domain (M1). Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


C . β - catenin - mediated reporter activity in HEK293T cells expressing the indicated RNF43 constructs . Cells were treated with control medium ( no Wnt3a ) or Wnt3a - conditioned medium ( Wnt3a ) overnight . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

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C. β-catenin-mediated reporter activity in HEK293T cells expressing the indicated RNF43 constructs. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


D . β - catenin - mediated reporter activity in HEK293T cells co - expressing oncogenic RNF43 ( R519X ) and the Dishevelled DEP - C tail . Cells were treated with control medium ( no Wnt3a ) or Wnt3a - conditioned medium ( Wnt3a ) overnight . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

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D. β-catenin-mediated reporter activity in HEK293T cells co-expressing oncogenic RNF43 (R519X) and the Dishevelled DEP-C tail. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


E . β - catenin - mediated reporter activity in HEK293T cells co - expressing oncogenic RNF43 ( R519X ) and dominant negative ∆ N - TCF4 . Cells were treated with control medium ( no Wnt3a ) or Wnt3a - conditioned medium ( Wnt3a ) overnight . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

E. β-catenin-mediated reporter activity in HEK293T cells co-expressing oncogenic RNF43 (R519X) and dominant negative ∆N-TCF4. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


A . Volcano plot showing proteins enriched after streptavidin pull - down of biotin - treated HEK293 cells stably expressing dox - inducible RNF43 - BirA * . Striped line demarcates the empirical 0 . 01 False discovery rate ( FDR ) cut off . Significantly enriched Wnt / β - catenin pathway components are highlighted in red and RNF43 in orange .

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A. Volcano plot showing proteins enriched after streptavidin pull-down of biotin-treated HEK293 cells stably expressing dox-inducible RNF43-BirA*. Striped line demarcates the empirical 0.01 False discovery rate (FDR) cut off. Significantly enriched Wnt/β-catenin pathway components are highlighted in red and RNF43 in orange.


B . Western blot analysis of endogenous destruction complex components that co - precipitated with the indicated RNF43 cancer variants expressed in HEK293T cells . Oncogenic RNF43 truncations are indicated in red . Data information : IP ; immunoprecipitation , IB ;

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B. Western blot analysis of endogenous destruction complex components that co-precipitated with the indicated RNF43 cancer variants expressed in HEK293T cells. Oncogenic RNF43 truncations are indicated in red. Data information: IP; immunoprecipitation, IB; immunoblot


Confocal microscopy analysis of the subcellular localization of Axin1 - GFP upon expression of WT or oncogenic RNF43 ( R519X ) . Scale bars represent 10 μm . RNF43 is visualised by Flag - staining . Asterisks indicate RNF43 - expressing cells .

Confocal microscopy analysis of the subcellular localization of Axin1-GFP upon expression of WT or oncogenic RNF43 (R519X). Scale bars represent 10 μm. RNF43 is visualised by Flag-staining. Asterisks indicate RNF43-expressing cells.


Confocal microscopy analysis of the subcellular localization of endogenous CK1α upon expression of WT or oncogenic RNF43 ( R519X ) . Scale bars represent 10 μm . RNF43 is visualised by Flag - staining . Asterisks indicate RNF43 - expressing cells .

Confocal microscopy analysis of the subcellular localization of endogenous CK1α upon expression of WT or oncogenic RNF43 (R519X). Scale bars represent 10 μm. RNF43 is visualised by Flag-staining. Asterisks indicate RNF43-expressing cells.


E . β - catenin - mediated reporter activity in HEK293T cells expressing non - mutated RNF43 R519X ( SLSS ) or the ALAA , DLDD and ∆ 486 - 89 mutants . Average β - catenin - mediated reporter activities ± s . d . in n = 2 independent wells are shown .

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E. β-catenin-mediated reporter activity in HEK293T cells expressing non-mutated RNF43 R519X (SLSS) or the ALAA, DLDD and ∆486-89 mutants. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.


A . Bright - field microscopy images of WT , TP53KO and onco - RNF43 / TP53KO human colon organoid lines grown in medium with high Wnt / Rspo ( 20 % conditioned medium ( CM ) ) or without Wnt / Rspo ( 20 , 2 or 0 . 2 % CM ) . Images were taken 6 days after splitting . Scale bars represent 1000 μm . Non - cystic , non - proliferative organoids are indicated with red asterisks .

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A. Bright-field microscopy images of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines grown in medium with high Wnt/Rspo (20% conditioned medium (CM)) or without Wnt/Rspo (20, 2 or 0.2% CM). Images were taken 6 days after splitting. Scale bars represent 1000 μm. Non-cystic, non-proliferative organoids are indicated with red asterisks.


C . Bright - field microscopy and fluorescence microscopy pictures of WT , TP53KO and onco - RNF43 / TP53KO human colon organoid lines grown in two different media and transduced with the TOP - GFP reporter . Images were taken 6 days after splitting . Scale bars represent 1000 μm .

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C. Bright-field microscopy and fluorescence microscopy pictures of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines grown in two different media and transduced with the TOP-GFP reporter. Images were taken 6 days after splitting. Scale bars represent 1000 μm.


D . Gene Set Enrichment Analysis of onco - RNF43 / P53KO compared to TP53KO organoids in medium without Wnt / low Rspo ( 0 . 2 % ) . Significantly changed intestinal cell - type gene sets from Haber et al . , 2017 ( ) are shown ( FDR < 0 . 05 ) .

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D. Gene Set Enrichment Analysis of onco-RNF43/P53KO compared to TP53KO organoids in medium without Wnt/low Rspo (0.2%). Significantly changed intestinal cell-type gene sets from Haber et al., 2017() are shown (FDR < 0.05).


A . Top : Schematic experimental set up of the clonogenic assay . Bottom : Bright - field microscopy images of WT , TP53KO and onco - RNF43 / TP53KO human colon organoid lines at day 2 and 14 . Scale bars represent 1000 μm .

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A. Top: Schematic experimental set up of the clonogenic assay. Bottom: Bright-field microscopy images of WT, TP53KO and onco-RNF43/TP53KO human colon organoid lines at day 2 and 14. Scale bars represent 1000 μm.


B . Relative outgrowth of WT , TP53KO and onco - RNF43 / TP53KO organoid lines treated with the PORCN inhibitor C59 ( 1 μM ) for 7 days . Graph shows the number of organoids at 5 days after splitting ( day 14 ) relative to the number of organoids at day 2 . Error bars represent ± s . d . of the mean of n = 3 experiments .

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B. Relative outgrowth of WT, TP53KO and onco-RNF43/TP53KO organoid lines treated with the PORCN inhibitor C59 (1 μM) for 7 days. Graph shows the number of organoids at 5 days after splitting (day 14) relative to the number of organoids at day 2. Error bars represent ±s.d. of the mean of n = 3 experiments.


( C , D ) Representative confocal images of expanded centrioles from RPE - 1 cells stained for α / β - tubulin ( αβTub , magenta ) and SFI1 ( green ) . Right panels show top view images across the centriolar length confirming the distal localization of SFI1 at centrioles . The white arrowhead indicates SFI1 distal dot at centrioles . Scale bars : 200 nm and 100 nm ( right panels ) . Average position of SFI1 alongside the centriole is shown in ( D ) .

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(C, D) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta) and SFI1 (green). Right panels show top view images across the centriolar length confirming the distal localization of SFI1 at centrioles. The white arrowhead indicates SFI1 distal dot at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of SFI1 alongside the centriole is shown in (D).


( E , F ) Representative confocal images of expanded centrioles from RPE - 1 cells stained for α / β - tubulin ( αβTub , magenta ) and Centrin 2 / 3 ( Cetn2 / 3 , grey ) . Right panels show top view images across the centriolar length confirming the distal localization of Centrin2 / 3 at centrioles . The white arrowhead indicates Centrin2 / 3 distal dot at centrioles . Scale bars : 200 nm and 100 nm ( right panels ) . Average position of Centrin2 / 3 alongside the centriole is shown in ( F ) .

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(E, F) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta) and Centrin 2/3 (Cetn2/3, grey). Right panels show top view images across the centriolar length confirming the distal localization of Centrin2/3 at centrioles. The white arrowhead indicates Centrin2/3 distal dot at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of Centrin2/3 alongside the centriole is shown in (F).


( G , H ) Representative confocal images of expanded centrioles from RPE - 1 cells stained for α / β - tubulin ( αβTub , magenta ) and Centrin 3 ( Cetn3 , cyan ) . Right panels show top view images across the centriolar length confirming the distal localization of Centrin 3 at centrioles . The white arrowhead indicates Centrin 3 distal dot at centrioles . Scale bars : 200 nm and 100 nm ( right panels ) . Average position of Centrin 3 alongside the centriole is shown in ( H ) .

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(G, H) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta) and Centrin 3 (Cetn3, cyan). Right panels show top view images across the centriolar length confirming the distal localization of Centrin 3 at centrioles. The white arrowhead indicates Centrin 3 distal dot at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of Centrin 3 alongside the centriole is shown in (H).


( I , J ) Representative confocal images of expanded centrioles from RPE - 1 cells stained for α / β - tubulin ( αβTub , magenta ) , SFI1 ( green ) and Centrin 2 / 3 ( Cetn3 , grey ) . Right panels show top view images confirming the distal localization of SFI1 and Centrin 2 / 3 at centrioles . Scale bars : 200 nm and 100 nm ( right panels ) . Average position of SFI1 and Centrin2 / 3 alongside the centriole is shown in ( J ) .

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(I, J) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta), SFI1 (green) and Centrin 2/3 (Cetn3, grey). Right panels show top view images confirming the distal localization of SFI1 and Centrin 2/3 at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of SFI1 and Centrin2/3 alongside the centriole is shown in (J).


( A - F ) Representative confocal images of expanded U2OS centrioles treated with siCT ( A , D ) , siSFI1 ( B , E ) and siPOC5 ( C , F ) stained for α / β - tubulin ( αβTub , magenta ) and SFI1 ( A - C , green ) or Centrin2 / 3 ( D - F , grey ) . Insets show top views of expanded centrioles at different positions along the centriole ( P = proximal , C = central and D = distal ) . Note that in the absence of SFI1 and Centrin staining at the distal tip , orientation of the centriole was decided based on the larger diameter of the proximal region compared to the distal one , as previously observed in cryo - tomography ( Greenan et al , 2020 ) . White arrowheads points to the distal dot of SFI1 and Centrin that disappear in SFI1 - depleted ( yellow arrowheads ) but not in POC5 - depleted centrioles . Scale bars : 200 nm and 100 nm ( inset ) . Longitudinal and radial localization of SFI1 and Centrin2 / 3 in siCT ( A , D ) , siSFI1 ( B , E ) and siPOC5 ( C , F ) are presented below the corresponding image . N = 25 , 40 and 60 centrioles from 3 independent experiments . N = 29 , 34 , 34 centrioles from 3 independent experiments .

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(A-F) Representative confocal images of expanded U2OS centrioles treated with siCT (A, D), siSFI1 (B, E) and siPOC5 (C, F) stained for α/β-tubulin (αβTub, magenta) and SFI1 (A-C, green) or Centrin2/3 (D-F, grey). Insets show top views of expanded centrioles at different positions along the centriole (P= proximal, C= central and D= distal). Note that in the absence of SFI1 and Centrin staining at the distal tip, orientation of the centriole was decided based on the larger diameter of the proximal region compared to the distal one, as previously observed in cryo-tomography (Greenan et al, 2020). White arrowheads points to the distal dot of SFI1 and Centrin that disappear in SFI1-depleted (yellow arrowheads) but not in POC5-depleted centrioles. Scale bars: 200 nm and 100 nm (inset). Longitudinal and radial localization of SFI1 and Centrin2/3 in siCT (A, D), siSFI1 (B, E) and siPOC5 (C, F) are presented below the corresponding image. N=25, 40 and 60 centrioles from 3 independent experiments. N=29, 34, 34 centrioles from 3 independent experiments.


( K - N ) Representative images of expanded U2OS cells expressing GFP alone or GFP + SFI1 - RR and treated with siCT or siSFI1 . Cells were stained for α / β - tubulin ( αβTub , magenta ) and SFI1 ( K , green ) or Centrin2 / 3 ( M , grey ) . Yellow asterisk indicates the centriole lacking SFI1 and Centrin distal dots . Scale bar : 250 nm . Expression of GFP + SFI1 - RR in siSFI1 treated cell rescues the presence of SFI1 and Centrin at the distal tip of the centriole .

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(K-N) Representative images of expanded U2OS cells expressing GFP alone or GFP+SFI1-RR and treated with siCT or siSFI1. Cells were stained for α/β-tubulin (αβTub, magenta) and SFI1 (K, green) or Centrin2/3 (M, grey). Yellow asterisk indicates the centriole lacking SFI1 and Centrin distal dots. Scale bar: 250 nm. Expression of GFP+SFI1-RR in siSFI1 treated cell rescues the presence of SFI1 and Centrin at the distal tip of the centriole.


( O , P ) Representative confocal images of expanded Cetn2 WT , Cetn KO or Cetn2 rescue RPE - 1 . Cells were stained for α / β - tubulin ( αβTub , magenta ) and Centrin2 ( O , grey ) or SFI1 ( P , green ) . Arrowheads indicate the presence ( white ) or the absence ( yellow ) of Centrin2 and SFI1 . Scale bar : 200nm . Quantification shows the total absence of Centrin2 in the KO cells correlating with the absence of SFI1 . Both Centrin2 and SFI1 localization are rescued when Centrin2 is re - expressed in KO cells .

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(O, P) Representative confocal images of expanded Cetn2 WT, Cetn KO or Cetn2 rescue RPE-1. Cells were stained for α/β-tubulin (αβTub, magenta) and Centrin2 (O, grey) or SFI1 (P, green). Arrowheads indicate the presence (white) or the absence (yellow) of Centrin2 and SFI1. Scale bar: 200nm. Quantification shows the total absence of Centrin2 in the KO cells correlating with the absence of SFI1. Both Centrin2 and SFI1 localization are rescued when Centrin2 is re-expressed in KO cells.


( A ) Representative confocal images of expanded duplicating centrioles from siCT - and siSFI1 - treated U2OS cells . Cells were stained for SFI1 ( green ) and α / β - tubulin ( αβTub , magenta ) . Inset shows a distal position of the mother centriole where SFI1 signal is visible . White arrowhead indicates the position of SFI1 distal dot in the procentriole of control cell , which is lost in SFI1 - depleted cells ( yellow arrowhead ) . Scale bars : 200 nm . ( B ) Quantification of the percentage of SFI1 - negative procentrioles . ( C ) Quantification of the percentage of duplicating centrioles .

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(A) Representative confocal images of expanded duplicating centrioles from siCT- and siSFI1- treated U2OS cells. Cells were stained for SFI1 (green) and α/β-tubulin (αβTub, magenta). Inset shows a distal position of the mother centriole where SFI1 signal is visible. White arrowhead indicates the position of SFI1 distal dot in the procentriole of control cell, which is lost in SFI1-depleted cells (yellow arrowhead). Scale bars: 200 nm. (B) Quantification of the percentage of SFI1-negative procentrioles. (C) Quantification of the percentage of duplicating centrioles.


( D ) Representative confocal image of expanded duplicating centrioles from siCT and siSFI1 U2OS treated cells . Cells were stained for HsSAS - 6 ( yellow ) and α / β - tubulin ( αβTub , magenta ) . Quantification shows no difference in the percentage of HsSAS - 6 - positive centrioles in siSFI1 - depleted cells compared to control cells . ( E ) Representative confocal image of expanded duplicating centrioles from siCT and siSFI1 U2OS treated cells . Cells were stained for STIL ( yellow ) and α / β - tubulin ( αβTub , magenta ) . Quantification shows no difference in the percentage of STIL - positive centrioles in siSFI1 compared to control cells .

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(D) Representative confocal image of expanded duplicating centrioles from siCT and siSFI1 U2OS treated cells. Cells were stained for HsSAS-6 (yellow) and α/β-tubulin (αβTub, magenta). Quantification shows no difference in the percentage of HsSAS-6-positive centrioles in siSFI1-depleted cells compared to control cells. (E) Representative confocal image of expanded duplicating centrioles from siCT and siSFI1 U2OS treated cells. Cells were stained for STIL (yellow) and α/β-tubulin (αβTub, magenta). Quantification shows no difference in the percentage of STIL-positive centrioles in siSFI1 compared to control cells.


( F ) Top views of expanded U2OS centrioles treated with siCT or siSFI1 stained for α / β - tubulin ( αβTub , magenta ) . Scale bars : 200 nm . ( G ) Roundness index of centrioles from siCT and siSFI1 - treated cells . ( H ) Length ( circle ) and diameter ( square ) of expanded centrioles in siCT or siSFI1 - treated cells .

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(F) Top views of expanded U2OS centrioles treated with siCT or siSFI1 stained for α/β-tubulin (αβTub, magenta). Scale bars: 200 nm. (G) Roundness index of centrioles from siCT and siSFI1-treated cells. (H) Length (circle) and diameter (square) of expanded centrioles in siCT or siSFI1-treated cells.


( I ) Representative confocal images of expanded U2OS centrioles from siCT and siSFI1 - treated cells stained for α / β - tubulin ( αβTub , magenta ) . White stars point to broken microtubule wall . Scale bars : 200 nm . ( J ) Percentage of abnormal centrioles in the indicated conditions .

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(I) Representative confocal images of expanded U2OS centrioles from siCT and siSFI1-treated cells stained for α/β-tubulin (αβTub, magenta). White stars point to broken microtubule wall. Scale bars: 200 nm. (J) Percentage of abnormal centrioles in the indicated conditions.


( K ) Top views of expanded procentrioles from U2OS cells treated with siCT or siSFI1 and stained for α / β - tubulin ( αβTub , magenta ) . ( L ) Roundness index of procentrioles from siCT and siSFI1 - treated cells .

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(K) Top views of expanded procentrioles from U2OS cells treated with siCT or siSFI1 and stained for α/β-tubulin (αβTub, magenta). (L) Roundness index of procentrioles from siCT and siSFI1-treated cells.


( M ) Representative confocal images of expanded U2OS centrioles from siCT - and siSFI1 - treated cells stained for α / β - tubulin ( αβTub , magenta ) and Centrin ( Cetn2 / 3 , grey ) . White dashed lines delimitate the proximal , central and distal regions . White star points to the broken microtubule wall . ( N ) Centrin coverage ( % of the total tubulin length ) along the centriole in the indicated conditions .

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(M) Representative confocal images of expanded U2OS centrioles from siCT- and siSFI1-treated cells stained for α/β-tubulin (αβTub, magenta) and Centrin (Cetn2/3, grey). White dashed lines delimitate the proximal, central and distal regions. White star points to the broken microtubule wall. (N) Centrin coverage (% of the total tubulin length) along the centriole in the indicated conditions.


( O ) Representative images of expanded U2OS expressing GFP alone or GFP + SFI1 - RR and treated with siCT or siSFI1 . Cells were stained for α / β - tubulin ( αβTub , magenta ) and SFI1 ( green ) or Centrin2 / 3 ( grey ) . Red arrowhead indicates abnormal centriole . ( P ) Percentage of abnormal centrioles in the indicated conditions .

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(O) Representative images of expanded U2OS expressing GFP alone or GFP+SFI1-RR and treated with siCT or siSFI1. Cells were stained for α/β-tubulin (αβTub, magenta) and SFI1 (green) or Centrin2/3 (grey). Red arrowhead indicates abnormal centriole. (P) Percentage of abnormal centrioles in the indicated conditions.


( F ) Representative widefield images of expanded centrioles during ciliogenesis from siCT and siSFI1 RPE - 1 treated cells . Cells were stained for α / β - tubulin ( magenta ) and SFI1 ( green ) . Yellow asterisks indicate the absence of SFI1 at the distal tip of the centrioles . Red arrowhead indicates abnormal centriole . Scale bar : 250 nm . ( G ) Percentage of RPE - 1 cells with centrioles SFI1 - positive ( intact SFI1 ) , partially depleted ( partial SFI1 ) or totally missing SFI1 ( no SFI1 ) at the distal dot in siSFI - treated cells . ( H ) Percentage of abnormal centrioles in RPE - 1 cells treated with siSFI1 .

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(F) Representative widefield images of expanded centrioles during ciliogenesis from siCT and siSFI1 RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and SFI1 (green). Yellow asterisks indicate the absence of SFI1 at the distal tip of the centrioles. Red arrowhead indicates abnormal centriole. Scale bar: 250 nm. (G) Percentage of RPE-1 cells with centrioles SFI1-positive (intact SFI1), partially depleted (partial SFI1) or totally missing SFI1 (no SFI1) at the distal dot in siSFI-treated cells. (H) Percentage of abnormal centrioles in RPE-1 cells treated with siSFI1.


( I ) Representative widefield images of expanded centrioles during ciliogenesis from siCT and siSFI1 RPE - 1 treated cells . Cells were stained for α / β - tubulin ( magenta ) and CP110 ( yellow ) . ( J ) Percentage of ciliated cells observed in U - ExM under the indicated conditions . ( K ) Percentage of CP110 capped / uncapped centrioles under the indicated conditions .

(I) Representative widefield images of expanded centrioles during ciliogenesis from siCT and siSFI1 RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and CP110 (yellow). (J) Percentage of ciliated cells observed in U-ExM under the indicated conditions. (K) Percentage of CP110 capped/uncapped centrioles under the indicated conditions.


( L ) Representative widefield images of expanded RPE - 1 during ciliogenesis , expressing GFP alone or GFP + SFI1 - RR and treated with siCT or siSFI1 . Cells were stained for α / β - tubulin ( αβTub , magenta ) and CP110 ( yellow ) . Arrowheads indicate a cilium . ( M ) Percentage of ciliated cells observed in U - ExM in the indicated conditions . ( N ) Percentage of CP110 capped / uncapped centrioles in the indicated conditions .

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

(L) Representative widefield images of expanded RPE-1 during ciliogenesis, expressing GFP alone or GFP+SFI1-RR and treated with siCT or siSFI1. Cells were stained for α/β-tubulin (αβTub, magenta) and CP110 (yellow). Arrowheads indicate a cilium. (M) Percentage of ciliated cells observed in U-ExM in the indicated conditions. (N) Percentage of CP110 capped/uncapped centrioles in the indicated conditions.


( A - D ) Representative widefield images of expanded centrioles during ciliogenesis from siCT ( A , B ) and siSFI1 ( C , D ) RPE - 1 treated cells . Cells were stained for α / β - tubulin ( magenta ) and CEP164 ( red hot ) . Note the abnormal organization of the distal appendage protein CEP164 in siSFI1 - treated cells . ( E - H ) Representative widefield images of expanded centrioles during ciliogenesis from siCT ( E , F ) and siSFI1 ( G , H ) RPE - 1 treated cells . Cells were stained for α / β - tubulin ( magenta ) and CEP90 ( cyan ) . ( I ) Frequency distribution of the number of dots per centriole formed by CEP164 in indicated conditions . ( J ) Frequency distribution of the number of dots per centriole formed by CEP90 under the indicated conditions . ( K ) Percentage of cells presenting abnormal CEP164 and CEP90 under the indicated conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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- D) Representative widefield images of expanded centrioles during ciliogenesis from siCT (A, B) and siSFI1 (C, D) RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and CEP164 (red hot). Note the abnormal organization of the distal appendage protein CEP164 in siSFI1-treated cells. (E-H) Representative widefield images of expanded centrioles during ciliogenesis from siCT (E, F) and siSFI1 (G, H) RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and CEP90 (cyan). (I) Frequency distribution of the number of dots per centriole formed by CEP164 in indicated conditions. (J) Frequency distribution of the number of dots per centriole formed by CEP90 under the indicated conditions. (K) Percentage of cells presenting abnormal CEP164 and CEP90 under the indicated conditions.


( D ) The 43 overlapping candidate genes are described in Figure 1C . Known driver genes listed in the Cancer Gene Census ( CGC ) or Oncology Knowledge Base ( OncoKB ) were removed from further analysis . Genes were assessed for a known association with the DDR pathway .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The 43 overlapping candidate genes are described in Figure 1C. Known driver genes listed in the Cancer Gene Census (CGC) or Oncology Knowledge Base (OncoKB) were removed from further analysis. Genes were assessed for a known association with the DDR pathway.


( H ) Immunofluorescence staining of γ . H2 . AX expression of cells described in Figure 1G .

(H) Immunofluorescence staining of γ.H2.AX expression of cells described in Figure 1G.


( D ) Enrichment of control - sgRNA ( n = 5 ) and Slf2 - sgRNA ( n = 6 ) transduced HSPCs over time .

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(D) Enrichment of control-sgRNA (n=5) and Slf2-sgRNA (n=6) transduced HSPCs over time.


( E ) Kaplan - Meier curves of overall survival of mice transplanted with Eµ - myc ; Rosa26Cas9 HSPCs transduced with control - sgRNA ( n = 15 ) or sgRNA targeting Slf2 ( n = 6 ) . p < 0 . 0001 , log - rank ( Mantel - Cox ) test .

(E) Kaplan-Meier curves of overall survival of mice transplanted with Eµ-myc;Rosa26Cas9 HSPCs transduced with control-sgRNA (n=15) or sgRNA targeting Slf2 (n=6). p < 0.0001, log-rank (Mantel-Cox) test.


( F ) Immunohistochemical analysis of Slf2 - sgRNA lymphomas described in Figure 2E . Representative example of three analyzed Slf2 - sgRNA lymphomas .

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(F) Immunohistochemical analysis of Slf2-sgRNA lymphomas described in Figure 2E. Representative example of three analyzed Slf2-sgRNA lymphomas.


( B , C ) Kaplan - Meier overall survival curves of the indicated cohorts of DLBCL patients ( Lenz , NEJM ) . DLBCL patients were classified according to SLF2 or SLF1 mRNA expression . P - value was determined by log - rank ( Mantel - Cox ) test .

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(B, C) Kaplan-Meier overall survival curves of the indicated cohorts of DLBCL patients (Lenz, NEJM). DLBCL patients were classified according to SLF2 or SLF1 mRNA expression. P-value was determined by log-rank (Mantel-Cox) test.


( E ) Mafosphamide dose - response curves of SLF2KO and control SU - DHL - 5 cells . Cells were treated with mafosphamide ( MAF ; 0 , 31 . 25 , 62 . 5 , 125 , 250 , 500 , 1000 , 2000 , 4000 ng / ml ) for 48 hours and viability was determined by DAPI staining and flow cytometry measurement . Error bars represent SD from three independent experiments .

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(E) Mafosphamide dose-response curves of SLF2KO and control SU-DHL-5 cells. Cells were treated with mafosphamide (MAF; 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000 ng/ml) for 48 hours and viability was determined by DAPI staining and flow cytometry measurement. Error bars represent SD from three independent experiments.


( G ) Representative plots of the flow cytometry experiment described in Figure 3F .

(G) Representative plots of the flow cytometry experiment described in Figure 3F.


( A ) Immunoblot analysis of U - 2 - OS cells following CRISPR / Cas9 mediated knockout of SLF2 upon DRB treatment ( 0 . 5 µM ) for indicated times with the indicated antibodies .

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(A) Immunoblot analysis of U-2-OS cells following CRISPR/Cas9 mediated knockout of SLF2 upon DRB treatment (0.5 µM) for indicated times with the indicated antibodies.


( B ) Immunoblot analysis of a panel of human DLBCL cell lines with a specific SLF2 antibody and quantification of the SLF2 western blots ( middle panel ) . Protein expression of SLF2 was normalized to β - Actin expression . The expression level of SLF2 in DB cells was set as 1 . Data are presented as mean + / - SD of n = 3 independent experiments .

(B) Immunoblot analysis of a panel of human DLBCL cell lines with a specific SLF2 antibody and quantification of the SLF2 western blots (middle panel). Protein expression of SLF2 was normalized to β-Actin expression. The expression level of SLF2 in DB cells was set as 1. Data are presented as mean +/- SD of n=3 independent experiments.


( E ) Immunoblot analysis of human SU - DHL - 5 control and SLF2KO cell lines upon DRB treatment ( 0 . 5 µM ) for indicated time points with the indicated antibodies .

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(E) Immunoblot analysis of human SU-DHL-5 control and SLF2KO cell lines upon DRB treatment (0.5 µM) for indicated time points with the indicated antibodies.


( A ) TMT - based MS results of chromatin fractions from SU - DHL - 5 control and SU - DHL - 5 SLF2KO cells . Volcano plot depicting depleted chromatin - associated proteins . Significant hits are shown by red dots ( depleted in SLF2KO cells ) . Proteins belonging to the DDR and cell cycle regulation are labeled . Experiments were performed in triplicates .

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(A) TMT-based MS results of chromatin fractions from SU-DHL-5 control and SU-DHL-5 SLF2KO cells. Volcano plot depicting depleted chromatin-associated proteins. Significant hits are shown by red dots (depleted in SLF2KO cells). Proteins belonging to the DDR and cell cycle regulation are labeled. Experiments were performed in triplicates.


( E ) Immunoblot analysis of human SU - DHL - 5 control and SLF2KO whole cell fractions with the indicated antibodies . ( F ) Immunoblot analysis of human chromatin fractions of SU - DHL - 5 control and SLF2KO cell lines with the indicated antibodies .

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(E) Immunoblot analysis of human SU-DHL-5 control and SLF2KO whole cell fractions with the indicated antibodies. (F) Immunoblot analysis of human chromatin fractions of SU-DHL-5 control and SLF2KO cell lines with the indicated antibodies.


( D ) Quantification of the SAE1 and SAE2 western blots from Figure 6C . Protein expression of SAE1 and SAE2 was normalized to β - Actin protein expression . Data are presented as mean + / - SD of n = 3 independent experiments . P - value determined by unpaired t - test .

(D) Quantification of the SAE1 and SAE2 western blots from Figure 6C. Protein expression of SAE1 and SAE2 was normalized to β-Actin protein expression. Data are presented as mean +/- SD of n=3 independent experiments. P-value determined by unpaired t-test.


( E ) SUMO inhibitor ( SUMOi ( ML - 093 ) ; 0 , 6 . 25 , 12 . 5 , 25 , 50 , 100 , 200 , 400 , 800 nM ) dose response curves of SLF2KO and control SU - DHL - 5 cells . Cells were treated with increasing concentrations of SUMOi for 72 hours and viability was determined by negative DAPI staining and flow cytometry measurement . Error bars represent SD from three independent experiments .

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(E) SUMO inhibitor (SUMOi (ML-093); 0, 6.25, 12.5, 25, 50, 100, 200, 400, 800 nM) dose response curves of SLF2KO and control SU-DHL-5 cells. Cells were treated with increasing concentrations of SUMOi for 72 hours and viability was determined by negative DAPI staining and flow cytometry measurement. Error bars represent SD from three independent experiments.


( F ) Quantification of flow cytometry results for DAPI and Annexin V staining in human SLF2KO and control SU - DHL - 5 cells after 12 . 5 nM of SUMOi treatment for 72 hours . P - value determined by unpaired t - test . Error bars represent SD from three independent experiments .

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(F) Quantification of flow cytometry results for DAPI and Annexin V staining in human SLF2KO and control SU-DHL-5 cells after 12.5 nM of SUMOi treatment for 72 hours. P-value determined by unpaired t-test. Error bars represent SD from three independent experiments.


( B ) Immunoblot analysis of human SU - DHL - 5 control and CLSPNKO cell lines upon DRB treatment ( 0 . 5µM ) for indicated time points with the indicated antibodies .

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(B) Immunoblot analysis of human SU-DHL-5 control and CLSPNKO cell lines upon DRB treatment (0.5µM) for indicated time points with the indicated antibodies.


( C ) Quantification of the p - CHK1 western blots from Figure 7B . Protein expression of p - CHK1 was normalized to β - Actin protein expression . p - CHK1 expression in control cells at 3 hours of doxorubicin treatment was arbitrarily set to 1 . Data are presented as mean + / - SD of n = 3 independent experiments . P - value determined by two - way ANOVA ; Šídák ' s multiple comparisons test .

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(C) Quantification of the p-CHK1 western blots from Figure 7B. Protein expression of p-CHK1 was normalized to β-Actin protein expression. p-CHK1 expression in control cells at 3 hours of doxorubicin treatment was arbitrarily set to 1. Data are presented as mean +/- SD of n=3 independent experiments. P-value determined by two-way ANOVA; Šídák's multiple comparisons test.


( F ) SUMO inhibitor ( SUMOi , TAK981 ; 0 , 6 . 25 , 12 . 5 , 25 , 50 , 100 , 200 , 400 , 800 nM ) dose - response curves of CLSPNKO and control SU - DHL - 5 cells . Cells were treated with increasing concentrations of SUMOi for 72 hours and viability was determined by negative of DAPI staining and flow cytometry measurement . Error bars represent SD from three independent experiments . ( G ) Mafosphamide dose - response curves of CLSPNKO and control SU - DHL - 5 cells . Cells were treated with mafosphamide ( MAF ; 0 , 31 . 25 , 62 . 5 , 125 , 250 , 500 , 1000 , 2000 , 4000 ng / ml ) for 48 hours and viability was determined by negative of DAPI staining and flow cytometry measurement . Error bars represent SD from three independent experiments .

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(F) SUMO inhibitor (SUMOi, TAK981; 0, 6.25, 12.5, 25, 50, 100, 200, 400, 800 nM) dose-response curves of CLSPNKO and control SU-DHL-5 cells. Cells were treated with increasing concentrations of SUMOi for 72 hours and viability was determined by negative of DAPI staining and flow cytometry measurement. Error bars represent SD from three independent experiments. (G) Mafosphamide dose-response curves of CLSPNKO and control SU-DHL-5 cells. Cells were treated with mafosphamide (MAF; 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000 ng/ml) for 48 hours and viability was determined by negative of DAPI staining and flow cytometry measurement. Error bars represent SD from three independent experiments.


( B ) Representative histograms of the flow cytometry experiment described in Figure 8A .

(B) Representative histograms of the flow cytometry experiment described in Figure 8A.


( G ) Quantification of cell viability measured by celltiterGlo from primary murine Eµ - Myc lymphoma derived cells co - treated with indicated concentrations of SUMOi and Rabusertib for 72 hours . Bar plots represent cell viability relative to the DMSO treated control cells . Error bars represent SD from three independent experiments . P - value was determined by ANOVA test ; Tukey ' s post hoc test .

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(G) Quantification of cell viability measured by celltiterGlo from primary murine Eµ-Myc lymphoma derived cells co-treated with indicated concentrations of SUMOi and Rabusertib for 72 hours. Bar plots represent cell viability relative to the DMSO treated control cells. Error bars represent SD from three independent experiments. P-value was determined by ANOVA test; Tukey's post hoc test.


( J ) Tumour size was measured over time ( n = 4 mice in each group ) . Error bars represent SEM . P - value determined by unpaired t - test .

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(J) Tumour size was measured over time (n=4 mice in each group). Error bars represent SEM. P-value determined by unpaired t-test.


C , D Growth rates as measured by the change in optical density ( OD595 ) over time for wild type ( C ) and Torin1 - resistant ( D ) cells upon 5 µM of Torin1 treatment ( at T = 0 min ) . Respective strains were treated with the DMSO vehicle alone to control for potential effects of the solvent on cell growth . Logarithmic values of the OD595 increase are plotted against time in minutes . Data aggregated from 3 independent experiments with error bars representing the standard deviations ( SD ) . Linear regressions were fitted using the curve fitting function on Prism9 . Ratios in the rate of mass doubling ( as calculated from gradient of regression lines detailed in Methods ) of Torin1 treated cells to DMSO control were 0 . 34 in wild type ( C ) and 0 . 97 for Torin1 - resistant ( D ) cells .

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C, D Growth rates as measured by the change in optical density (OD595) over time for wild type (C) and Torin1-resistant (D) cells upon 5 µM of Torin1 treatment (at T = 0 min). Respective strains were treated with the DMSO vehicle alone to control for potential effects of the solvent on cell growth. Logarithmic values of the OD595 increase are plotted against time in minutes. Data aggregated from 3 independent experiments with error bars representing the standard deviations (SD). Linear regressions were fitted using the curve fitting function on Prism9. Ratios in the rate of mass doubling (as calculated from gradient of regression lines detailed in Methods) of Torin1 treated cells to DMSO control were 0.34 in wild type (C) and 0.97 for Torin1-resistant (D) cells.


E Rate of protein synthesis as measured by the amount of HPG incorporation after 20 mins of differing time intervals ( 0 - 60 mins ) of 5 µM Torin1 for wild type ( green ) and tor1 ∆ ( yellow ) . Values are normalised to T = 0 minutes . Exponential one - phase decay non - linear regressions were fitted using the curve fitting function on Prism9 . Data aggregated from 3 independent experiments with error bars representing the standard deviations ( SD ) .

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E Rate of protein synthesis as measured by the amount of HPG incorporation after 20 mins of differing time intervals (0 - 60 mins) of 5 µM Torin1 for wild type (green) and tor1∆ (yellow). Values are normalised to T = 0 minutes. Exponential one-phase decay non-linear regressions were fitted using the curve fitting function on Prism9. Data aggregated from 3 independent experiments with error bars representing the standard deviations (SD).


C - F Phosphorylation changes of the conserved phosphosites , S235 and S236 , on Rps601 ( C ) and Rps602 ( D - F ) , over 85 minutes of Torin1 ( 5 µM ) treatment wild type ( green ) and Torin1 - resistant ( brown ) cells . M - values ( in brackets ) represent the different multiplicities of the same phosphosites . Lines were fitted with the curve fitting function on Prism9 using the the non - linear regressions exponential one - phase decay ( C , D ) or plateau followed by one - phase decay ( E , F ) functions respectively . Grey dashed lines indicate 2 - fold threshold .

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C - F Phosphorylation changes of the conserved phosphosites, S235 and S236, on Rps601(C) and Rps602 (D - F), over 85 minutes of Torin1 (5 µM) treatment wild type (green) and Torin1-resistant (brown) cells. M-values (in brackets) represent the different multiplicities of the same phosphosites. Lines were fitted with the curve fitting function on Prism9 using the the non-linear regressions exponential one-phase decay (C, D) or plateau followed by one-phase decay (E, F) functions respectively. Grey dashed lines indicate 2-fold threshold.


A Relative protein expression levels of the 3 S6 kinase homologues , Psk1 , Sck1 and Sck2 , plotted against time of Torin1 addition . Respective proteins are represented by the different coloured lines as detailed in the legend .

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A Relative protein expression levels of the 3 S6 kinase homologues, Psk1, Sck1 and Sck2, plotted against time of Torin1 addition. Respective proteins are represented by the different coloured lines as detailed in the legend.


B Relative phosphorylation level of the T632 phosphosite identified on Sck1 in the 40 - minute time course study . Phosphorylation levels of both wild type ( green ) and tor1 ∆ ( yellow ) are presented relative to the value for wild type cells at T = 0 minutes . Grey dashed line indicates 2 - fold threshold .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative phosphorylation level of the T632 phosphosite identified on Sck1 in the 40-minute time course study. Phosphorylation levels of both wild type (green) and tor1∆ (yellow) are presented relative to the value for wild type cells at T = 0 minutes. Grey dashed line indicates 2-fold threshold.


A , B Phosphosites identified on ( A ) Tif212 and ( B ) Erf1 respectively showing 2 - fold phosphorylation change within 20 minutes of Torin1 ( 5 µM ) addition . Both wild type ( green ) and tor1 ∆ ( yellow ) phosphorylation values are relative to the starting phosphorylation levels before Torin1 addition in wild type cells ( T = 0 minutes ) . Grey dashed lines indicate 2 - fold threshold .

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A, B Phosphosites identified on (A) Tif212 and (B) Erf1 respectively showing 2-fold phosphorylation change within 20 minutes of Torin1 (5 µM) addition. Both wild type (green) and tor1∆ (yellow) phosphorylation values are relative to the starting phosphorylation levels before Torin1 addition in wild type cells (T=0 minutes). Grey dashed lines indicate 2-fold threshold.


E , F Changes in rates of protein synthesis of the tif471 _ 18A mutant compared to the wild type control ( tif471 + ) upon Torin1 ( 5 µM ) treatment . Residual rates of protein synthesis after 60 minutes of Torin1 treatment was 17 % for the tif471 _ 18A phosphomutant and 4 . 6 % for the tif471 + control relative to starting levels in the respective strains . The two graphs represent the untransformed ( E ) and log2 transformed ( F ) rates respectively . Error bars represent the standard deviation ( SD ) of data aggregated from 3 independent experiments .

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

E, F Changes in rates of protein synthesis of the tif471_18A mutant compared to the wild type control (tif471+) upon Torin1 (5 µM) treatment. Residual rates of protein synthesis after 60 minutes of Torin1 treatment was 17% for the tif471_18A phosphomutant and 4.6% for the tif471+ control relative to starting levels in the respective strains. The two graphs represent the untransformed (E) and log2 transformed (F) rates respectively. Error bars represent the standard deviation (SD) of data aggregated from 3 independent experiments.


A . Representative bright field micrographs of netrin - 1 immunohistochemistry in formalin - fixed paraffin - embedded human ovarian , endometrial and hepatocellular carcinoma ( HCC ) tumor sections . Scale bar 50 µm . B . Quantification by immunoblots of netrin - 1 in the ECM of cancer cells of human tumors ( n = 3 , error bars are SD ) . The vertical line corresponds to the intercellular

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A. Representative bright field micrographs of netrin-1 immunohistochemistry in formalin-fixed paraffin-embedded human ovarian, endometrial and hepatocellular carcinoma (HCC) tumor sections. Scale bar 50 µm. B. Quantification by immunoblots of netrin-1 in the ECM of cancer cells of human tumors(n=3, error bars are SD). The vertical line corresponds to the intercellular zone


F . Analysis of h - netrin - 1 ( human recombinant netrin - 1 ) binding to ECM components : mouse laminin - I ( m - laminin - I ) , recombinant human vitronectin , recombinant human lumican , recombinant human integrin α6β4 , recombinant human glypican - 1 and recombinant human FLTR1 by bio - layer interferometry

F. Analysis of h-netrin-1 (human recombinant netrin-1) binding to ECM components: mouse laminin-I (m-laminin-I), recombinant human vitronectin, recombinant human lumican, recombinant human integrin α6β4, recombinant human glypican-1 and recombinant human FLTR1 by bio-layer interferometry assays


C . NP137 fragments were produced by enzymatic cleavage and subjected to electrophoresis in denaturing or non - denaturing conditions .

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C. NP137 fragments were produced by enzymatic cleavage and subjected to electrophoresis in denaturing or non-denaturing conditions.


D . Bio - layer interferometry analysis of netrin - 1 / NP137 and derivates after NODAGA bioconjugation . Numbers indicate the concentration in nM of NP137 and derivates .

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D. Bio-layer interferometry analysis of netrin-1/NP137 and derivates after NODAGA bioconjugation. Numbers indicate the concentration in nM of NP137 and derivates.


D . Biodistribution properties of NP137 - NODAGA - 111In in a Balb / cJ mouse bearing 4T1 xenografts at 48 h ( 4 mice ) , 72 h ( 4 mice ) and 96 h ( 3 mice ) . Radioactivity incorporation was quantified by the percentage of the injected dose per gram of organ . Error bars indicate s . d . .

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D. Biodistribution properties of NP137-NODAGA-111In in a Balb/cJ mouse bearing 4T1 xenografts at 48 h (4 mice), 72 h (4 mice) and 96 h (3 mice). Radioactivity incorporation was quantified by the percentage of the injected dose per gram of organ. Error bars indicate s.d..


G . Whole body autoradiography of NP137 - NODAGA - 111In in Balb / cJ mice bearing a 4T1 ( top ) or an EMT6 ( bottom ) tumor using a beta imager system .

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G. Whole body autoradiography of NP137-NODAGA-111In in Balb/cJ mice bearing a 4T1 (top) or an EMT6 (bottom) tumor using a beta imager system.


H . Analysis of the autoradiography ratio of NP137 - NODAGA - 111In in tumor versus bone in Balb / cJ mice bearing EMT6 ( n = 14 relative values ) or 4T1 ( n = 24 relative values ) tumors . Whiskers indicate min to max , Central band of the box indicates the median , bottom of the box indicates Q1 and top of the box indicates Q3 .

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H. Analysis of the autoradiography ratio of NP137-NODAGA-111In in tumor versus bone in Balb/cJ mice bearing EMT6 (n= 14 relative values) or 4T1 (n=24 relative values) tumors. Whiskers indicate min to max, Central band of the box indicates the median, bottom of the box indicates Q1 and top of the box indicates Q3.


A . ( Top ) Balb / cJ mice were engrafted with EMT6 cells by subcutaneous injection of 1 million cells . After 5 days , animals were treated by IV injection of PBS , DOTA - NP137 or NP137 - 177Lu . n = 9 animals / group for PBS and DOTA - NP137 ; n = 12 animals / group for NP137 - 177Lu ; p < 0 . 001 between PBS and NP137 - 177Lu and between DOTA - NP137 and NP137 - 177Lu ( Two - way ANOVA ) between PBS and NP137 - 177Lu and between DOTA - NP137 and NP137 - 177Lu . Error bars indicate s . e . m . . ( Bottom ) NP137 - 177Lu survival of mice engrafted with EMT6 cells . Kaplan - Meier survival curves of mice treated or not with DOTA - NP137 . Mantel Cox test ; n = 9 animals / group for PBS and DOTA - NP137 ; n = 12 animals / group considered as dead at a tumor volume of 2 , 000mm3 for NP137 - 177Lu ; p < 0 . 001 between PBS and NP137 - 177Lu and between DOTA - NP137 and NP137 - 177Lu . B . ( Top ) Balb / c mice were engrafted with 4T1 cells by subcutaneous injection of 1 million cells . After 8 days , animals received IV injections of PBS , DOTA - NP137 or NP137 - 177Lu ; n = 10 animals / group for PBS and DOTA - NP137 ; n = 9 animals / group for NP137 - 177Lu ; p < 0 . 001 between PBS and NP137 - 177Lu and between DOTA - NP137 and NP137 - 177Lu ( Two - way ANOVA ) . Error bars indicate s . e . m . . ( Bottom ) NP137 - 177Lu survival of mice engrafted with 4T1 cells . Kaplan - Meier survival curves of mice treated or not with DOTA - NP137 . Mantel Cox test ; n = 10 animals / group for PBS and DOTA - NP137 ; n = 9 animals / group considered as dead at a tumor volume of 2 , 000 mm3 for NP137 - 177Lu ; p = 0 . 002 between PBS and NP137 - 177Lu and between DOTA - NP137 and NP137 - 177Lu . C . ( Top ) NMRI nude mice were engrafted with H358 cells by subcutaneous injection of 5 million cells and considered as dead when tumor volume reached 2 , 000mm3 . After 15 days , animals were treated by IV injection of PBS , DOTA - NP137 or NP137 - 177Lu ; n = 8 animals / group for PBS and DOTA - NP137 ; n = 9 animals / group for NP137 - 177Lu ; p < 0 . 001 between PBS and NP137 - 177Lu and between DOTA - NP137 and NP137 - 177Lu ( 2 - way ANOVA ) between PBS and NP137 - 177Lu and between DOTA - NP137 and NP137 - 177Lu . Error bars indicate s . e . m . ( Bottom ) NP137 - 177Lu survival of mice engrafted with H358 cells . . Kaplan - Meier survival curves of mice treated or not with DOTA - NP137 . Mantel Cox test ; n = 8 animals / group for PBS and DOTA - NP137 ; n = 9 animals / group for NP137 - 177Lu ; p = 0 . 025 between PBS and NP137 - 177Lu and between DOTA - NP137 and NP137 - 177Lu .

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A. (Top) Balb/cJ mice were engrafted with EMT6 cells by subcutaneous injection of 1 million cells. After 5 days, animals were treated by IV injection of PBS, DOTA-NP137 or NP137-177Lu. n = 9 animals/group for PBS and DOTA-NP137; n = 12 animals/group for NP137-177Lu; p < 0.001 between PBS and NP137-177Lu and between DOTA-NP137 and NP137-177Lu (Two-way ANOVA) between PBS and NP137-177Lu and between DOTA-NP137 and NP137-177Lu. Error bars indicate s.e.m.. (Bottom) NP137-177Lu survival of mice engrafted with EMT6 cells. Kaplan-Meier survival curves of mice treated or not with DOTA-NP137. Mantel Cox test; n = 9 animals/group for PBS and DOTA-NP137; n = 12 animals/group considered as dead at a tumor volume of 2,000mm3 for NP137-177Lu; p < 0.001 between PBS and NP137-177Lu and between DOTA-NP137 and NP137-177Lu. B. (Top) Balb/c mice were engrafted with 4T1 cells by subcutaneous injection of 1 million cells. After 8 days, animals received IV injections of PBS, DOTA-NP137 or NP137-177Lu; n = 10 animals/group for PBS and DOTA-NP137; n = 9 animals/group for NP137-177Lu; p < 0.001 between PBS and NP137-177Lu and between DOTA-NP137 and NP137-177Lu (Two-way ANOVA). Error bars indicate s.e.m.. (Bottom) NP137-177Lu survival of mice engrafted with 4T1 cells. Kaplan-Meier survival curves of mice treated or not with DOTA-NP137. Mantel Cox test; n = 10 animals/group for PBS and DOTA-NP137; n = 9 animals/group considered as dead at a tumor volume of 2,000 mm3 for NP137-177Lu; p = 0.002 between PBS and NP137-177Lu and between DOTA-NP137 and NP137-177Lu. C. (Top) NMRI nude mice were engrafted with H358 cells by subcutaneous injection of 5 million cells and considered as dead when tumor volume reached 2,000mm3. After 15 days, animals were treated by IV injection of PBS, DOTA-NP137 or NP137-177Lu; n = 8 animals/group for PBS and DOTA-NP137; n = 9 animals/group for NP137-177Lu; p < 0.001 between PBS and NP137-177Lu and between DOTA-NP137 and NP137-177Lu (2-way ANOVA) between PBS and NP137-177Lu and between DOTA-NP137 and NP137-177Lu. Error bars indicate s.e.m. (Bottom) NP137-177Lu survival of mice engrafted with H358 cells.. Kaplan-Meier survival curves of mice treated or not with DOTA-NP137. Mantel Cox test; n = 8 animals/group for PBS and DOTA-NP137; n = 9 animals/group for NP137-177Lu; p = 0.025 between PBS and NP137-177Lu and between DOTA-NP137 and NP137-177Lu.


D . ( Left ) Quantification of Ki67 expression in EMT6 tumors harvested in Balb / cJ mice 7 days post injection of PBS , DOTA - NP137 or NP137 - 177Lu . ( Right ) Representative bright field micrographs of Ki67 immunohistochemistry in formalin - fixed paraffin - embedded EMT6 tumors harvested in Balb / cJ mice 7 days post injection of PBS , DOTA - NP137 or NP137 - 177Lu ; n = 4 mice per groups ; One - way ANOVA . Error bars indicate s . e . m . Scale bar : 50μm . E . ( Left ) Quantification of cleaved caspase - 3 expression in EMT6 tumors harvested in Balb / cJ mice 7 days post injection of PBS , DOTA - NP137 or NP137 - 177Lu . ( Right ) Representative bright field micrographs of cleaved caspase - 3 immunohistochemistry in formalin - fixed paraffin - embedded EMT6 tumor harvested in Balb / cJ mice 7 days post injection of PBS , DOTA - NP137 or NP137 - 177Lu ; n = 4 mice per groups ; One - way ANOVA . Error bars indicate s . e . m . Scale bar : . 50μm .

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D. (Left) Quantification of Ki67 expression in EMT6 tumors harvested in Balb/cJ mice 7 days post injection of PBS, DOTA-NP137 or NP137-177Lu. (Right) Representative bright field micrographs of Ki67 immunohistochemistry in formalin-fixed paraffin-embedded EMT6 tumors harvested in Balb/cJ mice 7 days post injection of PBS, DOTA-NP137 or NP137-177Lu; n = 4 mice per groups; One-way ANOVA. Error bars indicate s.e.m. Scale bar: 50μm. E. (Left) Quantification of cleaved caspase-3 expression in EMT6 tumors harvested in Balb/cJ mice 7 days post injection of PBS, DOTA-NP137 or NP137-177Lu. (Right) Representative bright field micrographs of cleaved caspase-3 immunohistochemistry in formalin-fixed paraffin-embedded EMT6 tumor harvested in Balb/cJ mice 7 days post injection of PBS, DOTA-NP137 or NP137-177Lu; n = 4 mice per groups; One-way ANOVA. Error bars indicate s.e.m. Scale bar:.50μm.


B . Detection of netrin - 1 expression in the MMTV / neuT model by immunoblots ( Tumor 1 to 3 ) .

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B. Detection of netrin-1 expression in the MMTV/neuT model by immunoblots (Tumor 1 to 3).


E . Biodistribution properties of NP137 - NODAGA - 111In in MMTV / neuT mice at 96 h ( 3 mice ) and measured for all organs . Radioactivity incorporation was quantified by the percentage of the injected dose per gram of organ . Error bars indicate s . d .

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E. Biodistribution properties of NP137-NODAGA-111In in MMTV/neuT mice at 96 h (3 mice) and measured for all organs. Radioactivity incorporation was quantified by the percentage of the injected dose per gram of organ. Error bars indicate s.d.


F . NP137 - 177Lu enhances MMTV / neuT mouse survival . Mice were treated at 3 . 5 months with PBS , DOTA - NP137 or NP137 - 177Lu . Kaplan - Meier survival curves of mice treated or not with NP137 and considered as dead when cumulative tumors volume reached 2 , 000 mm3 . Mantel Cox test ; n = 10 animals / group for PBS ; n = 14 animals / group for DOTA - NP137 ; n = 8 animals / group for NP137 - 177Lu ; p < 0 . 001 between PBS and NP137 - 177Lu and between DOTA - NP137 and NP137 - 177Lu .

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F. NP137-177Lu enhances MMTV/neuT mouse survival. Mice were treated at 3.5 months with PBS, DOTA-NP137 or NP137-177Lu. Kaplan-Meier survival curves of mice treated or not with NP137 and considered as dead when cumulative tumors volume reached 2,000 mm3. Mantel Cox test; n = 10 animals/group for PBS; n = 14 animals/group for DOTA-NP137; n = 8 animals/group for NP137-177Lu; p < 0.001 between PBS and NP137-177Lu and between DOTA-NP137 and NP137-177Lu.


( a ) Miz1ΔPOZNes mice move slowly and lack motor coordination . The panel shows the average speed of movement and time on a rotarod ( maximum : 300 s ) maintained at constant speed of control and age - matched Miz1ΔPOZNes mice . The average age of mice tested was 16 months . P - values were calculated using an unpaired two - tailed Student ' s t - test ( the number of animals tested is shown below each graph ) .

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(a) Miz1ΔPOZNes mice move slowly and lack motor coordination. The panel shows the average speed of movement and time on a rotarod (maximum: 300 s) maintained at constant speed of control and age-matched Miz1ΔPOZNes mice. The average age of mice tested was 16 months. P-values were calculated using an unpaired two-tailed Student's t-test (the number of animals tested is shown below each graph).


( b ) Defect of cerebellar growth in Miz1ΔPOZNes mice . The panels show sagittal sections of brains of control and Miz1ΔPOZNes mice at the indicated age . Dotted lines show the circumference of the cerebellum . For each mouse , the cerebellar surface area was determined ; numbers show the ratio between control and Miz1ΔPOZNes mice . The number of mice analysed for each genotype is indicated on the right . Scale bar , 1 mm .

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(b) Defect of cerebellar growth in Miz1ΔPOZNes mice. The panels show sagittal sections of brains of control and Miz1ΔPOZNes mice at the indicated age. Dotted lines show the circumference of the cerebellum. For each mouse, the cerebellar surface area was determined; numbers show the ratio between control and Miz1ΔPOZNes mice. The number of mice analysed for each genotype is indicated on the right. Scale bar, 1 mm.


( c , d ) Histology of the cerebellum . Sections of the cerebellum of 11 - month - old control ( c ) and Miz1ΔPOZNes ( d , please note different scale ) mice were stained with antibodies directed against calbindin , a marker for PCs , and glial fibrillary acid protein ( GFAP ) , a marker for Bergman glial cells . Boxes indicate the area of a detail picture in the overviews . GFAP staining is shown for adjacent section ( arrowheads : regions of missing PCs ) . Scale bar , 500 μm ( upper panels ) and 100 μm ( lower panels ) .

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(c,d) Histology of the cerebellum. Sections of the cerebellum of 11-month-old control (c) and Miz1ΔPOZNes (d, please note different scale) mice were stained with antibodies directed against calbindin, a marker for PCs, and glial fibrillary acid protein (GFAP), a marker for Bergman glial cells. Boxes indicate the area of a detail picture in the overviews. GFAP staining is shown for adjacent section (arrowheads: regions of missing PCs). Scale bar, 500 μm (upper panels) and 100 μm (lower panels).