C . Prognostic parameters in ACC patients . Results are presented as hazard ratios ( HR ) including 95 % confidence interval ( CI ) . The significance level was set at α = 5 % for all comparisons . The results for SF - 1 , FATE1 and combined SF - 1 / FATE1 expression levels are shown both in univariate and mutivariate ( adjusted for age , sex and ENSAT tumour stage ) analysis .

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C. Prognostic parameters in ACC patients. Results are presented as hazard ratios (HR) including 95% confidence interval (CI). The significance level was set at α=5% for all comparisons. The results for SF-1, FATE1 and combined SF-1/FATE1 expression levels are shown both in univariate and mutivariate (adjusted for age, sex and ENSAT tumour stage) analysis.


D . Overall survival of ACC patients according to FATE1 expression in their tumours , as measured by quantitative immunohistochemistry ( low , green curve ; high , red curve ) in mutivariate analysis . p = 0 . 008 .

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D. Overall survival of ACC patients according to FATE1 expression in their tumours, as measured by quantitative immunohistochemistry (low, green curve; high, red curve) in mutivariate analysis. p=0.008.


A ) Quantification of the effect of individual BCL - 2 - family proteins on HCT AKO cell death , measured as percentage of cells showing released cyt c or pyknotic nuclei . Data correspond to at least three independent experiments , n > 30 cells per condition per experiment . * * * p < 0 , 001 and * p < 0 , 05 with respect to GFP condition .

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A) Quantification of the effect of individual BCL-2-family proteins on HCT AKO cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n>30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to GFP condition.


C ) Western blot analysis of SMAC and cyt c release in GFP - BAX , tBID - GFP and GFP - BCLXL expressing cells at 12 h , 16 h and 24 h after transfection in HCT AKO . SN ( supernatant , cytosolic fraction ) , P ( pellet , membrane fraction ) and PS ( Ponceau Staining ) .

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C) Western blot analysis of SMAC and cyt c release in GFP-BAX, tBID-GFP and GFP-BCLXL expressing cells at 12 h, 16 h and 24 h after transfection in HCT AKO. SN (supernatant, cytosolic fraction), P (pellet, membrane fraction) and PS (Ponceau Staining).


F ) Kinetics of the expression of tBID - GFP ( blue ) and PI intake upon tBID expression ( purple ) and unstransfected HCT116 AKO cells ( in grey ) . Plots show the average fluorescence intensity ( dark lines ) and its standard deviation ( shaded areas ) from 3 technical replicates and 3 independent experiments . Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min .

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F) Kinetics of the expression of tBID-GFP (blue) and PI intake upon tBID expression (purple) and unstransfected HCT116 AKO cells (in grey). Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 3 technical replicates and 3 independent experiments. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.


G ) Quantification of PI + cells from total transfected cells with GFP - BAX , tBID - GFP , GFP - BCLXL and GFP . n = 3 independent experiments with > 10000 cells per condition per experiment . * * * p < 0 , 001 with respect to untransfected condition .

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G) Quantification of PI+ cells from total transfected cells with GFP-BAX, tBID-GFP, GFP-BCLXL and GFP. n=3 independent experiments with >10000 cells per condition per experiment. *** p<0,001 with respect to untransfected condition.


H ) Cell death induced by tBID - GFP in HCT AKO cells in the absence and presence of ZVAD , NSA and Ferrostatin - 1 . Data correspond to three independent experiments , n > 30 cells per condition per experiment . * * * p < 0 , 001 with respect to MOCK condition .

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H) Cell death induced by tBID-GFP in HCT AKO cells in the absence and presence of ZVAD, NSA and Ferrostatin-1. Data correspond to three independent experiments, n>30 cells per condition per experiment. *** p<0,001 with respect to MOCK condition.


A ) Representative confocal immunofluorescence and bright field ( BF ) images of HCT DKO cells expressing tBID - GFP ( in green ) treated or not with COMBO ( ABT737 + S63586 ) . Cyt c shown in magenta and nuclei in cyan . Scale bar 10 µm . B ) Effect of tBID - GFP and mCherry - BCLXL on cell death , measured as percentage of cells showing released cyt c or pyknotic nuclei . Data correspond to at least three independent experiments , n > 30 cells per condition per experiment . * * * p < 0 , 001 , * * p < 0 , 025 and * p < 0 , 05 with respect to tBID - GFP in HCT AKO condition .

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A) Representative confocal immunofluorescence and bright field (BF) images of HCT DKO cells expressing tBID-GFP (in green) treated or not with COMBO (ABT737 + S63586). Cyt c shown in magenta and nuclei in cyan. Scale bar 10 µm. B) Effect of tBID-GFP and mCherry-BCLXL on cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n> 30 cells per condition per experiment. *** p<0,001, ** p<0,025 and * p<0,05 with respect to tBID-GFP in HCT AKO condition.


C ) Representative confocal immunofluorescence images of HCT DKO cells upon indicated treatments . Nuclei ( cyan ) and cyt c ( magenta ) are shown . Scale bar 10µm . Arrowheads represent cells showing released cytochrome c and pyknotic nuclei .

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C) Representative confocal immunofluorescence images of HCT DKO cells upon indicated treatments. Nuclei (cyan) and cyt c (magenta) are shown. Scale bar 10µm. Arrowheads represent cells showing released cytochrome c and pyknotic nuclei.


E ) % of cell death induced by TRAIL ( 1µg / ml ) , COMBO and their combination at different treatment durations on HCT DKO measured as in panel B . Paired Student ' s t - test * * p < 0 , 025 and * p < 0 , 05 with respect to MOCK condition .

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E) % of cell death induced by TRAIL (1µg/ml), COMBO and their combination at different treatment durations on HCT DKO measured as in panel B. Paired Student's t-test ** p<0,025 and * p<0,05 with respect to MOCK condition.


I ) Effect of TRAIL ( 1µg / ml ) , COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells . Each dot corresponds to the average of 3 technical replicates from 4 independent experiments . * * * p < 0 , 001 .

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I) Effect of TRAIL (1µg/ml), COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells. Each dot corresponds to the average of 3 technical replicates from 4 independent experiments. *** p<0,001.


J ) Representative HyVolution - superresolution images of TFAM - RFP expressing U2OS BAX / BAK DKO cells in the in the absence ( top ) and presence of tBID - GFP ( bottom ) . TFAM appears in red , TOM20 / mitochondria in grey and tBID - GFP in green .

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J) Representative HyVolution - superresolution images of TFAM-RFP expressing U2OS BAX/BAK DKO cells in the in the absence (top) and presence of tBID-GFP (bottom). TFAM appears in red, TOM20/mitochondria in grey and tBID-GFP in green.


K ) Quantification of cells with TFAM release on U2OS BAX / BAK DKO cells transfected with GFP - BAX / tBID - GFP and TFAM - RFP in the presence / absence of COMBO . Dots correspond to individual experiments ( n = 3 individual experiments with more than 10 cells per condition per experiment ) . Unpaired Student ' s t - test * * * p < 0 , 001 with respect to non - treated conditions .

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K) Quantification of cells with TFAM release on U2OS BAX/BAK DKO cells transfected with GFP-BAX/tBID-GFP and TFAM-RFP in the presence/ absence of COMBO. Dots correspond to individual experiments (n=3 individual experiments with more than 10 cells per condition per experiment). Unpaired Student's t-test *** p<0,001 with respect to non-treated conditions.


B ) Cell death induced by tBID - GFP and its variants on HCT AKO , measured as percentage of cells showing released cyt c or pyknotic nuclei . Data correspond to three independent experiments , with n > 30 cells per condition per experiment . * * * p < 0 , 001 and * p < 0 , 05 with respect to tBID - GFP WT condition .

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B) Cell death induced by tBID-GFP and its variants on HCT AKO, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to three independent experiments, with n> 30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to tBID-GFP WT condition.


D ) Effect of tBID wt and α6 - mutants overexpression on HCT AKO cells , measured as DRAQ7 + / GFP + cells and normalized to tBID wt condition . n = 3 independent experiments , * * p < 0 , 025 and * p < 0 , 05 with respect to tBID - GFP wt condition . E ) Effect of tBID wt and α6 - mutants overexpression on MEF BID KO cells , measured as DRAQ7 + / GFP + cells and normalized to tBID wt condition . Effect of GFP overexpression on BID KO cells relative to tBID wt was assessed by pyknotic nuclei formation quantification . n = 3 independent experiments * * * p < 0 , 001 with respect to tBID - GFP wt condition .

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D) Effect of tBID wt and α6-mutants overexpression on HCT AKO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. n=3 independent experiments, ** p<0,025 and * p<0,05 with respect to tBID-GFP wt condition. E) Effect of tBID wt and α6-mutants overexpression on MEF BID KO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. Effect of GFP overexpression on BID KO cells relative to tBID wt was assessed by pyknotic nuclei formation quantification. n=3 independent experiments *** p<0,001 with respect to tBID-GFP wt condition.


A ) Representative CLEM images of mitochondria in HCT AKO cells ( control ) and HCT AKO cells expressing GFP - BAX or tBID - GFP . Black arrows indicate MOM disruptions . B - D ) Effect of GFP - BAX and tBID - GFP on HCT AKO mitochondrial structure . ( B ) Number of disruptions at the MOM per mitochondrial area , each dot represents an individual cell . ( C ) Mean area per mitochondrion at the condition tested , each dot represents an individual mitochondrion . ( D ) Analysis of cristae morphology , each dot represents an individual crista . Cristae shape calculated as length / width . Data represent n > 50 mitochondria from 3 - 5 independent cells . In B - D ) , boxes represent 96 % confidence interval , the average is represented by the line inside the box and whiskers correspond to S . D . The statistical significance was assessed by unpaired Student ' s t - test . * * * p < 0 , 001 and * * p < 0 , 025 with respect to HCT AKO or untransfected condition .

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A) Representative CLEM images of mitochondria in HCT AKO cells (control) and HCT AKO cells expressing GFP-BAX or tBID-GFP. Black arrows indicate MOM disruptions. B-D) Effect of GFP -BAX and tBID-GFP on HCT AKO mitochondrial structure. (B) Number of disruptions at the MOM per mitochondrial area, each dot represents an individual cell. (C) Mean area per mitochondrion at the condition tested, each dot represents an individual mitochondrion. (D) Analysis of cristae morphology, each dot represents an individual crista. Cristae shape calculated as length/width. Data represent n>50 mitochondria from 3-5 independent cells. In B-D), boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to S.D. The statistical significance was assessed by unpaired Student's t-test. *** p<0,001 and ** p<0,025 with respect to HCT AKO or untransfected condition.


E ) Representative confocal and STED images of GFP - BAX and tBID - GFP expressed in HCT AKO . Scale bar 1 µm for BAX images , 5 µm for tBID images . F ) Zoomed - in structures of GFP - BAX supramolecular assemblies with the shape of lines ( * double lines ) , arcs and rings in HCT AKO cells . Scale bar 100 nm .

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E) Representative confocal and STED images of GFP-BAX and tBID-GFP expressed in HCT AKO. Scale bar 1 µm for BAX images, 5 µm for tBID images. F) Zoomed-in structures of GFP-BAX supramolecular assemblies with the shape of lines (*double lines), arcs and rings in HCT AKO cells. Scale bar 100 nm.


A ) representative western blot of cytosolic cyt c and SMAC in HeLa cells . B , C ) Western Blot analysis of active BAX ( B ) and BAK ( C ) , in HeLa cells treated with STS or infected with Shigella flexneri ( MOI 30 ) at different time points after infection .

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A) representative western blot of cytosolic cyt c and SMAC in HeLa cells. B,C) Western Blot analysis of active BAX (B) and BAK (C), in HeLa cells treated with STS or infected with Shigella flexneri (MOI 30) at different time points after infection.


E ) Representative Western Blot analysis of cytosolic and total ( lysate ) cyt c and SMAC in HCT wt , HCT DKO and HCT TKO cells infected with Shigella flexneri at the indicated time points after treatment / infection .

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E) Representative Western Blot analysis of cytosolic and total (lysate) cyt c and SMAC in HCT wt, HCT DKO and HCT TKO cells infected with Shigella flexneri at the indicated time points after treatment/infection.


A ) Caspase - 3 / 7 activity induced by ABT199 and etoposide on Nalm6 ( venetoclax - sensitive ) and Nalm6 199R ( venetoclax - resistant ) cells , normalized to untreated cells . Unpaired Student ' s t - test * * * p < 0 , 001 and * * p < 0 , 025 ( Nalm6 with respect to Nalm6 199R at different conditions ) . Dots correspond to technical replicates of n = 3 independent experiments .

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A) Caspase-3/7 activity induced by ABT199 and etoposide on Nalm6 (venetoclax-sensitive) and Nalm6 199R (venetoclax-resistant) cells, normalized to untreated cells. Unpaired Student's t-test *** p<0,001 and ** p<0,025 (Nalm6 with respect to Nalm6 199R at different conditions). Dots correspond to technical replicates of n=3 independent experiments.


D ) Representative kinetics of cell death , represented as DRAQ7 + cells in Nalm6 199R cells ( grey ) , Nalm6 199R BID KO # 1 cells ( blue ) and Nalm6 199R BID KO # 5 cells ( purple ) upon 250 ng / ml TRAIL treatment . Plots show the average fluorescence intensity ( dark lines ) and its standard deviation ( shaded areas ) from 5 technical replicates . Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min .

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D) Representative kinetics of cell death, represented as DRAQ7+ cells in Nalm6 199R cells (grey), Nalm6 199R BID KO #1 cells (blue) and Nalm6 199R BID KO #5 cells (purple) upon 250 ng/ml TRAIL treatment. Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 5 technical replicates. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.E


H ) Cell death induced by GFP - BAK and GFP - BAK R127H on HCT AKO , measured as DRAQ7 + / GFP + cells . Unpaired Student ' s t - test . * * * p < 0 , 001 with respect to GFP BAK wt .

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H) Cell death induced by GFP-BAK and GFP-BAK R127H on HCT AKO, measured as DRAQ7+/GFP+ cells. Unpaired Student's t-test. *** p<0,001 with respect to GFP BAK wt.


I ) Effect of 24 h incubation with 250 ng / ml TRAIL on Nalm6 199R , Nalm6 199R BAK KO and Nalm6 199R BOK KO , measured as DRAQ7 + cells with respect of total cell surface and normalized to untreated conditions . Each dot corresponds to the average normalized value of 3 - 4 technical replicates in n = 3 independent experiments .

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I) Effect of 24 h incubation with 250 ng/ml TRAIL on Nalm6 199R, Nalm6 199R BAK KO and Nalm6 199R BOK KO, measured as DRAQ7+ cells with respect of total cell surface and normalized to untreated conditions. Each dot corresponds to the average normalized value of 3-4 technical replicates in n=3 independent experiments.


( I ) Representative image showing changes in colon morphology and length following DSS - induced colitis with concurrent treatment with MBELNs . ( J ) Column graph showing changes in colon length . Data are mean ± SEM from seven biological replicates , * * P < 0 . 01 using one - way ANOVA . (

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(I) Representative image showing changes in colon morphology and length following DSS-induced colitis with concurrent treatment with MBELNs. (J) Column graph showing changes in colon length. Data are mean ± SEM from seven biological replicates, **P<0.01 using one-way ANOVA. (


Hematoxylin and eosin ( HE ) staining to show histological changes in colon tissue Scale bar 200 μm .

Hematoxylin and eosin (HE) staining to show histological changes in colon tissue Scale bar 200 μm.


( M ) Enzyme - linked immunosorbent assay ( ELISA ) for interleukin ( IL ) - 6 and IL - 1β from colon tissue . Data are mean ± SEM from seven biological replicates , * * P < 0 . 01 using one - way ANOVA .

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(M) Enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-6 and IL-1β from colon tissue. Data are mean ± SEM from seven biological replicates, **P<0.01 using one-way ANOVA.


( A ) Mass spectrometry ( MS ) analysis of aryl hydrocarbon receptor ( AhR ) pulldown protein from Mulberry bark derived exosome - like nanoparticles ( MBELNs ) and 2 , 3 , 7 , 8 - tetrachlorodibenzo - p - dioxin ( TCDD ) treated MC38 cell lysate . Data from three technical replicates are shown .

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(A) Mass spectrometry (MS) analysis of aryl hydrocarbon receptor (AhR) pulldown protein from Mulberry bark derived exosome-like nanoparticles (MBELNs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated MC38 cell lysate. Data from three technical replicates are shown.


( D ) Representative images showing effect of MBELNs with and without TCDD ( 1 nm ) / carbobenzoxy - Leu - Leu - leucinal ( MG132 ; 20 µM ) on co - expression of AhR and COPS8 using immunofluorescence . Scale bar 10 μm , images are from three biological replicates .

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(D) Representative images showing effect of MBELNs with and without TCDD (1 nm)/ carbobenzoxy-Leu-Leu-leucinal (MG132; 20 µM) on co-expression of AhR and COPS8 using immunofluorescence. Scale bar 10 μm, images are from three biological replicates.


( E ) Western blot showing MBELNs , TCDD and MG132 effect on co - expression of AhR and COPS8 . The data represent as mean ± SEM from three biological replicates * * P < 0 . 01 , * * * P < 0 . 001 using one - way ANOVA .

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(E) Western blot showing MBELNs, TCDD and MG132 effect on co-expression of AhR and COPS8. The data represent as mean ± SEM from three biological replicates **P<0.01, ***P<0.001 using one-way ANOVA.


( F ) Heat map showing MS analysis of AhR pulldown protein from MC38 cells treated with MBELNs in combination with proteasome inhibitor , MG132 . Data are represented from three technical replicates .

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(F) Heat map showing MS analysis of AhR pulldown protein from MC38 cells treated with MBELNs in combination with proteasome inhibitor, MG132. Data are represented from three technical replicates.


( C ) Transmission electron microscopy ( TEM ) of crypts of COPS8fl / fl and COPS8ΔIEC mice . The base of the crypt in COPS8ΔIEC mice is occupied by poorly differentiated columnar epithelial cells that lack secretory granules , rudimentary electron - dense granules ( black arrows ) , granules in the lumen ( blue arrows ) and have a contracted endoplasmic reticulum ( ER ) ( white arrows ) . Scale bar 5μm , data are from seven biological replicates per genotype .

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(C) Transmission electron microscopy (TEM) of crypts of COPS8fl/fl and COPS8ΔIEC mice. The base of the crypt in COPS8ΔIEC mice is occupied by poorly differentiated columnar epithelial cells that lack secretory granules, rudimentary electron-dense granules (black arrows), granules in the lumen (blue arrows) and have a contracted endoplasmic reticulum (ER) (white arrows). Scale bar 5μm, data are from seven biological replicates per genotype.


( G - H ) Real - time quantitative reverse transcription Polymerase chain reaction ( PCR ) ( qRT - PCR ) analysis of the expression of genes encoding AMPs in the ileum ( G ) and colon ( H ) in COPS8fl / fl and COPS8ΔIEC mice . Error bars indicate the mean ± SD from three biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 using Student ' s t - test .

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(G-H) Real-time quantitative reverse transcription Polymerase chain reaction (PCR) (qRT-PCR) analysis of the expression of genes encoding AMPs in the ileum (G) and colon (H) in COPS8fl/fl and COPS8ΔIEC mice. Error bars indicate the mean ± SD from three biological replicates. *P<0.05, **P<0.01, ***P<0.001 using Student's t-test.


( A ) Real - time quantitative reverse transcription polymerase chain reaction ( PCR ) ( qRT - PCR ) analysis of the differential abundance of selected bacterial taxa in stool samples of ileum and colon of Villin - Cre and COPS8 - lox alleles expressing ( COPS8fl / fl ) and COPS8 knockout ( KO ) ( COPS8ΔIEC ) mice . Error bars indicate the mean ± SD from three biological replicates per genotype . * * P < 0 . 01 , NS - nonsignificant using Student ' s t - test . (

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(A) Real-time quantitative reverse transcription polymerase chain reaction (PCR) (qRT-PCR) analysis of the differential abundance of selected bacterial taxa in stool samples of ileum and colon of Villin-Cre and COPS8-lox alleles expressing (COPS8fl/fl ) and COPS8 knockout (KO) (COPS8ΔIEC) mice. Error bars indicate the mean ± SD from three biological replicates per genotype. **P<0.01, NS- nonsignificant using Student's t-test. (


Mice were treated by 2 % dextran sodium sulfate ( DSS ) in drinking water for 7 days . ( F ) Representative hematoxylin and eosin ( HE ) stained sections of colon and cecum from COPS8fl / fl and COPS8ΔIEC mice after DSS - indcued colitis ( day 9 ) . Scale bar 100μm , representative data from five biological replicates per genotype . ( G ) Histopathologically scored sections of distal colon ; cecum and colon length were analyzed from COPS8fl / fl and COPS8ΔIEC mice after DSS - indcued colitis ( day 7 ) . Data are represented as mean ± SEM from five biological replicates per genotype . * * P < 0 . 01 using Student ' s t - test . (

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Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (F) Representative hematoxylin and eosin (HE) stained sections of colon and cecum from COPS8fl/fl and COPS8ΔIEC mice after DSS-indcued colitis (day 9). Scale bar 100μm, representative data from five biological replicates per genotype. (G) Histopathologically scored sections of distal colon; cecum and colon length were analyzed from COPS8fl/fl and COPS8ΔIEC mice after DSS-indcued colitis (day 7). Data are represented as mean ± SEM from five biological replicates per genotype. **P<0.01 using Student's t-test. (


Mice were treated by 2 % dextran sodium sulfate ( DSS ) in drinking water for 7 days . ( H ) Cytokine levels ( interleukin ( IL ) - 1β , IL - 6 and tumor necrosis factor - α ( TNF - α ) ) in the colon collected on day 7 in colitis induced COPS8fl / fl and COPS8ΔIEC mice . Data are represented as mean ± SEM from five biological replicates per genotype . * P < 0 . 05 , * * P < 0 . 01 , NS - non - significant using Student ' s t - test .

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Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (H) Cytokine levels (interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α)) in the colon collected on day 7 in colitis induced COPS8fl/fl and COPS8ΔIEC mice. Data are represented as mean ± SEM from five biological replicates per genotype. *P<0.05, **P<0.01, NS- non-significant using Student's t-test.


Mice were treated by 2 % dextran sodium sulfate ( DSS ) in drinking water for 7 days . ( I ) The frequency of CD11b + Ly6C + and CD11b + Ly6G + cells in colonic lamina propria ( cLP ) of COPS8fl / fl and or COPS8ΔIEC mice with DSS - induced colitis . The column graph represents percentage of Ly6C + and Ly6G + cells , presented as mean ± SEM from three biological replicates per genotype . * P < 0 . 05 , NS - non - significant using Student ' s t - test . ( J ) Representative FACS plots and percentage of intracellular staining of Forkhead box protein P3 ( FOXP3 ) , Interferon gamma ( IFN - γ ) and IL - 17A in CD3 + CD4 + T cells from colonic lamina propria of COPS8fl / fl or COPS8ΔIEC mice with DSS - induced colitis . The column graph represents percentage of Treg + and ratio of Th1 / Th17 cells , represented as mean ± SEM from three biological replicates per genotype . * P < 0 . 05 using Student ' s t - test .

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Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (I) The frequency of CD11b+Ly6C+ and CD11b+Ly6G+ cells in colonic lamina propria (cLP) of COPS8fl/fl and or COPS8ΔIEC mice with DSS-induced colitis. The column graph represents percentage of Ly6C+ and Ly6G+ cells, presented as mean ± SEM from three biological replicates per genotype. *P<0.05, NS- non-significant using Student's t-test. (J) Representative FACS plots and percentage of intracellular staining of Forkhead box protein P3 (FOXP3), Interferon gamma (IFN-γ) and IL-17A in CD3+CD4+ T cells from colonic lamina propria of COPS8fl/fl or COPS8ΔIEC mice with DSS-induced colitis. The column graph represents percentage of Treg+ and ratio of Th1/Th17 cells, represented as mean ± SEM from three biological replicates per genotype. *P<0.05 using Student's t-test.


( H ) Flow cytometric analysis of CD45 + infiltrating immune cells in the lamina propria of the ileum . Data are represented as mean ± SEM from three biological replicates per genotype . * * P < 0 . 01 , NS - non - significant using one - way ANOVA .

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(H) Flow cytometric analysis of CD45+ infiltrating immune cells in the lamina propria of the ileum. Data are represented as mean ± SEM from three biological replicates per genotype. **P<0.01, NS- non-significant using one-way ANOVA.


( 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 .

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

(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 .

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

(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 .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(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 .

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

(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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O O O O O O B-MEASURED_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

(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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_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

(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 ) .

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

(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 B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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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 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 .

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(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 B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 ) .

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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 - animal lysates from wild - type , Atg5 - and park - / - animals harvested at the 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 - 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 - .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 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 .

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(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 .

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(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 ) .

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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 ) 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 .

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(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 ) .

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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 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 .

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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 ) 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 .

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(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 ) .

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(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 .

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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 ± 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 ) .

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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 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

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(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 .

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

(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.