( F ) Catalytically active HOIP decreases the number of cells with TDP - 43 aggregates dependent on proteasomal degradation . SH - SY5Y cells were transfected with TDP - 43 Q331K and either control vector ( CO ) , HOIP or HOIP C885A . Cells were treated 48 h after transfection with the proteasomal inhibitor MG132 ( 1 µM ) or the p97 / VCP inhibitor NSM - 873 ( 1 µM ) for 3 h . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Dunnett ' s Multiple Comparison Test , n = 5 . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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

(F) Catalytically active HOIP decreases the number of cells with TDP-43 aggregates dependent on proteasomal degradation. SH-SY5Y cells were transfected with TDP-43 Q331K and either control vector (CO), HOIP or HOIP C885A. Cells were treated 48 h after transfection with the proteasomal inhibitor MG132 (1 µM) or the p97/VCP inhibitor NSM-873 (1 µM) for 3 h. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Dunnett's Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.


( G ) Inhibition of the proetasome and p97 / VCP do not have additive effects on the degradation of misfolded TDP - 43 species . SH - SY5Y cells expressing TDP - 43 Q331K were treated 48 h after transfection with MG132 ( 1 µM ) and / or NSM - 873 ( 1 µM ) for 3 h . All data are mean ± SD , data were analyzed by one - way ANOVA followed by Bonferroni ' s Multiple Comparison Test , n = 5 . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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

(G) Inhibition of the proetasome and p97/VCP do not have additive effects on the degradation of misfolded TDP-43 species. SH-SY5Y cells expressing TDP-43 Q331K were treated 48 h after transfection with MG132 (1 µM) and/or NSM-873 (1 µM) for 3 h. All data are mean ± SD, data were analyzed by one-way ANOVA followed by Bonferroni's Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.


( h ) The cellular levels of Ca2 + ions after ligand ( C10ORF99 ) stimulation of GPR15 in human , mouse , Japanese quail , painted turtle . YM . 254890 inhibits Ca2 + ion levels in cells transfected with GPR15 receptors of vertebrates . The fold - change in calcium levels was calculated based on the fluorescence intensity ( excitation and emission wavelengths : 490 and 520 nm , respectively ) .

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

(h) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in human, mouse, Japanese quail, painted turtle. YM.254890 inhibits Ca2+ ion levels in cells transfected with GPR15 receptors of vertebrates. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively).


( i , j ) The cellular levels of Ca2 + ions after ligand ( C10ORF99 ) stimulation of GPR15 in coelacanth , Asian bony tongue , and their mutants . ( k , l ) The cellular levels of Ca2 + ions after ligand ( C10ORF99 ) stimulating GPR15 in Japanese quail and its mutants . The fold - change in calcium levels was calculated based on the fluorescence intensity ( excitation and emission wavelengths : 490 and 520 nm , respectively ) .

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

(i, j) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in coelacanth, Asian bony tongue, and their mutants. (k, l) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulating GPR15 in Japanese quail and its mutants. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively).


( a - h ) The migration of HEK293 cells expressing mGPR15 , qGPR15 , tGPR15 , cGPR15 , aGPR15 , and their mutants induced by ligand C10ORF99 . Data information : All data shown are generated from at least three technical replicates . Error bars indicate SD ( n = 3 ) . * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ( t - test ) .

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

(a-h) The migration of HEK293 cells expressing mGPR15, qGPR15, tGPR15, cGPR15, aGPR15, and their mutants induced by ligand C10ORF99. Data information: All data shown are generated from at least three technical replicates. Error bars indicate SD (n=3). ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05 (t-test).


( i - p ) The migration of CD4 + cells from GPR15 - knockout mice , engineered to express mGPR15 , qGPR15 , tGPR15 , cGPR15 , aGPR15 , and their mutants induced by ligand C10ORF99 . Data information : All data shown are generated from at least three technical replicates . Error bars indicate SD ( n = 3 ) . * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ( t - test ) . Ad - NC represents adeno - virus - based gene - delivery vector as negative control .

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

(i-p) The migration of CD4+ cells from GPR15-knockout mice, engineered to express mGPR15, qGPR15, tGPR15, cGPR15, aGPR15, and their mutants induced by ligand C10ORF99. Data information: All data shown are generated from at least three technical replicates. Error bars indicate SD (n=3). ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05 (t-test). Ad-NC represents adeno-virus-based gene-delivery vector as negative control.


( a ) CCR9 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( b ) CCR9 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( c ) CCR9 expression in the small intestine ( SI ) and colon of Rana catesbiana Shaw and Rana nigromaculata . ( d ) GPR15 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( e ) GPR15 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( f ) C10ORF99 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( g ) C10ORF99 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( h ) GPR15 and C10ORF99 expression in the small intestine ( SI ) and colon of Rana catesbeiana Shaw and Rana nigromaculata . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) .

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 B-MEASURED_VAR 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 B-MEASURED_VAR 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 B-MEASURED_VAR 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 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

(a) CCR9 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (b) CCR9 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (c) CCR9 expression in the small intestine (SI) and colon of Rana catesbiana Shaw and Rana nigromaculata. (d) GPR15 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (e) GPR15 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (f) C10ORF99 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (g) C10ORF99 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (h) GPR15 and C10ORF99 expression in the small intestine (SI) and colon of Rana catesbeiana Shaw and Rana nigromaculata. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).


( i ) GPR15 expression in CD4 + T cell from chicken small intestine ( SI ) and colon . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) .

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

(i) GPR15 expression in CD4+ T cell from chicken small intestine (SI) and colon. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).


( j ) The migration of CD4 + cells from chicken small intestine ( SI ) and colon induced by ligand C10ORF99 . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) .

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 O O O O O O O O O O O O O O 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) The migration of CD4+ cells from chicken small intestine (SI) and colon induced by ligand C10ORF99. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).


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 ) .

O O 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 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).


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 ) .

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

O O O O O B-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 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

(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 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 ) .

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

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

(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 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 ) .

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

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 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 .

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

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

O O O O O O 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) 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 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 ) .

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

(C) 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 ) 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 .

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

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 B-MEASURED_VAR O O O O O O

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 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 .

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

(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 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 .

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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 ) . 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 .

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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 ) 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 .

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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 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 .

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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 and 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 ) 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 ) .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 -

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 and 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 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 . ) .

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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 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 .

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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 and HRD1 LKO livers .

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


( 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 .

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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 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 .

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

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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 and 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 and 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 ) 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 .

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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 white adipose tissue WT and HRD1 LKO mice 14 weeks after HFD feeding .

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


( 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 .

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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 proteins between WT and HRD1 LKO livers .

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


( 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 ) .

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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 protein levels in the WT and 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 ) 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 . ) .

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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 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 .

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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 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 .

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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 analysis interaction of CREBH and 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 and HRD1 in liver .

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


( G ) Western Blot analysis 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 the WT and L - HRD1 KO

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


( 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 .

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

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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 - length and 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 and 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 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 .

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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 analysis of the 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 analysis of the CREBH K6 , K11 , K27 , K29 and 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 analysis of the CREBH WT , K27R ubiquitination level after HRD1 full - length 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 analysis of the CREBH WT , K27 and 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 protein levels 5 weeks 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 the WT and L - HRD1 KO mice 5 weeks 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 protein levels in the WT and L - HRD1 KO mice 5 weeks 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 ) Body height and tibia length of the WT and L - HRD1 KO mice 5 weeks 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 and Cyp4a12 mRNA in the WT and L - HRD1 KO mice 5 weeks 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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

O O B-CONTROLLED_VAR O B-CONTROLLED_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 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 O O O B-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 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 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 ) , 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 .

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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 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 .

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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 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 .

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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ( 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 .

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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 ( 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 .

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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 ( IHC ) staining in humanbrain tissue . G - I Hematoxylin and eosin ( H & amp ; 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 .

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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 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 )

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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 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 )

(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 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 )

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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 ) The ALP staining ( B ) and ALP quantification ( C ) of Cdc20f / f and 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 ) The ALP staining ( E ) and ALP quantification ( F ) of NC and 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 ) 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 )

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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 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 .

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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 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 .

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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 cells .

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


( 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 .

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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 cells .

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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 the degradation of p65 protein in NC and APC11si HEK293T cells .

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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 cells after the 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 ) The ALP staining ( H ) and ALP quantification ( I ) of NC and 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 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 .

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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 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 .

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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 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 )

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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 ) 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 )

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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 ) 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 .

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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 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 .

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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.