expression of GPR15 , C10ORF99 , FOXP3 in mice fed with corn , chicken , mixture of corn plus chicken , commercial food chow a positive control ( propionate added to commercial food chow ) at different times ( weanling mice were defined as w - 1 ) in colonic tissue were determined by q - RT - PCR . Each symbol or bar represents pooled from colon tissues from three mice . Data information : data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

The expression levels of GPR15, C10ORF99, FOXP3 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


expression of IL - 10 in mice fed with corn , chicken , mixture of corn plus chicken , commercial food chow a positive control ( propionate added to commercial food chow ) at different times ( weanling mice were defined as w - 1 ) in colonic tissue were determined by q - RT - PCR . Each symbol or bar represents pooled from colon tissues from three mice . Data information : data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

The expression levels of IL-10 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


( f , g ) SCFAs acetic acid content in dietary experiments . Faecal samples from mice on indicated diets were analysed for SCFAs by chromatography . Data information : data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

(f, g) SCFAs and acetic acid content in dietary experiments. Faecal samples from mice on the indicated diets were analysed for SCFAs by gas chromatography. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


( h ) TNF - α IL - 6 in faecal samples from mice on indicated diet demonstrated no colitis in mouse groups . IL - 6 TNF - α production , as determined by performing enzyme - linked immunosorbent assays . Data information : data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

(h) TNF-α and IL-6 levels in faecal samples from mice on the indicated diet demonstrated no colitis in all mouse groups. IL-6 and TNF-α protein production levels, as determined by performing enzyme-linked immunosorbent assays. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


( i ) Luciferase activities of constructs with mouse C10ORF99 promoter in HEK293 . Data information : data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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(i) Luciferase activities of constructs with the mouse C10ORF99 promoter in HEK293 cells. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


Luciferase activities of constructs with Japanese quail C10ORF99 promoter in HEK293 . Data information : data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

Luciferase activities of constructs with the Japanese quail C10ORF99 promoter in HEK293 cells. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


( A ) Single confocal sections of HeLa infected with GFP - expressing S . Typhimurium strains ( blue ) for 10 h , fixed immunolabelled for LAMP1 ( green ) ubiquitin ( Ub , red ) ( false coloured , scale bars , 5 µm ) . far right panels show merged of LAMP1 , ubiquitin Salmonella . Arrows indicate SCV - associated ubiquitinated .

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(A) Single confocal sections of HeLa cells infected with GFP-expressing S. Typhimurium strains (blue) for 10 h, fixed and immunolabelled for LAMP1 (green) and ubiquitin (Ub, red) (false coloured, scale bars, 5 µm). The far right panels show merged images of LAMP1, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitinated aggregates.


B ) of of infected containing SCV - associated ubiquitinated . were processed as in ( A ) analysed by microscopy .

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B) Quantification of the percentage of infected cells containing SCV-associated ubiquitinated aggregates. Cells were processed as in (A) and analysed by fluorescence microscopy.


( C ) Kinetics of formation of SCV - associated ubiquitinated . were infected with GFP - expressing S . Typhimurium strains for indicated amount of time , processed as in ( A ) .

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(C) Kinetics of the formation of SCV-associated ubiquitinated aggregates. Cells were infected with GFP-expressing S. Typhimurium strains for the indicated amount of time, and processed as in (A).


( D ) of of infected with SCV - associated ubiquitinated in HeLa infected with S . Typhimurium strains for 14 h . were processed as in ( A ) analysed by microscopy . For quantifications , a minimum of 50 infected were counted per experiment values are mean ± from at least 3 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(D) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates in HeLa cells infected with S. Typhimurium strains for 14 h. Cells were processed as in (A) and analysed by fluorescence microscopy. For all quantifications, a minimum of 50 infected cells were counted per experiment and values are the mean ± SEM from at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.


A ) Immunoelectron microscopy of HeLa infected with S . Typhimurium strains for 12 h ( ubiquitin - 10 nm gold particles ; scale bars , 0 . 5 µm ) . Arrows indicate ubiquitin accumulations near within vacuoles .

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A) Immunoelectron microscopy of HeLa cells infected with S. Typhimurium strains for 12 h (ubiquitin - 10 nm gold particles; scale bars, 0.5 µm). Arrows indicate ubiquitin accumulations near bacteria within vacuoles.


( B ) Single confocal sections of HeLa infected with GFP - expressing ΔsseL mutant ( blue ) for 10 h immunolabelled for ubiquitin ( chain types - Ub , red ) lysine - 48 - linked ( K48 - Ub , green ) or lysine - 63 - linked ( K63 - Ub , green ) ubiquitin chains ( false coloured , scale bars , 5 µm ) . Arrows indicate SCV - associated ubiquitin K63 - Ub labelling .

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(B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 10 h and immunolabelled for ubiquitin (all chain types - Ub, red) and lysine-48-linked (K48-Ub, green) or lysine-63-linked (K63-Ub, green) ubiquitin chains (false coloured, scale bars, 5 µm). Arrows indicate SCV-associated ubiquitin and K63-Ub labelling.


SseL DUB activity reduces recruitment of ubiquitin autophagic markers to Salmonella microcolonies . Single confocal sections of HeLa infected with GFP - expressing ΔsseL mutant ( blue ) for 12 h immunolabelled for ubiquitin ( Ub , red ) ( A ) p62 ( green ) or ( B ) LC3 ( green ) ( false coloured , scale bars , 5 µm ) . far right panels show merged of p62 or LC3 , ubiquitin Salmonella . Arrows indicate SCV - associated ubiquitin labelling with p62 or LC3 .

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SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to Salmonella microcolonies.Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 12 h and immunolabelled for ubiquitin (Ub, red) and (A) p62 (green) or (B) LC3 (green) (false coloured, scale bars, 5 µm). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3.


( C - E ) . of relative of ( C ) ubiquitin , ( D ) p62 ( E ) LC3 per microcolony ( see Materials Methods ) . were processed as in ( A ) a minimum of 30 microcolonies analysed for each infection per experiment . Results from representative experiments showing relative mean ± are shown . Similar results were obtained in at least 3 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(C-E). Quantification of the relative fluorescence intensity of (C) ubiquitin, (D) p62 and (E) LC3 per microcolony (see Materials and Methods). Cells were processed as in (A) and a minimum of 30 microcolonies analysed for each bacterial infection per experiment. Results from representative experiments showing the relative mean fluorescence intensity ± SEM are shown. Similar results were obtained in at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.


( A ) of of infected with SCV - associated ubiquitinated . HeLa were infected with ΔsseL mutant for 12 h . At 8 h post - infection , were subjected to indicated treatments for 4 h before fixation immunolabelling . A minimum of 50 were counted per condition in each experiment values are mean ± of at least 3 independent experiments . p values are relative to mean value of mock - treated . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(A) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates. HeLa cells were infected with ΔsseL mutant bacteria for 12 h. At 8 h post-infection, cells were subjected to the indicated treatments for 4 h before fixation and immunolabelling. A minimum of 50 cells were counted per condition in each experiment and values are the mean ± SEM of at least 3 independent experiments. p values are relative to the mean value of mock-treated cells. * p<0.05; ** p<0.01; *** p<0.001.


( B ) Single confocal sections of HeLa infected with GFP - expressing ΔsseL mutant ( blue ) , treated processed as in ( A ) analysed by microscopy for ubiquitin ( Ub , red ) LC3 ( green ) ( scale bars , 5 µm ) . far right panels show merged of LC3 , ubiquitin Salmonella .

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(B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue), treated and processed as in (A) and analysed by fluorescence microscopy for ubiquitin (Ub, red) and LC3 (green) (scale bars, 5 µm). The far right panels show merged images of LC3, ubiquitin and Salmonella.


( A ) Single confocal sections of RAW264 . 7 macrophages infected with GFP - expressing strains of S . Typhimurium ( green ) for 10 h immunolabelled for ubiquitin ( Ub , red ) ( scale bars , 5 µm ) .

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(A) Single confocal sections of RAW264.7 macrophages infected with GFP-expressing strains of S. Typhimurium (green) for 10 h and immunolabelled for ubiquitin (Ub, red) (scale bars, 5 µm).


( B ) of infected macrophages containing ubiquitinated 10 h after uptake . were processed as in ( A ) analysed by microscopy . A minimum of 50 were counted for each infection per experiment values are mean ± of at least 3 independent experiments . * p < 0 . 05 ; * * * p < 0 . 001 . Uninfected ( uninf ) were from same wells as infected therefore were exposed to extracellular . ( C ) Immunoelectron microscopy of RAW264 . 7macrophages infected with ΔsseL mutant for 12 h . Arrows arrowheads indicate ubiquitin ( 15 nm gold particles ) p62 ( 10 nm gold particles ) , respectively ( scale bar , 0 . 5 µm ) .

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(B) Quantification of infected macrophages containing ubiquitinated aggregates 10 h after bacterial uptake. Cells were processed as in (A) and analysed by fluorescence microscopy. A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of at least 3 independent experiments. * p<0.05; *** p<0.001. Uninfected cells (uninf) were from the same wells as infected and therefore were exposed to extracellular bacteria. (C) Immunoelectron microscopy of RAW264.7macrophages infected with the ΔsseL mutant bacteria for 12 h. Arrows and arrowheads indicate ubiquitin (15 nm gold particles) and p62 (10 nm gold particles), respectively (scale bar, 0.5 µm).


( D E ) Single confocal sections of RAW264 . 7 macrophages infected with GFP - expressing strains of S . Typhimurium ( blue ) immunolabelled for ubiquitin ( Ub , red ) ( D ) p62 ( green ) or ( E ) LC3 ( green ) . far right panels show merged of p62 or LC3 , ubiquitin Salmonella . Arrows indicate Arrows indicate SCV - associated ubiquitin labelling with p62 or LC3 ( scale bars , 5 µm ) . outlines nuclei are delineated by lines .

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(D and E) Single confocal sections of RAW264.7 macrophages infected with GFP-expressing strains of S. Typhimurium (blue) and immunolabelled for ubiquitin (Ub, red) and (D) p62 (green) or (E) LC3 (green). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3 (scale bars, 5 µm). Cell outlines and nuclei are delineated by white lines.


( A ) of large ( > 2 µm ) , ubiquitinated aggresome - like induced structures ( ALIS ) in macrophages . bone marrow - derived macrophages ( BMM ) were infected with indicated GFP - expressing S . Typhimurium strains for 10 h , immunolabelled for ubiquitin analysed by confocal microscopy . A minimum of 50 were counted for each infection per experiment values are mean ± of 3 independent experiments . Uninfected ( uninf ) were from same wells as infected therefore were exposed to extracellular . * p < 0 . 05 ; * * p < 0 . 01 .

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(A) Quantification of large (>2 µm), ubiquitinated aggresome-like induced structures (ALIS) in macrophages. Primary bone marrow-derived macrophages (BMM) were infected with the indicated GFP-expressing S. Typhimurium strains for 10 h, immunolabelled for ubiquitin and analysed by confocal microscopy. A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of 3 independent experiments. Uninfected cells (uninf) were from the same wells as infected and therefore were exposed to extracellular bacteria. * p<0.05; ** p<0.01.


( B ) of number of puromycin - induced ubiquitin inclusions in . HeLa were transfected with a vector expressing myc - SseL or myc - SseLC / A for 16 h followed by treatment with puromycin ( 5 µg / ml ) for 4 h immunolabelled with anti - myc , anti - p62 anti - ubiquitin . 50 were counted per condition in each experiment . Values are means ± of at least 3 independent experiments . * * p < 0 . 01 .

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(B) Quantification of the number of puromycin-induced ubiquitin inclusions in cells. HeLa cells were transfected with a vector expressing myc-SseL or myc-SseLC/A for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc, anti-p62 and anti-ubiquitin. 50 individual cells were counted per condition in each experiment. Values are the means ± SEM of at least 3 independent experiments. ** p<0.01.


( C ) Single confocal sections of HeLa transfected with a vector expressing myc - SseL for 16 h followed by treatment with puromycin ( 5 µg / ml ) for 4 h immunolabelled with anti - myc ( blue ) , anti - p62 ( green ) anti - ubiquitin ( Ub , red ) ( scale bars , 5 µm ) . lower panels show a merged image of p62 , ubiquitin myc - SseL . Arrows indicate ubiquitin p62 present in untransfected arrowheads indicate expressing myc -

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(C) Single confocal sections of HeLa cells transfected with a vector expressing myc-SseL for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc (blue), anti-p62 (green) and anti-ubiquitin (Ub, red) (scale bars, 5 µm). The lower panels show a merged image of p62, ubiquitin and myc-SseL. Arrows indicate ubiquitin and p62 aggregates present in untransfected cells and arrowheads indicate cells expressing myc-SseL


SseL interacts with p62 autophagic substrates . ( A B ) Co - immunoprecipitation from RAW264 . 7 macrophages infected with ΔsseL mutant expressing SseL - HA or SseLC / A - HA for 10 h . ( A ) Infectedcell lysates immunoprecipated fractions were probed with anti - HA , anti - ubiquitin anti - p62 antibodies . ( B ) At 7 h post - infection , infected were subjected to mock , 3 - MA or starvation ( Stv ) treatments for 3 h before harvesting processing as described in ( A ) . lysates immunoprecipiations were probed with anti - HA anti - ubiquitin antibodies .

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SseL interacts with p62 and autophagic substrates.(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.


( A ) Lipidated LC3B ( LC3 - II ) in RAW264 . 7 macrophages infected with indicated strains of S . Typhimurium for 12 h . were left untreated ( − ) or subjected to 10 mM NH4Cl treatment ( + ) for 2 h before harvesting .

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(A) Lipidated LC3B (LC3-II) levels in RAW264.7 macrophages infected with the indicated strains of S. Typhimurium for 12 h. Cells were left untreated (−) or subjected to 10 mM NH4Cl treatment (+) for 2 h before harvesting.


( B ) of relative LC3 II in infected . Denistometry of bands was analysed using Image J software . of LC3 - II / tubulin signal was normalized to 1 for wild - type untreated infections . graph shows mean relative LC3 II / tubulin ratios ± for 3 independent experiments .

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(B) Quantification of relative LC3 II levels in infected cells. Denistometry of bands was analysed using Image J software. The ratio of LC3-II/tubulin signal intensity was normalized to 1 for wild-type untreated infections. The graph shows the mean relative LC3 II/tubulin ratios ± SEM for 3 independent experiments.


C ) RAW264 . 7 or ( D ) murine bone marrow - derived macrophages were infected with indicated strains of S . Typhimurium for ( C ) 10 h or ( D ) 16 h replication was monitored using flow cytometry . Results are represented as fold replication normalized to wt ; values are means ± of at least 3 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 .

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C) RAW264.7 or (D) murine bone marrow-derived primary macrophages were infected with the indicated strains of S. Typhimurium for (C) 10 h or (D) 16 h and bacterial replication was monitored using flow cytometry. Results are represented as fold replication normalized to wt; values are the means ± SEM of at least 3 independent experiments. * p<0.05; ** p<0.01.


( B , C ) of WT L - HRD KO mice at of 12 . ( n = 5 for each group ) Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(B, C) Body weight of the WT and L-HRD KO mice at the age of 12 weeks. (n=5 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) heights tibia lengths of WT L - HRD KO mice . ( n = 5 for each group ) Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(D) Body heights and tibia lengths of the WT and L-HRD KO mice. (n=5 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( E ) Phosphorylation of STAT5 in WT HRD1 LKO liver .

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(E) Phosphorylation of STAT5 in WT and HRD1 LKO liver.


( E ) Hepatic growth hormone signaling target genes mRNA in WT L - HRD1 KO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) .

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(E) Hepatic growth hormone signaling target genes mRNA levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.).


Hepatic growth hormone signaling target genes mRNA in WT L - HRD1 KO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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Hepatic growth hormone signaling target genes mRNA levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( A ) Volcano plot of differential genes between WT HRD1 LKO livers .

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(A) Volcano plot of differential genes between WT and HRD1 LKO livers.


( B ) Hepatic Fgf21 mRNA in WT L - HRD1 KO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(B) Hepatic Fgf21 mRNA in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( C ) Serum FGF21 in WT L - HRD1 KO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(C) Serum FGF21 protein levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) Correlation of gene expression ( Red : Upregulated genes Blue : Downrelated genes ) between HRD1 LKO mice FGF21 transgenic mice .

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(D) Correlation analysis of the gene expression (Red: Upregulated genes and Blue: Downrelated genes) between HRD1 LKO mice and FGF21 transgenic mice.


( E ) of mated with proven stud at onset of puberty in WT L - HRD KO mice . ( n = 10 for each group ) Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(E) Percentage of mated with proven stud males and age at onset of puberty in the female WT and L-HRD KO mice. (n=10 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( F ) Examples of ovarian histology from WT HRD1 LKO mice .

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(F) Examples of ovarian histology from WT and HRD1 LKO mice.


( G ) Representative examples of estrus cycles in WT L - HRD1 KO mice as determined by vaginal cytology .

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(G) Representative examples of estrus cycles in WT and L-HRD1 KO mice as determined by vaginal cytology.


( H ) of feeding , water intake activity in time . ( n = 8 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(H) Percentage of feeding, water intake and activity in the light time. (n=8 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( I ) H & E stain of adipose tissue WT HRD1 LKO mice 14 after HFD feeding .

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(I) H&E stain of white adipose tissue WT and HRD1 LKO mice 14 weeks after HFD feeding.


( J ) Ucp1 Dio2 mRNA of adipose tissue from ( I ) ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(J) Ucp1 and Dio2 mRNA levels of adipose tissue from (I) (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( B ) Volcano plot of differential between WT HRD1 LKO livers .

(B) Volcano plot of differential proteins between WT and HRD1 LKO livers.


( F ) of IRE1α , NRF1 , RXRβ , NOTCH1 , SIRT3 GAPDH in liver of WT HRD1 LKO mice under refed condition . ( n = 6 for each group ) .

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(F) Protein levels of IRE1α, NRF1, RXRβ, NOTCH1, SIRT3 and GAPDH in the liver of WT and HRD1 LKO mice under the refed condition. (n=6 for each group).


Hepatic CREBH in WT L - HRD1 KO mice . ( n = 6 for each group ) .

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Hepatic CREBH protein levels in the WT and L-HRD1 KO mice. (n=6 for each group).


Hepatic CREBH mRNA ( B ) in WT L - HRD1 KO mice . ( n = 6 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) .

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Hepatic CREBH mRNA (B) levels in the WT and L-HRD1 KO mice. (n=6 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.).


( C ) Western Blot CREBH stability after HRD1 over - expression . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(C) Western Blot analysis CREBH protein stability after HRD1 over-expression. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) Western Blot of hepatic CREBH HRD1 in WT HRD1 LKO mice . mice were fasted 16 hours refed for another 4 hours .

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(D) Western Blot analysis of hepatic CREBH and HRD1 protein levels in WT and HRD1 LKO mice. The mice were fasted 16 hours and refed for another 4 hours.


( E ) Western Blot interaction of CREBH HRD1 after immunoprecipitates of Flag - agarose in transfected HEK293T .

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(E) Western Blot analysis interaction of CREBH and HRD1 after immunoprecipitates of Flag-agarose in transfected HEK293T.


( F ) Endogenous interaction between CREBH HRD1 in liver .

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(F) Endogenous interaction between CREBH and HRD1 in liver.


( G ) Western Blot ubiquitination of CREBH after immunoprecipitates of Flag - agarose in transfected HEK293T .

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(G) Western Blot analysis ubiquitination of CREBH after immunoprecipitates of Flag-agarose in transfected HEK293T.


( H ) Hepatic CREBH ubiquitination level in WT L - HRD1 KO

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(H) Hepatic CREBH ubiquitination level in the WT and L-HRD1 KO mice


( I ) Relative of CREBH , HRD1 ubiquitination of CREBH . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(I) Relative protein levels of CREBH, HRD1 and ubiquitination of CREBH. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( J ) hepatocytes were isolated from WT HRD1 LKO mice Western Blot CREBH stability .

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(J) Primary hepatocytes were isolated from WT and HRD1 LKO mice and Western Blot analysis CREBH protein stability.


( L ) Interactions of full - C - terminal of HRD1 with CREBH were measured by Co - IP . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(L) Interactions of full-length and C-terminal of HRD1 with CREBH were measured by Co-IP. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( B ) Interactions of full - activated form of CREBH with HRD1 were measured by Co - IP .

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(B) Interactions of full-length and activated form of CREBH with HRD1 were measured by Co-IP.


( C , D ) Ubiquitination of CREBH CREBH K294R were measured after co - transfected C - terminal of HRD1 .

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(C, D) Ubiquitination of CREBH and CREBH K294R were measured after co-transfected C-terminal of HRD1.


( E ) Western Blot CREBH or CREBH - K294R stability after HRD1 over - expression . Data information : data are representative of three independent experiments ( mean ± s . d .

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(E) Western Blot analysis CREBH or CREBH-K294R protein stability after HRD1 protein over-expression. Data information: The data are representative of three independent experiments (mean ± s.d.


( F ) Western Blot of CREBH WT or K48 only ubiquitination level after HRD1 C - terminal co - expression .

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(F) Western Blot analysis of the CREBH WT or K48 only ubiquitination level after HRD1 C-terminal co-expression.


( H ) Western Blot of CREBH K6 , K11 , K27 , K29 K33 only ubiquitination level after HRD1 co - expression .

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(H) Western Blot analysis of the CREBH K6, K11, K27, K29 and K33 only ubiquitination level after HRD1 co-expression.


( I ) Western Blot of CREBH WT , K27R ubiquitination level after HRD1 full - co - expression .

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(I) Western Blot analysis of the CREBH WT, K27R ubiquitination level after HRD1 full-length co-expression.


( J ) Western Blot of CREBH WT , K27 K48 only ubiquitination level after HRD1 C - terminal co - expression .

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(J) Western Blot analysis of the CREBH WT, K27 and K48 only ubiquitination level after HRD1 C-terminal co-expression.


( B ) Hepatic CREBH 5 after AAV - shCrebh injection . ( n = 5 for each group ) .

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(B) Hepatic CREBH protein levels 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( C ) Hepatic Fgf21 mRNA in WT L - HRD1 KO mice 5 after AAV - shCrebh injection . ( n = 5 for each group ) .

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(C) Hepatic Fgf21 mRNA in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( D ) Serum FGF21 in WT L - HRD1 KO mice 5 after AAV - shCrebh injection . ( n = 5 for each group ) .

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(D) Serum FGF21 protein levels in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( E ) height tibia of WT L - HRD1 KO mice 5 after AAV - shCrebh injection ( n = 5 for each group ) .

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(E) Body height and tibia length of the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection (n=5 for each group).


( F ) Hepatic Igf1 , Cyp2d9 Cyp4a12 mRNA in WT L - HRD1 KO mice 5 after AAV - shCrebh injection . ( n = 5 for each group ) .

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(F) Hepatic Igf1, Cyp2d9 and Cyp4a12 mRNA in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( G , H ) of mated estrus cycle of WT L - HRD KO 5 after AAV - shCrebh injection . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(G, H) Percentage of the mated and estrus cycle of the WT and L-HRD KO 5 weeks after AAV-shCrebh injection. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( A ) Examples of sizes of WT , L - HRD KO HRD1 / FGF21 DKO mice . ( B , C ) height of WT , L - HRD KO HRD1 / FGF21 DKO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(A) Examples of the body sizes of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (B, C) Body weight and body height of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) Tibia lengths of WT , L - HRD KO HRD1 / FGF21 DKO mice fed with normal chow diet . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(D) Tibia lengths of the WT, L-HRD KO and HRD1/FGF21 DKO mice fed with normal chow diet. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( E ) Hepatic Hsd3b5 , Cyp2d9 Cyp4a12 mRNA in WT , L - HRD1 KO HRD1 / FGF21 DKO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(E) Hepatic Hsd3b5, Cyp2d9 and Cyp4a12 mRNA in the WT, L-HRD1 KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( F ) Estrus cycle of L - HRD KO HRD1 / FGF21 DKO mice at of 4 months . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(F) Estrus cycle of L-HRD KO and HRD1/FGF21 DKO mice at the age of 4 months. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( G ) of Activity , feeding water intake of WT , L - HRD KO HRD1 / FGF21 DKO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(G) Percentage of the Activity, feeding and water intake of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


Immunoblot of p62 LC3 I / II in KRIT1 wt KRIT1 - KO endothelial . Actin was used as a loading control . of total LC3 on actin is reported ( * P = 0 . 02712 ) . results are representative of three independent experiments .

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Immunoblot analysis of p62 and LC3 I/II in KRIT1 wt and KRIT1-KO endothelial cells. Actin was used as a loading control. Quantification of total LC3 on actin is reported (*P = 0.02712). The results are representative of three independent experiments.


Representative of p62 dots in KRIT1 wt KRIT1 - KO endothelial . Scale bar , 20 μm . Magnifications in insets . Right , quantitative of p62 distribution of dots is reported ( four independent experiments ; n = 35 per group ) . * P = 0 . 00542 ( dotted ) ; * P = 0 . 00014 ( ) .

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Representative images of p62 dots in KRIT1 wt and KRIT1-KO endothelial cells. Scale bar, 20 μm. Magnifications in insets. Right, quantitative analysis of p62 distribution of dots is reported (four independent experiments; n = 35 cells per group). *P = 0.00542 (dotted); *P = 0.00014 (nuclear).


Immunoblot of p62 LC3 I / II in KRIT1 - KO KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) MEFs . Left , immunoblot showing KRIT1 in KRIT1 - KO KO + KRIT1 . Right , immunoblots for p62 LC3 I / II . Actin was used as a loading marker . of total LC3 on actin is reported ( * P = 0 . 01248 ) . results are representative of three independent experiments .

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Immunoblot analysis of p62 and LC3 I/II in KRIT1-KO and KRIT1-KO re-expressing KRIT1 (KO+KRIT1) MEFs. Left, immunoblot showing KRIT1 levels in KRIT1-KO and KO+KRIT1 cells. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.01248). The results are representative of three independent experiments.


Representative of p62 dots in KO + KRIT1 ( top ) KRIT1 - KO ( bottom ) . Scale bar , 50 μm . Magnifications in insets . Right , quantitative of number of p62 dots per is shown ( four independent experiments ; n = 50 per group ) . * P = 7 . 18e − 14 .

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

Representative images of p62 dots in KO+KRIT1 (top) and KRIT1-KO cells (bottom). Scale bar, 50 μm. Magnifications in insets. Right, quantitative analysis of the number of p62 dots per cell is shown (four independent experiments; n = 50 cells per group). *P = 7.18e−14.


Immunoblot of hBMECs transiently transfected with control siRNA or KRIT1 siRNA . Left , evaluation of siRNA efficiency with antibody directed against KRIT1 . Right , immunoblots for p62 LC3 I / II . Actin was used as a loading marker . of total LC3 on actin is reported ( * P = 0 . 03071 ) . results are representative of three independent experiments .

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

Immunoblot analysis of hBMECs transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.03071). The results are representative of three independent experiments.


Immunoblot of EA . hy926 transiently transfected with control siRNA or KRIT1 siRNA . Left , evaluation of siRNA efficiency with antibody directed against KRIT1 . Right , immunoblot for p62 LC3 I / II . Actin was used as a loading marker . of total LC3 on actin is reported ( * P = 0 . 02527 ) . results are representative of three independent experiments .

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

Immunoblot analysis of EA.hy926 cells transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblot for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.02527). The results are representative of three independent experiments.


Immunofluorescence of p62 ( green ) ProteoStat Aggresome detection reagent ( red ) in KRIT1 wt KRIT1 - KO lung endothelial . yellow signal in merged represents an overlapping spatial relationship between green red . Magnification in insets . Scale bar , 50 μm . are representative of four independent experiments .

O O B-MEASURED_VAR O O O 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

Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1 wt and KRIT1-KO lung endothelial cells. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.


Immunofluorescence of p62 ( green ) ProteoStat Aggresome detection reagent ( red ) in KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) KRIT1 - KO MEFs . yellow signal in merged represents an overlapping spatial relationship between green red . Magnification in insets . Scale bar , 50 μm . are representative of four independent experiments .

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

Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.


Immunoblot with antibodies directed against phosphorylated mTOR ( Ser 2448 ) , total mTOR , phosphorylated p70 S6 Kinase ( Ser 371 ) , total p70 S6 Kinase , phosphorylated 4E - BP1 ( Thr 37 / 46 ) , total 4E - BP1 ; actin was used as a loading marker . Where indicated , KRIT1 wt KRIT1 - KO endothelial were treated with 100 nM Torin1 for 4 h . results are representative of three independent experiments .

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

Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot of total ULK1 actin in KRIT1 wt KRIT1 - KO endothelial . Where indicated , were treated with 100 nM Torin1 for 4 h . results are representative of three independent experiments .

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

Immunoblot analysis of total ULK1 and actin in KRIT1 wt and KRIT1-KO endothelial cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot of p62 , LC3 I / II , actin in KRIT1 wt KRIT1 - KO endothelial treated with 100 nM Torin1 or 500 nM rapamycin for 4 h . results are representative of three independent experiments .

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

Immunoblot analysis of p62, LC3 I/II, and actin in KRIT1 wt and KRIT1-KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.


Immunoblot with antibodies directed against phosphorylated mTOR ( Ser 2448 ) , total mTOR , phosphorylated p70 S6 Kinase ( Ser 371 ) , total p70 S6 Kinase , phosphorylated 4E - BP1 ( Thr 37 / 46 ) , total 4E - BP1 ; actin was used as a loading marker . Where indicated , KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) KRIT1 - KO MEFs were treated with 100 nM Torin1 for 4 h . results are representative of three independent experiments .

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

Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot of phosphorylated ULK1 ( Ser 757 ) , total ULK1 , actin in KRIT1 KO + KRIT1 , KRIT1 KO MEFs . Where indicated , were treated with 100 nM Torin1 for 4 h . results are representative of three independent experiments .

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

Immunoblot analysis of phosphorylated ULK1 (Ser 757), total ULK1, and actin in KRIT1 KO+KRIT1, and KRIT1 KO MEFs. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot of p62 , actin , LC3 I / II in KO + KRIT1 KRIT1 - KO . Where indicated , were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h . results are representative of three independent experiments .

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

Immunoblot analysis of p62, actin, LC3 I/II in KO+KRIT1 and KRIT1-KO cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.


KRIT1 wt KRIT1 - KO endothelial were transiently transfected with mRFP - GFP - LC3 . Where indicated , were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h . differences in autophagic flux were evaluated by counting yellow LC3 I / II dots / ( RFP + GFP + ) red LC3 dots / ( RFP + GFP − ) for each condition . Yellow dots : autophagosomes ; red dots : autophagolysosomes . * P = 5 . 74e − 5 ( red dots , WT ctrl vs . WT Tor1 ) ; * P = 9 . 62e − 5 ( red dots , WT ctrl vs . WT xesto ) ; * P = 0 . 00727 ( red dots , WT ctrl vs . KO ctrl ) ; # P = 0 . 00046 ( red dots , KO ctrl vs . KO Tor1 ) . data are expressed as mean ± s . e . m .

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

KRIT1 wt and KRIT1-KO endothelial cells were transiently transfected with mRFP-GFP-LC3. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 5.74e−5 (red dots, WT ctrl vs. WT Tor1); *P = 9.62e−5 (red dots, WT ctrl vs. WT xesto); *P = 0.00727 (red dots, WT ctrl vs. KO ctrl); #P = 0.00046 (red dots, KO ctrl vs. KO Tor1). The data are expressed as the mean ± s.e.m.


KO + KRIT1 KRIT1 - KO MEFs were transiently transfected with mRFP - GFP - LC3 tandem construct . Where indicated , were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h . differences in autophagic flux were evaluated by counting yellow LC3 I / II dots / ( RFP + GFP + ) red LC3 dots / ( RFP + GFP − ) for each condition . Yellow dots : autophagosomes ; red dots : autophagolysosomes . * P = 0 . 00023 ( red dots , KO + KRIT1 ctrl vs . KO + KRIT1 Tor1 ) ; * P = 0 . 00045 ( red dots , KO + KRIT1 ctrl vs . KO + KRIT1 xesto ) ; # P = 3 . 08e − 6 ( red dots , KO ctrl vs . KO Tor1 ) ; # # P = 6 . 73e − 5 ( yellow dots , KO + KRIT1 ctrl vs . KO ctrl ) . data are expressed as mean ± s . e . m . of four independent experiments .

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

KO+KRIT1 and KRIT1-KO MEFs were transiently transfected with the mRFP-GFP-LC3 tandem construct. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 0.00023 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 Tor1); *P = 0.00045 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 xesto); #P = 3.08e−6 (red dots, KO ctrl vs. KO Tor1); ##P = 6.73e−5 (yellow dots, KO+KRIT1 ctrl vs. KO ctrl). The data are expressed as the mean ± s.e.m. of four independent experiments.


Cd44 , PAI1 ( also known as Serpine1 ) , Id1 mRNA expression in KRIT1 wt KRIT1 - KO endothelial were assessed by quantitative real - time PCR . Where indicated , KRIT1 wt KRIT1 - KO endothelial were treated with 100 nM Torin1 or 500 nM rapamycin for 16h . data are expressed as mean ± s . e . m . Cd44 : * P = 0 . 02848 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 02605 ( KO ctrl vs . KO Tor1 ) . PAI1 : * P = 0 . 04446 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 03996 ( KO ctrl vs . KO Tor1 ) . Id1 : * P = 0 . 00266 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 01554 ( KO ctrl vs . KO Tor1 ) . n = 3 independent experiments .

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

Cd44, PAI1 (also known as Serpine1), and Id1 mRNA expression levels in KRIT1 wt and KRIT1-KO endothelial cells were assessed by quantitative real-time PCR. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h. The data are expressed as the mean ± s.e.m. Cd44: *P = 0.02848 (KO ctrl vs. KO Rapa); *P = 0.02605 (KO ctrl vs. KO Tor1). PAI1: *P = 0.04446 (KO ctrl vs. KO Rapa); *P = 0.03996 (KO ctrl vs. KO Tor1). Id1: *P = 0.00266 (KO ctrl vs. KO Rapa); *P = 0.01554 (KO ctrl vs. KO Tor1). n = 3 independent experiments.


Immunoblot of CD31 / Pecam - 1 , vascular endothelial cadherin ( VE - cadherin ) , actin in KRIT1 - KO endothelial that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h . results are representative of three independent experiments .

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

Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), and actin in KRIT1-KO endothelial cells that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h. The results are representative of three independent experiments.


Immunoblot of CD31 / Pecam - 1 , vascular endothelial cadherin ( VE - cadherin ) , N - cadherin , alpha - smooth muscle actin ( alpha - SMA ) , actin in HUVECs transfected with control siRNA or ATG7 siRNA .

Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), N-cadherin, alpha-smooth muscle actin (alpha-SMA), and actin in HUVECs transfected with control siRNA or ATG7 siRNA.


Formation of capillary - like structures measured by tube formation assays . HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h . Representative phase - contrast microscopy ( Scale bar , 100 μm ) calcein - ( Scale bar , 50 μm ) were reported . data are presented as ± s . e . m from three different experiments performed in duplicate . * P = 1 . 29e − 11 .

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

Formation of capillary-like structures measured by tube formation assays. HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h. Representative phase-contrast microscopy (Scale bar, 100 μm) and calcein-fluorescent (Scale bar, 50 μm) images were reported. All data are presented as percentage ± s.e.m from three different experiments performed in duplicate. *P = 1.29e−11.


Immunoblot of p62 , LC3I / II , rapamycin in CCM3 wt CCM3 - KO rapamycin treated with 100 nM Torin1 or 500 nM rapamycin for 4 h . results are representative of three independent experiments .

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

Immunoblot analysis of p62, LC3I/II, and rapamycin in CCM3 wt and CCM3-KO rapamycin treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.


Representative immunostaining of retina sections from wt a model of inducible endothelial - specific CCM3 - KO ( CCM3 - ECKO ) mice at postnatal day 14 . Endothelium was stained with isolectin B4 ( ISOB4 ) ( blue ) . A , artery ; V , vein . p62 can be observed in endothelial forming retinal lesions in CCM3 - ECKO ( scale bar : 200 μm ) . Scale bar of magnifications : 100 μm .

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

Representative immunostaining of retina sections from wt and a model of inducible and endothelial-specific CCM3-KO (CCM3-ECKO) mice at postnatal day 14. Endothelium was stained with isolectin B4 (ISOB4) (blue). A, artery; V, vein. p62 aggregates can be observed in endothelial cells forming retinal lesions in CCM3-ECKO animals (scale bar: 200 μm). Scale bar of magnifications: 100 μm.


Representative immunostaining of brain sections from wt a model of inducible endothelial - specific CCM3 - knockout mice ( CCM3 - ECKO ) at postnatal day 9 . p62 can be observed in proximity of CCM lesions ( arrows ) . nuclei ( DAPI ) are in blue . Scale bar , 30 μm . Smaller panel shows magnifications of blood vessels ( green ) . Scale bar , 10 μm .

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 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

Representative immunostaining of brain sections from wt and a model of inducible and endothelial-specific CCM3-knockout mice (CCM3-ECKO) at postnatal day 9. p62 aggregates can be observed in the proximity of CCM lesions (arrows). Cell nuclei (DAPI) are in blue. Scale bar, 30 μm. Smaller panel shows the magnifications of blood vessels (green). Scale bar, 10 μm.


p62 immunohistochemistry ( ) in humanbrain tissue . A , B Normal vascular endothelium of autoptic brain parenchyma samples is lacking typical autophagic p62 granules as shown by negative for p62 . Scale bars : ( A ) 200 μm ; ( B ) 100 μm .

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 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

p62 immunohistochemistry (IHC) staining in humanbrain tissue.A, B Normal vascular endothelium of autoptic brain parenchyma samples is lacking the typical autophagic p62 granules as shown by the negative staining for p62. Scale bars: (A) 200 μm; (B) 100 μm.


p62immunohistochemistry ( ) in humanbrain tissue . C - F Two different representative samples of CCM lesions with a thin , single layer brain endothelium displaying either moderate ( C , D ) or marked ( E , F ) positive perinuclear " pearl necklace - like " immunostaining for p62 granules . ( C , D ) , case n ° 4 ( p62 + + ) , ( E , F ) , case n ° 8 ( p62 + + + ) are representative of CCM cases listed in Appendix Table S1 . Scale bars : ( C , E ) 200 μm ; ( D , F ) 100 μm . Arrows indicate endothelial p62 positive .

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 B-MEASURED_VAR 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 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 B-MEASURED_VAR O O

p62immunohistochemistry (IHC) staining in humanbrain tissue.C-F Two different representative samples of CCM lesions with a thin, single layer brain endothelium displaying either moderate (C, D) or marked (E, F) positive perinuclear "pearl necklace-like" immunostaining for p62 granules. (C, D), case n° 4 (p62++), and (E, F), case n° 8 (p62+++) are representative of CCM cases listed in Appendix Table S1. Scale bars: (C, E) 200 μm; (D, F) 100 μm. Arrows indicate endothelial p62 positive staining.


p62immunohistochemistry ( ) in humanbrain tissue . G - I Hematoxylin eosin ( H & amp ; amp ; E ) ( G ) p62immunohistochemistry ( H , I ) of a CCM surgical sample ( case n ° 6 in Appendix Table S1 ) containing normal vessels in peri - lesional , which served as an internal control . Notice marked p62 - positive in endothelial lining a CCM lesion ( H , arrows ) p62 - negative in endothelial lining a normal peri - lesional vessel ( I , arrows ) . Scale bars : ( G ) 300 μm ; ( H , I ) 100 μm . Background in brainparenchyma surrounding CCM lesions may be attributed to either debris or p62 immunoreactivity in neuronal glial .

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 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 B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O

p62immunohistochemistry (IHC) staining in humanbrain tissue.G-I Hematoxylin and eosin (H&amp;E) staining (G) and p62immunohistochemistry analysis (H, I) of a CCM surgical sample (case n° 6 in Appendix Table S1) containing normal vessels in the peri-lesional area, which served as an internal control. Notice marked p62-positive staining in endothelial cells lining a CCM lesion (H, arrows) and p62-negative staining in endothelial cells lining a normal peri-lesional vessel (I, arrows). Scale bars: (G) 300 μm; (H, I) 100 μm. Background staining in brainparenchyma surrounding CCM lesions may be attributed to either cell debris or p62 immunoreactivity in neuronal cells and glial cells.


( A ) Representative micro - CT H & E of trabecular bone from femoral metaphysis of 6 - week - old Sp7 - Cre ; Cdc20f / f littermate control mice . Scale bar , 500 μm . ( B ) Histomorphometric analyses of 6 - week - old femurs . ( n = 6 )

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(A) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old male Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm. (B) Histomorphometric analyses of 6-week-old male femurs. (n=6)


( C ) Representative micro - CT H & E of trabecular bone from femoral metaphysis of 6 - week - old Sp7 - Cre ; Cdc20f / f littermate control mice . Scale bar , 500 μm . ( D ) Histomorphometric analyses of 6 - week - old femurs . ( n = 6 )

(C) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old female Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm. (D) Histomorphometric analyses of 6-week-old female femurs. (n=6)


( E ) Representative micro - CT of tibial cortical bone defects in Sp7 - Cre ; Cdc20f / f littermate control mice . green lines show position of original defect position . green circle represents regenerated bone . Scale bar , 500 μm . ( F ) Histomorphometric of regenerated bone in tibial cortical gaps . ( n = 5 )

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(E) Representative micro-CT images of tibial cortical bone defects in Sp7-Cre;Cdc20f/f and littermate control mice. The green lines show the position of the original defect position. The green circle represents the regenerated bone. Scale bar, 500 μm. (F) Histomorphometric analysis of the regenerated bone in tibial cortical gaps. (n=5)


( B , C ) ALP ( B ) ALP ( C ) of Cdc20f / f Sp7 - Cre ; Cdc20f / f BMSCs after 7 days osteogenic differentiation . ( n = 6 )

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(B, C) The ALP staining (B) and ALP quantification (C) of Cdc20f/f and Sp7-Cre;Cdc20f/f BMSCs after 7 days osteogenic differentiation. (n=6)


( E , F ) ALP ( E ) ALP ( F ) of NC CDC20sh hBMSCs after 7 days osteogenic differentiation . ( n = 5 )

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(E, F) The ALP staining (E) and ALP quantification (F) of NC and CDC20sh hBMSCs after 7 days osteogenic differentiation. (n=5)


( H , I ) ALP ( H ) ALP ( I ) of Vector truncated fragments overexpression of CDC20 in CDC20sh hBMSCs after 7 days osteogenic differentiation . ( n = 5 )

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(H, I) The ALP staining (H) and ALP quantification (I) of Vector and truncated fragments overexpression of CDC20 in CDC20sh hBMSCs after 7 days osteogenic differentiation. (n=5)


( E ) Western blot analyses of degradation of p65 after overexpression of CDC20 . of overexpressed Myc - CDC20 Vector HEK293T were extracted after TNF - α treatment for 30 minutes .

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(E) Western blot analyses of the degradation of nuclear p65 protein after the overexpression of CDC20. The cytoplasmic and nuclear protein of overexpressed Myc-CDC20 and Vector HEK293T cells were extracted after TNF-α treatment for 30 minutes.


( G ) Western blot analyses of degradation of p65 after overexpression of Myc - CDC20 . hBMSCs were transfected with Vector Myc - CDC20 plasmids for 36 h , were treated with or without 10 μM MG132 for 6 h before collected .

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(G) Western blot analyses of the degradation of p65 protein after the overexpression of Myc-CDC20. hBMSCs were transfected with Vector and Myc-CDC20 plasmids for 36 h, cells were treated with or without 10 μM MG132 for 6 h before collected.


( E , F ) Co - immunoprecipitation of endogenous CDC20 with endogenous p65 in HEK293T .

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(E, F) Co-immunoprecipitation of endogenous CDC20 with endogenous p65 in HEK293T cells.


( G ) Co - immunoprecipitation of purified GST - CDC20 with endogenous p65 in HEK293T construct of GST - CDC20 . Whole lysates ( WCL ) were subjected to perform GST pull - down analyses with purified GST - CDC20 GST as a negative control .

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(G) Co-immunoprecipitation of purified GST-CDC20 protein with endogenous p65 in HEK293T cells and the construct of GST-CDC20. Whole cell lysates (WCL) were subjected to perform GST pull-down analyses with purified GST-CDC20 and GST as a negative control.


( G , H ) Co - immunoprecipitation of ectopically expressed Myc - CDC20 171 - 499 with ectopically expressed Flag - p65 1 - 310 fragment in HEK293T .

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(G, H) Co-immunoprecipitation of ectopically expressed Myc-CDC20 171-499 with ectopically expressed Flag-p65 1-310 fragment in HEK293T cells.


( C ) Western blot analyses of degradation of p65 in NC APC11si HEK293T .

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(C) Western blot analyses of the degradation of p65 protein in NC and APC11si HEK293T cells.


( F ) Western blot analyses of p65 expression in NC or CDC20sh HEK293T after overexpression of Vector or HA - APC11 plasmids .

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(F) Western blot analyses of p65 expression in NC or CDC20sh HEK293T cells after the overexpression of Vector or HA-APC11 plasmids.


( H , I ) ALP ( H ) ALP ( I ) of NC APC11si hBMSCs after 7 days osteogenic differentiation . ( n = 6 )

(H, I) The ALP staining (H) and ALP quantification (I) of NC and APC11si hBMSCs after 7 days osteogenic differentiation. (n=6)


( A ) Immunoblot of Flag - p65 - linked ubiquitination promoted by Myc - CDC20 . HEK293T were co - transfected with Flag - p65 , HA - Ubiquitin , with or without Myc - CDC20 or Vector plasmids for 36 h . Transfected were then treated with 10 μM MG132 for 6 h before collection . ( B ) Immunoblot of Flag - p65 - linked ubiquitination promoted by HA - APC11 . HEK293T were transfected with Flag - p65 , His - Ubiquitin , with or without HA - APC11 or Vector plasmids for 36 h . Transfected were then treated with 10 μM MG132 for 6 h before collection .

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(A) Immunoblot of Flag-p65-linked ubiquitination promoted by Myc-CDC20. HEK293T cells were co-transfected with Flag-p65, HA-Ubiquitin, with or without Myc-CDC20 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection. (B) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11. HEK293T cells were transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.


( F ) Immunoblot of Flag - p65 - linked ubiquitination promoted by HA - APC11 in NC CDC20sh . NC or CDC20sh HEK293T were co - transfected with Flag - p65 , His - Ubiquitin , with or without HA - APC11 Vector plasmids . Transfected were treated with 10 μM MG132 for 6 h before collection .

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(F) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11 in NC and CDC20sh cells. NC or CDC20sh HEK293T cells were co-transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 and Vector plasmids. Transfected cells were treated with 10 μM MG132 for 6 h before collection.


( A ) Representative micro - CT of trabecular bone from femoral metaphysis of Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with p65sh lentivirus . Scale bar , 500 μm . ( B ) Histomorphometric analyses of Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with p65sh lentivirus . ( n = 6 )

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(A) Representative micro-CT images of trabecular bone from the femoral metaphysis of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. Scale bar, 500 μm. (B) Histomorphometric analyses of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. (n=6)


( C , D ) ALP ( C ) ALP ( D ) of BMSCs from Cdc20f / f mice Sp7 - Cre ; Cdc20f / f mice after 7 days osteogenic differentiation treated with negative control or p65si . ( n = 5 )

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(C, D) The ALP staining (C) and ALP quantification (D) of BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si. (n=5)


( G ) expression of RUNX2 in BMSCs from Cdc20f / f mice Sp7 - Cre ; Cdc20f / f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses .

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(G) The expression of RUNX2 in BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses.


F : IMAF for CSF , plasma urine of with samples collected at 6 months follow - up ( n = 3 ) . Matched MRI scans are added for annotation .

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F: IMAF for the CSF, plasma and urine of the patients with samples collected at 6 months follow-up (n=3). Matched MRI scans are added for annotation.


A : Mutant allele fraction ( MAF ) measured in , CSF , plasma urine samples ( on y axis ) for detected mutations , in 52 genes that are most frequently mutated in gliomas on TCGA databases ( on x axis ) , for 8 analysed by targeted sequencing panels . MAF from are derived from exome sequencing . MAF from CSF , plasma urine samples are derived from capture panels sequencing .

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A: Mutant allele fraction (MAF) measured in tumor, CSF, plasma and urine samples (on the y axis) for the detected mutations, in the 52 genes that are most frequently mutated in gliomas based on the TCGA databases (on the x axis), for the 8 patients analysed by targeted sequencing panels. MAF from tumor are derived from exome sequencing. MAF from CSF, plasma and urine samples are derived from the capture panels sequencing.


A - C : Fragment distributions for mutant ( blue ) non - mutant ( red ) cfDNA reads , determined from capture sequencing data for CSF samples ( A ) , plasma samples ( B ) urine samples ( C ) .

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A-C: Fragment size distributions for mutant (blue) and non-mutant (red) cfDNA reads, determined from the capture sequencing data for CSF samples (A), plasma samples (B) and urine samples (C).


( B ) SEC - MALLS traces of different TRIM25

(B) SEC-MALLS traces of different TRIM25 and


( C ) TRIM32 constructs . are colour coded domain architecture is reported next to respective MALLS curve . constructs were analysed over different ranges data are reported for : T25R , 4 mg / mL ; T25RB1 , 3 . 2 mg / mL ; T25RB1B2 , 3 . 6 mg / mL ; T25RBCC , 0 . 5 mg / mL ; T32R , 3 mg / mL ; T32RB , 4 mg / mL ; T32RBCC , 4 mg / mL .

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(C) TRIM32 constructs. The proteins are colour coded and the domain architecture is reported next to the respective MALLS curve. The constructs were analysed over different concentration ranges and the data are reported for: T25R, 4 mg/mL; T25RB1, 3.2 mg/mL; T25RB1B2, 3.6 mg/mL; T25RBCC, 0.5 mg/mL; T32R, 3 mg/mL; T32RB, 4 mg/mL; T32RBCC, 4 mg/mL.


( A , C ) UBE2D3 ~ Ub discharge assays with different TRIM25 TRIM32 constructs , respectively . Assays were carried out with TRIM constructs as indicated reaction was monitored over 30 min . asterisk indicates band for TRIM construct .

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(A, C) UBE2D3~Ub discharge assays with different TRIM25 and TRIM32 constructs, respectively. Assays were carried out with TRIM constructs as indicated and the reaction was monitored over 30 min. The asterisk indicates the band for the TRIM construct.


( B , D ) of discharge assays using UBE2D1 ~ UbAtto TRIM25 or TRIM32 , respectively . loss of UBE2D1 ~ UbAtto is plotted as average of experimental triplicates ( ± s . d . ) .

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(B, D) Quantification of the discharge assays using UBE2D1~UbAtto and TRIM25 or TRIM32, respectively. The loss of UBE2D1~UbAtto is plotted as the average of experimental triplicates (± s.d.).


( E , G ) K63 poly - ubiquitination assays using UBE2N / UBE2V1 TRIM25 TRIM32 , respectively . Reactions were incubated for 30 minutes samples taken at indicated times . asterisk indicates band for TRIM construct .

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(E, G) K63 poly-ubiquitination assays using UBE2N/UBE2V1 and TRIM25 and TRIM32, respectively. Reactions were incubated for 30 minutes and samples taken at indicated times. The asterisk indicates the band for the TRIM construct.


( F , H ) Assays were quantified ( see Materials Methods ) by supplementing reaction with 1 uM UbAtto integrating loss of free UbAtto for TRIM25 TRIM32 , respectively . Assays were carried out in triplicate .

(F, H) Assays were quantified (see Materials and Methods) by supplementing the reaction with 1 uM UbAtto and integrating the loss of free UbAtto for TRIM25 and TRIM32, respectively. Assays were carried out in triplicate.


( A ) Structure of TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan blue Zn2 + ions as grey spheres .

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(A) Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn2+ ions as grey spheres.


( B ) Overlap of RING dimers of TRIM32 ( cyan , 5FEY . pdb ) , TRIM25 ( yellow , 5FER . pdb ) TRIM5 ( orange , 4TKP . pdb ) . structures were overlapped on circled RING domain . This overlap shows that structures of RINGs are very similar but that there are differences in relative orientations of two RINGs .

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(B) Overlap of the RING dimers of TRIM32 (cyan, 5FEY.pdb), TRIM25 (yellow, 5FER.pdb) and TRIM5 (orange, 4TKP.pdb). The structures were overlapped on the circled RING domain. This overlap shows that the structures of the RINGs are very similar but that there are differences in the relative orientations of the two RINGs.


( C ) Structure of TRIM25 RING / E2 ~ Ub with RING domains in same colour scheme as in ( A ) , UBE2D1 in grey ubiquitin molecules in salmon red .

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(C) Structure of the TRIM25 RING/E2~Ub complex with the RING domains in the same colour scheme as in (A), UBE2D1 in grey and the ubiquitin molecules in salmon and red.


( A ) Close - up of TRIM25 RING dimer interface highlighting hydrophobic interactions made between four helices ( left ) . Close - up of interface between each RING monomer proximal ubiquitin ( right ) .

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(A) Close-up of the TRIM25 RING dimer interface highlighting the hydrophobic interactions made between the four helices (left). Close-up of the interface between each RING monomer and the proximal ubiquitin (right).


( B ) UBE2D1 ~ UbAtto discharge assays with TRIM25 wild - type RING , fused RING constructs different mutants important for dimerization or interaction with ubiquitin . Time point zero for T25 - R Linker T25 - R Linker V72R samples was taken before addition of E3 as discharge is very fast . loss of UBE2D1 ~ UbAtto is plotted as average of experimental duplicates ( ± s . d . ) .

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(B) UBE2D1~UbAtto discharge assays with TRIM25 wild-type RING, the fused RING constructs and different mutants important for dimerization or the interaction with ubiquitin. Time point zero for the T25-R Linker and T25-R Linker V72R samples was taken before the addition of E3 as discharge is very fast. The loss of UBE2D1~UbAtto is plotted as the average of experimental duplicates (± s.d.).


( C ) UBE2D3 ~ Ub discharge assays with same mutants as in ( B ) , stained with InstantBlue . ( D ) K63 poly - ubiquitination assays using UBE2N / UBE2V1 . asterisk indicates band for TRIM construct .

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(C) UBE2D3~Ub discharge assays with the same mutants as in (B), stained with InstantBlue. (D) K63 poly-ubiquitination assays using UBE2N/UBE2V1. The asterisk indicates the band for the TRIM construct.


( A ) Role of E10 in RING of TRIM25 ( blue ) in stabilizing closed E2 ~ Ub conformation by contacting K11 from proximal ubiquitin ( salmon ) N71 of opposite RING ( cyan ) which in turn contacts K33 of same ubiquitin .

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(A) Role of E10 in the RING of TRIM25 (blue) in stabilizing the closed E2~Ub conformation by contacting K11 from the proximal ubiquitin (salmon) and N71 of the opposite RING (cyan) which in turn contacts K33 of the same ubiquitin.


( B ) E2 ~ Ub discharge K63 poly - ubiquitination assays to role of E9 E10 in TRIM25 activity . Substitution of Glu9 with Arg has no significant effect on activity , whereas E10R mutation almost completely abolishes catalytic activity .

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(B) E2~Ub discharge and K63 poly-ubiquitination assays to test the role of E9 and E10 in TRIM25 activity. Substitution of Glu9 with Arg has no significant effect on activity, whereas the E10R mutation almost completely abolishes catalytic activity.


( C ) Discharge K63 poly - ubiquitination assays to role of equivalent residue E16 in TRIM32 role of I85R . Mutation of E16R abolishes catalytic activity indicating that role of glutamate is conserved .

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(C) Discharge and K63 poly-ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R. Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved.


( D E ) of UBE2D1 ~ UbAtto discharge assays . loss of E2 ~ Ub is plotted as average of experimental duplicates ( ± s . d . ) .

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(D and E) Quantification of UBE2D1~UbAtto discharge assays. The loss of E2~Ub is plotted as the average of experimental duplicates (± s.d.).


A subplantar injection of 5 % of formalin ( 20 µl ) was administered in right hind paw of Balb / c mice an equal volume of PBS was injected in left hind paw . After 1 hour of formalin injection , Balb / c mice were treated with a single dose of either PBS ( vehicle control ) or Diclofenac ( 10 mg / kg ) or different concentrations of rPPE2 ( 2 mg / kg 3 mg / kg ) via intraperitoneal route after three hours of treatment , mice were sacrificed paw sections were prepared stained with Toluidine blue to quantify mast population . ( A ) of representative sections were visualized at 20X magnification ( scale bar = 50 µm ) . ( B ) Counting of mast was performed in Toluidine blue stained paw sections using ImageJ software were normalized per unit ( mm2 ) . Data information : Data shown are Mean ± of 8 mice . For ( B , unpaired t - was applied to calculate p values .

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A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice and an equal volume of PBS was injected in left hind paw. After 1 hour of formalin injection, Balb/c mice were treated with a single dose of either PBS (vehicle control) or Diclofenac (10 mg/kg) or different concentrations of rPPE2 (2 mg/kg and 3 mg/kg) via intraperitoneal route and after three hours of treatment, mice were sacrificed and the paw sections were prepared and stained with Toluidine blue to quantify mast cell population. (A) Photographs of representative sections were visualized at 20X magnification (scale bar = 50 µm). (B) Counting of mast cells was performed in Toluidine blue stained paw sections using ImageJ software and were normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. For (B, unpaired t-test was applied to calculate p values.


( B - E ) In another experiment , NIH - 3T3 ( B , C ) or MEFs ( D , E ) were incubated with either PBS or rPPE2 ( 3 µg / ml ) . After 45 minutes of rPPE2 treatment , were fixed , permeabilized , stained with anti - PPE2 Ab . DAPI was used to stain nucleus . localization of rPPE2 in NIH - 3T3 ( C ) MEFs ( E ) was measured by calculating colocalization coefficient . Data information : Data represent Mean ± of more than 50 of three independent experiments . For Figure C , E unpaired t - was applied to calculate p values .

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(B-E) In another experiment, NIH-3T3 cells (B, C) or MEFs (D, E) were incubated with either PBS or rPPE2 (3 µg/ml). After 45 minutes of rPPE2 treatment, cells were fixed, permeabilized, and stained with anti-PPE2 Ab. DAPI was used to stain the nucleus. Nuclear localization of rPPE2 in NIH-3T3 (C) and MEFs (E) was measured by calculating the colocalization coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure C, E unpaired t-test was applied to calculate p values.


( F - G ) ( F ) NIH - 3T3 were transiently transfected with either pEGFP or pEGFP - PPE2 . After 24 hours of transfection , were fixed observed under a confocal microscope . DAPI was used to stain nucleus . ( G ) localization of rPPE2 was measured by calculating colocalization coefficient . Data information : Data represent Mean ± of more than 50 of three independent experiments . For Figure G , unpaired t - was applied to calculate p values .

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(F-G) (F) NIH-3T3 cells were transiently transfected with either pEGFP or pEGFP-PPE2. After 24 hours of transfection, cells were fixed and observed under a confocal microscope. DAPI was used to stain the nucleus. (G) Nuclear localization of rPPE2 was measured by calculating colocalization coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure G, unpaired t-test was applied to calculate p values.


( D ) Lysates prepared from same samples used in C were resolved on SDS - PAGE checked for GFP expression by Western blotting using anti - GFP Ab . GAPDH was used as a loading control .

(D) Lysates prepared from the same samples used in C were resolved on SDS-PAGE and checked for GFP expression by Western blotting using anti-GFP Ab. GAPDH was used as a loading control.


( E ) Varying concentrations of rPPE2 were incubated with [ γ - 32P ] - ATP oligonucleotides of 60 bp spanning SCF promoter . - complexes were resolved on 7 % native polyacrylamide gel . In cold competition reactions , equimolar cold / unlabeled 60 bp oligonucleotides were used ( arrow represents PPE2 - oligo ) . data shown are representative of 3 independent experiments .

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(E) Varying concentrations of rPPE2 protein were incubated with [γ-32P]-ATP oligonucleotides of 60 bp spanning SCF promoter. DNA-protein complexes were resolved on 7% native polyacrylamide gel. In cold competition reactions, equimolar cold/unlabeled 60 bp oligonucleotides were used (arrow represents PPE2-oligo complex). The data shown are representative of 3 independent experiments.