( A ) Western blots showing expression of AhR in MC38 induced by different fractions ( , lipid ) 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 ) effect of different fractions of mulberry bark derived exosome - like nanoparticles ( MBELNs ) on 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 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 stimulated with either control , Wnt3a , Wnt3a RSPO1 - 4 conditioned media , or by transfection with indicated constructs . Where indicated , were co - transfected with 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 that were transfected with GFP - USP42 or indicated GFP - tagged control , together with 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 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 transfected with constructs shown in ( A ) . In ( F ) 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 , surface biotinylation performed in HEK293T transfected as indicated treated with 10 μM MG132 to prevent proteasomal degradation . Consecutive immunoprecipitations were carried out as indicated in scheme . Avidin pulldown was carried under denaturing conditions ( 1st IP ) . unrelated receptor CD147 was used as loading control for avidin pull down , CUL1 for total input of 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 transfected with indicated constructs . 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 upon knockdown of indicated genes using single siRNAs . were stimulated with control or Wnt3a conditioned medium . Data is displayed as mean ± SD , 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 of USP42 , LGR5 AXIN2 expression in HCT116 . Data are displayed as mean ± SD 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 in wt WLS ( Evi ) KO HCT116 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 ) , of signal ( Alexa - 488 ) in n ≥ 4 biological replicates with n ≥ 15 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 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 , were treated with 5 μM IWP - 2 or 20 μM LGK - 974 ( PORCN inhibitors ) . median 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 of mouse small intestinal organoids generated with empty ( Non - edited ) or USP42gRNA containing CRISPR - concatemers , cultured for 3 days in matrigel with indicated growth factors , or Wnt secretion inhibitor IWP - 2 . EN , EGF noggin . Note that knockout of Usp42 ( Usp42 gRNAs ) rendered 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 of Lgr5 Axin2 expression 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 of H & E of colon from 3m C57Bl ( n = 4 ) mdx ( n = 4 ) mice . High magnification ( scale bar : 20 μm ) 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 , between yellow dash lines ; crypt , yellow headed arrow . Scale bar : 100 μm . ( C ) Mucus layer thickness crypt were quantified for n = 4 mice per group ( with pooled samples of n = 60 for mucus layer thickness n = 80 for crypt ) .

(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 of 3m C57Bl ( n = 3 ) 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 - ) .

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

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

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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 of SCFAs in 3m mdx 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 - ) .

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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 of spleen muscle homogenates from 3m C57Bl ( n = 4 ) , mdx ( n = 5 ) mdx + ABX ( n = 7 ) mice demonstrates no significant alteration of CD45 + CD11b + CD4 - CD8 - myeloid ( A C ) few differences in CD4 + or CD8 + naïve ( CD62L + CD44 - ) , central memory ( CD62L + CD44 + ) effector ( CD62L - CD44 + ) T ( B 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 .

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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 of spleen of 3m C57Bl ( n = 7 ) , mdx ( n = 9 ) GFmdx ( n = 5 ) mice revealed similar proportions of CD4 + CD8 + T but reduced activated CD44 + T 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 .

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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 + CD8 + T on live of 3m C57Bl ( n = 7 ) , mdx ( n = 4 ) GFmdx ( n = 5 ) mice . Representative dot plots cumulative frequencies of splenic CD4 + GITR + CD25 + Treg . Frequencies of effector CD44 + T 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 .

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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 proportion of muscle - infiltrating CD45 + of 3m C57Bl ( n = 6 ) , mdx ( n = 6 ) GFmdx ( n = 5 ) mice . Cumulative frequencies of muscle - infiltrating CD45 + 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 .

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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 of TA from 3m mdx GFmdx mice stained for CD45 ( in green ) , isolectin ( in red ) 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 ( arrows ) were quantified in n = 12 of TA of 3m C57Bl , 3m mdx 3m GFmdx mice ( n = 6 each ) . CD3 is shown in green 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 comparisons among averages of CD3 + were evaluated using unpaired t - .

(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 of TA of two independent experiments with 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) GFmdx ( n = 5 ) mice determined 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 non - parametric followed by Kruskal - Wallis 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 of myofiber relative frequency of myofiber cross - sectional ( CSA ) expressed as frequency distribution of TA of 3m C57Bl ( n = 4 ) , 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) 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 , 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

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

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(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 of of SDH + myofibers of TAs from 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) GFmdx mice ( n = 5 ) ( n = 12 per ) . Scale bars : 50 μm .

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

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

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

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(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 ) isolectin ( in red ) their in TA from mdx ( n = 5 ) ABX - mdxFMT _ C57Bl ( n = 6 ) mice . Scale bar : 500 μm . Data information : Data for are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , one - way ANOVA , Tukey ' s multiple comparison ) .

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

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

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

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

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

(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 - lysates from untreated flies of 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 - / - .

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(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 of ATP5A of pS65 - Ub - positive ( + ve ) versus - negative ( - ve ) mitochondrial structures from 3 biological replicates ( 4 - 7 per ; average per shown in open circles ) .

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(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 - lysates from wild - type , Atg5 - park - / - harvested at indicated ages .

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(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 - lysates from young of following genotypes : wild - type , Pink1 - , park - / - , park RNAi ( daG4 > UAS - park RNAi ) , Atg1 RNAi ( daG4 > UAS - Atg1 RNAi ) , Atg8a - .

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

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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 - lysates following paraquat - chase 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 returned to normal food .

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 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

(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 ( RevertTM stain ) , of n = 3 independent replicates of ( A ) , expressed as pS65 - Ub relative to 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

(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 C . elegans strains with neuronal expression of human WT SOD1 or ALS - linked mutant SOD1G85R , in presence ( M1 / M1 ) or absence ( + / + ) of suppressor mutation ( n = 16 ) .

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(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 ) western ( middle , bottom panels ) blot analyses of total from SOD1G85R strains , with ( M1 / M1 ) or without ( + / + ) suppressor mutations , demonstrating that of SOD1G85R mRNA total are not changed by suppressor mutation ( M1 / M1 ) . western blot lanes are from same gel exposure .

(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 UFD - 2 their mammalian homologs lysine - specific demethylase 1 ( LSD1 ) ubiquitination factor E4 B ( UBE4B ) share major domains . These include Swi3 - Rsc8 - Moira ( SWIRM ) , amine oxidase - like ( AOL ) , TOWER domains in LSD1 / SPR - 5 Ufd2 U - box domains in UBE4B / UFD - 2 . Positions of missense , nonsense , 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 C . elegans carrying SOD1G85R transgene on normal background ( WT ) , with null mutation of either ufd - 2 ( tm1380 ) or spr - 5 ( by134 ) , double mutation ufd - 2 ( tm1380 ) ; spr - 5 ( by134 ) , or M1 suppressor ufd - 2 ( W824X ) ; spr - 5 ( R646Q ) ( n = 16 ) .

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

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

(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 ventral neurons in context of C . elegans plan . Left : show SOD1G85R - YFP expressed in WT or spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) mutant background . double - mutant worms show a marked decrease in aggregation in neurons . Enlarged sections of headneurons ( red framed ) ventral cord neurons ( framed ) are shown . Middle : of locomotion in spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) WT C . elegans with neuronal expression of SOD1G85R - YFP ( n = 30 ) . Right : a decrease in of SOD1G85R - YFP in presence of spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) is shown by western blots of supernatant ( S ) pellet ( P ) fractions . ( B C ) Analyses of spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) 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 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 knockdown of Drosophila homologs of ufd - 2 / UBE4B spr - 5 / LSD1 - CG9934 Su ( Var ) 3 - 3 , respectively , compared to control ( CTRL ) . Eye - specific expression of TDP - 43M337V , FUSR521C , interference ( RNAi ) was driven by GMR - Gal4 .

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(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 ± . numerical data used to make this figure can be found in S1 Data .

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

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(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 half - lives in double UBE4B LSD1 knockdown versus controls . ( D ) of SOD1G85R clearance , as analyzed by western blotting in ( C ) . graph indicates relative band of SOD1G85R at each chase time point . n = 5 ; Overall p = 0 . 02 ( paired t , CTRL versus UBE4B LSD1 double knockdown ) . p = 0 . 03 ( 3 h ) , p = 0 . 003 ( 6 h ) , p = 0 . 06 ( 9 h ) , 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 ) half - life of SOD1G85R is reduced from 8 . 5 h to 5 h upon knockdown of UBE4B LSD1 . Data represent means ± . 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 ) volcano scatter plot indicates fold changes in of gene transcripts affected differentially by UBE4B LSD1 double knockdown versus control shRNA . Gray spots represent 22 , 148 annotated transcripts . Red spots are predicted p53 - activated targets , blue spots are predicted p53 - inhibited targets . enrichment of red spots in up - regulated genes ( right upper quadrant , > 1 . 2 - fold change , p ≤ 0 . 04 ) blue spots in down - regulated genes ( left upper quadrant ) indicates that p53 - mediated transcription is activated by UBE4B 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 expression of representative p53 target genes in UBE4B 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 ) p53 level is significantly increased by UBE4B LSD1 double knockdown . Left : representative western blots showing p53 , knockdown of UBE4B LSD1 , actin control in HEK293T . Right : of p53 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 ) p53 - mediated transcriptional activity is measured by a luciferase reporter under control of a p53 - response element promoter , which was transfected into HEK293T 72 - 96 h after initiation of UBE4B LSD1 single or double knockdowns ( n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O 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) 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 LSD1 knockdown . Left : representative western blots of 53BP1 co - immunoprecipitation . Right : of 53BP1 co - immunoprecipitation ( n = 2 ) . Data represent means ± .

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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 expression of FOXO3a , FOXO4 , PSMD11 ( n = 6 ) .

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


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

(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 of proteasome subunits upon UBE4B LSD1 double knockdown in HEK293T

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


. ( B ) UBE4B LSD1 single or double knockdowns increase proteasomal degradation of a reporter substrate , Suc - LLVY - Luciferin , whose degradation is measured in a luciferase release . double knockdown shows synergistic proteasomal activation when compared with 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 ) autophagy activity is significantly increased in with UBE4B LSD1 double knockdown . in vivo activity of ATG4B protease , which cleaves LC3 precursors , was quantified via a Gaussia luciferase release ( see S5D Fig . , S5E Fig . , Materials Methods for details ) . HCT116 were analyzed 48 h after initiation of 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 ) of LC3 - II in HEK293T with UBE4B LSD1 double knockdown or mock control . were treated with or without 10 mM 3 - methyladenine ( 3 - MA ) for 48h before lysis . Western blots indicate an increase in LC3 ( top panels ) in double UBE4B LSD1 knockdown ( middle panels ) , while actin control is unchanged ( bottom panels ) . graph shows of LC3 - II as normalized to actin ( n = 3 ) . A one - way ANOVA with matched multiple comparisons Tukey correction was used for statistics . Data represent means ± . 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 CP - 31398 reduce of misfolded SOD1 , as determined by SOD1G85R solubility in HEK293 . Increasing concentrations of p53 activators significantly decrease of SOD1G85R but not endogenous WT SOD1 in western blots of both supernatant 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 result of shRNA knockdown increases of SOD1G85R but not WT SOD1 in SOD1G85R aggregation , as shown by western blots of both supernatant ( S ) ( n = 2 ) 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 accumulation of SOD1G85R mutant in p53 - / - HCT116 when compared with controls . Representative western blots ( left panels ) of SOD1G85R in supernatant lysates are shown . middle graph indicates of G85R to WT SOD1 in presence or absence of p53 with varying amounts of transfected mutant SOD1 . right graph panel shows same data as shown in middle panel , but normalized to average SOD1G85R level for each amount of transfected plasmid . Data represent means ± . numerical data used to make this figure can be found in S1 Data .

(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 enhanced clearance of SOD1G85R conferred by UBE4B LSD1 double knockdown . Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs . Right : of insoluble aggregated SOD1G85R in pellet fractions from HEK293T 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 ) degenerative TDP - 43M337V eye phenotype is exacerbated by knockdown of Drosophila homolog of p53 ( p53 RNAi ) , or by overexpression of a dominant - negative p53 mutant ( p53 . R155H ) . Expression of p53 RNAi , p53 . R155H , 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 ) 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 Methods treated with 0 . 8 μM TEN1 or vehicle ( VEH ) DMSO , followed by infection of HSV - SOD1G85R or HSV - LacZ control after 24 h . At day 5 post - infection , were fixed stained with a motor neuron - specific anti - neurofilament H ( NF - H ) antibody . Left : representative of motor neurons in each condition . Scale bar = 50 μm . Right : of motor neuron survival ( one - way ANOVA with multiple comparisons Tukey correction ) . Data represent means ± .

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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 . plot shows relative S9 . 6 values for transformed with each of 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 of HeLa immunostained with S9 . 6 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 per nucleus after nucleolus signal removal in HeLa transfected with indicated siRNAs . More than 250 total from three independent experiments were considered . median of each population is shown . Boxes whiskers indicate 25 - 75 10 - 90 percentiles , respectively . * * * p < 0 . 001 ( Mann - Whitney U ) .

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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 RHOT2 genes in HeLa transfected with indicated siRNAs treated in vitro with RNase H pre - immunoprecipitation where indicated . mean ± from at least three independent experiments is shown . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - paired t - ) . 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 of immunostaining with S9 . 6 anti - nucleolin antibodies in HeLa transfected with indicated

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

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(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 of single - alkaline gel electrophoresis ( comet ) of HeLa transfected with indicated siRNAs 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 - alkaline gel electrophoresis ( comet ) of HeLa transfected with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 250 total were considered . mean ± of median from at least three independent experiments is shown , except for RAD1 , RAD17 UBE2B ( n = 2 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - unpaired t - ) . 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 of HeLa with more than 5 H3S10 - P foci after transfection with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . Mitotic were excluded for by DAPI . More than 300 total were considered . Data represent mean ± from three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - paired t - ) . 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 H3K9me2 signal of HeLa transfected with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . At least three experiments were performed . median of each population in a representative experiment with at least 100 per condition is shown . Boxes whiskers indicate 25 - 75 10 - 90 percentiles , respectively . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - paired t - ) . 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 combing in HeLa transfected with indicated siRNAs either pEGFP - C1 ( RNH1 - ) or pEGFP - M27 ( RNH1 + ) . More than 200 tracks were considered except for RAD1 + RNH1 ( n = 125 ) , ATR + RNH1 ( n = 182 ) RAD18 + RNH1 ( n = 146 ) . Median values are indicated . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Mann - Whitney U ) . 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.


( B ) Fork asymmetry as measured by combing in HeLa transfected with indicated siRNAs 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 ) . Black stars denote significant increases whereas red stars denote significant decreases .

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

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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 ) - hybrids , damage fork stalling in 9 - 1 - 1 / ATR / CHK1 , ATM / CHK2 PRR deficient . - hybrids represent mean ± of DRIP data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Paired t - ) . damage represents mean of medians ± of comet tail moment data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( paired t - ) . Fork stalling represents median ± of fork asymmetry data * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( Mann - Whitney U ) . 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 . was performed with anti - HA ( Alexa - 488 , green ) , anti - HSPA5 ( Alexa - 647 , red ) 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 previously transfected with ΔDUF3669 - ZNF398 - GFP WT - ZNF398 - GFP followed by detection of ZNF398 constructs in IPs through Western - Blot using an anti - GFP antibody . Bottom : Western - Blot using an anti - HA antibody on IPs . Input = 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 KRAB domain constructs used . Right : HA immunoprecipitation in previously co - transfected with ZNF398 - DUF3669 - HA either ZNF282 - KRAB - Flag or ZNF282 - DUF3669 - Flag followed by detection of either of these constructs in IPs through Western - Blot using an anti - Flag antibody . Bottom : Western - Blot using an anti - HA antibody on 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 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 L1PA6 . Top : boxes containing interactors of KZFPs found enriched on L1PA5 , 6 . Middle : plot showing average ChIP - exo signals ( scaled between 0 1 ) for each selected KZFP plotted on top of L1PA5 L1PA6 multiple 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 phylogenetic tree associated with their corresponding acid sequences colored according to Clustal ZAPPO color scheme ( residues sharing common physicochemical properties display same color : http : / / www . jal - view . org / help / html / colourSchemes / zappo . html ) . This figure also displays a zoom - in on variant KRAB domain cluster ( right ) . On this zoom - in , tested vKZFPs , whose interactomes were defined in our study , were marked by an asterisk . D . Box plot representing KAP1FC values in function of KZFPs . On a superimposed swarm plot , vKZFPs corresponding KAP1FC values were represented by red dots for their sKZFPs counterparts , by blue dots . Mann - Whitney two - sided rank .

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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 ) HA - tagged ZNF398 DUF3669 domain negative control , H2ZYM4 M3XKG1 cKRAB domains ZNF93 hKRAB domain in KAP1 KO HAP1 followed by HA - immunoprecipitation . cKAP1 hKAP1 presence was revealed by Western - Blot using an anti - Flag antibody . Input = lysate , IP = Immunoprecipitate . Western - Blot using an HA antibody on IPs at bottom .

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

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 of mouse intestinal crypts treated with Wnt3a ( 60 ng / mL ) for indicated time . relative level was quantified 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 . right panels show distributions of Ythdf1 Actb in polysome fractions . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - ) .

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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 of SW620 with or without APC overexpression . relative level was quantified shown under each band .

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

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

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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 expression in intestinal tissue from 4 pairs of Apc + / + Apcmin / + mice . N , normal tissue . T , tissue . A , 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 in matched samples of human CRC tissue adjacent non - tissue . Scale bar , 450 μm . right panel shows statistics of scores . Chi - square ; 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.


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

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

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


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

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

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

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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 Apcmin / + ; Ythdf1cKO mice .

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


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


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

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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 Ythdf1cKO mice on day 84 of AOM / DSS induction .

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


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

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


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

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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).


in situ hybridization for Ythdf1 mRNA in Ythdf1CTL 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 of fractionated murine intestine enriched for villus crypt .

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


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

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


of organoids from Lgr5 - creERT2 : Ythdf1fl / fl mice in Wnt3a - conditioned medium without or with 4 - OHT induction 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.


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

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


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

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 ribosome profiling data from control YTHDF1 knockdown . upregulated ( red ) 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.


relative m6A peak coverage in control for transcripts grouped according to TE changes after YTHDF1 knockdown . POI : peak over input . Down , genes significantly downregulated in YTHDF1 knockdown ( < 0 . 5 fold ) . Unchanged , genes not significantly changed . Up , genes significantly upregulated ( > 2 fold ) . upper lower quartiles median are shown for each group . Mann - Whitney . * * p < 0 . 01 . * * * p < 0 . 001 .

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The relative m6A peak coverage in control cells for transcripts grouped according to TE changes after YTHDF1 knockdown. POI: peak over input. Down, genes significantly downregulated in YTHDF1 knockdown cells (<0.5 fold). Unchanged, genes not significantly changed. Up, genes significantly upregulated (>2 fold). The upper and lower quartiles and the median are shown for each group. Mann-Whitney test. **p<0. 01. ***p<0. 001.


Violin plots showing TE change between control YTHDF1 knockdown for not - methylated ( non - m6A ) methylated ( m6A ) transcripts . upper lower quartiles median are indicated for each group . Mann - Whitney .

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Violin plots showing TE change between control and YTHDF1 knockdown cells for not-methylated (non-m6A) and methylated (m6A) transcripts. The upper and lower quartiles and the median are indicated for each group. Mann-Whitney test.


Cumulative distributions of TE change for non - m6A m6A transcripts as in ( C ) . Kolmogorov - Smirnov .

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Cumulative distributions of TE change for non-m6A and m6A transcripts as in (C). Kolmogorov-Smirnov test.


showing TE of Wnt signaling components in control YTHDF1 knockdown . values show fold change of indicated color of .

Heatmap showing the TE of Wnt signaling components in control and YTHDF1 knockdown cells. The values show the fold change of the indicated color of the heatmap.


Immunoblot of HCT116 with YTHDF1 knockdown . Two major bands were detected for human TCF7L2 due to alternative splicing .

Immunoblot analysis of HCT116 cells with YTHDF1 knockdown. Two major bands were detected for human TCF7L2 due to alternative splicing.


Polysome profiles of HCT116 with or without YTHDF1 knockdown . right panels show distributions of TCF7L2 , ACTB in polysome fractions . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - ) .

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Polysome profiles of HCT116 cells with or without YTHDF1 knockdown. The right panels show the distributions of TCF7L2, and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).


Immunoblot of crypts from Ythdf1CTL Ythdf1cKO mice . Multiple bands were detected for mouse TCF7L2 representing different isoforms .

Immunoblot analysis of crypts from Ythdf1CTL and Ythdf1cKO mice. Multiple bands were detected for mouse TCF7L2 representing different isoforms.


Immunoblot of mouse intestinal crypts treated with Wnt3a for 60 min .

Immunoblot analysis of mouse intestinal crypts treated with Wnt3a for 60 min.


MeRIP - qPCR of m6A of TCF7L2 in crypts treated with or without Wnt3a . Data are represented as mean ± . * p < 0 . 05 ( 3 biological replicates , t - ) .

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MeRIP-qPCR analysis of m6A levels of TCF7L2 in crypts treated with or without Wnt3a. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).


RIP of interaction of YTHDF1 with TCF7L2 mRNA . enrichment was measured by qPCR normalized to input . Data are represented as mean ± . * p < 0 . 05 ( 3 biological replicates , t - ) .

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RIP analysis of the interaction of YTHDF1 with TCF7L2 mRNA. The enrichment was measured by qPCR and normalized to input. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).


β - catenin / TCF4 reporter activity in YTHDF1 knockdown infected with lentivirus expressing TCF7L2 . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( 3 biological replicates , t - ) .

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β-catenin/TCF4 reporter activity in YTHDF1 knockdown cells infected with lentivirus expressing TCF7L2. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01, ***p<0.001 (3 biological replicates, t-test).


of organoids from Lgr5 - creERT2 : Ythdf1fl / fl mice in Wnt3a - conditioned medium without or with 4 - OHT induction infected with lentivirus expressing TCF7L2 . Scale bar , 250 μm . of differentiated versus undifferentiated organoids from ( H ) . Data are represented as mean ± . * * p < 0 . 01 , * * * p < 0 . 001 ( 3 biological replicates , t - ) .

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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 TCF7L2. Scale bar, 250 μm. Quantification of differentiated versus undifferentiated organoids from (H). Data are represented as mean ± SEM. **p<0. 01, ***p<0. 001 (3 biological replicates, t-test).


Colons from mice in ( A ) treated with or without TAM on day 80 of AOM / DSS induction .

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Colons from mice in (A) treated with or without TAM on day 80 of AOM/DSS induction.


Colon number from mice treated with or without TAM on day 80 of AOM / DSS induction . Data are shown as mean ± ( 7 mice for each group ) . * * * * p < 0 . 0001 ( t - ) .

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Colon tumor number from mice treated with or without TAM on day 80 of AOM/DSS induction. Data are shown as mean ± SEM (7 mice for each group). ****p<0. 0001 (t-test).


Immunoblot of 2 pairs of colon tumors from mice treated with or without TAM .

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Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM.


Kaplan - Meier survival plots for Apcmin / + ; Lgr5 - creERT2 : Ythdf1fl / fl mice ( 8 mice for each group ) treated with or without TAM . Log - rank ( Mantel Cox ) . * * p < 0 . 01 ( t - ) .

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Kaplan-Meier survival plots for Apcmin/+; Lgr5-creERT2:Ythdf1fl/fl mice (8 mice for each group) treated with or without TAM. Log-rank (Mantel Cox) test. **p<0. 01 (t-test).


( A ) Motility of C . c . ΔpseI expressing predicted Leg or Pse synthases ( LegI or PseI , in yellow or red , respectively ) from Plac on pSRK - Gm . Overnight cultures were spotted on PYE soft ( 0 . 3 % ) agar plates with gentamycin IPTG ( 0 . 5 mM ) incubated for 3 days at 30 ° C . Only predicted PseI - like synthase coding sequences ( CDSs ) restore motility of C . c . ΔpseI . Note that predicted synthases labeled in do not share similarities with LegI or PseI of C . jejuni . Motility assays are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Motility assay of C.c. ΔpseI cells expressing predicted Leg or Pse synthases (LegI or PseI, in yellow or red, respectively) from Plac on pSRK-Gm. Overnight cultures were spotted on PYE soft (0.3%) agar plates with gentamycin and IPTG (0.5 mM) and incubated for 3 days at 30°C. Only predicted PseI-like synthase coding sequences (CDSs) restore motility of the C.c. ΔpseI cells. Note that the predicted synthases labeled in white do not share similarities with LegI or PseI of C. jejuni. Motility assays are representative of three independent experiments.