Figure 3BsAb 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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Figure 3BsAb 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.


Figure 4B 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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Figure 4B 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.


Figure 5C - 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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Figure 5C-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.


Figure 6B , 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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Figure 6B, 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.


Figure 7The 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 .

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Figure 7The 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.


Figure 1B . 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 . 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 . 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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Figure 1B. 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.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.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).


Figure 2A . 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 . 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 .

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Figure 2A. 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.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.


Figure 3A . 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 ; immunoblotConfocal 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 . 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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Figure 3A. 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; immunoblotConfocal 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.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.


Figure 4A . 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 . 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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Figure 4A. 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.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).


Figure 5A . 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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Figure 5A. 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.


Figure 1 ( 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 ) . ( 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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Figure 1(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).(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).


Figure 3 ( 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 . ( 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 .

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

Figure 3(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.(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.


Figure 4 ( 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 . ( 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 . ( 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 . ( 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 .

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

Figure 4(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.(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.(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.(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.


Figure 5 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.


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

Figure 6(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.


Figure 1 ( 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 .

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

Figure 1(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.


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

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

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


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

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

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


Figure 4 ( 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 . ( 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 .

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

Figure 4(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.(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.


Figure 5 ( 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 .

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

Figure 5(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.


Figure 6 ( 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 . ( 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 .

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

Figure 6(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.(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.


Figure 7 ( 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 . ( 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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Figure 7(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.(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.


Figure 8 ( 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 . ( 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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Figure 8(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.(J) Tumour size was measured over time (n=4 mice in each group). Error bars represent SEM. P-value determined by unpaired t-test.


Figure 1C , 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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Figure 1C, 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).


Figure 2C - 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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Figure 2C - 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.


Figure 4A 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 .

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Figure 4A 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.


Figure 5A , 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 .

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Figure 5A, 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.


Figure 1A . 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 zoneF . 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

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Figure 1A. 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 zoneF. 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


Figure 2C . 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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Figure 2C. 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.


Figure 3D . 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 . 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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Figure 3D. 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.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.


Figure 4A . ( 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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Figure 4A. (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.


Figure 5B . 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 . 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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Figure 5B. 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.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.


f1 ( 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 . ( 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 ) . ( e ) Enhanced apoptosis in the granular layer of cerebella in Miz1ΔPOZNes mice . The upper panel shows representative TUNEL ( terminal deoxynucleotidyl transferase dUTP nick end labelling ) staining of cerebella of age - matched control and Miz1ΔPOZNes mice . The lower panel shows a quantification of staining observed in four control and five Miz1ΔPOZNes mice . Error bars show s . e . m . P - value was calculated using an unpaired two - tailed Student ' s t - test .

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f1(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.(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).(e) Enhanced apoptosis in the granular layer of cerebella in Miz1ΔPOZNes mice. The upper panel shows representative TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining of cerebella of age-matched control and Miz1ΔPOZNes mice. The lower panel shows a quantification of staining observed in four control and five Miz1ΔPOZNes mice. Error bars show s.e.m. P-value was calculated using an unpaired two-tailed Student's t-test.


f2 ( a ) ISH documenting the expression of Miz1 mRNA in PCs of the cerebellum in wild - type mice . Left panel shows ISH , right panel shows haematoxylin and eosin staining . Scale bar , 50 μm . ( b ) IHC documenting the presence of Miz1 in the nuclei of PCs . The panel on the left shows staining with α Miz1 ( H190 ) antibody , staining on the right is a control staining lacking primary antibody . Scale bar , 50 μm .

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f2(a) ISH documenting the expression of Miz1 mRNA in PCs of the cerebellum in wild-type mice. Left panel shows ISH, right panel shows haematoxylin and eosin staining. Scale bar, 50 μm. (b) IHC documenting the presence of Miz1 in the nuclei of PCs. The panel on the left shows staining with α Miz1 (H190) antibody, staining on the right is a control staining lacking primary antibody. Scale bar, 50 μm.


f3 ( a ) Representative examples for ChIP - sequencing traces obtained from NPCs using an α - Miz1 antibody . Input traces are shown as negative control . For each gene , the intron / exon structure is shown below the traces with exons indicated as vertical bars and the transcriptional start site ( TSS ) at the left . An enlarged picture of the peak region is shown on the right . ( b ) Miz1 binds predominantly to core promoters . The panel shows a histogram of the percentage of promoters with the indicated distances between the peak of the Miz1 - binding site and the next TSS . Of all the peaks , 140 / 261 ( 54 % ) are located in core promoters ( ± 1 . 5 kb ) . ( c ) Miz1 binding is conserved across different cell types and species . The plot shows a Venn diagram documenting the overlap in Miz1 - bound promoters between murine NPCs and human MDA - MB231 cells ( a mammary epithelial tumour cell line ) . The P - value was calculated using a hypergeometric test . Note that different α - Miz1 antibodies ( H190 and G18 ) were used for the analysis of mouse and human Miz1 - binding sites . ( e ) Electrophoretic mobility shift assay documenting binding of Miz1 to the conserved sequence element ( ' Miz1 - binding motif ' ) . Nuclear extracts from control HeLa cells ( ' Mock ' ) or cells ectopically expressing Miz1 were incubated with radioactive oligonucleotides spanning the Miz1 - binding motif of the Ambra1 and Vps28 promoters . Where indicated , binding reactions included a 25 - fold excess of non - labelled oligonucleotides ( ' Competitor ' ) , the indicated mutants thereof or of an oligonucleotide containing an E - box sequence from the Ubap2 promoter . Supershift assays were performed using α - Miz1 ( H190 , G18 and C19 ) , α - Myc ( N262 ) or control antibodies . * Non - specific band . The sequences of the oligonucleotides used are shown above the panel ; mutated nucleotides are shown in bold .

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

f3(a) Representative examples for ChIP-sequencing traces obtained from NPCs using an α-Miz1 antibody. Input traces are shown as negative control. For each gene, the intron/exon structure is shown below the traces with exons indicated as vertical bars and the transcriptional start site (TSS) at the left. An enlarged picture of the peak region is shown on the right.(b) Miz1 binds predominantly to core promoters. The panel shows a histogram of the percentage of promoters with the indicated distances between the peak of the Miz1-binding site and the next TSS. Of all the peaks, 140/261 (54%) are located in core promoters (±1.5 kb).(c) Miz1 binding is conserved across different cell types and species. The plot shows a Venn diagram documenting the overlap in Miz1-bound promoters between murine NPCs and human MDA-MB231 cells (a mammary epithelial tumour cell line). The P-value was calculated using a hypergeometric test. Note that different α-Miz1 antibodies (H190 and G18) were used for the analysis of mouse and human Miz1-binding sites.(e) Electrophoretic mobility shift assay documenting binding of Miz1 to the conserved sequence element ('Miz1-binding motif'). Nuclear extracts from control HeLa cells ('Mock') or cells ectopically expressing Miz1 were incubated with radioactive oligonucleotides spanning the Miz1-binding motif of the Ambra1 and Vps28 promoters. Where indicated, binding reactions included a 25-fold excess of non-labelled oligonucleotides ('Competitor'), the indicated mutants thereof or of an oligonucleotide containing an E-box sequence from the Ubap2 promoter. Supershift assays were performed using α-Miz1 (H190, G18 and C19), α-Myc (N262) or control antibodies. *Non-specific band. The sequences of the oligonucleotides used are shown above the panel; mutated nucleotides are shown in bold.


f4 ( a ) Miz1 activates the Rorc and Ambra1 promoters , and the POZ domain is required for activation . HEK293 cells were transiently transfected with plasmids containing promoter sequences of Ambra1 or Rorc in front of luciferase and with expression vectors encoding Miz1 or MizΔPOZ . Empty vectors were used as control . Values were normalized with CMV - βGal . Error bars are s . d . of biological triplicates . ( b ) Direct target genes of Miz1 are downregulated in cerebella of Miz1ΔPOZNes mice . Microarray analysis was performed from cerebella of mice at the indicated ages . The panels show box plots ( line : median , box : 50 % of data points , whisker : 1 . 5 interquantile range , outliers are not shown ) of relative gene expression ( plotted as log2 - fold change ) of the 140 Miz1 direct target genes comparing control and Miz1ΔPOZNes mice . The P - value was calculated using a Mann - Whitney - Wilcoxon test . Microarrays were performed as technical duplicates from RNA pooled from two animals for each genotype . ( d ) qRT - PCR documenting the expression of selected Miz1 target genes in cerebella of control and of Miz1ΔPOZNes mice at the indicated ages . Data represent the mean of two independent biological experiments each with three technical replicates . Error bars are s . e . m .

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f4(a) Miz1 activates the Rorc and Ambra1 promoters, and the POZ domain is required for activation. HEK293 cells were transiently transfected with plasmids containing promoter sequences of Ambra1 or Rorc in front of luciferase and with expression vectors encoding Miz1 or MizΔPOZ. Empty vectors were used as control. Values were normalized with CMV-βGal. Error bars are s.d. of biological triplicates.(b) Direct target genes of Miz1 are downregulated in cerebella of Miz1ΔPOZNes mice. Microarray analysis was performed from cerebella of mice at the indicated ages. The panels show box plots (line: median, box: 50% of data points, whisker: 1.5 interquantile range, outliers are not shown) of relative gene expression (plotted as log2-fold change) of the 140 Miz1 direct target genes comparing control and Miz1ΔPOZNes mice. The P-value was calculated using a Mann-Whitney-Wilcoxon test. Microarrays were performed as technical duplicates from RNA pooled from two animals for each genotype.(d) qRT-PCR documenting the expression of selected Miz1 target genes in cerebella of control and of Miz1ΔPOZNes mice at the indicated ages. Data represent the mean of two independent biological experiments each with three technical replicates. Error bars are s.e.m.


f5 ( a , b ) Immunostaining using an α - ubiquitin antibody of ( a ) PCs and ( b ) the molecular layer of Miz1ΔPOZNes and age - matched control mice ( dotted area : higher magnification ) . Arrowheads designate ubiquitin - positive structures . Scale bar , ( a ) 25 μm ; ( b ) 50 μm . ( c ) Immunofluorescence staining using an α - p62 and an α - ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age - matched control mice . Nuclei were counterstained with DAPI ( 4 ' , 6 - diamidino - 2 - phenylindole ) and overviews of the cerebella clarifying the selected areas are shown . Scale bar , 25 μm . ( a , b ) Immunostaining using an α - ubiquitin antibody of ( a ) PCs and ( b ) the molecular layer of Miz1ΔPOZNes and age - matched control mice ( dotted area : higher magnification ) . Arrowheads designate ubiquitin - positive structures . Scale bar , ( a ) 25 μm ; ( b ) 50 μm . ( c ) Immunofluorescence staining using an α - p62 and an α - ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age - matched control mice . Nuclei were counterstained with DAPI ( 4 ' , 6 - diamidino - 2 - phenylindole ) and overviews of the cerebella clarifying the selected areas are shown . Scale bar , 25 μm . ( d ) Quantification of a , b and c . Error bars represent s . e . m . derived from the indicated numbers of animals . P - values were calculated using an unpaired two - tailed Student ' s t - test . ( e ) Immunoblots of Triton X - 100 soluble ( ' TX100 ' ) and insoluble ( ' 1 % SDS ' ) fractions of cerebella obtained from control and Miz1ΔPOZNes mice of the indicated ages . Blots were probed with antibodies recognizing either mono - or polyubiquitinated proteins . Staining of the membrane with amido black was used as loading control . ( f ) Transmission electron microscopy documenting the accumulation of large multilamellar bodies in PC bodies ( ' PC ' ) and Purkinje dendrites ( ' D ' ) of Miz1ΔPOZNes mice ( N , nucleus ) . Such structures were not observed in age - matched control mice ( as quantified in Supplementary Fig . S7b ) . Mean age of mice for all electron microscopy pictures is 13 monthsfor Miz1ΔPOZNes and 12 . 5 months for control mice . Morphologically similar structures accumulate in neurons of mice deficient in late steps of autophagy . Scale bars , 2 , 0 . 2 , 0 . 5 and 1 μm , respectively .

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

f5(a,b) Immunostaining using an α-ubiquitin antibody of (a) PCs and (b) the molecular layer of Miz1ΔPOZNes and age-matched control mice (dotted area: higher magnification). Arrowheads designate ubiquitin-positive structures. Scale bar, (a) 25 μm; (b) 50 μm.(c) Immunofluorescence staining using an α-p62 and an α-ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age-matched control mice. Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole) and overviews of the cerebella clarifying the selected areas are shown. Scale bar, 25 μm.(a,b) Immunostaining using an α-ubiquitin antibody of (a) PCs and (b) the molecular layer of Miz1ΔPOZNes and age-matched control mice (dotted area: higher magnification). Arrowheads designate ubiquitin-positive structures. Scale bar, (a) 25 μm; (b) 50 μm. (c) Immunofluorescence staining using an α-p62 and an α-ubiquitin antibody of the white matter of cerebella from Miz1ΔPOZNes and age-matched control mice. Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole) and overviews of the cerebella clarifying the selected areas are shown. Scale bar, 25 μm. (d) Quantification of a, b and c. Error bars represent s.e.m. derived from the indicated numbers of animals. P-values were calculated using an unpaired two-tailed Student's t-test.(e) Immunoblots of Triton X-100 soluble ('TX100') and insoluble ('1% SDS') fractions of cerebella obtained from control and Miz1ΔPOZNes mice of the indicated ages. Blots were probed with antibodies recognizing either mono- or polyubiquitinated proteins. Staining of the membrane with amido black was used as loading control.(f) Transmission electron microscopy documenting the accumulation of large multilamellar bodies in PC bodies ('PC') and Purkinje dendrites ('D') of Miz1ΔPOZNes mice (N, nucleus). Such structures were not observed in age-matched control mice (as quantified in Supplementary Fig. S7b). Mean age of mice for all electron microscopy pictures is 13 monthsfor Miz1ΔPOZNes and 12.5 months for control mice. Morphologically similar structures accumulate in neurons of mice deficient in late steps of autophagy. Scale bars, 2, 0.2, 0.5 and 1 μm, respectively.


f6 ( a ) Genomic PCR documenting recombination of the floxed Miz1 alleles in Cre - ER - positive and - negative MEFs . 4 - OHT was added to the media for 4 days . ( b ) qRT - PCR assays of Miz1 target genes in Miz1flox / floxCre - ER − and Miz1flox / floxCre - ER + MEFs . The experiment was performed independently from four embryos with identical results . Error bars depict s . d . of technical triplicates from one experiment . ( c ) Immunoblots using an α - Lc3 antibody detecting Lc3 - I and conjugated Lc3 - II . 4 - OHT - treated and control - treated MEFs of the indicated genotype were incubated with leupeptin and ammonium chloride ( lys PI ) for 2 h in DMEM ( + amino acids ) or in EBSS ( − amino acids ) medium . The expression of α - tubulin ( upper panels ) or α - vinculin ( lower panels ) was used as loading control . ( d ) qRT - PCR assays of Miz1 target genes in control and Miz1ΔPOZ MEFs after incubation in DMEM or in EBSS . Error bars depict s . d . of technical triplicates from one experiment . ( e ) qRT - PCR assays documenting expression of the indicated Miz1 target genes in control and Miz1ΔPOZ neurospheres ; assays were performed at passage three . s . e . m . values are derived from six biological replicates , each with technical triplicates . ( f ) Recombination of Miz1flox / floxCre - ER + neurospheres was tested by genomic PCR . 4 - OHT was added to the media for 3 days . ( g ) Immunoblots using an α - Lc3 antibody detecting Lc3 - I and Lc3 - II . 4 - OHT - treated and non - treated Miz1flox / floxCre - ER + neurospheres were incubated with leupeptin and ammonium chloride ( lys PI ) for 30 min . The expression of α - tubulin was used as loading control . ( h ) Fluorescent microscopy of SHEP cells stably infected with lentiviruses expressing Lc3 - GFP and Lamp1 - RFP ( to mark lysosomes ) . Vps28 was depleted with siRNAs and a non - targeting pool ( siNTP ) was used as a control . Note the accumulation of Lc3 in vesicles that do not overlap with lysosomes upon depletion of Vps28 . Scale bar , 25 μm .

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

f6(a) Genomic PCR documenting recombination of the floxed Miz1 alleles in Cre-ER-positive and -negative MEFs. 4-OHT was added to the media for 4 days.(b) qRT-PCR assays of Miz1 target genes in Miz1flox/floxCre-ER− and Miz1flox/floxCre-ER+ MEFs. The experiment was performed independently from four embryos with identical results. Error bars depict s.d. of technical triplicates from one experiment.(c) Immunoblots using an α-Lc3 antibody detecting Lc3-I and conjugated Lc3-II. 4-OHT-treated and control-treated MEFs of the indicated genotype were incubated with leupeptin and ammonium chloride (lys PI) for 2 h in DMEM (+amino acids) or in EBSS (−amino acids) medium. The expression of α-tubulin (upper panels) or α-vinculin (lower panels) was used as loading control.(d) qRT-PCR assays of Miz1 target genes in control and Miz1ΔPOZ MEFs after incubation in DMEM or in EBSS. Error bars depict s.d. of technical triplicates from one experiment.(e) qRT-PCR assays documenting expression of the indicated Miz1 target genes in control and Miz1ΔPOZ neurospheres; assays were performed at passage three. s.e.m. values are derived from six biological replicates, each with technical triplicates.(f) Recombination of Miz1flox/floxCre-ER+ neurospheres was tested by genomic PCR. 4-OHT was added to the media for 3 days.(g) Immunoblots using an α-Lc3 antibody detecting Lc3-I and Lc3-II. 4-OHT-treated and non-treated Miz1flox/floxCre-ER+ neurospheres were incubated with leupeptin and ammonium chloride (lys PI) for 30 min. The expression of α-tubulin was used as loading control.(h) Fluorescent microscopy of SHEP cells stably infected with lentiviruses expressing Lc3-GFP and Lamp1-RFP (to mark lysosomes). Vps28 was depleted with siRNAs and a non-targeting pool (siNTP) was used as a control. Note the accumulation of Lc3 in vesicles that do not overlap with lysosomes upon depletion of Vps28. Scale bar, 25 μm.