( I ) Real - time quantitative reverse transcription polymerase chain reaction ( qRT - PCR ) analysis of the expression of genes encoding inflammatory cytokines in the ileum . COPS8fl / fl and COPS8ΔIEC mice were either untreated ( phosphate buffered saline ( PBS ) ) or treated with the antibiotic cocktail ( Abx ) for 3 months . Data are mean ± SEM from three biological replicates . * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(I) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression of genes encoding inflammatory cytokines in the ileum. COPS8fl/fl and COPS8ΔIEC mice were either untreated (phosphate buffered saline (PBS)) or treated with the antibiotic cocktail (Abx) for 3 months. Data are mean ± SEM from three biological replicates. **P<0.01, NS- non-significant using one-way ANOVA.


( B ) Representative image showing changes in colon morphology and length following DSS - induced colitis with concurrent treatment with MBELNs COPS8fl / fl and COPS8ΔIEC mice . Representative data from seven biological replicates per genotype .

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(B) Representative image showing changes in colon morphology and length following DSS-induced colitis with concurrent treatment with MBELNs COPS8fl/fl and COPS8ΔIEC mice. Representative data from seven biological replicates per genotype.


Hematoxylin and eosin staining ( HE ) to show histological changes Scale bar

Hematoxylin and eosin staining (HE) to show histological changes Scale bar 200μm


( F ) Expression of anti - microbial peptide ( AMPs ) in the colon of COPS8fl / fl and COPS8ΔIEC mice while being treated with MBELNs by oral administration . Data are mean ± SEM from three biological replicates per group . * P < 0 . 05 , * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(F) Expression of anti-microbial peptide (AMPs) in the colon of COPS8fl/fl and COPS8ΔIEC mice while being treated with MBELNs by oral administration. Data are mean ± SEM from three biological replicates per group. *P<0.05, **P<0.01, NS- non-significant using one-way ANOVA.


( I - J ) Transcriptional expression of nuclear factor kappa B ( NF - κB ) ( I ) and inhibitor of nuclear factor kappa - β kinase ( IKK - β ) ( J ) mRNA using Real - time quantitative reverse transcription Polymerase chain reaction ( qRT - PCR ) in colonic epithelial cells upon treatment with MBELNs in addition to DSS . Data are mean ± SEM from three biological replicates per group . * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(I-J) Transcriptional expression of nuclear factor kappa B (NF-κB) (I) and inhibitor of nuclear factor kappa-β kinase (IKK-β) (J) mRNA using Real-time quantitative reverse transcription Polymerase chain reaction (qRT-PCR) in colonic epithelial cells upon treatment with MBELNs in addition to DSS. Data are mean ± SEM from three biological replicates per group. **P<0.01, NS- non-significant using one-way ANOVA.


( A ) Western blots showing expression of AhR in MC38 cells induced by different fractions ( protein , lipid and RNA ) derived from Mulberry bark derived exosome - like nanoparticles ( MBELNs ) . Data shown are from three biological replicates .

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(A) Western blots showing expression of AhR in MC38 cells induced by different fractions (protein, lipid and RNA) derived from Mulberry bark derived exosome-like nanoparticles (MBELNs). Data shown are from three biological replicates.


( B ) The effect of different size protein fractions of mulberry bark derived exosome - like nanoparticles ( MBELNs ) on the expression of aryl hydrocarbon receptor ( AhR ) demonstrated in Western blots . Data shown are from three biological replicates .

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(B) The effect of different size protein fractions of mulberry bark derived exosome-like nanoparticles (MBELNs) on the expression of aryl hydrocarbon receptor (AhR) demonstrated in Western blots. Data shown are from three biological replicates.


( F ) SPR experiment showing recombinant HSPA8 protein interaction with recombinant hAhR on a SPR chip . Data shown are from three biological replicates .

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(F) SPR experiment showing recombinant HSPA8 protein interaction with recombinant hAhR on a SPR chip. Data shown are from three biological replicates.


TOPflash reporter assays in HEK293T cells stimulated with either control , Wnt3a , Wnt3a and RSPO1 - 4 conditioned media , or by transfection with the indicated constructs . Where indicated , cells were co - transfected with the indicated siRNAs .

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TOPflash reporter assays in HEK293T cells stimulated with either control, Wnt3a, Wnt3a and RSPO1-4 conditioned media, or by transfection with the indicated constructs. Where indicated, cells were co-transfected with the indicated siRNAs.


K , Representative co - immunoprecipitation experiments in HEK293T cells that were transfected with GFP - USP42 or the indicated GFP - tagged control , together with the stated Wnt signalling component ( N = 3 independent experiments ) . IgG heavy chain is indicated with an asterisk . Dvl1 functions as a positive control that binds both ZNRF3 and Axin1 .

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K, Representative co-immunoprecipitation experiments in HEK293T cells that were transfected with GFP-USP42 or the indicated GFP-tagged control, together with the stated Wnt signalling component (N = 3 independent experiments). IgG heavy chain is indicated with an asterisk. Dvl1 functions as a positive control that binds both ZNRF3 and Axin1.


E , F , Co - immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in ( A ) . In ( F ) cells were co - transfected with DVL2 as indicated . Unspecific bands resulting from antibody cross - reaction are marked with asterisks . Representative blots of n ≥ 3 independent experiments are shown .

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E,F, Co-immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in (A). In (F) cells were co-transfected with DVL2 as indicated. Unspecific bands resulting from antibody cross-reaction are marked with asterisks. Representative blots of n ≥ 3 independent experiments are shown.


A , Cell surface biotinylation assay performed in HEK293T cells transfected as indicated and treated with 10 μM MG132 to prevent proteasomal degradation . Consecutive immunoprecipitations were carried out as indicated in the scheme . The Avidin pulldown was carried under denaturing conditions ( 1st IP ) . The unrelated receptor CD147 was used as loading control for the avidin pull down , and CUL1 for the total input of cell lysate .

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A, Cell surface biotinylation assay performed in HEK293T cells transfected as indicated and treated with 10 μM MG132 to prevent proteasomal degradation. Consecutive immunoprecipitations were carried out as indicated in the scheme. The Avidin pulldown was carried under denaturing conditions (1st IP). The unrelated receptor CD147 was used as loading control for the avidin pull down, and CUL1 for the total input of cell lysate.


F , G , Western blots of lysates from HEK293T cells transfected with the indicated constructs . The asterisk marks an unspecific band . Representative blots from at least three independent experiments are shown .

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F,G, Western blots of lysates from HEK293T cells transfected with the indicated constructs. The asterisk marks an unspecific band. Representative blots from at least three independent experiments are shown.


D , TOPflash reporter assays in HCT116 cells upon knockdown of the indicated genes using single siRNAs . Cells were stimulated with control or Wnt3a conditioned medium . Data is displayed as mean ± SD , and show one representative of n = 3 independent experiments with three biological replicates .

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D, TOPflash reporter assays in HCT116 cells upon knockdown of the indicated genes using single siRNAs. Cells were stimulated with control or Wnt3a conditioned medium. Data is displayed as mean ± SD, and show one representative of n = 3 independent experiments with three biological replicates.


E , F , qPCR analysis of USP42 , LGR5 and AXIN2 expression levels in HCT116 cells . Data are displayed as mean ± SD and show n = 4 ( E ) or n = 3 ( F ) independent experiments .

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E,F, qPCR analysis of USP42, LGR5 and AXIN2 expression levels in HCT116 cells. Data are displayed as mean ± SD and show n = 4 (E) or n = 3 (F) independent experiments.


E - H , Immunofluorescence microscopy showing endogenous E - cadherin levels in wt and WLS ( Evi ) KO HCT116 cells upon transfection with siUSP42 ( E ) , or electroporation with eSpCas9 empty plasmid ( non - edited ) or eSpCas9 - USP42 gRNAs ( G ) . Scale bar = 20 μm . In ( F , G ) , quantification of the fluorescent signal ( Alexa - 488 ) in n ≥ 4 biological replicates with n ≥ 15 cells per replicate are shown .

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E-H, Immunofluorescence microscopy showing endogenous E-cadherin levels in wt and WLS (Evi) KO HCT116 cells upon transfection with siUSP42 (E), or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs(G). Scale bar = 20 μm. In (F,G), quantification of the fluorescent signal (Alexa-488) in n ≥ 4 biological replicates with n ≥ 15 cells per replicate are shown.


I - L , Spheroid formation experiments with HCT116 cells upon transfection with siUSP42 ( I - K ) or electroporation with eSpCas9 empty plasmid ( non - edited ) or eSpCas9 - USP42 gRNAs ( L ) . Scale bar = 500 μm . Where indicated , cells were treated with 5 μM IWP - 2 or 20 μM LGK - 974 ( PORCN inhibitors ) . The median size was quantified for n ≥ 13 spheroids from a representative experiment repeated independently n ≥ 3 is shown .

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I-L, Spheroid formation experiments with HCT116 cells upon transfection with siUSP42 (I-K) or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs (L). Scale bar = 500 μm. Where indicated, cells were treated with 5 μM IWP-2 or 20 μM LGK-974 (PORCN inhibitors). The median size was quantified for n ≥ 13 spheroids from a representative experiment repeated independently n ≥ 3 is shown.


A , Representative images of mouse small intestinal organoids generated with empty ( Non - edited ) or USP42gRNA containing CRISPR - concatemers , and cultured for 3 days in matrigel with the indicated growth factors , or the Wnt secretion inhibitor IWP - 2 . EN , EGF and noggin . Note that knockout of Usp42 ( Usp42 gRNAs ) rendered the organoids insensitive to RSPO1 withdrawal . Scale bar = 1 mm .

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A, Representative images of mouse small intestinal organoids generated with empty (Non-edited) or USP42gRNA containing CRISPR-concatemers, and cultured for 3 days in matrigel with the indicated growth factors, or the Wnt secretion inhibitor IWP-2. EN, EGF and noggin. Note that knockout of Usp42 (Usp42 gRNAs) rendered the organoids insensitive to RSPO1 withdrawal. Scale bar = 1 mm.


C , qPCR analysis of Lgr5 and Axin2 expression levels in first passage mouse intestinal organoids cultured in EN + 5 % RSPO1 .

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C, qPCR analysis of Lgr5 and Axin2 expression levels in first passage mouse intestinal organoids cultured in EN + 5% RSPO1.


( A ) Representative images of H & E staining of colon from 3m C57Bl ( n = 4 ) and mdx ( n = 4 ) mice . High magnification ( scale bar : 20 μm ) and low magnification ( scale bar : 200 μm ) .

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(A) Representative images of H&E staining of colon from 3m C57Bl (n=4) and mdx (n=4) mice. High magnification (scale bar: 20 μm) and low magnification (scale bar: 200 μm).


( B ) Mucus layer , area between yellow dash lines ; crypt length , yellow headed arrow . Scale bar : 100 μm . ( C ) Mucus layer thickness and crypt length were quantified for n = 4 mice per group ( with pooled samples of n = 60 for mucus layer thickness and n = 80 for crypt length ) .

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(B) Mucus layer, area between yellow dash lines; crypt length, yellow headed arrow. Scale bar: 100 μm. (C) Mucus layer thickness and crypt length were quantified for n=4 mice per group (with pooled samples of n=60 for mucus layer thickness and n=80 for crypt length).


( D ) SCFA faecal quantification of 3m C57Bl ( n = 3 ) and mdx ( n = 4 - 5 ) mice . Data information : Data are presented as mean ± SD ( * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; Student t - test ) .

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(D) SCFA faecal quantification of 3m C57Bl (n=3) and mdx (n=4-5) mice. Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).


( E ) Colon images captured with the iMScope TRIO described altered pattern of expression of different Phosphatidylcholines ( PC ) and Lysophosphatidylcholines ( LysoPC ) ( as indicated by m / z values ) in 3m mdx mice ( n = 3 ) . Scale bar : 50 μm . For each lipid , the mean intensities measured at 12 positions throughout colon images are shown on the right side where bars are mean ± SEM ( n = 3 ) . Data information : Data are presented as mean ± SD ( * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; Student t - test ) .

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(E) Colon images captured with the iMScope TRIO described altered pattern of expression of different Phosphatidylcholines (PC) and Lysophosphatidylcholines (LysoPC) (as indicated by m/z values) in 3m mdx mice (n=3). Scale bar: 50 μm. For each lipid, the mean intensities measured at 12 positions throughout colon images are shown on the right side where bars are mean ± SEM (n = 3). Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).


( F ) Cropped images of representative WB showing the expression of proteins involved in inflammation and fibrosis in colon tissues of 3m C57Bl ( n = 3 ) and 3m mdx ( n = 5 ) . Densitometric analyses of protein expression was shown as ratio to actin . Data information : Data are presented as mean ± SD ( * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; Student t - test ) .

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(F) Cropped images of representative WB showing the expression of proteins involved in inflammation and fibrosis in colon tissues of 3m C57Bl (n=3) and 3m mdx (n=5). Densitometric analyses of protein expression was shown as ratio to actin. Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).


( E ) Faecal content quantification of SCFAs in 3m mdx and 3m mdx + ABX mice ( n = 5 per group ) . Data equal to 0 . Data are presented as mean ± SD ( * * p < 0 . 01 , * * * p < 0 . 001 ; Student t - test ) .

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(E) Faecal content quantification of SCFAs in 3m mdx and 3m mdx+ABX mice (n=5 per group). Data equal to 0. Data are presented as mean ± SD (**p< 0.01, ***p< 0.001; Student t-test).


( A - D ) FACS analysis of spleen and muscle homogenates from 3m C57Bl ( n = 4 ) , mdx ( n = 5 ) and mdx + ABX ( n = 7 ) mice demonstrates no significant alteration of CD45 + CD11b + CD4 - CD8 - myeloid cells ( A and C ) and few differences in CD4 + or CD8 + naïve ( CD62L + CD44 - ) , central memory ( CD62L + CD44 + ) and effector ( CD62L - CD44 + ) T cells ( B and D ) . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test .

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(A-D) FACS analysis of spleen and muscle homogenates from 3m C57Bl (n=4), mdx (n=5) and mdx+ABX (n=7) mice demonstrates no significant alteration of CD45+CD11b+CD4-CD8- myeloid cells (A and C) and few differences in CD4+ or CD8+ naïve (CD62L+ CD44-), central memory (CD62L+ CD44+) and effector (CD62L- CD44+) T cells (B and D). Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.


( E ) FACS analysis of spleen of 3m C57Bl ( n = 7 ) , mdx ( n = 9 ) and GFmdx ( n = 5 ) mice revealed similar proportions of CD4 + and CD8 + T cells but reduced activated CD44 + T cells in GFmdx mice . Representative plots are depicted . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test .

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(E) FACS analysis of spleen of 3m C57Bl (n=7), mdx (n=9) and GFmdx (n=5) mice revealed similar proportions of CD4+ and CD8+ T cells but reduced activated CD44+ T cells in GFmdx mice. Representative plots are depicted. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.


( F ) Graphs show cumulative frequencies of CD4 + and CD8 + T cells on live cells of 3m C57Bl ( n = 7 ) , mdx ( n = 4 ) and GFmdx ( n = 5 ) mice . Representative dot plots and cumulative frequencies of splenic CD4 + GITR + CD25 + Treg . Frequencies of effector CD44 + T cells were significantly decreased in spleen of GFmdx mice . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test .

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(F) Graphs show cumulative frequencies of CD4+ and CD8+ T cells on live cells of 3m C57Bl (n=7), mdx (n=4) and GFmdx (n=5) mice. Representative dot plots and cumulative frequencies of splenic CD4+GITR+CD25+ Treg. Frequencies of effector CD44+ T cells were significantly decreased in spleen of GFmdx mice. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.


( G ) Representative dot - plots showing the proportion of muscle - infiltrating CD45 + cells of 3m C57Bl ( n = 6 ) , mdx ( n = 6 ) and GFmdx ( n = 5 ) mice . Cumulative frequencies of muscle - infiltrating CD45 + cells are shown . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ordinary one - way ANOVA , Tukey ' s multiple comparison test .

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(G) Representative dot-plots showing the proportion of muscle-infiltrating CD45+ cells of 3m C57Bl (n=6), mdx (n=6) and GFmdx (n=5) mice. Cumulative frequencies of muscle-infiltrating CD45+ cells are shown. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 ordinary one-way ANOVA, Tukey's multiple comparison test.


( H ) Representative images of TA muscles from 3m mdx and GFmdx mice stained for CD45 ( in green ) , isolectin ( in red ) and phalloidin ( in purple ) . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 10 μm .

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(H) Representative images of TA muscles from 3m mdx and GFmdx mice stained for CD45 (in green), isolectin (in red) and phalloidin (in purple). Nuclei were counterstained with DAPI (in blue). Scale bar: 10 μm.


( I ) Absolute number of CD3 + inflammatory cells ( white arrows ) were quantified in n = 12 images of TA of 3m C57Bl , 3m mdx and 3m GFmdx mice ( n = 6 each ) . CD3 staining is shown in green and DAPI in blue . Scale bars : 50 μm . Data information : Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 The comparisons among the averages of CD3 + cells were evaluated using unpaired t - test .

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(I) Absolute number of CD3+ inflammatory cells (white arrows) were quantified in n=12 images of TA of 3m C57Bl, 3m mdx and 3m GFmdx mice (n=6 each). CD3 staining is shown in green and DAPI in blue. Scale bars: 50 μm. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 The comparisons among the averages of CD3+ cells were evaluated using unpaired t-test.


( D ) RT - qPCR analysis of TA muscles of two independent experiments with 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx ( n = 5 ) mice determined the expression of myogenic markers . Data information : Data are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , ordinary one - way ANOVA and non - parametric test followed by Kruskal - Wallis test for RT - qPCR ) .

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(D) RT-qPCR analysis of TA muscles of two independent experiments with 3m mdx (n=4), mdx+ABX (n=4) and GFmdx (n=5) mice determined the expression of myogenic markers. Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA and non-parametric test followed by Kruskal-Wallis test for RT-qPCR).


( E ) Representative Gomori modified staining and quantification of myofiber area and relative frequency of the myofiber cross - sectional area ( CSA ) expressed as the frequency distribution of the TA muscles of 3m C57Bl ( n = 4 ) , 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx mice ( n = 5 ) . Pooled samples for each group with n = 6240 for 3m C57Bl ; n = 6001 for 3m mdx ; n = 10556 for 3m mdx + ABX ; n = 23059 for GFmdx . For morphometric analysis , images were quantified with Image J software for each mouse . Scale bars : 50 μm . Data information : Data are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , ordinary one - way

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

(E) Representative Gomori modified staining and quantification of myofiber area and relative frequency of the myofiber cross-sectional area (CSA) expressed as the frequency distribution of the TA muscles of 3m C57Bl (n=4), 3m mdx (n=4), mdx+ABX (n=4) and GFmdx mice (n=5). Pooled samples for each group with n=6240 for 3m C57Bl; n=6001 for 3m mdx; n=10556 for 3m mdx+ABX; n=23059 for GFmdx. For morphometric analysis, images were quantified with Image J software for each mouse. Scale bars: 50 μm. Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA


( G ) Representative images of skeletal muscle showed the distribution and composition of the myosin heavy chain ( MyHC ) isoforms ( Type IIa , Type IIx and Type IIb ) . ( H , I ) Graph portrays ( H ) the percentage of myofibers expressing different MyHC isoforms and ( I ) myofibers area per type of MyHC in TAs of 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx ( n = 5 ) mice ( n = 12 images per animal ) . Data information : Data are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , ordinary one - way

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of skeletal muscle showed the distribution and composition of the myosin heavy chain (MyHC) isoforms (Type IIa, Type IIx and Type IIb). (H, I) Graph portrays (H) the percentage of myofibers expressing different MyHC isoforms and (I) myofibers area per type of MyHC in TAs of 3m mdx (n=4), mdx+ABX (n=4) and GFmdx (n=5) mice (n=12 images per animal). Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA


( J ) Representative SDH staining and quantification of percentage of SDH + myofibers of TAs from 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx mice ( n = 5 ) ( n = 12 images per animal ) . Scale bars : 50 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

(J) Representative SDH staining and quantification of percentage of SDH+ myofibers of TAs from 3m mdx (n=4), mdx+ABX (n=4) and GFmdx mice (n=5) (n=12 images per animal). Scale bars: 50 μm.


( D , E ) Representative H & E staining ( D ) and quantification of myofiber area with Image J software ( E ) of TA muscles from 3m C57Bl ( n = 5 ) , mdx ( n = 5 ) and ABX - mdxFMT _ C57Bl ( n = 6 ) . Scale bars for H & E : 200 μm . Data information : Data for staining quantification are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , one - way ANOVA , Tukey ' s multiple comparison test ) .

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(D, E) Representative H&E staining (D) and quantification of myofiber area with Image J software (E) of TA muscles from 3m C57Bl (n=5), mdx (n=5) and ABX-mdxFMT_C57Bl (n=6). Scale bars for H&E: 200 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).


( H ) Representative images of skeletal muscle showed the distribution and composition of MyHC isoforms ( Type IIa , Type IIx and Type IIb ) . Scale bar : 50 μm . ( I ) Graph portrays the percentage of myofibers expressing different MyHC isoforms . n = 12 images were analysed for each mouse .

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

(H) Representative images of skeletal muscle showed the distribution and composition of MyHC isoforms (Type IIa, Type IIx and Type IIb). Scale bar: 50 μm. (I) Graph portrays the percentage of myofibers expressing different MyHC isoforms. n=12 images were analysed for each mouse.


( J - K ) Representative SDH staining and quantification of percentage of SDH + myofibers of TA muscles from mdx ( n = 5 ) and ABX - mdxFMT _ C57Bl ( n = 6 ) ( n = 12 images per mouse ) . Scale bar : 200 μm . Data information : Data for staining quantification are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , one - way ANOVA , Tukey ' s multiple comparison test ) .

(J-K) Representative SDH staining and quantification of percentage of SDH+ myofibers of TA muscles from mdx (n=5) and ABX-mdxFMT_C57Bl (n=6) (n=12 images per mouse). Scale bar: 200 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).


( L - M ) Representative image of CD31 ( in cyan ) , α - SMA ( in green ) and isolectin ( in red ) staining and their quantification in TA muscles from mdx ( n = 5 ) and ABX - mdxFMT _ C57Bl ( n = 6 ) mice . Scale bar : 500 μm . Data information : Data for staining quantification are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , one - way ANOVA , Tukey ' s multiple comparison 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

(L-M) Representative image of CD31 (in cyan), α-SMA (in green) and isolectin (in red) staining and their quantification in TA muscles from mdx (n=5) and ABX-mdxFMT_C57Bl (n=6) mice. Scale bar: 500 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).


( C ) pS65 - Ub ( left ) and total Ub ( right ) immunoblots of mitochondria - enriched fractions from wild - type flies treated 3 days with either paraquat or vehicle ( sucrose ) .

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

(C) pS65-Ub (left) and total Ub (right) immunoblots of mitochondria-enriched fractions from wild-type flies treated 3 days with either paraquat or vehicle (sucrose).


( D ) Immunoblots for the indicated antibodies following subcellular fractionation of flies treated with paraquat for 3 days . T , total lysate ; N , nuclear - enriched fraction ; M , mitochondria - enriched fraction ; S , post - mitochondrial supernatant .

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

(D) Immunoblots for the indicated antibodies following subcellular fractionation of flies treated with paraquat for 3 days. T, total lysate; N, nuclear-enriched fraction; M, mitochondria-enriched fraction; S, post-mitochondrial supernatant.


( E ) Immunoblots for the indicated antibodies of ( T ) total lysates or ( M ) mitochondria - enriched fractions from fly heads ( with proboscises removed ) and bodies ( thoraces and abdomens ) .

O O O O O O O O O O O O O O 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) Immunoblots for the indicated antibodies of (T) total lysates or (M) mitochondria-enriched fractions from fly heads (with proboscises removed) and bodies (thoraces and abdomens).


( C ) pS65 - Ub immunoblot of mitochondria - enriched fractions following the paraquat pulse - chase assay in wild - type and park - / - flies . UT , untreated ; PQ , 1 - day paraquat treatment ; Recovery , flies removed from paraquat and returned to normal food . Asterisk ( * ) denotes non - specific band . ( D ) pS65 - Ub lane densitometry analysis of n = 3 independent replicates from ( C ) , expressed relative to the most intense lane signal in each blot .

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

(C) pS65-Ub immunoblot of mitochondria-enriched fractions following the paraquat pulse-chase assay in wild-type and park-/- flies. UT, untreated; PQ, 1-day paraquat treatment; Recovery, flies removed from paraquat and returned to normal food. Asterisk (*) denotes non-specific band. (D) pS65-Ub lane densitometry analysis of n = 3 independent replicates from (C), expressed relative to the most intense lane signal in each blot.


( A - B ) pS65 - Ub immunoblot of whole - animal lysates from untreated flies of the indicated genotypes . park RNAi was induced by da - GAL4 ( ubiquitous ) , Mef2 - GAL4 ( muscle ) or nSyb - GAL4 ( neurons ) . ( C ) pS65 - Ub immunoblot of lysates from bodies or heads ( without proboscises ) , as indicated , from park - / - animals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) pS65-Ub immunoblot of whole-animal lysates from untreated flies of the indicated genotypes. park RNAi was induced by da-GAL4 (ubiquitous), Mef2-GAL4 (muscle) or nSyb-GAL4 (neurons). (C) pS65-Ub immunoblot of lysates from bodies or heads (without proboscises), as indicated, from park-/- animals.


( E ) Higher magnification image of pS65 - Ub positive structures in flight muscle counterstained for ATP5A . Scale bar = 5 μm . Chart shows quantification of ATP5A levels of pS65 - Ub - positive ( + ve ) versus - negative ( - ve ) mitochondrial structures from 3 biological replicates ( 4 - 7 images per animal ; average per animal shown in open circles ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Higher magnification image of pS65-Ub positive structures in flight muscle counterstained for ATP5A. Scale bar = 5 μm. Chart shows quantification of ATP5A levels of pS65-Ub-positive (+ve) versus -negative (-ve) mitochondrial structures from 3 biological replicates (4-7 images per animal; average per animal shown in open circles).


( A ) pS65 - Ub immunoblot of whole - animal lysates from wild - type , Atg5 - and park - / - animals harvested at the indicated ages .

O O O O O O O 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) pS65-Ub immunoblot of whole-animal lysates from wild-type, Atg5- and park-/- animals harvested at the indicated ages.


( B ) pS65 - Ub immunoblot of whole - animal lysates from young animals of the following genotypes : wild - type , Pink1 - , park - / - , park RNAi ( daG4 > UAS - park RNAi ) , Atg1 RNAi ( daG4 > UAS - Atg1 RNAi ) , Atg8a - .

(B) pS65-Ub immunoblot of whole-animal lysates from young animals of the following genotypes: wild-type, Pink1-, park-/-, park RNAi (daG4>UAS-park RNAi), Atg1 RNAi (daG4>UAS-Atg1 RNAi), Atg8a-.


( A - G ) Representative images of pS65 - Ub immunostaining false - coloured for intensity ( Fire LUT ) in muscle segments 6 / 7 from wandering L3 larvae of the following genotypes : ( A ) wild type , ( B ) Pink1 - , ( C ) park - / - , ( D ) Atg5 - , ( E ) Atg8a - , ( F ) Atg5 - ; park - / - , ( G ) Atg8a - ; park - / - . ( H ) Quantification of number of pS65 - Ub puncta from A - G displaying mean + / - SEM from the indicated number of animals ( n = 3 - 5 as indicated ) . ( I ) Puncta volume from C - G expressed as mean + / - SEM ( n = 4 - 5 animals ) .

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(A-G) Representative images of pS65-Ub immunostaining false-coloured for intensity (Fire LUT) in muscle segments 6/7 from wandering L3 larvae of the following genotypes: (A) wild type, (B) Pink1-, (C) park-/-, (D) Atg5-, (E) Atg8a-, (F) Atg5-; park-/-, (G) Atg8a-; park-/-. (H) Quantification of number of pS65-Ub puncta from A-G displaying mean +/- SEM from the indicated number of animals (n = 3-5 as indicated). (I) Puncta volume from C-G expressed as mean +/- SEM (n = 4-5 animals).


( A ) pS65 - Ub immunoblot of whole - animal lysates following paraquat pulse - chase assay of Atg5 - flies with parkin overexpression ( OE ) ( Atg55cc5 daG4 > UAS - park ) compared with Atg5 - flies overexpressing mitochondrially targeted GFP ( Atg55cc5 daG4 > UAS - mito - HA - GFP ) . UT , untreated flies ; PQ , 1 - day paraquat treatment ; Recovery , flies removed from paraquat and returned to normal food .

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

(A) pS65-Ub immunoblot of whole-animal lysates following paraquat pulse-chase assay of Atg5- flies with parkin overexpression (OE) (Atg55cc5 daG4>UAS-park) compared with Atg5- flies overexpressing mitochondrially targeted GFP (Atg55cc5 daG4>UAS-mito-HA-GFP). UT, untreated flies; PQ, 1-day paraquat treatment; Recovery, flies removed from paraquat and returned to normal food.


( B ) pS65 - Ub lane densitometry , normalised to total protein ( RevertTM stain ) , of n = 3 independent replicates of ( A ) , expressed as pS65 - Ub intensity relative to the most intense band in each blot .

O O O O O O O O O O O O O O O O O O O O O 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) pS65-Ub lane densitometry, normalised to total protein (RevertTM stain), of n = 3 independent replicates of (A), expressed as pS65-Ub intensity relative to the most intense band in each blot.


( B ) Locomotor behavior , measured by thrashing rates in liquid medium , in the C . elegans strains with neuronal expression of human WT SOD1 or ALS - linked mutant SOD1G85R , in the presence ( M1 / M1 ) or absence ( + / + ) of the suppressor mutation ( n = 16 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Locomotor behavior, measured by thrashing rates in liquid medium, in the C. elegans strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (n = 16).


( C ) Northern ( top panel ) and western ( middle , bottom panels ) blot analyses of total RNA and protein from SOD1G85R strains , with ( M1 / M1 ) or without ( + / + ) suppressor mutations , demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation ( M1 / M1 ) . The western blot lanes are from the same gel and exposure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure.


( E ) C . elegans SPR - 5 and UFD - 2 and their mammalian homologs lysine - specific demethylase 1 ( LSD1 ) and ubiquitination factor E4 B ( UBE4B ) share all major protein domains . These include the Swi3 - Rsc8 - Moira ( SWIRM ) , amine oxidase - like ( AOL ) , and TOWER domains in LSD1 / SPR - 5 and the Ufd2 Core and U - box domains in UBE4B / UFD - 2 . Positions of the missense , nonsense , and deletion mutations in mutant C . elegans are indicated .

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(E) C. elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated.


( F ) Locomotor behavior , measured by thrashing rates , of the C . elegans carrying the SOD1G85R transgene on the normal background ( WT ) , with the null mutation of either ufd - 2 ( tm1380 ) or spr - 5 ( by134 ) , the double mutation ufd - 2 ( tm1380 ) ; spr - 5 ( by134 ) , or the M1 suppressor ufd - 2 ( W824X ) ; spr - 5 ( R646Q ) ( n = 16 ) .

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

(F) Locomotor behavior, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the null mutation of either ufd-2(tm1380) or spr-5(by134), the double mutation ufd-2(tm1380);spr-5(by134), or the M1 suppressor ufd-2(W824X);spr-5(R646Q) (n = 16).


( G ) Western blotting of the supernatant ( S ) and pellet ( P ) protein fractions from the C . elegans carrying the SOD1G85R transgene with the double mutation ufd - 2 ( tm1380 ) ; spr - 5 ( by134 ) compared with controls . While SOD1G85R protein levels are unchanged in the S fraction , those in the P fraction are decreased in the double suppressor mutant . The P fraction represents only about 1 . 7 % of total SOD1G85R in the WT sample ; therefore , a higher ratio of the P fraction relative to the S fraction ( approximately 2 : 1 ) is used for the western analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C. elegans carrying the SOD1G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis.


( H ) The overexpression ( OE ) of WT UFD - 2 or SPR - 5 in the C . elegans nervous system blocks the protection conferred by the double mutation ufd - 2 ( tm1380 ) ; spr - 5 ( by134 ) ( n = 16 ) . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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

(H) The overexpression (OE) of WT UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) (n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Top : schematic drawing depicts pan - neuronal expression of YFP in head and ventral neurons in the context of the C . elegans body plan . Left : micrographs show the SOD1G85R - YFP proteins expressed in WT or the spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) mutant background . The double - mutant worms show a marked decrease in protein aggregation in neurons . Enlarged sections of headneurons ( red framed ) and ventral cord neurons ( white framed ) are shown . Middle : quantification of locomotion in the spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) and the WT C . elegans with neuronal expression of SOD1G85R - YFP ( n = 30 ) . Right : a decrease in the protein levels of SOD1G85R - YFP in the presence of spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) is shown by western blots of the supernatant ( S ) and the pellet ( P ) fractions . ( B and C ) Analyses of the spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) and the WT C . elegans with neuronal expression of TDP - 43 - c25 - YFP ( n = 30 ) or PolyQ - YFP ( n = 12 ) as in ( A ) .

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

(A) Top: schematic drawing depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the C. elegans body plan. Left: micrographs show the SOD1G85R-YFP proteins expressed in WT or the spr-5(by134);ufd-2(tm1380) mutant background. The double-mutant worms show a marked decrease in protein aggregation in neurons. Enlarged sections of headneurons (red framed) and ventral cord neurons (white framed) are shown. Middle: quantification of locomotion in the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of SOD1G85R-YFP (n = 30). Right: a decrease in the protein levels of SOD1G85R-YFP in the presence of spr-5(by134);ufd-2(tm1380) is shown by western blots of the supernatant (S) and the pellet (P) fractions. (B and C) Analyses of the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of TDP-43-c25-YFP (n = 30) or PolyQ-YFP (n = 12) as in (A).


( D ) Neurodegenerative rough - eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd - 2 / UBE4B and spr - 5 / LSD1 - CG9934 and Su ( Var ) 3 - 3 , respectively , compared to the control ( CTRL ) . Eye - specific expression of TDP - 43M337V , FUSR521C , and RNA interference ( RNAi ) was driven by GMR - Gal4 .

(D) Neurodegenerative rough-eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd-2/UBE4B and spr-5/LSD1-CG9934 and Su(Var)3-3, respectively, compared to the control (CTRL). Eye-specific expression of TDP-43M337V, FUSR521C, and RNA interference (RNAi) was driven by GMR-Gal4.


( E ) Drosophila eye pigment quantitation . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(E) Drosophila eye pigment quantitation. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Western blots of cell lysates derived from mock ( CTRL ) , single UBE4B or LSD1 , or double UBE4B and LSD1 knockdowns . Supernatant ( S ) and pellet ( P ) fractions were probed with indicated antibodies . While the LSD1 or UBE4B single - knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions , the combined knockdown produces the strongest reduction in the aggregates . ( B ) Quantification of SOD1G85R protein levels by western blotting ( A ) . n = 3 ( supernatant ) ; n = 8 ( pellet ) .

(A) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSD1 or UBE4B single-knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. (B) Quantification of SOD1G85R protein levels by western blotting (A). n = 3 (supernatant); n = 8 (pellet).


( C ) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half - lives in the double UBE4B and LSD1 knockdown cells versus controls . ( D ) Quantification of SOD1G85R clearance , as analyzed by western blotting in ( C ) . The graph indicates the relative band intensity of SOD1G85R at each chase time point . n = 5 ; Overall p = 0 . 02 ( paired t test , CTRL versus UBE4B and LSD1 double knockdown ) . Individual p = 0 . 03 ( 3 h ) , p = 0 . 003 ( 6 h ) , p = 0 . 06 ( 9 h ) , and p = 0 . 004 ( 12 - 21 h ) .

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(C) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. (D) Quantification of SOD1G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1G85R at each chase time point. n = 5; Overall p = 0.02 (paired t test, CTRL versus UBE4B and LSD1 double knockdown). Individual p = 0.03 (3 h), p = 0.003 (6 h), p = 0.06 (9 h), and p = 0.004 (12-21 h).


( E ) The half - life of SOD1G85R is reduced from 8 . 5 h to 5 h upon knockdown of UBE4B and LSD1 . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(E) The half-life of SOD1G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( B ) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA . Gray spots represent 22 , 148 annotated transcripts . Red spots are predicted p53 - activated targets , and blue spots are predicted p53 - inhibited targets . The enrichment of red spots in the up - regulated genes ( right upper quadrant , > 1 . 2 - fold change , p ≤ 0 . 04 ) and blue spots in the down - regulated genes ( left upper quadrant ) indicates that the p53 - mediated transcription is activated by the UBE4B and LSD1 double knockdown .

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(B) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA. Gray spots represent 22,148 annotated transcripts. Red spots are predicted p53-activated targets, and blue spots are predicted p53-inhibited targets. The enrichment of red spots in the up-regulated genes (right upper quadrant, >1.2-fold change, p ≤ 0.04) and blue spots in the down-regulated genes (left upper quadrant) indicates that the p53-mediated transcription is activated by the UBE4B and LSD1 double knockdown.


C ) RT - qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double - knockdown samples . n = ( 2 to 6 ) , p ≤ 0 . 04 .

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C) RT-qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double-knockdown samples. n = (2 to 6), p ≤ 0.04.


( D ) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown . Left : representative western blots showing p53 levels , the knockdown of UBE4B and LSD1 , and the actin control in HEK293T cells . Right : quantification of the p53 protein levels normalized against actin ( n = 3 ) .

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(D) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown. Left: representative western blots showing p53 levels, the knockdown of UBE4B and LSD1, and the actin control in HEK293T cells. Right: quantification of the p53 protein levels normalized against actin (n = 3).


E ) The p53 - mediated transcriptional activity is measured by a luciferase reporter under the control of a p53 - response element promoter , which was transfected into HEK293T cells 72 - 96 h after the initiation of the UBE4B and LSD1 single or double knockdowns ( n = 6 ) .

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E) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72-96 h after the initiation of the UBE4B and LSD1 single or double knockdowns (n = 6).


( F ) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown . Left : representative western blots of 53BP1 co - immunoprecipitation . Right : quantification of the 53BP1 co - immunoprecipitation ( n = 2 ) . Data represent means ± SEM .

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(F) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown. Left: representative western blots of 53BP1 co-immunoprecipitation. Right: quantification of the 53BP1 co-immunoprecipitation (n = 2). Data represent means ± SEM.


( G ) RT - qPCR validation of the expression levels of FOXO3a , FOXO4 , and PSMD11 ( n = 6 ) .

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(G) RT-qPCR validation of the expression levels of FOXO3a, FOXO4, and PSMD11 (n = 6).


( H ) The FOXO3a - mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO - response element promoter . HEK293T cells were transfected with shRNAs for control , LSD1 , UBE4B , or both LSD1 and UBE4B , followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a ( TM ) 72 - 96 h later ( n = 6 ) . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(H) The FOXO3a-mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO-response element promoter. HEK293T cells were transfected with shRNAs for control, LSD1, UBE4B, or both LSD1 and UBE4B, followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a (TM) 72-96 h later (n = 6). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T

(A) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells


. ( B ) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate , Suc - LLVY - Luciferin , whose degradation is measured in a luciferase release assay . The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns ( n = 6 ) .

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. (B) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY-Luciferin, whose degradation is measured in a luciferase release assay. The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns (n = 6).


( C ) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown . The in vivo activity of the ATG4B protease , which cleaves LC3 precursors , was quantified via a Gaussia luciferase release assay ( see S5D Fig . , S5E Fig . , and Materials and Methods for details ) . The HCT116 cells were analyzed 48 h after the initiation of the knockdown .

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(C) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown. The in vivo activity of the ATG4B protease, which cleaves LC3 precursors, was quantified via a Gaussia luciferase release assay (see S5D Fig., S5E Fig., and Materials and Methods for details). The HCT116 cells were analyzed 48 h after the initiation of the knockdown.


( D ) Quantification of LC3 - II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control . The cells were treated with or without 10 mM 3 - methyladenine ( 3 - MA ) for 48h before lysis . Western blots indicate an increase in LC3 levels ( top panels ) in the double UBE4B and LSD1 knockdown cells ( middle panels ) , while the actin control is unchanged ( bottom panels ) . The graph shows the quantification of LC3 - II levels as normalized to actin levels ( n = 3 ) . A one - way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(D) Quantification of LC3-II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control. The cells were treated with or without 10 mM 3-methyladenine (3-MA) for 48h before lysis. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD1 knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels (n = 3). A one-way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) p53 small molecule activators Tenovin - 1 and CP - 31398 reduce the levels of misfolded SOD1 proteins , as determined by the SOD1G85R solubility assay in HEK293 cells . Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions .

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(A) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SOD1 proteins, as determined by the SOD1G85R solubility assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions.


( B ) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SOD1 proteins in the SOD1G85R aggregation assay , as shown by western blots of both supernatant ( S ) ( n = 2 ) and pellet ( P ) ( n = 3 ) fractions .

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(B) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SOD1 proteins in the SOD1G85R aggregation assay, as shown by western blots of both supernatant (S) (n = 2) and pellet (P) (n = 3) fractions.


( C ) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53 - / - HCT116 cells when compared with controls . Representative western blots ( left panels ) and quantification of SOD1G85R levels in the supernatant lysates are shown . The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1 . The right graph panel shows the same data as shown in the middle panel , but normalized to the average SOD1G85R level for each amount of the transfected plasmid . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(C) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53-/- HCT116 cells when compared with controls. Representative western blots (left panels) and quantification of SOD1G85R levels in the supernatant lysates are shown. The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1. The right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1G85R level for each amount of the transfected plasmid. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Left : p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown . Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs . Right : quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control , double ( UBE4B / LSD1 ) , or triple ( UBE4B / LSD1 / p53 ) shRNAs ( n = 2 ) .

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(A) Left: p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (n = 2).


( B ) The degenerative TDP - 43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 ( p53 RNAi ) , or by the overexpression of a dominant - negative p53 mutant ( p53 . R155H ) . Expression of p53 RNAi , p53 . R155H , and TDP - 43M337V are driven by GMR - Gal4 .

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(B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4.


( C ) The p53 - activating drug Tenovin - 1 ( TEN1 ) protects spinal cord motor neurons from SOD1G85R - induced proteotoxicity . Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0 . 8 μM TEN1 or vehicle ( VEH ) DMSO , followed by infection of HSV - SOD1G85R or the HSV - LacZ control after 24 h . At day 5 post - infection , cells were fixed and stained with a motor neuron - specific anti - neurofilament H ( NF - H ) antibody . Left : representative images of motor neurons in each condition . Scale bar = 50 μm . Right : quantification of motor neuron survival ( one - way ANOVA with multiple comparisons and Tukey correction ) . Data represent means ± SEM .

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(C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM.


( A ) Top hit selected candidates are listed . The plot shows relative S9 . 6 nuclear intensity values for cells transformed with each of the indicated pool of siRNAs .

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(A) Top hit selected candidates are listed. The plot shows relative S9.6 nuclear intensity values for cells transformed with each of the indicated pool of siRNAs.


( B ) Representative images of HeLa cells immunostained with S9 . 6 and nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool .

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(B) Representative images of HeLa cells immunostained with S9.6 and nucleolin antibodies after transfection with at least a pool of two siRNAs of each original pool.


( C ) Relative S9 . 6 signal intensity per nucleus after nucleolus signal removal in HeLa cells transfected with the indicated siRNAs . More than 250 total cells from three independent experiments were considered . The median of each population is shown . Boxes and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . * * * p < 0 . 001 ( Mann - Whitney U test ) .

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(C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells transfected with the indicated siRNAs. More than 250 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. ***p<0.001 (Mann-Whitney U test).


( A ) Relative DRIP - qPCR signal values at RPL13A , APOE , MIB2 and RHOT2 genes in HeLa cells transfected with the indicated siRNAs and treated in vitro with RNase H pre - immunoprecipitation where indicated . The mean ± SEM from at least three independent experiments is shown . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed paired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Relative DRIP-qPCR signal values at RPL13A, APOE, MIB2 and RHOT2 genes in HeLa cells transfected with the indicated siRNAs and treated in vitro with RNase H pre-immunoprecipitation where indicated. The mean ± SEM from at least three independent experiments is shown. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Representative images of immunostaining with S9 . 6 and anti - nucleolin antibodies in HeLa cells transfected with the indicated

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(B) Representative images of immunostaining with S9.6 and anti-nucleolin antibodies in HeLa cells transfected with the indicated siRNAs


( C ) Relative S9 . 6 signal intensity per nucleus after nucleolus signal removal in HeLa cells after cytoplasm pre - extraction ( CE ) and treated in vitro with RNaseIII and RNase H where indicated . More than 500 total cells from three independent experiments were considered . The median of each population is shown . Boxes and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U test ) .

(C) Relative S9.6 signal intensity per nucleus after nucleolus signal removal in HeLa cells after cytoplasm pre-extraction (CE) and treated in vitro with RNaseIII and RNase H where indicated. More than 500 total cells from three independent experiments were considered. The median of each population is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. **p<0.01, ***p<0.001 (Mann-Whitney U test).


( A ) Representative images of single - cell alkaline gel electrophoresis ( comet assay ) of HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) .

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(A) Representative images of single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+).


( B ) Comet tail moment from single - cell alkaline gel electrophoresis ( comet assay ) of HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 250 total cells were considered . The mean ± SEM of the median from at least three independent experiments is shown , except for RAD1 , RAD17 and UBE2B ( n = 2 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed unpaired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(B) Comet tail moment from single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 250 total cells were considered. The mean ± SEM of the median from at least three independent experiments is shown, except for RAD1, RAD17 and UBE2B (n=2). *p<0.05, **p<0.01, ***p<0.001 (one-tailed unpaired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Representative images and percentage of HeLa cells with more than 5 H3S10 - P foci after transfection with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . Mitotic cells were excluded for the analysis by DAPI staining . More than 300 total cells were considered . Data represent mean ± SEM from three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed paired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Representative images and percentage of HeLa cells with more than 5 H3S10-P foci after transfection with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). Mitotic cells were excluded for the analysis by DAPI staining. More than 300 total cells were considered. Data represent mean ± SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Representative images and H3K9me2 nuclear signal intensity of HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . At least three experiments were performed . The median of each population in a representative experiment with at least 100 cells per condition is shown . Boxes and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - tailed paired t - test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(B) Representative images and H3K9me2 nuclear signal intensity of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). At least three experiments were performed. The median of each population in a representative experiment with at least 100 cells per condition is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Fork velocity as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 200 tracks were considered except for RAD1 + RNH1 ( n = 125 ) , ATR + RNH1 ( n = 182 ) and RAD18 + RNH1 ( n = 146 ) . Median values are indicated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) Fork velocity as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 200 tracks were considered except for RAD1 +RNH1 (n=125), ATR +RNH1 (n=182) and RAD18 +RNH1 (n=146). Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


( B ) Fork asymmetry as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . From 40 to 200 total measurements were considered for each candidate . Median values are indicated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U test ) . Black stars denote significant increases whereas red stars denote significant decreases .

(B) Fork asymmetry as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). From 40 to 200 total measurements were considered for each candidate. Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


( A ) Left column : Flow cytometry profiles showing DNA content of the indicated RNase III - treated cells . G1 ( red ) , S ( blue ) and G2 ( green ) phases were calculated from the profile . Right column : Flow cytometry histograms depicting intensity of S9 . 6 signals in each phase of the cell cycle for the indicated cells . ( B ) Quantification of panel B . The mean ± SD of the S9 . 6 mean intensity of five experiments is shown . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( repeated measures ANOVA test with Bonferroni ' s post - test ) . ( C ) Graph represents fold change of S9 . 6 mean signal in G2 with respect to G1 cells . Values for five independent experiments together with the mean are shown . * p < 0 . 05 ( one - tailed paired t - test ) .

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(A) Left column: Flow cytometry profiles showing DNA content of the indicated RNase III-treated cells. G1 (red), S (blue) and G2 (green) phases were calculated from the profile. Right column: Flow cytometry histograms depicting intensity of S9.6 signals in each phase of the cell cycle for the indicated cells. (B) Quantification of panel B. The mean ± SD of the S9.6 mean intensity of five experiments is shown. ***p<0.001, **p<0.01, *p<0.05 (repeated measures ANOVA test with Bonferroni's post-test). (C) Graph represents fold change of S9.6 mean signal in G2 with respect to G1 cells. Values for five independent experiments together with the mean are shown. *p<0.05 (one-tailed paired t-test).


( A ) DNA - RNA hybrids , DNA damage and fork stalling in 9 - 1 - 1 / ATR / CHK1 , ATM / CHK2 and PRR deficient cells . DNA - RNA hybrids represent the mean ± SEM of all the DRIP data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Paired t - test ) . DNA damage represents the mean of medians ± SEM of all the comet tail moment data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( paired t - test ) . Fork stalling represents the median ± SEM of all the fork asymmetry data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Mann - Whitney U test ) . Black stars denote significant increases whereas red stars denote significant decreases .

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(A) DNA-RNA hybrids, DNA damage and fork stalling in 9-1-1/ATR/CHK1, ATM/CHK2 and PRR deficient cells. DNA-RNA hybrids represent the mean ± SEM of all the DRIP data ***p<0.001, **p<0.01, *p<0.05 (Paired t-test). DNA damage represents the mean of medians ± SEM of all the comet tail moment data ***p<0.001, **p<0.01, *p<0.05 (paired t-test). Fork stalling represents the median ± SEM of all the fork asymmetry data ***p<0.001, **p<0.01, *p<0.05 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.


IF by confocal microscopy performed on KRAB - HA overexpressing cells . Staining was performed with anti - HA ( Alexa - 488 , green ) , anti - HSPA5 ( Alexa - 647 , red ) and DNA was stained with Dapi ( blue ) .

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IF by confocal microscopy performed on KRAB-HA overexpressing cells. Staining was performed with anti-HA (Alexa-488, green), anti-HSPA5 (Alexa-647, red) and DNA was stained with Dapi (blue).


HA immunoprecipitation of stably expressed ZNF282 - HA in cells previously transfected with ΔDUF3669 - ZNF398 - GFP and WT - ZNF398 - GFP followed by detection of ZNF398 constructs in the IPs through Western - Blot using an anti - GFP antibody . Bottom : Western - Blot using an anti - HA antibody on the IPs . Input = Cellular lysate , IP =

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HA immunoprecipitation of stably expressed ZNF282-HA in cells previously transfected with ΔDUF3669-ZNF398-GFP and WT-ZNF398-GFP followed by detection of ZNF398 constructs in the IPs through Western-Blot using an anti-GFP antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs. Input = Cellular lysate, IP = Immunoprecipitate


Left : DUF3669 - only and KRAB domain constructs used . Right : HA immunoprecipitation in cells previously co - transfected with ZNF398 - DUF3669 - HA and either ZNF282 - KRAB - Flag or ZNF282 - DUF3669 - Flag followed by detection of either of these protein constructs in the IPs through Western - Blot using an anti - Flag antibody . Bottom : Western - Blot using an anti - HA antibody on the IPs .

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Left: DUF3669-only and KRAB domain constructs used. Right: HA immunoprecipitation in cells previously co-transfected with ZNF398-DUF3669-HA and either ZNF282-KRAB-Flag or ZNF282-DUF3669-Flag followed by detection of either of these protein constructs in the IPs through Western-Blot using an anti-Flag antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs.


Histograms representing the percentage of KZFPs ( identified in Fig . 3B ) binding sites falling in TEs ( red ) or TSSs ( blue ) .

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Histograms representing the percentage of KZFPs (identified in Fig. 3B) binding sites falling in TEs (red) or TSSs (blue).


KZFPs binding sites on L1PA5 and L1PA6 . Top : boxes containing the interactors of KZFPs found enriched on L1PA5 , 6 . Middle : plot showing the average ChIP - exo signals ( scaled between 0 and 1 ) for each selected KZFP plotted on top of L1PA5 and L1PA6 multiple sequence alignment ( MSA ) . Bottom : schematic representation of L1PA5 , 6 different domains .

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KZFPs binding sites on L1PA5 and L1PA6. Top: boxes containing the interactors of KZFPs found enriched on L1PA5,6. Middle: plot showing the average ChIP-exo signals (scaled between 0 and 1) for each selected KZFP plotted on top of L1PA5 and L1PA6 multiple sequence alignment (MSA). Bottom: schematic representation of L1PA5,6 different domains.


Human KZFPs KRAB domain A - boxes protein phylogenetic tree associated with their corresponding amino acid sequences colored according to the Clustal ZAPPO color scheme ( residues sharing common physicochemical properties display the same color : http : / / www . jal - view . org / help / html / colourSchemes / zappo . html ) . This figure also displays a zoom - in on the variant KRAB domain cluster ( right ) . On this zoom - in , the tested vKZFPs , whose interactomes were defined in our study , were marked by an asterisk . D . Box plot representing KAP1FC values in function of KZFPs age . On a superimposed swarm plot , the individual vKZFPs corresponding KAP1FC values were represented by red dots and for their sKZFPs counterparts , by blue dots . Mann - Whitney two - sided rank test .

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Human KZFPs KRAB domain A-boxes protein phylogenetic tree associated with their corresponding amino acid sequences colored according to the Clustal ZAPPO color scheme (residues sharing common physicochemical properties display the same color: http://www.jal- view.org/ help/html/colourSchemes/zappo.html). This figure also displays a zoom-in on the variant KRAB domain cluster (right). On this zoom-in, the tested vKZFPs, whose interactomes were defined in our study, were marked by an asterisk. D. Box plot representing KAP1FC values in function of KZFPs age. On a superimposed swarm plot, the individual vKZFPs corresponding KAP1FC values were represented by red dots and for their sKZFPs counterparts, by blue dots. Mann-Whitney two-sided rank test.


Immunoprecipitations of HA - tagged KRAB domains in order to check interaction with cKAP1 . Co - transfection of Flag - tagged cKAP1 ( upper panel ) or Flag - tagged hKAP1 ( lower panel ) and HA - tagged ZNF398 DUF3669 domain negative control , H2ZYM4 and M3XKG1 cKRAB domains and ZNF93 hKRAB domain in KAP1 KO HAP1 cells followed by HA - immunoprecipitation . cKAP1 and hKAP1 presence was revealed by Western - Blot using an anti - Flag antibody . Input = Cellular lysate , IP = Immunoprecipitate . Western - Blot using an HA antibody on the IPs at the bottom .

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Immunoprecipitations of HA-tagged KRAB domains in order to check
 interaction with cKAP1. Co-transfection of Flag-tagged cKAP1 (upper panel) or Flag-tagged hKAP1 (lower panel) and HA-tagged ZNF398 DUF3669 domain negative control, H2ZYM4 and M3XKG1 cKRAB domains and ZNF93 hKRAB domain in KAP1 KO HAP1 cells followed by HA-immunoprecipitation. cKAP1 and hKAP1 presence was revealed by Western-Blot using an anti-Flag antibody. Input = Cellular lysate, IP = Immunoprecipitate. Western-Blot using an HA antibody on the IPs at the bottom.


Immunoblot analysis of mouse intestinal crypts treated with Wnt3a ( 60 ng / mL ) for indicated time . The relative protein level was quantified and shown under each band .

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Immunoblot analysis of mouse intestinal crypts treated with Wnt3a (60 ng/mL) for indicated time. The relative protein level was quantified and shown under each band.


Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min . The right panels show the distributions of Ythdf1 and Actb in polysome fractions . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) .

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Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min. The right panels show the distributions of Ythdf1 and Actb in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


Immunoblot analysis of SW620 cells with or without APC overexpression . The relative protein level was quantified and shown under each band .

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Immunoblot analysis of SW620 cells with or without APC overexpression. The relative protein level was quantified and shown under each band.


Polysome profiles of SW620 cells with or without APC overexpression . The right panels show the distributions of YTHDF1 and ACTB in polysome fractions . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) .

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Polysome profiles of SW620 cells with or without APC overexpression. The right panels show the distributions of YTHDF1 and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


Dual - Luciferase assay wih a construct bearing the 5 ' UTR of YTHDF1 in SW620 cells with or without APC overexpression . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) .

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Dual-Luciferase assay wih a construct bearing the 5'UTR of YTHDF1 in SW620 cells with or without APC overexpression. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc + / + and Apcmin / + mice . N , normal tissue . T , tumor tissue . A , tumor adjacent tissue .

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YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc+/+ and Apcmin/+ mice. N, normal tissue. T, tumor tissue. A, tumor adjacent tissue.


YTHDF1 staining in matched samples of human CRC tissue and adjacent non - tumor tissue . Scale bar , 450 μm . The right panel shows the statistics of IHC scores . Chi - square test ; n = 75 for each group .

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YTHDF1 staining in matched samples of human CRC tissue and adjacent non-tumor tissue. Scale bar, 450 μm. The right panel shows the statistics of IHC scores. Chi-square test; n = 75 for each group.


The representative jejunum from wildtype ( Ythdf1CTL ) and Ythdf1cKO mice . Left : hematoxylin and eosin staining ( H & E ) ; middle : BrdU staining ; right : quantification of crypt height and BrdU + cells . Scale bar , 50 μm . Data are represented as mean ± SEM . ( 9 biological replicates , t - test ) .

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The representative jejunum from wildtype (Ythdf1CTL) and Ythdf1cKO mice. Left: hematoxylin and eosin staining (H&E); middle: BrdU staining; right: quantification of crypt height and BrdU+ cells. Scale bar, 50 μm. Data are represented as mean ± SEM. (9 biological replicates, t-test).


Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR .

Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR.


YTHDF1 ( left ) and Ki67 ( right ) staining of small intestine from wildtype mice 72 hr after 12 Gy IR . Scale bar , 50 μm . Red arrows indicate the proliferating cells .

YTHDF1 (left) and Ki67 (right) staining of small intestine from wildtype mice 72 hr after 12 Gy IR. Scale bar, 50 μm. Red arrows indicate the proliferating cells.


Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR . Left : H & E staining ; middle : Ki67 staining ; right : quantification of crypt height and Ki67 + cells . Scale bar , 50 μm . Vertical bars indicate the length of the crypts . Data are represented as mean ± SEM . * * * * p < 0 . 0001 ( 9 biological replicates , t - test ) .

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Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Left: H&E staining; middle: Ki67 staining; right: quantification of crypt height and Ki67+ cells. Scale bar, 50 μm. Vertical bars indicate the length of the crypts. Data are represented as mean ± SEM. ****p<0. 0001 (9 biological replicates, t-test).


RT - qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 5 biological replicates , t - test ) .

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RT-qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (5 biological replicates, t-test).


Small intestines from 24 - week - old Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice .

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Small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice.


H & E staining of small intestines from 24 - week - old Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice . Scale bar , 1 mm . The black arrows indicate the tumors .

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H&E staining of small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Scale bar, 1 mm. The black arrows indicate the tumors.


Tumor number ( C ) and size distribution ( D ) from the small intestines of 24 - week - old Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice . Data are shown as mean ± SEM ( 6 mice for each group ) . * * p < 0 . 01 , * * * p < 0 . 001 ( t - test ) .

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Tumor number (C) and size distribution (D) from the small intestines of 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01, ***p<0. 001 (t-test).


Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM / DSS induction .

Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM/DSS induction.


H & E staining of colons from Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice after AOM / DSS induction . Scale bar , 200 μm .

H&E staining of colons from Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice after AOM/DSS induction. Scale bar, 200 μm.


Colon tumor number ( H ) and size distribution ( I ) from Ythdf1CTL and Ythdf1cKO mice on day 84 of induction . Data are shown as mean ± SEM ( 6 mice for each group ) . * * p < 0 . 01 ( t - test ) .

Colon tumor number (H) and size distribution (I) from Ythdf1CTL and Ythdf1cKO mice on day 84 of induction. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01 (t-test).


Fluorescent in situ hybridization for Ythdf1 mRNA in Ythdf1CTL and Ythdf1cKO small intestines . Scale bar , 50 μm .

Fluorescent in situ hybridization for Ythdf1 mRNA in Ythdf1CTL and Ythdf1cKO small intestines. Scale bar, 50 μm.


Immunoblot analysis of fractionated murine intestine enriched for villus cells and crypt cells .

Immunoblot analysis of fractionated murine intestine enriched for villus cells and crypt cells.


Flowcytometry analysis of YTHDF1 protein expression in GFPhigh / low / neg populations from Lgr5 - GFP - IRES - creERT2 mice .

Flowcytometry analysis of YTHDF1 protein expression in GFPhigh/low/neg populations from Lgr5-GFP-IRES-creERT2 mice.


Morphology of organoids from Lgr5 - creERT2 : Ythdf1fl / fl mice in Wnt3a - conditioned medium without or with 4 - OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant . Scale bar , 250 μm .

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Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant. Scale bar, 250 μm.


Quantification of differentiated versus undifferentiated organoids from ( D ) . Data are represented as mean ± SEM . * * p < 0 . 01 ( 4 biological replicates , t - test ) .

Quantification of differentiated versus undifferentiated organoids from (D). Data are represented as mean ± SEM. **p<0. 01 (4 biological replicates, t-test).


Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in ( D ) . Data are represented as mean ± SEM . 4 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - test ) .

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Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in (D). Data are represented as mean ± SEM. 4 biological replicates. *p<0. 05, **p<0. 01, ***p<0. 001 (t-test).


Scatterplot of mRNA expression and ribosome profiling data from control and YTHDF1 knockdown cells . The upregulated ( red ) and downregulated ( blue ) genes in translational efficiency ( TE ) are highlighted .

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Scatterplot of mRNA expression and ribosome profiling data from control and YTHDF1 knockdown cells. The upregulated (red) and downregulated (blue) genes in translational efficiency (TE) are highlighted.