Duolink analysis ( A ) and its quantification ( B ) of the interaction by assessing the PLA complex in mitotic cells after BCL9 or Axin1 knockdown ( n = 20 ) . Scale bars represent 5 μm .

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Duolink analysis (A) and its quantification (B) of the interaction by assessing the PLA complex in mitotic cells after BCL9 or Axin1 knockdown (n=20). Scale bars represent 5 μm.


Duolink analysis of the interaction of BCL9 and CLTC in mitotic cells . Scale bars represent 5 μm .

Duolink analysis of the interaction of BCL9 and CLTC in mitotic cells. Scale bars represent 5 μm.


Immunoprecipitation analysis of the interaction of truncated BCL9 forms in mitosis ; the purple stars indicate the domain required for clathrin complex binding , and the green stars indicate the domains for β - catenin complex binding . The domain required for both clathrin and β - catenin complexes binding is indicated by a red line .

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

Immunoprecipitation analysis of the interaction of truncated BCL9 forms in mitosis; the purple stars indicate the domain required for clathrin complex binding, and the green stars indicate the domains for β-catenin complex binding. The domain required for both clathrin and β-catenin complexes binding is indicated by a red line.


Quantification of the relative binding of the truncated forms with the clathrin complex or β - catenin complex ; the red frame indicates the common domains of BCL9 for binding of both complexes .

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

Quantification of the relative binding of the truncated forms with the clathrin complex or β-catenin complex; the red frame indicates the common domains of BCL9 for binding of both complexes.


Immunoprecipitation analysis of the BCL9 - MF interaction in CLTC knockdown mitotic cells .

Immunoprecipitation analysis of the BCL9-MF interaction in CLTC knockdown mitotic cells.


Immunoprecipitation analysis of the endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF - overexpressing mitotic cells .

Immunoprecipitation analysis of the endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF-overexpressing mitotic cells.


Immunoblotting analysis of signalosome components by immunoblotting in BCL9 , CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX . Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX .

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

Immunoblotting analysis of signalosome components by immunoblotting in BCL9, CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX. Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX.


Immunofluorescence confocal analysis of Ser1490 - phosphorylated LRP6 and CLTC co - localization in mitotic HeLa cells ; white arrows indicate the colocalization . Scale bars represent 5 μm .

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Immunofluorescence confocal analysis of Ser1490-phosphorylated LRP6 and CLTC co-localization in mitotic HeLa cells; white arrows indicate the colocalization. Scale bars represent 5 μm.


Immunofluorescence analysis of α - tubulin , Axin1 and CLTC triple - staining in mitotic cells . The white arrows indicate the colocalization . Scale bars represent 5 μm .

Immunofluorescence analysis of α-tubulin, Axin1 and CLTC triple-staining in mitotic cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.


Immunofluorescence analysis of colocalization of LRP6CAGFP and CLTC or CD63 in interphase cells . The white arrows indicate the colocalization . Scale bars represent 5 μm .

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Immunofluorescence analysis of colocalization of LRP6CAGFP and CLTC or CD63 in interphase cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.


Quantification of signalosome components and mitotic Wnt target expression level by immunoblotting in natural mitotic cells treated for 1 hour with DMSO or MDC , an inhibitor of clathrin - mediated - endocytosis .

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

Quantification of signalosome components and mitotic Wnt target expression level by immunoblotting in natural mitotic cells treated for 1 hour with DMSO or MDC, an inhibitor of clathrin-mediated-endocytosis.


A representative image of soft agar oncogenic colony formation assays for stable knockdown cells .

A representative image of soft agar oncogenic colony formation assays for stable knockdown cells.


A representative image of the mouse xenograft tumours for stable knockdown cells at the end point of the experiment .

A representative image of the mouse xenograft tumours for stable knockdown cells at the end point of the experiment.


In vitro CDK1 / cyclin B1 kinase assay for immunoprecipitation - purified BCL9 - Flag or T172A - Flag proteins .

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In vitro CDK1/cyclin B1 kinase assay for immunoprecipitation-purified BCL9-Flag or T172A-Flag proteins.


Immunoprecipitation analysis of transiently - expressed BCL9 - MF or T172A - MF interactors in synchronised cells , from G1S to G2 M phase . The relative level of BCL9 - MF or T172A - MF immunoprecipitation is determined ; red numbers indicate up - regulation , and green numbers indicate down - regulation compared to the starting time point .

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

Immunoprecipitation analysis of transiently-expressed BCL9-MF or T172A-MF interactors in synchronised cells, from G1S to G2 M phase. The relative level of BCL9-MF or T172A-MF immunoprecipitation is determined; red numbers indicate up-regulation, and green numbers indicate down-regulation compared to the starting time point.


Immunoprecipitation analysis of the transiently - expressed BCL9 - MF interaction in mitotic cells treated with RO3306 .

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Immunoprecipitation analysis of the transiently-expressed BCL9-MF interaction in mitotic cells treated with RO3306.


Immunoprecipitation analysis of the interaction of the T172 inactive mutant T172A , the activation mutant T172D , and the double inactive mutant T172 \ T144 ( 2A ) or activation mutant ( 2D ) transiently - expressed in mitotic cells .

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Immunoprecipitation analysis of the interaction of the T172 inactive mutant T172A, the activation mutant T172D, and the double inactive mutant T172\T144 (2A) or activation mutant (2D) transiently-expressed in mitotic cells.


Quantification for the inactive or activation mutant interactions indicated in Fig 6F . The wild type BCL9 interaction is normalised to 1 , indicated by a green dashed line with an arrow .

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Quantification for the inactive or activation mutant interactions indicated in Fig 6F. The wild type BCL9 interaction is normalised to 1, indicated by a green dashed line with an arrow.


Immunohistochemical staining of p - T172 - BCL9 expression in a human HCC tissue . MN : matched normal tissues , T : tumour tissues . Scale bar represents1000 μm .

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Immunohistochemical staining of p-T172-BCL9 expression in a human HCC tissue. MN: matched normal tissues, T: tumour tissues. Scale bar represents1000 μm.


IHC staining and the expression scored in a tissue array of 75 pairs of human lung cancer patient tissues . Scale bar represents 1000 μm .

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IHC staining and the expression scored in a tissue array of 75 pairs of human lung cancer patient tissues. Scale bar represents 1000 μm.


A representative figure of p - T172 - BCL9 IHC staining in HCC to indicate the expression in mitotic or in interphase cells . Scale bar represents 100 μm .

A representative figure of p-T172-BCL9 IHC staining in HCC to indicate the expression in mitotic or in interphase cells. Scale bar represents 100 μm.


Representative figures of p - T172 - BCL9 IHC staining in interphase cells of breast cancer ( D ) or lung cancer ( E ) patient tissue . Scale bars represent 100 μm .

Representative figures of p-T172-BCL9 IHC staining in interphase cells of breast cancer (D) or lung cancer (E) patient tissue. Scale bars represent 100 μm.


Immunoprecipitation analysis of the interaction in 5 pairs of human HCC patient tissues by a p - T172 - BCL9 antibody or a BCL9 antibody . CLTC relative binding with BCL9 was determined .

Immunoprecipitation analysis of the interaction in 5 pairs of human HCC patient tissues by a p-T172-BCL9 antibody or a BCL9 antibody. CLTC relative binding with BCL9 was determined.


Log - rank ( Mantel - Cox ) survival analysis based on high and low p - T172 - BCL9 expression scored by immunohistochemical staining in two tissue arrays with ( G ) 75 lung cancer samples or ( H ) 145 breast cancer samples .

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

Log-rank (Mantel-Cox) survival analysis based on high and low p-T172-BCL9 expression scored by immunohistochemical staining in two tissue arrays with (G) 75 lung cancer samples or (H) 145 breast cancer samples.


WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin - expressing STm Cell pellets collected at the indicated time points after STm infection were lysed , fractionated by SDS - PAGE and immunoblotted for caspase - 1 ( E , F ) ( E Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) bands ( E ) ( F , Quantification of band intensities for caspase - 1 p20 normalized to pro - caspase - 1 and actin ( E ) from three independent experiments are shown as fold change ( F Data information : Data represent mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test .

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WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin-expressing STm Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for caspase-1 (E, F) (E Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20) bands (E) (F, Quantification of band intensities for caspase-1 p20 normalized to pro-caspase-1 and actin (E) from three independent experiments are shown as fold change (F Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test.


WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin - expressing STm Cell pellets collected at the indicated time points after STm infection were lysed , fractionated by SDS - PAGE and immunoblotted for GSDMD or LC - 3 G ) . Representative immunoblots showing GSDMD full length ( GSDMD FL ) , cleaved GSDMD N - terminal fragment ( GSDMD N - ter ) , LC3 - II and LC3 - I bands in cell lysates ( G ) . H , Quantification of band intensities for GSDMD N - ter normalized to actin ( G ) and LC3 - II normalized to LC3 - I and actin ( H ) from three independent experiments are shown as fold change , H ) Data information : Data represent mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test .

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

WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin-expressing STm Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for GSDMD or LC-3 G). Representative immunoblots showing GSDMD full length (GSDMD FL), cleaved GSDMD N-terminal fragment (GSDMD N-ter), LC3-II and LC3-I bands in cell lysates (G). H, Quantification of band intensities for GSDMD N-ter normalized to actin (G) and LC3-II normalized to LC3-I and actin (H) from three independent experiments are shown as fold change , H) Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test.


Intestinal CD11c + DCs were isolated from colons of WT and SLC15A4feeble mice on day 16 and cultured overnight . B . Cell pellets from three independent experiments were immunoblotted for LC3 and actin . Upper panel . Representative immunoblot . Lower panel . Quantification of band intensities for LC3 - II normalized to LC3 - I and actin are shown as fold induction relative to samples from untreated mice . Note that LC3 - I signal from ex vivo samples is almost undetectable . Data information : Mean is indicated . * * p < 0 . 01 . Mann - Whitney non - parametric statistical test .

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

Intestinal CD11c+ DCs were isolated from colons of WT and SLC15A4feeble mice on day 16 and cultured overnight. B. Cell pellets from three independent experiments were immunoblotted for LC3 and actin. Upper panel. Representative immunoblot. Lower panel. Quantification of band intensities for LC3-II normalized to LC3-I and actin are shown as fold induction relative to samples from untreated mice. Note that LC3-I signal from ex vivo samples is almost undetectable. Data information: Mean is indicated. **p<0.01. Mann-Whitney non-parametric statistical test.


WT or SLC15A4feeble BMDCs expressing ASC - GFP and mCherry - LC3 ( A , B were infected with flagellin - expressing STm and fixed A . Cells were analyzed by fluorescence microscopy . Representative images showing ASC speck ( green ) relative to mCherry - LC3 ( red ) in two infected WT and SLC15A4feeble DCs each . B . Quantification of LC3 fluorescence per unit area in a radius of 1 μm surrounding the ASC speck in 20 cells per cell type in each of 3 independent experiments . N , nucleus . Corresponding DIC images show nuclear position . Scale bar , 8 μm . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - tailed Student ' s t - test .

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WT or SLC15A4feeble BMDCs expressing ASC-GFP and mCherry-LC3 (A, B were infected with flagellin-expressing STm and fixed A. Cells were analyzed by fluorescence microscopy. Representative images showing ASC speck (green) relative to mCherry-LC3 (red) in two infected WT and SLC15A4feeble DCs each. B. Quantification of LC3 fluorescence per unit area in a radius of 1 μm surrounding the ASC speck in 20 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.


WT or SLC15A4feeble BMDCs non - transduced ( C ) were infected with flagellin - expressing STm and fixed C . Cells were stained for endogenous ASC and p62 , labeled with DAPI and analyzed by fluorescence microscopy . Representative images showing ASC speck ( red ) relative to p62 ( green ) in two infected WT and SLC15A4feeble DCs each . Note p62 staining surrounding ASC specks in SLC15A4feeble DCs . D . Quantification of perinuclear ( within a radius of 3 μm from the nucleus ) ASC specks in at least 30 cells per cell type in each of 3 independent experiments . N , nucleus . Corresponding DIC images show nuclear position . Scale bar , 8 μm . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - tailed Student ' s t - test .

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

WT or SLC15A4feeble BMDCs non-transduced (C) were infected with flagellin-expressing STm and fixed C. Cells were stained for endogenous ASC and p62, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) relative to p62 (green) in two infected WT and SLC15A4feeble DCs each. Note p62 staining surrounding ASC specks in SLC15A4feeble DCs. D. Quantification of perinuclear (within a radius of 3 μm from the nucleus) ASC specks in at least 30 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.


SLC15A4feeble BMDCs transduced with SLC15A4 - GFP ( E were infected with flagellin - expressing STm and fixed ( E , Cells were stained for endogenous ASC , labeled with DAPI and analyzed by fluorescence microscopy . Representative images showing ASC speck ( red ) and SLC15A4 - GFP ( green ) in SLC15A4feeble DCs together with non - transduced SLC15A4feeble DCs . Note the perinuclear positioning of ASC specks in the transduced cells ( E ) . N , nucleus . Dotted white lines , cell outlines . Corresponding DIC images show nuclear position . Scale bar , 8 μm .

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

SLC15A4feeble BMDCs transduced with SLC15A4-GFP (E were infected with flagellin-expressing STm and fixed (E, Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) and SLC15A4-GFP (green) in SLC15A4feeble DCs together with non-transduced SLC15A4feeble DCs. Note the perinuclear positioning of ASC specks in the transduced cells (E). N, nucleus. Dotted white lines, cell outlines. Corresponding DIC images show nuclear position. Scale bar, 8 μm.


SLC15A4feeble BMDCs transduced with SLC15A4 - GFP F , were infected with flagellin - expressing STm and fixed Quantification of perinuclear ( within a radius of 3 μm from the nucleus ) and non - perinuclear ASC specks in at least 30 cells per cell type in each of three independent experiments ( F ) . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - tailed Student ' s t - test .

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

SLC15A4feeble BMDCs transduced with SLC15A4-GFP F, were infected with flagellin-expressing STm and fixed Quantification of perinuclear (within a radius of 3 μm from the nucleus) and non-perinuclear ASC specks in at least 30 cells per cell type in each of three independent experiments (F). Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.


SLC15A4feeble BMDCs transduced with SLC15A4 - GFP G ) were infected with flagellin - expressing STm and lysed ( G ) 1 h after infection . G . Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) , LC3 - II and LC3 - I , and actin bands .

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

SLC15A4feeble BMDCs transduced with SLC15A4-GFP G) were infected with flagellin-expressing STm and lysed (G) 1 h after infection. G. Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), LC3-II and LC3-I, and actin bands.


WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) were infected with flagellin - expressing STm . ( A , B ) . Cell pellets collected 1 h after STm infection were lysed , fractionated by SDS - PAGE and immunoblotted for phospho ( P ) and total mTOR , ULK1 kinase , p70 kinase and S6 , FLAG and actin . Representative immunoblots . Non - specific band right above FLAG - RagBQ99L is indicated with an asterisk ( A ) . Quantification of band intensities for P - mTOR normalized to total mTOR , P - ULK1 normalized to total ULK1 , P - p70 normalized to total p70 and P - S6 normalized to S6 , in transduced cells from 3 independent experiments are shown as fold change relative to WT control ( B ) . Data information : Data represent mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test .

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

WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) were infected with flagellin-expressing STm. (A, B). Cell pellets collected 1 h after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for phospho (P) and total mTOR, ULK1 kinase, p70 kinase and S6, FLAG and actin. Representative immunoblots. Non-specific band right above FLAG-RagBQ99L is indicated with an asterisk (A). Quantification of band intensities for P-mTOR normalized to total mTOR, P-ULK1 normalized to total ULK1, P-p70 normalized to total p70 and P-S6 normalized to S6, in transduced cells from 3 independent experiments are shown as fold change relative to WT control (B). Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.


WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) ( E , Cell pellets were probed for caspase - 1 , GSDMD , actin ( E , Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) , GSDMD full length ( GSDMD FL ) and cleaved GSDMD N - terminal fragment ( GSDMD N - ter ) bands .

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WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) (E, Cell pellets were probed for caspase-1, GSDMD, actin (E, Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands.


WT or SLC15A4feeble BMDCs transduced with non - target control , ATG5 or ATG7 shRNAs G ) G ) . Cell pellets were probed for caspase - 1 , GSDMD , actin , ATG5 and ATG 7 ( G ) . Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) and cleaved p20 ( casp . - 1 p20 ) , GSDMD full length ( GSDMD FL ) and cleaved GSDMD N - terminal fragment ( GSDMD N - ter ) bands .

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WT or SLC15A4feeble BMDCs transduced with non-target control, ATG5 or ATG7 shRNAs G) G). Cell pellets were probed for caspase-1, GSDMD, actin , ATG5 and ATG 7 (G). Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands.


WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) H , I ) and mcherry - LC3 ( H , I ) , ( H , I ) . Cells were stained for endogenous ASC , labeled with DAPI and analyzed by fluorescence microscopy . Representative images showing ASC speck ( green ) and LC3 ( red ) in SLC15A4feeble DCs ( H ) . Quantification of perinuclear ( within a radius of 3 μm from the nucleus ) in at least 30 cells per cell type in each of three independent experiments ( I ) . N , nucleus . Corresponding DIC images show nuclear position . Scale bar , 8 μ Data information : Data represent mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . Two - tailed Student ' s t - test .

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WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) H, I) and mcherry-LC3 (H, I), (H, I). Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (green) and LC3 (red) in SLC15A4feeble DCs (H). Quantification of perinuclear (within a radius of 3 μm from the nucleus) in at least 30 cells per cell type in each of three independent experiments (I). N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μ Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.


F . Microscopic images of the Q97 - GFP fluorescence . Bars , 5 µm . G . The percentage of cells containing or lacking detectable aggregates was quantified . Cell boundaries are indicated by dashed lines .

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F. Microscopic images of the Q97-GFP fluorescence. Bars, 5 µm. G. The percentage of cells containing or lacking detectable aggregates was quantified. Cell boundaries are indicated by dashed lines.


E . Q97 - GFP was expressed in mia40 - 3 and corresponding wild type cells for the times indicated . Cells were harvested , washed and survivors were counted after plating on glucose plates . Mean values and standard deviations of three replicates are shown .

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E. Q97-GFP was expressed in mia40-3 and corresponding wild type cells for the times indicated. Cells were harvested, washed and survivors were counted after plating on glucose plates. Mean values and standard deviations of three replicates are shown.


B . Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h . 25 , Q25 - GFP ; 97 , Q97 - GFP ; ev , empty vector .

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

B. Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h. 25, Q25-GFP; 97, Q97-GFP; ev, empty vector.


C . Microscopy images of the indicated strains 12 h after shifting them to galactose . Note that in GAL - Mia40 cells the form and number of aggregates is very different to WT cells . Bar , 5 µm .

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C. Microscopy images of the indicated strains 12 h after shifting them to galactose. Note that in GAL-Mia40 cells the form and number of aggregates is very different to WT cells. Bar, 5 µm.


E . PolyQ proteins were expressed for 24 h before survivors were counted . Mean values and standard deviations from three independent experiments are shown .

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E. PolyQ proteins were expressed for 24 h before survivors were counted. Mean values and standard deviations from three independent experiments are shown.


B . Still frames after different times of growth indicate a uniform fluorescence in Q25 - GFP - expressing cells . Q97 - GFP expression in wild type leads to many small and scattered aggregates per cell . In GAL - Mia40 cells , Q97 - GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell . The arrow depicts aggregate formation . Bars , 4 µm . C . Quantified signal intensities in cells of the indicated mutants . In top and middle graphs , each curve corresponds to a single cell . Averaged signals ( mean + SEM ) in bottom graph ( N = 20 for WT , N = 17 cells for GAL - Mia40 ) .

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B. Still frames after different times of growth indicate a uniform fluorescence in Q25-GFP-expressing cells. Q97-GFP expression in wild type leads to many small and scattered aggregates per cell. In GAL-Mia40 cells, Q97-GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell. The arrow depicts aggregate formation. Bars, 4 µm. C. Quantified signal intensities in cells of the indicated mutants. In top and middle graphs, each curve corresponds to a single cell. Averaged signals (mean + SEM) in bottom graph (N=20 for WT, N=17 cells for GAL-Mia40).


B . Q97 - GFP was expressed for 4 . 5 h in wild type and GAL - Mia40 cells in two biological replicates each . Cells were lysed with NP - 40 before soluble proteins were quantified . Mitochondrial proteins ( mito . ) and members of the mitochondrial carrier family ( carrier ) are visualized in a volcano plot . P - values were derived from a moderated t - test . Mitochondrial ( mito . ) and carrier proteins are indicated by yellow and red dots , respectively . Rnq1 is indicated in blue .

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B. Q97-GFP was expressed for 4.5 h in wild type and GAL-Mia40 cells in two biological replicates each. Cells were lysed with NP-40 before soluble proteins were quantified. Mitochondrial proteins (mito.) and members of the mitochondrial carrier family (carrier) are visualized in a volcano plot. P-values were derived from a moderated t-test. Mitochondrial (mito.) and carrier proteins are indicated by yellow and red dots, respectively. Rnq1 is indicated in blue.


I . Cells of the newly made strains were grown on galactose for 1 or 7 days , respectively . Cells were lysed before the distribution of Rnq1 in the soluble and pellet fractions was quantified . Shown are mean values and standard deviations of three independent replicates .

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I. Cells of the newly made strains were grown on galactose for 1 or 7 days, respectively. Cells were lysed before the distribution of Rnq1 in the soluble and pellet fractions was quantified. Shown are mean values and standard deviations of three independent replicates.


C . ∆ ura3 cells expressing the Oxa1 - Ura3 reporter for the cytosolic accumulation of mitochondrial precursor proteins ( Hansen et al . , 2018 ) were transformed with plasmids expressing the proteins TDP - 43 , the TDP - 43Q331K mutant , Q25 - GFP , Q97 - GFP , α - synuclein , FUS or GFP from a constitutive glyceraldehyde - 3 - phosphate dehydrogenase ( GPD ) promoter . Note that the expression of all aggregation - prone proteins ( indicated by red arrows ) allows cells to grow on uracil - deficient plates indicating the cytosolic accumulation of the Oxa1 - Ura3 fusion protein .

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C. ∆ura3 cells expressing the Oxa1-Ura3 reporter for the cytosolic accumulation of mitochondrial precursor proteins (Hansen et al., 2018) were transformed with plasmids expressing the proteins TDP-43, the TDP-43Q331K mutant, Q25-GFP, Q97-GFP, α-synuclein, FUS or GFP from a constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. Note that the expression of all aggregation-prone proteins (indicated by red arrows) allows cells to grow on uracil-deficient plates indicating the cytosolic accumulation of the Oxa1-Ura3 fusion protein.


E . Wild type cells were transformed with plasmids for the expression of the indicated proteins . Q97 - GFPnF is a non - fluorescent version of Q97 - GFP . Close proximity of the two split GFP parts results in fluorescence which was visualized by microscopy . Shown are mean values and standard deviations of three replicates . Bar , 5 µm .

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E. Wild type cells were transformed with plasmids for the expression of the indicated proteins. Q97-GFPnF is a non-fluorescent version of Q97-GFP. Close proximity of the two split GFP parts results in fluorescence which was visualized by microscopy. Shown are mean values and standard deviations of three replicates. Bar, 5 µm.


B . The open reading frames of the indicated proteins were cloned downstream of a GAL promoter in plasmids and transformed into cells harboring the Q97 - GFP expression plasmid . Cells harboring an empty vector ( ev ) were used for control . Cells were shifted to galactose medium overnight before they were dropped onto glucose or galactose plates . If cells are directly dropped from lactate medium onto galactose plates , only individual cells ( indicated by red protein names ) escaped the polyQ toxicity ( see Fig . EV4C ) .

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B. The open reading frames of the indicated proteins were cloned downstream of a GAL promoter in plasmids and transformed into cells harboring the Q97-GFP expression plasmid. Cells harboring an empty vector (ev) were used for control. Cells were shifted to galactose medium overnight before they were dropped onto glucose or galactose plates. If cells are directly dropped from lactate medium onto galactose plates, only individual cells (indicated by red protein names) escaped the polyQ toxicity (see Fig. EV4C).


D . Cells expressing the indicated carrier proteins from GAL promoters were dropped onto galactose plates . Cell growth was quantified by densitometry from one representative plate . OM refers to carrier proteins that were expressed as fusions with an N - terminal outer membrane anchor . See Fig . EV4D , E for scans of the respective plates .

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D. Cells expressing the indicated carrier proteins from GAL promoters were dropped onto galactose plates. Cell growth was quantified by densitometry from one representative plate. OM refers to carrier proteins that were expressed as fusions with an N-terminal outer membrane anchor. See Fig. EV4D, E for scans of the respective plates.


B . SH - SY5Y cells co - expressing Q97 - GFP with MIA40 ( middle row ) , ∆ N - MIA40 ( lower row ) or a mock - transfected control ( upper row ) were analyzed by immunocytochemistry and super - resolution microscopy . The magnification shows an enlarged section of the TOM20 staining . TOM20 and TRAP1 serve as markers for the mitochondrial outer membrane and the matrix , respectively . Bars , 20 µm . Magnifications : 40fold of a 63x objective field .

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B. SH-SY5Y cells co-expressing Q97-GFP with MIA40 (middle row), ∆N-MIA40 (lower row) or a mock-transfected control (upper row) were analyzed by immunocytochemistry and super-resolution microscopy. The magnification shows an enlarged section of the TOM20 staining. TOM20 and TRAP1 serve as markers for the mitochondrial outer membrane and the matrix, respectively. Bars, 20 µm. Magnifications: 40fold of a 63x objective field.


D . SH - SY5Y cells co - expressing Q97 - GFP with MIA40 ( upper row ) or ∆ N - MIA40 ( lower row ) were analyzed by immunocytochemistry and super - resolution microscopy . The magnification shows an enlarged section of the TRAP1 staining used as a marker for the mitochondrial matrix , respectively . Bars , 20 µm . Magnifications : 40fold of a 63x objective field .

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D. SH-SY5Y cells co-expressing Q97-GFP with MIA40 (upper row) or ∆N-MIA40 (lower row) were analyzed by immunocytochemistry and super-resolution microscopy. The magnification shows an enlarged section of the TRAP1 staining used as a marker for the mitochondrial matrix, respectively. Bars, 20 µm. Magnifications: 40fold of a 63x objective field.


Immunoblot analysis of presenilin expression and endoproteolysis in cell lysates of untransfected HEK293 / sw cells endogenously expressing presenilin and single cell clones overexpressing PS1 WT or the indicated PS1 mutants .

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Immunoblot analysis of presenilin expression and endoproteolysis in cell lysates of untransfected HEK293/sw cells endogenously expressing presenilin and single cell clones overexpressing PS1 WT or the indicated PS1 mutants.


Immunoblot analysis of total Aβ secretion and the production of different Aβ species ( shown in low and high immunoblot exposures ) in conditioned media of cell

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Immunoblot analysis of total Aβ secretion and the production of different Aβ species (shown in low and high immunoblot exposures) in conditioned media of cell lines


Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of cell lines


Immunoblot analysis of C99 - PS1 NTF or full - length ( FL ) crosslinks ( CL ) of the indicated mutants . Samples that were not UV - irradiated were loaded to control for specificity .

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Immunoblot analysis of C99-PS1 NTF or full-length (FL) crosslinks (CL) of the indicated mutants. Samples that were not UV-irradiated were loaded to control for specificity.


Changes in the ratio of crosslink efficiencies at positions T48 and L49 compared to corresponding controls ( n = 4 - 5 biological replicates ) . Changes in the ratio of crosslink efficiencies at positions K54 and V44 compared to corresponding controls ( n = 4 - 5 biological replicates ) .

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Changes in the ratio of crosslink efficiencies at positions T48 and L49 compared to corresponding controls (n = 4-5 biological replicates). Changes in the ratio of crosslink efficiencies at positions K54 and V44 compared to corresponding controls (n = 4-5 biological replicates).


ELISA analysis of Aβ species in conditioned medium of HEK293 / sw cells treated with RO7019009 or vehicle ( DMSO ) . Aβ levels are shown relative to DMSO vehicle control ( n = 4 biological replicates ) .

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ELISA analysis of Aβ species in conditioned medium of HEK293/sw cells treated with RO7019009 or vehicle (DMSO). Aβ levels are shown relative to DMSO vehicle control (n = 4 biological replicates).


Dose response curve of RO7019009 in HEK293 / sw cells measured using the MSD sandwich - immunoassay ( n = 3 biological replicates ) .

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Dose response curve of RO7019009 in HEK293/sw cells measured using the MSD sandwich-immunoassay (n = 3 biological replicates).


Dose response curves for inhibition of Aβ42 generation and Notch cleavage by RO7019009 in different cell lines . Comparison of in vitro potencies for Aβ42 inhibition in HEK293 / sw cells , IC50 = 14 nM ( n = 3 ) ( as in ( C ) ) with those in H4 / sw cells , IC50 = 5 nM ( n = 7 ) and N2a / sw cells , IC50 = 2 nM ( n = 6 ) together with that of Notch cleavage inhibition in a HEK293 - based Notch1 luciferase reporter cell line , IC50 > 10 µM ( n = 2 ) ( all n numbers represent biological replicates ) .

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Dose response curves for inhibition of Aβ42 generation and Notch cleavage by RO7019009 in different cell lines. Comparison of in vitro potencies for Aβ42 inhibition in HEK293/sw cells, IC50 = 14 nM (n = 3) (as in (C)) with those in H4/sw cells, IC50 = 5 nM (n = 7) and N2a/sw cells, IC50 = 2 nM (n = 6) together with that of Notch cleavage inhibition in a HEK293-based Notch1 luciferase reporter cell line, IC50 > 10 µM (n = 2) (all n numbers represent biological replicates).


Left panel : Immunoblot analysis of total Aβ in conditioned media of HEK293 / sw cells treated with RO7019009 or vehicle ( DMSO ) . Total APPs levels were analyzed to control for normal APP secretion and equal sample loading . Right panel : Quantification of relative Aβ amounts in ( E ) ( n = 2 biological replicates ) .

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Left panel: Immunoblot analysis of total Aβ in conditioned media of HEK293/sw cells treated with RO7019009 or vehicle (DMSO). Total APPs levels were analyzed to control for normal APP secretion and equal sample loading. Right panel: Quantification of relative Aβ amounts in (E) (n = 2 biological replicates).


Immunoblot analysis of Aβ species in conditioned media of RO7019009 treated HEK293 / sw cells after separation by Tris - Bicine urea SDS PAGE ; synthetic Aβ peptides were loaded as size markers .

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Immunoblot analysis of Aβ species in conditioned media of RO7019009 treated HEK293/sw cells after separation by Tris-Bicine urea SDS PAGE; synthetic Aβ peptides were loaded as size markers.


ELISA analysis of Aβ species in conditioned media of HEK293 / sw cells overexpressing WT or mutant PS1 treated with RO7019009 or vehicle ( DMSO ) showing the secretion of Aβ38 ( A ) , Aβ43 ( B ) and Aβ42 ( C ) compared to total Aβ ( Aβ38 + Aβ40 + Aβ42 + Aβ43 ) ( n = 5 biological replicates ) . Note that in some experiments Aβ38 generation was below the detection limit for some mutants . Data are presented as mean ± SD .

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ELISA analysis of Aβ species in conditioned media of HEK293/sw cells overexpressing WT or mutant PS1 treated with RO7019009 or vehicle (DMSO) showing the secretion of Aβ38 (A), Aβ43 (B) and Aβ42 (C) compared to total Aβ (Aβ38 + Aβ40 + Aβ42 + Aβ43) (n = 5 biological replicates). Note that in some experiments Aβ38 generation was below the detection limit for some mutants. Data are presented as mean ± SD.


Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of HEK293 / sw cells overexpressing WT or mutant PS1 treated with 500 nM RO7019009 or vehicle ( DMSO ) .

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Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of HEK293/sw cells overexpressing WT or mutant PS1 treated with 500 nM RO7019009 or vehicle (DMSO).


Immunoblot analysis of C99 V44Bpa crosslinking ( CL ) to PS1 WT or NTFs ( circle ) of normally endoproteolysed mutants in the presence of vehicle ( DMSO ) or RO7019009 . Quantitation of crosslink efficiencies of C99 V44Bpa to the WT or mutant NTF in ( A ) . Immunoblot analysis of C99 V44Bpa crosslinking to full - length ( FL ) PS1 ( triangle ) of endoproteolysis - deficient mutants and the corresponding M292D control in the presence of vehicle ( DMSO ) or RO7019009 . Quantitation of crosslink efficiencies of C99 V44Bpa to the M292 or FAD mutant FL PS1 in ( C ) .

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Immunoblot analysis of C99 V44Bpa crosslinking (CL) to PS1 WT or NTFs (circle) of normally endoproteolysed mutants in the presence of vehicle (DMSO) or RO7019009. Quantitation of crosslink efficiencies of C99 V44Bpa to the WT or mutant NTF in (A). Immunoblot analysis of C99 V44Bpa crosslinking to full-length (FL) PS1 (triangle) of endoproteolysis-deficient mutants and the corresponding M292D control in the presence of vehicle (DMSO) or RO7019009. Quantitation of crosslink efficiencies of C99 V44Bpa to the M292 or FAD mutant FL PS1 in (C).


IES retention PCR ( cropped inverted images ) . Four maternally - controlled IES and four non - maternally controlled IESs are shown . The IES + band represents retained IES ; the IES - band represents an excised IES ; additional bands are likely PCR artefacts . IRS is IES retention Score for the IESs calculated after whole genome sequencing .

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IES retention PCR (cropped inverted images). Four maternally-controlled IES and four non-maternally controlled IESs are shown. The IES+ band represents retained IES; the IES- band represents an excised IES; additional bands are likely PCR artefacts. IRS is IES retention Score for the IESs calculated after whole genome sequencing.


( A ) Schematic drawing of the life cycle stages of Paramecium tetraurelia . MIC and parental MAC are represented in red , representing the DAPI signal , and developing MAC ( dm ) is represented in green until fully developed , representing the GFP signal . ( B ) ISWI1 - tagged C - terminally with GFP localizes in the developing MAC as soon as developing new MACs ( panel Early Development ) become visible and remain there throughout late MAC development ( panel Late Development ) . Red : DAPI , Green : ISWI1 - GFP . Blue arrows identify developing MAC ;

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(A) Schematic drawing of the life cycle stages of Paramecium tetraurelia. MIC and parental MAC are represented in red, representing the DAPI signal, and developing MAC (dm) is represented in green until fully developed, representing the GFP signal. (B) ISWI1-tagged C-terminally with GFP localizes in the developing MAC as soon as developing new MACs (panel Early Development) become visible and remain there throughout late MAC development (panel Late Development). Red: DAPI, Green: ISWI1-GFP. Blue arrows identify developing MAC;


( C ) Western blot analysis using anti - HA antibody after coimmunoprecipitation of ISWI1 - 3XFlagHA fusion protein . Non - transformed cells ( WT ) of the same strain were used as the negative control . 1 % of the total lysate was loaded as Input and 20 % of co - immunoprecipitated samples were loaded on 12 % SDS gel .

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(C) Western blot analysis using anti-HA antibody after coimmunoprecipitation of ISWI1-3XFlagHA fusion protein. Non-transformed cells (WT) of the same strain were used as the negative control. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 12% SDS gel.


( D ) Volcano plot illustrating the distribution of proteins identified in label - free MS in WT Vs ISWI1 - 3XFlagHA . Significantly abundant proteins ( fold change ≥ 4 ) are highlighted in orange .

(D) Volcano plot illustrating the distribution of proteins identified in label-free MS in WT Vs ISWI1-3XFlagHA. Significantly abundant proteins (fold change ≥ 4) are highlighted in orange.


( E ) Western blot analysis using anti - HA and anti - GFP antibodies after coimmunoprecipitation of Ptiwi01 - 3XFlagHA fusion protein co - transformed with ISWI1 - GFP . Non - transformed cells ( WT ) of the same strain and ISWI1 - GFP fusion protein transformation were used as negative controls . 1 % of the total lysate was loaded as Input and 20 % of co - immunoprecipitated samples were loaded on 10 % SDS gel .

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(E) Western blot analysis using anti-HA and anti-GFP antibodies after coimmunoprecipitation of Ptiwi01-3XFlagHA fusion protein co-transformed with ISWI1-GFP. Non-transformed cells (WT) of the same strain and ISWI1-GFP fusion protein transformation were used as negative controls. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 10%SDS gel.


B . Internalization ( cytoplasm ) and recycling ( plasma membrane ) of FGF7 - and FGF10 - stimulated endogenous FGFR2 ( green ) for 0 , 15 , 40 , and 120 min . in T47D . TRITC - Tf is a marker of recycling ( red ) . Nuclei are stained in blue . * , cells with receptor recycled to the plasma membrane . Scale bar , 5 μm . C . Red and green pixels overlap fraction representing the co - localization of FGFR2b with the recycling endosomes marker Tf ( above ) and the proportion of green over total pixels representing FGFR2b in the cytoplasm ( below ) upon stimulation with FGF7 ( dark green ) or FGF10 ( burgundy ) for 15 , 40 , or 120 min . . Values represent the median ± SD of at least 3 independent experiments . Representative pictures are shown in B ( cytoplasm ) . * , p value < 0 . 005 ( Student ' s t - test ) . D

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B. Internalization (cytoplasm) and recycling (plasma membrane) of FGF7- and FGF10-stimulated endogenous FGFR2 (green) for 0, 15, 40, and 120 min. in T47D. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm. C. Red and green pixels overlap fraction representing the co-localization of FGFR2b with the recycling endosomes marker Tf (above) and the proportion of green over total pixels representing FGFR2b in the cytoplasm (below) upon stimulation with FGF7 (dark green) or FGF10 (burgundy) for 15, 40, or 120 min.. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in B (cytoplasm). *, p value<0.005 (Student's t-test). D


H . Internalization ( cytoplasm ) and recycling ( plasma membrane ) of FGF10 - stimulated HA - FGFR2b ( green ) transfected in BT549 stimulated for 0 , 40 and 120 min . . Cells were depleted or not of TTP or RCP by a pool of siRNAs . TRITC - Tf is a marker of recycling ( red ) . Nuclei are stained in blue . * , cells with receptor recycled to the plasma membrane . Scale bar , 5 μm .

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H. Internalization (cytoplasm) and recycling (plasma membrane) of FGF10-stimulated HA-FGFR2b (green) transfected in BT549 stimulated for 0, 40 and 120 min.. Cells were depleted or not of TTP or RCP by a pool of siRNAs. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm.


( J ) control or 40 min . FGF10 - or TGFα - stimulated T47D left untreated or depleted of TTP , followed or not by transfection with siRNA resistant Flag - TTP ( Flag - TTP * ) or depleted of RCP followed or not by transfection with siRNA resistant RCP - GFP ( RCP - GFP * ) were immunoblotted with the indicated antibodies . * * , non - specific band ( J ) .

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(J) control or 40 min. FGF10- or TGFα-stimulated T47D left untreated or depleted of TTP, followed or not by transfection with siRNA resistant Flag-TTP (Flag-TTP*) or depleted of RCP followed or not by transfection with siRNA resistant RCP-GFP (RCP-GFP*) were immunoblotted with the indicated antibodies. **, non-specific band (J).


F , G Lysates from T47D or BT20 treated with DMSO , PD173074 , AG1478 , or the MEK inhibitors U0126 and MEK162 and stimulated or not with either FGF10 or TGFα ( F ) ; T47D or BT20 treated with DMSO , or the p38 inhibitor BMS582949 and stimulated or not with either FGF10 or TGFα ( G ) were immunoblotted with the indicated antibodies .

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F, G Lysates from T47D or BT20 treated with DMSO, PD173074, AG1478, or the MEK inhibitors U0126 and MEK162 and stimulated or not with either FGF10 or TGFα (F); T47D or BT20 treated with DMSO, or the p38 inhibitor BMS582949 and stimulated or not with either FGF10 or TGFα (G) were immunoblotted with the indicated antibodies.


Lysates from T47D stimulated or not with FGF10 , EGF for different time periods or treated with PD173074 ( C ) , MEK162 ( D ) , AG1478 ( E ) before stimulation were immunoblotted with the indicated antibodies .

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Lysates from T47D stimulated or not with FGF10, EGF for different time periods or treated with PD173074 (C), MEK162 (D), AG1478 (E) before stimulation were immunoblotted with the indicated antibodies.


G , H Percentage of EdU incorporation in T47D pre - treated or not with FGF10 for 40 min . and stimulated or not with FGF10 or EGF ( G ) or incubated with MEK162 , PD173074 , or AG1478 before pre - treatment ( H ) . N = 6 . P value = < 0 . 05 * , < 0 . 01 * * , < 0 . 001 * * * ( one - way ANOVA with Tukey test ) .

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

G, H Percentage of EdU incorporation in T47D pre-treated or not with FGF10 for 40 min. and stimulated or not with FGF10 or EGF (G) or incubated with MEK162, PD173074, or AG1478 before pre-treatment (H). N= 6. P value = <0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).


A . Co - localization of FGFR2 ( red ) , EGFR ( green ) , and the recycling marker Tf ( blue ) in T47D stimulated or not with FGF10 or TGFα for 40 min . Scale bar , 5 μm . C . Co - localization of EGFR ( green ) , T693 phosphorylated EGFR ( blue ) , and the recycling marker Tf ( red ) in T47D stimulated or not with FGF10 or TGFα for 40 min . Scale bar , 5 μm .

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A. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm. C. Co-localization of EGFR (green), T693 phosphorylated EGFR (blue), and the recycling marker Tf (red) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm.


E . Co - localization of FGFR2 ( red ) , EGFR ( green ) , and the recycling marker Tf ( blue ) in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A and stimulated or not with either FGF10 or TGFα for the indicated time periods . Scale bar , 5 μm .

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E. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A and stimulated or not with either FGF10 or TGFα for the indicated time periods. Scale bar, 5 μm.


E , F Lysates from T47D ( E ) and BT20 ( F ) depleted of EGFR , transfected with wt or T693A , and stimulated or not with either FGF10 or TGFα for the indicated time intervals were immunoblotted with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O 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 Lysates from T47D (E) and BT20 (F) depleted of EGFR, transfected with wt or T693A, and stimulated or not with either FGF10 or TGFα for the indicated time intervals were immunoblotted with the indicated antibodies.


A - D Percentage of EdU incorporation in T47D ( A , C ) or BT20 ( B , D ) depleted of EGFR or not , transfected with wt or T693A , and stimulated or not with FGF10 ( A , B ) or with TGFα ( C , D ) and pre - treated ( C , D ) or not ( A , B ) with FGF10 for 40 min . Values represent the median ± SD of N = 4 . P value = < 0 . 05 * , < 0 . 01 * * , < 0 . 001 * * * ( one - way ANOVA with Tukey test ) .

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A-D Percentage of EdU incorporation in T47D (A, C) or BT20 (B, D) depleted of EGFR or not, transfected with wt or T693A, and stimulated or not with FGF10 (A, B) or with TGFα (C, D) and pre-treated (C, D) or not (A, B) with FGF10 for 40 min. Values represent the median ± SD of N= 4. P value =<0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).


A , B Temporal expression of Snail genes in the intestinal tract in ( A ) embryonic E12 . 5 to E18 . 5 and ( B ) postnatal P0 to P20 measured by qRT - PCR . Shown are means ± SD .

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

A, B Temporal expression of Snail genes in the intestinal tract in (A) embryonic E12.5 to E18.5 and (B) postnatal P0 to P20 measured by qRT-PCR. Shown are means ± SD.


C , E , G , I Immunohistochemical staining for Snai1 / 2 ( Abcam ab85931 ) ( C ) E14 . 5midgut , ( E ) E14 . 5hind gut , ( G ) postnatal day 14 SI , ( I ) postnatal day 14 colon . Snai1immunohistochemistry peptide competition controls are shown as inserts ( C ' , E ' , G ' , I ' ) . Scale bars : 20 mm . D , F , H , J , K X ‐ gal staining for β ‐ galactosidase activity in tissue from Snai2LacZ mice stained in whole mount , sectioned and counterstained with nuclear fast red ( D ) E14 . 5midgut , ( F ) E14 . 5hind gut , ( H ) postnatal day 14 SI , ( J ) postnatal day 14 colon , ( K ) adult SI . Controls for X ‐ gal staining are shown as inserts ( D ' , F ' , H ' , J ' ) . L , M , N Double immunostaining of small intestinalcrypts for Snai1 / 2 ( red ) and ( M ) Lgr5 ‐ GFP ( green ) ; ( N ) merge shows Snai1 is present in both CBCstem cells and proliferating transit amplifying cells . Scale bars : 10 mm . O , P Immunohistochemistry for Snai1 using a different primary antibody ( Genetex GTX125918 ) shows minimal expression of Snai1 in villi . ( P ) Expression of Snai1 is detected at the base of crypts ( P ) with strong nuclear expression in CBCstem cells indicated by arrows . Scale bars : 20 mm ( O ) , 10 mm ( P ) . Q Schematic diagram showing Snai1 expression ( red ) within different cell types in the crypt . SI = small intestine . See also Supplementary Fig S1 .

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C, E, G, I Immunohistochemical staining for Snai1/2 (Abcam ab85931) (C) E14.5midgut, (E) E14.5hind gut, (G) postnatal day 14 SI, (I) postnatal day 14 colon. Snai1immunohistochemistry peptide competition controls are shown as inserts (C', E', G', I'). Scale bars: 20 mm.D, F, H, J, K X‐gal staining for β‐galactosidase activity in tissue from Snai2LacZ mice stained in whole mount, sectioned and counterstained with nuclear fast red (D) E14.5midgut, (F) E14.5hind gut, (H) postnatal day 14 SI, (J) postnatal day 14 colon, (K) adult SI. Controls for X‐gal staining are shown as inserts (D', F', H', J').L, M, N Double immunostaining of small intestinalcrypts for Snai1/2 (red) and (M) Lgr5‐GFP (green); (N) merge shows Snai1 is present in both CBCstem cells and proliferating transit amplifying cells. Scale bars: 10 mm.O, P Immunohistochemistry for Snai1 using a different primary antibody (Genetex GTX125918) shows minimal expression of Snai1 in villi. (P) Expression of Snai1 is detected at the base of crypts (P) with strong nuclear expression in CBCstem cells indicated by arrows. Scale bars: 20 mm (O), 10 mm (P).Q Schematic diagram showing Snai1 expression (red) within different cell types in the crypt. SI = small intestine. See also Supplementary Fig S1.


Periodic Acid Schiff ( PAS ) staining was used to detect goblet cells in sections of small intestine from control and Snai1 KO mice . Graph shows number of goblet cells per crypt - villus axes ( n = 3 ) . Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells ( marked by arrows ) . Quantification of enteroendocrine cells per crypt - villus axes in control and Snai1 KO tissue revealed a significant increase in the number of enteroendocrine cells following Snai1 loss ( n = 3 , P = 0 . 004 ) . Immunohistochemistry for lysozyme was used to detect Paneth cells . Quantification of Paneth cells in control and Snai1 KO indicates a significant increase in Paneth cell numbers following knockout of Snai1 ( n = 4 , P = 0 . 002 ) .

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

Periodic Acid Schiff (PAS) staining was used to detect goblet cells in sections of small intestine from control and Snai1 KO mice. Graph shows number of goblet cells per crypt-villus axes (n = 3).Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells (marked by arrows). Quantification of enteroendocrine cells per crypt-villus axes in control and Snai1 KO tissue revealed a significant increase in the number of enteroendocrine cells following Snai1 loss (n = 3, P = 0.004).Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cells in control and Snai1 KO indicates a significant increase in Paneth cell numbers following knockout of Snai1 (n = 4, P = 0.002).


Measurement of villus length ( μm ) ( n = 3 , P = 0 . 013 ) and quantification of enterocytes per crypt - villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss ( n = 3 , P = 0 . 036 ) . Measurement of villus length ( μm ) ( n = 3 , P = 0 . 003 ) 8 days after β ‐ NF induction demonstrates a significant shortening of villi . Bars represent mean ± SD . Two ‐ tailed Student ' s t ‐ test was used to assess significance . Scale bars : 20 μm .

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Measurement of villus length (μm) (n = 3, P = 0.013) and quantification of enterocytes per crypt-villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss (n = 3, P = 0.036).Measurement of villus length (μm) (n = 3, P = 0.003) 8 days after β‐NF induction demonstrates a significant shortening of villi. Bars represent mean ± SD. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 20 μm.


Sections from control and Snai1 KO small intestine were stained for PCNA to detect proliferating cells ( arrows depicting proliferating CBC stem cells at the base of crypts ) . Quantification of total PCNA ‐ positive cells per crypt and PCNA ‐ positive cells in the base of crypts both revealed a significant reduction in the number of proliferating cells after conditional depletion of Snai1 ( n = 4 , P = 0 . 049 , 0 . 026 ) . Scale bars : 20 μm .

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Sections from control and Snai1 KO small intestine were stained for PCNA to detect proliferating cells (arrows depicting proliferating CBC stem cells at the base of crypts). Quantification of total PCNA‐positive cells per crypt and PCNA‐positive cells in the base of crypts both revealed a significant reduction in the number of proliferating cells after conditional depletion of Snai1 (n = 4, P = 0.049, 0.026). Scale bars: 20 μm.


The expression of 2 CBCstem cell markers Olfm4 and Lgr5 were examined by in situ hybridisation in control and Snai1KO small intestine . Lgr5 and Olfm4in situ hybridisation staining shows a loss of CBCstem cell marker expression after loss of Snai1 ( high magnification inserts depict a representative crypt demarcated by a dotted line ) . Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry . Scale bars : 20 μm .

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The expression of 2 CBCstem cell markers Olfm4 and Lgr5 were examined by in situ hybridisation in control and Snai1KO small intestine. Lgr5 and Olfm4in situ hybridisation staining shows a loss of CBCstem cell marker expression after loss of Snai1 (high magnification inserts depict a representative crypt demarcated by a dotted line). Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry. Scale bars: 20 μm.


The analysis of sorted and EGFP ‐ positive cells from Lgr5 ‐ EGFP ‐ IRES ‐ creERT2AhCreSnai1fl / fl and Lgr5 ‐ EGFP ‐ IRES ‐ creERT2AhCreSnai1 + / + mice 5 days after βNF induction show an ˜ tenfold reduction in Lgr5GFP ‐ positive CBCstem cells .

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

The analysis of sorted and EGFP‐positive cells from Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1fl/fl and Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1+/+mice 5 days after βNF induction show an ˜tenfold reduction in Lgr5GFP‐positive CBCstem cells.


Immunohistochemistry for active caspase ‐ 3 to detect apoptotic cells revealed a significant increase in the number of apoptotic cells in both crypt ( n = 5 , P = 0 . 0001 ) and villus ( n = 5 , P = 0 . 005 ) regions in Snai1KO tissue ( arrows mark active caspase ‐ 3 ‐ positive cells ) . Scale bars : 20 μm .

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Immunohistochemistry for active caspase‐3 to detect apoptotic cells revealed a significant increase in the number of apoptotic cells in both crypt (n = 5, P = 0.0001) and villus (n = 5, P = 0.005) regions in Snai1KO tissue (arrows mark active caspase‐3‐positive cells). Scale bars: 20 μm.


A lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1 . Tissue from Lgr5 ‐ EGFP ‐ IRES ‐ creERT2Snai1 + / + ( control ) and Lgr5 ‐ EGFP ‐ IRES ‐ creERT2Snai1fl / fl ( Snai1KO ) that also contained the ROSA26LacZCre reporter allele was stained with X ‐ Gal 5 days after tamoxifen injection . Quantification of X ‐ Gal ‐ positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells . Bars represent mean ± SD ( n = 3 , P = 0 . 0001 ) . Scale bars : 0 . 50 mm . Two ‐ tailed Student ' s t ‐ test was used to assess significance . See also Supplementary Figure S2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1. Tissue from Lgr5‐EGFP‐IRES‐creERT2Snai1+/+ (control) and Lgr5‐EGFP‐IRES‐creERT2Snai1fl/fl (Snai1KO) that also contained the ROSA26LacZCre reporter allele was stained with X‐Gal 5 days after tamoxifen injection. Quantification of X‐Gal‐positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells. Bars represent mean ± SD (n = 3, P = 0.0001). Scale bars: 0.50 mm. Two‐tailed Student's t‐test was used to assess significance. See also Supplementary Figure S2.


VillinCreERT2Snai1fl / florganoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls . Scale bars : 50 μm . Quantification of viable cell growth in VillinCreERT2Snai1fl / florganoids treated with tamoxifen compared to controls over 4 days . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 002 .

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VillinCreERT2Snai1fl/florganoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cell growth in VillinCreERT2Snai1fl/florganoids treated with tamoxifen compared to controls over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.


Assessment of apoptosis in control and VillinCreERT2 Snai1fl / fl organoids after 4 days of growth . Photographs show an overlay of bright ‐ field and fluorescent images with propidium iodide and Hoechst staining . Quantification of apoptotic organoids . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 00001 . Two ‐ tailed Student ' s t ‐ test was used to assess significance . Scale bars : 50 μm . See also Supplementary Figure S3 .

Assessment of apoptosis in control and VillinCreERT2 Snai1fl/fl organoids after 4 days of growth. Photographs show an overlay of bright‐field and fluorescent images with propidium iodide and Hoechst staining. Quantification of apoptotic organoids. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.00001. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 50 μm. See also Supplementary Figure S3.


The consequences of Snai1 knockout were analysed over a time course . Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control . The epithelium is re ‐ populated by wild ‐ type cells as shown on day 60 . Scale bars : 20 μm .

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The consequences of Snai1 knockout were analysed over a time course. Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control. The epithelium is re‐populated by wild‐type cells as shown on day 60. Scale bars: 20 μm.


A PAS staining was used to detect goblet cells in the small intestine , and no differences in cell numbers were observed in test versus control mice ( n = 4 , NS ) .

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A PAS staining was used to detect goblet cells in the small intestine, and no differences in cell numbers were observed in test versus control mice (n = 4, NS).


B Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells . Quantification of the number of enteroendocrine cells per crypt - villus axes revealed a significant decrease in enteroendocrine cell numbers when Snai1 levels are elevated ( n = 4 , P = 0 . 003 ) .

B Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells. Quantification of the number of enteroendocrine cells per crypt-villus axes revealed a significant decrease in enteroendocrine cell numbers when Snai1 levels are elevated (n = 4, P = 0.003).


C Immunohistochemistry for lysozyme was used to detect Paneth cells . Quantification of Paneth cell numbers indicated a significant decrease in Paneth cells in the base of crypts in mice with elevated Snai1 levels ( n = 5 , P = 0 . 027 ) .

O O O O O O O O O O O O O O O O O O O O O O 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 Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cell numbers indicated a significant decrease in Paneth cells in the base of crypts in mice with elevated Snai1 levels (n = 5, P = 0.027).


D , E Quantification of total PCNA ‐ positive cells per crypt revealed a significant increase when Snai1 levels were elevated ( n = 4 , P = 0 . 009 ) . This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of ( E ) PCNA ‐ positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue ( n = 4 , P = 0 . 0005 ) . Bars represent mean ± SD .

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D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.


F PCNAimmunostaining ( green ) and DAPIstaining ( blue ) of VillinCreERT2ROSA26Snai1 compared to control VillinCreERT2 littermates . An expansion of proliferating CBCcells ( indicated by arrows ) is observed at the base of crypts . Scale bars : 10 μm .

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F PCNAimmunostaining (green) and DAPIstaining (blue) of VillinCreERT2ROSA26Snai1 compared to control VillinCreERT2 littermates. An expansion of proliferating CBCcells (indicated by arrows) is observed at the base of crypts. Scale bars: 10 μm.


G qRT - PCR analysis of stem cell marker expression in the small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T littermates . Bars represent mean ± SD . n = 3 experiments per group , Olfm4 P = 0 . 013 , Cdh1 / E ‐ cadherin P = 0 . 003 , Lgr5 P = 0 . 013 .

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G qRT-PCR analysis of stem cell marker expression in the small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T littermates. Bars represent mean ± SD. n = 3 experiments per group, Olfm4 P = 0.013, Cdh1/E‐cadherin P = 0.003, Lgr5 P = 0.013.


H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue . A significant increase in CD44v6 ‐ positive cells localised at the base of crypts ( depicted by arrows ) was observed . Bars represent mean ± SD . n = 3 mice per group , * P = 0 . 00023 . Two ‐ tailed Student ' s t ‐ test was used to assess significance . Scale bars : 10 μm .

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H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue. A significant increase in CD44v6‐positive cells localised at the base of crypts (depicted by arrows) was observed. Bars represent mean ± SD. n = 3 mice per group, *P = 0.00023. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 10 μm.


VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls . Scale bars : 50 μm . Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 002 .

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VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.


Assessment of apoptosis in control and VillinCreERT2 RosaSnai1 organoids after 4 days of growth . Photographs show an overlay of bright ‐ field , propidium iodide and Hoechst fluorescence . Scale bars : 50 μm . Quantification of apoptotic organoids with and without Wnt3A treatment shown as mean ± SD . n = 3 experiments per group , * * P = 0 . 00003 . Two ‐ tailed Student ' s t ‐ test was used to assess significance .

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

Assessment of apoptosis in control and VillinCreERT2 RosaSnai1 organoids after 4 days of growth. Photographs show an overlay of bright‐field, propidium iodide and Hoechst fluorescence. Scale bars: 50 μm.Quantification of apoptotic organoids with and without Wnt3A treatment shown as mean ± SD. n = 3 experiments per group, **P = 0.00003. Two‐tailed Student's t‐test was used to assess significance.


A - C Droplet digital PCR analysis of Bmf , Bbc3 / Puma and Bcl2l 11 / Bim after conditional Snai1 loss in in vitro organoid culture . VillinCreERT2 Snai1 + / + ( control ) and VillinCreERT2 Snai1fl / fl SI organoids were treated for 24 h with tamoxifen . No differences in gene expression were observed ( n = 3 ) .

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A-C Droplet digital PCR analysis of Bmf, Bbc3/Puma and Bcl2l 11/Bim after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 h with tamoxifen. No differences in gene expression were observed (n = 3).


D qRT - PCR analysis of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture . VillinCreERT2 Snai1 + / + ( control ) and VillinCreERT2 Snai1fl / fl SI organoids were treated for 24 ( P = 0 . 0001 ) and 72 h ( P = 0 . 006 ) with tamoxifen . Bars represent mean ± SD . n = 3 mice per group .

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D qRT-PCR analysis of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 (P = 0.0001) and 72 h (P = 0.006) with tamoxifen. Bars represent mean ± SD. n = 3 mice per group.


E SW480 cells , which transiently express GFP ‐ SNAI1 , were used to detect direct binding of SNAI1 to the promoters of known target genes CDH1 , IL8 and a novel target SERINC3 revealed by microarray analysis . An unrelated DNA region was used as a negative control . Anti ‐ GFP antibody was used to immunoprecipitate GFP ‐ tagged SNAI1 , while IgG control antibody was used as a negative control . The results are given as relative enrichment compared to input material and are representative of three independent experiments .

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E SW480 cells, which transiently express GFP‐SNAI1, were used to detect direct binding of SNAI1 to the promoters of known target genes CDH1, IL8 and a novel target SERINC3 revealed by microarray analysis. An unrelated DNA region was used as a negative control. Anti‐GFP antibody was used to immunoprecipitate GFP‐tagged SNAI1, while IgG control antibody was used as a negative control. The results are given as relative enrichment compared to input material and are representative of three independent experiments.


Metaphysis region in the BM is rich in endomucin + capillaries expressing HO - 1 . mp - metaphysis ; gp - growth plate ; scale bar 100 μm . The HO - 1 positive small capillaries in metaphysis express endomucin and CD31 . Shown maximum intensity projection , scale bar 20 μm . HO - 1 is expressed by smaller endomucin + CD31 + capillaries ( # ) as well as in bigger endomucin - / lowCD31 + arteries ( * ) . CD31 - pericytes wrapping the artery also express HO - 1 ( * ) ; scale bar 20 μm . HO - 1 positive capillaries in the metaphysis expressed CD31 and Sca - 1 . The capillaries are enveloped by HO - 1 expressing pericytes . Part of the HO - 1 + pericytes express Sca - 1 ( # ) while others show no or low Sca - 1 signal ( * ) ; scale bar 20 μm .

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Metaphysis region in the BM is rich in endomucin+ capillaries expressing HO-1. mp - metaphysis; gp - growth plate; scale bar 100 μm. The HO-1 positive small capillaries in metaphysis express endomucin and CD31. Shown maximum intensity projection, scale bar 20 μm. HO-1 is expressed by smaller endomucin+CD31+ capillaries (#) as well as in bigger endomucin-/lowCD31+ arteries (*). CD31- pericytes wrapping the artery also express HO-1 (*); scale bar 20 μm. HO-1 positive capillaries in the metaphysis expressed CD31 and Sca-1. The capillaries are enveloped by HO-1 expressing pericytes. Part of the HO-1+ pericytes express Sca-1 (#) while others show no or low Sca-1 signal (*); scale bar 20 μm.


Flow cytometry analysis revealed the highest expression of HO - 1 in CD31 + Sca - 1 + ECs . CAR and PαS populations also express HO - 1 , while most of non - hematopoietic CD45 - Ter119 - are HO - 1 - negative in steady - state conditions .

Flow cytometry analysis revealed the highest expression of HO-1 in CD31+Sca-1+ ECs. CAR and PαS populations also express HO-1, while most of non-hematopoietic CD45-Ter119- are HO-1-negative in steady-state conditions.


BM macrophages ( MQ ) express HO - 1 . The MHCIIhigh MQ express higher levels of HO - 1 than MHCIIlow MQ . Cells within whole HSPC compartment ( LKS ) express no or low levels of HO - 1 in comparison to MQ .

BM macrophages (MQ) express HO-1. The MHCIIhigh MQ express higher levels of HO-1 than MHCIIlow MQ. Cells within whole HSPC compartment (LKS) express no or low levels of HO-1 in comparison to MQ.


HO - 1 expression on mRNA level quantified by qPCR qPCR analysis based on two independent experiments n = 10 - 11 /

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HO-1 expression on mRNA level quantified by qPCR qPCR analysis based on two independent experiments n = 10-11/group


HO - 1 expression on mRNA level quantified by RNA - seq . RNAseq analysis has n = 3 - 4 / group , two - tailed t - test for two groups comparison , and one way ANOVA with Bonferroni post - test for multiple group comparison . * p < 0 . 05 , * * * * p < 0 . 0001 . Data are shown as mean ± SEM .

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HO-1 expression on mRNA level quantified by RNA-seq. RNAseq analysis has n = 3-4/group, two-tailed t-test for two groups comparison, and one way ANOVA with Bonferroni post-test for multiple group comparison. *p< 0.05, ****p< 0.0001. Data are shown as mean ± SEM.


ECs and CARs from middle - aged animals ( 11 - 12 months ) express lower levels of HO - 1 protein . Two independent experiments , n = 5 - 10 / group . Data are shown as mean ± SEM . * p < 0 . 05 , * * * p < 0 . 001 , two - tailed unpaired t - test .

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ECs and CARs from middle-aged animals (11-12 months) express lower levels of HO-1 protein. Two independent experiments, n = 5-10/group. Data are shown as mean ± SEM. *p< 0.05, ***p<0.001, two-tailed unpaired t-test.


Middle - aged animals have lower frequency of ECs . Two independent experiments , n = 10 - 11 / group . Data are shown as mean ± SEM . * * p < 0 . 01 , two - tailed unpaired t - test . The control staining of HO - 1 on HO - 1 - / - bone marrow section is provided in Appendix Fig S1 .

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Middle-aged animals have lower frequency of ECs. Two independent experiments, n = 10-11/group. Data are shown as mean ± SEM. **p<0.01, two-tailed unpaired t-test. The control staining of HO-1 on HO-1-/- bone marrow section is provided in Appendix Fig S1.


RNA - seq revealed 111 DEGs in HO - 1 - / - ECs .

RNA-seq revealed 111 DEGs in HO-1-/- ECs.


The identified DEGs separated HO - 1 - / - and HO - 1 + / + ECs by ( B ) hierarchical clustering and ( C ) PCA .

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The identified DEGs separated HO-1-/- and HO-1+/+ ECs by (B) hierarchical clustering and (C) PCA.


Heatmap of selected ( E ) hematopoietic factors and ( F ) adhesion molecules expression in HO - 1 - / - and HO - 1 + / + ECs .

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Heatmap of selected (E) hematopoietic factors and (F) adhesion molecules expression in HO-1-/- and HO-1+/+ ECs.


RNA - seq revealed 100 DEGs in HO - 1 - / - CARs .

RNA-seq revealed 100 DEGs in HO-1-/- CARs.


The identified DEGs separated HO - 1 - / - and HO - 1 + / + CARs by ( H ) hierarchical clustering and ( I ) PCA .

The identified DEGs separated HO-1-/- and HO-1+/+ CARs by (H) hierarchical clustering and (I) PCA.


Heatmap of selected ( K ) skeletal biology and ( L ) hematopoietic factors expression in HO - 1 - / - and HO - 1 + / + CARs . Correlation similarity metric and average linkage clustering were used in the presented heatmaps .

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Heatmap of selected (K) skeletal biology and (L) hematopoietic factors expression in HO-1-/- and HO-1+/+ CARs. Correlation similarity metric and average linkage clustering were used in the presented heatmaps.


HO - 1 - / - mice have more ECs and less CARs ( p = 0 . 053 ) in BM , while frequency of PaS does not differ .

HO-1-/- mice have more ECs and less CARs (p = 0.053) in BM, while frequency of PaS does not differ.


Young HO - 1 - / - mice have lower frequency of Sca - 1high fraction among BM ECs , what resembles phenotype of BM ECs in old mice .

Young HO-1-/- mice have lower frequency of Sca-1high fraction among BM ECs, what resembles phenotype of BM ECs in old mice.


Intracellular flow cytometry reveals ( C ) lower levels of SDF - 1α in HO - 1 - / - CARs and ( D ) lower levels of LAP protein in HO - 1 - / - ECs .

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Intracellular flow cytometry reveals (C) lower levels of SDF-1α in HO-1-/- CARs and (D) lower levels of LAP protein in HO-1-/- ECs.


Analysis of cell surface SCF revealed lower frequency of SCF + ECs in HO - 1 - / - while no differences among CARs .

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Analysis of cell surface SCF revealed lower frequency of SCF+ ECs in HO-1-/- while no differences among CARs.


The frequency of ( F ) CD163 + MQs and ( G ) CD163 - MQs in BM of HO - 1 - / - mice is altered .

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The frequency of (F) CD163+ MQs and (G) CD163- MQs in BM of HO-1-/- mice is altered.


Gating strategy of hematopoietic stem and progenitor cells .

Gating strategy of hematopoietic stem and progenitor cells.


Young HO - 1 - / - mice possess higher number of ( B ) LT - HSCs , ( C ) ST - HSCs and ( D ) MPPs .

Young HO-1-/- mice possess higher number of (B) LT-HSCs, (C) ST-HSCs and (D) MPPs.


Exemplary analysis of cell cycle with Ki67 and nuclear dye .

Exemplary analysis of cell cycle with Ki67 and nuclear dye.


More young HO - 1 - / - LT - HSCs are in G1 and S / G2 / M cell cycle phases . The presented cell cycle analysis is from two independent experiments . Young HO - 1 - / - MPPs do not differ in cell cycling from young HO - 1 + / + MPPs . The presented cell cycle analysis is from two independent experiments . More old HO - 1 - / - LT - HSCs are in G1 phase in comparison to old HO - 1 + / + LT - HSCs , but not in S / G2 / M phase . The presented cell cycle analysis is from two independent experiments . Old MPPs do not differ in cell cycling between genotypes . The presented cell cycle analysis is from two independent experiments .

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More young HO-1-/- LT-HSCs are in G1 and S/G2/M cell cycle phases. The presented cell cycle analysis is from two independent experiments. Young HO-1-/- MPPs do not differ in cell cycling from young HO-1+/+ MPPs. The presented cell cycle analysis is from two independent experiments. More old HO-1-/- LT-HSCs are in G1 phase in comparison to old HO-1+/+ LT-HSCs, but not in S/G2/M phase. The presented cell cycle analysis is from two independent experiments. Old MPPs do not differ in cell cycling between genotypes. The presented cell cycle analysis is from two independent experiments.


Young HO - 1 - / - LT - HSCs contain more γH2aXhigh cells than young HO - 1 + / + , 378 - 991 cells analyzed from 7 mice / group . Young HO - 1 - / - MPPs contain more γH2aXhigh cells than young HO - 1 + / + , 1693 - 2790 cells analyzed from 7 mice / group .

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Young HO-1-/- LT-HSCs contain more γH2aXhigh cells than young HO-1+/+, 378-991 cells analyzed from 7 mice/group. Young HO-1-/- MPPs contain more γH2aXhigh cells than young HO-1+/+, 1693-2790 cells analyzed from 7 mice/group.


Alkaline comet assay revealed that HO - 1 - / - LT - HSCs possess ( L ) higher oil tail moment and ( M ) more DNA in the comet tail . 1068 - 1189 cells analyzed from 6 mice / group .

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Alkaline comet assay revealed that HO-1-/- LT-HSCs possess (L) higher oil tail moment and (M) more DNA in the comet tail. 1068-1189 cells analyzed from 6 mice/group.


HSCs from young HO - 1 - / - mice provide worse hematopoietic reconstitution after transplantation than HO - 1 + / + HSCs . Data are shown as mean ± SEM , n = 8 - 9 mice / group .

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HSCs from young HO-1-/- mice provide worse hematopoietic reconstitution after transplantation than HO-1+/+ HSCs. Data are shown as mean ± SEM, n = 8-9 mice/group.


RNA - seq revealed 1067 DEGs between young HO - 1 - / - and HO - 1 + / + LT - HSCs . RNA - seq revealed 595 DEGs between old HO - 1 - / - and HO - 1 + / + LT - HSCs .

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RNA-seq revealed 1067 DEGs between young HO-1-/- and HO-1+/+ LT-HSCs. RNA-seq revealed 595 DEGs between old HO-1-/- and HO-1+/+ LT-HSCs.


PC1 in PCA analysis on selected 1148 genes separated young HO - 1 + / + LT - HSCs from other groups , clustering the young HO - 1 - / - LT - HSCs together with old LT - HSCs .

PC1 in PCA analysis on selected 1148 genes separated young HO-1+/+ LT-HSCs from other groups, clustering the young HO-1-/- LT-HSCs together with old LT-HSCs.


Hierarchical clustering on the 20 % of most correlated 297 genes with PC1 showed 2 major clusters that includes young HO - 1 + / + LT - HSCs in one and old LT - HSCs with young HO - 1 - / - LT - HSCs in second .

Hierarchical clustering on the 20% of most correlated 297 genes with PC1 showed 2 major clusters that includes young HO-1+/+ LT-HSCs in one and old LT-HSCs with young HO-1-/- LT-HSCs in second.


Deletion of HO - 1 in myeloid lineage did not cause expansion of LT - HSCs and did not alter their cell cycle status .

Deletion of HO-1 in myeloid lineage did not cause expansion of LT-HSCs and did not alter their cell cycle status.


Long - term PB chimerism derived from HSCs transplanted to HO - 1 - / - recipients is lower than from HSCs transplanted to HO - 1 + / + recipients .

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Long-term PB chimerism derived from HSCs transplanted to HO-1-/- recipients is lower than from HSCs transplanted to HO-1+/+ recipients.


Chimerism among BM HSPCs fractions transplanted to HO - 1 - / - recipients are lower than from HSCs transplanted to HO - 1 + / + recipients . Data are shown as mean ± SEM , n = 5 - 12 /

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Chimerism among BM HSPCs fractions transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/group


total number of cells derived from HSCs transplanted to HO - 1 - / - recipients are lower than from HSCs transplanted to HO - 1 + / + recipients . Data are shown as mean ± SEM , n = 5 - 12 /

total number of cells derived from HSCs transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/group


Short term homing of HSPC to HO - 1 + / + or HO - 1 - / - BM did not differ . Data are shown as mean ± SEM , n = 5 / group , unpaired t - test .

Short term homing of HSPC to HO-1+/+ or HO-1-/- BM did not differ. Data are shown as mean ± SEM, n = 5/group, unpaired t-test.


Transplantation of HO - 1 - / - HSCs provides lower chimerism among HSPC fractions in primary recipients . Data are shown as mean ± SEM , n = 7 - 8 mice / group .

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Transplantation of HO-1-/- HSCs provides lower chimerism among HSPC fractions in primary recipients. Data are shown as mean ± SEM, n = 7-8 mice/group.


Transplantation of the same number of donor - derived BM cells from primary recipients provides the same ( C ) PB

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Transplantation of the same number of donor-derived BM cells from primary recipients provides the same (C) PB chimerism


Transplantation of the same number of donor - derived BM cells from primary recipients provides the same BM LT - HSC chimerism .

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Transplantation of the same number of donor-derived BM cells from primary recipients provides the same BM LT-HSC chimerism.


Comparison of gene log fold changes in non - transplanted HO - 1 - / - LT - HSCs and HO - 1 - / - LT - HSCs transplanted twice to the wild - type HO - 1 + / + showed that transplantation of HO - 1 - / - LT - HSCs twice to the wild type niche reverses their transcriptional alterations .

Comparison of gene log fold changes in non-transplanted HO-1-/- LT-HSCs and HO-1-/- LT-HSCs transplanted twice to the wild-type HO-1+/+ showed that transplantation of HO-1-/- LT-HSCs twice to the wild type niche reverses their transcriptional alterations.


Frequency of colonies appearing at day 8 and day 12 from HSCs co - cultured with HO - 1 + / + or HO - 1 - / - MSCs . * p < 0 . 05 , * * * p < 0 . 001 , Fisher exact test .

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Frequency of colonies appearing at day 8 and day 12 from HSCs co-cultured with HO-1+/+ or HO-1-/- MSCs. *p< 0.05, ***p<0.001, Fisher exact test.


PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed ( C ) 4 distinct clusters of colonies , that differ by frequency of ( D ) CD11b + Gr1 - , ( E ) CD11b + Gr1 +

PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (C) 4 distinct clusters of colonies, that differ by frequency of (D) CD11b+Gr1-, (E) CD11b+Gr1+