( a ) 3D reconstruction from wholemount lightsheet imaging of an E5 . 75 Hex - GFP ( green , anterior marker ) embryo , stained for nuclei ( DAPI , grey ) on the left , mitosis ( Phh3 , magenta ) and F - actin ( Phalloidin , grey ) on the right . ( b ) Z - slice ( left ) and 3D reconstruction ( right ) from wholemount lightsheet imaging of an E6 . 25 embryo stained for a posterior marker ( Eomesodermin , cyan ) , mitosis ( Phh3 , magenta ) and F - actin ( Phalloidin , grey ) . ( a ' , b ' ) Mitotic Index ( Phh3 / DAPI ) in percentage at the anterior and posterior regions of E5 . 75 ( a ' ) and ( b ' ) E6 . 25 embryos . Posterior region includes the PS region , as it could not be precisely discriminated . ( a " , b " ) Ratio of non - apical mitosis ( non - apical Phh3 over total Phh3 , normalized by number of nuclei ) in percentage in the anterior and posterior region of E5 . 75 ( a " ) and E6 . 25 ( b " ) embryos .

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(a) 3D reconstruction from wholemount lightsheet imaging of an E5.75 Hex-GFP (green, anterior marker) embryo, stained for nuclei (DAPI, grey) on the left, mitosis (Phh3, magenta) and F-actin (Phalloidin, grey) on the right. (b) Z-slice (left) and 3D reconstruction (right) from wholemount lightsheet imaging of an E6.25 embryo stained for a posterior marker (Eomesodermin, cyan), mitosis (Phh3, magenta) and F-actin (Phalloidin, grey). (a', b') Mitotic Index (Phh3/DAPI) in percentage at the anterior and posterior regions of E5.75 (a') and (b') E6.25 embryos. Posterior region includes the PS region, as it could not be precisely discriminated. (a", b") Ratio of non-apical mitosis (non-apical Phh3 over total Phh3, normalized by number of nuclei) in percentage in the anterior and posterior region of E5.75 (a") and E6.25 (b") embryos.


( b , c ) 3D reconstruction ( b ) and confocal Z - slice ( c ) of E6 . 25 WT ( left ) and RhoAVE - deleted ( right ) embryos stained for an AVE marker ( Cerberus 1 , yellow ) , F - actin ( Phalloidin , grey ) and mitosis ( Phh3 , magenta ) . Scale bar : 25 μm . Blue dotted line delimitates the pro - amniotic cavity ( apical side of the epiblast ) . Red arrowheads point to non - apical mitosis . White lines delimitates the boundary between extraembryonic ( up ) and embryonic ( down ) regions of the embryo .

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(b, c) 3D reconstruction (b) and confocal Z-slice (c) of E6.25 WT (left) and RhoAVE-deleted (right) embryos stained for an AVE marker (Cerberus 1, yellow), F-actin (Phalloidin, grey) and mitosis (Phh3, magenta). Scale bar: 25 μm. Blue dotted line delimitates the pro-amniotic cavity (apical side of the epiblast). Red arrowheads point to non-apical mitosis. White lines delimitates the boundary between extraembryonic (up) and embryonic (down) regions of the embryo.


( d ) Percentage of AVE migration in WT and RhoAVE - deleted mutants . Data information : Values are shown as Mean ± SEM . WT : n = 31 embryos ; RhoAVE - deleted : n = 17 embryos .

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(d) Percentage of AVE migration in WT and RhoAVE-deleted mutants. Data information: Values are shown as Mean ± SEM. WT: n=31 embryos; RhoAVE-deleted: n=17 embryos.


( e ) Ratio of non - apical mitosis ( non - apical Phh3 over total Phh3 , normalised by the volume in μm3 ) in percentage , in AVE - adjacent and AVE - opposed regions from WT and RhoAVE - deleted embryos .

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(e) Ratio of non-apical mitosis (non-apical Phh3 over total Phh3, normalised by the volume in μm3) in percentage, in AVE-adjacent and AVE-opposed regions from WT and RhoAVE-deleted embryos.


( C ) T2 - weighted MR images in coronal plane show abscess formation in the left lower pulmonary lobe ( upper panel ) and in spleen and left axilla ( lower panel ) .

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(C) T2-weighted MR images in coronal plane show abscess formation in the left lower pulmonary lobe (upper panel) and in spleen and left axilla (lower panel).


( D ) Histological sections of patient biopsies stained with hematoxylin and eosin ( left panel ) or with an actin antibody ( right panel ) . Scale bars : skin : left 1000µm , right 100µm ; lung : left 500µm , right 250µm ; spleen : left 1000µm , right 75µm .

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(D) Histological sections of patient biopsies stained with hematoxylin and eosin (left panel) or with an actin antibody (right panel). Scale bars: skin: left 1000µm, right 100µm; lung: left 500µm, right 250µm; spleen: left 1000µm, right 75µm.


( A ) Expression of indicated proteins was determined by western blot in patient - derived and control fibroblasts and B cells and A549 OTULIN WT and KO cells . One representative out of three independent experiments is shown .

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(A) Expression of indicated proteins was determined by western blot in patient-derived and control fibroblasts and B cells and A549 OTULIN WT and KO cells. One representative out of three independent experiments is shown.


( B ) FLAG - tagged tandem ubiquitin binding entity ( TUBE ) assay was performed to pull down linear ubiquitin linkages in patient - derived and control fibroblasts and B cells . One representative out of three independent experiments is shown .

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(B) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed to pull down linear ubiquitin linkages in patient-derived and control fibroblasts and B cells. One representative out of three independent experiments is shown.


Biochemical characterization of recombinant OTULIN variants by means of differential scanning fluorimetry ( DSF ) A ) DSF measurements with OTULINWT , OTULINM86I or OTULINW167S . Melting temperatures ( Tm ) are calculated from five independent experiments with standard deviations . * , P = 0 . 011 ; * * * * , P = 6 . 64x10 - 18 , unpaired t - test .

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Biochemical characterization of recombinant OTULIN variants by means of differential scanning fluorimetry (DSF) A) DSF measurements with OTULINWT, OTULINM86I or OTULINW167S. Melting temperatures (Tm) are calculated from five independent experiments with standard deviations. *, P = 0.011; ****, P = 6.64x10-18, unpaired t-test.


Biochemical characterization of recombinant OTULIN variants by means of surface plasmon resonance ( SPR ) measurements ( B ) SPR measurements and steady - state binding curves with calculated dissociation constants ( Kd ) after injection of a concentration series of OTULINC129A , OTULINC129A / M86I or OTULINC129A / W167S to CM5 - immobilized di - ubiquitin chains .

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

Biochemical characterization of recombinant OTULIN variants by means of surface plasmon resonance (SPR) measurements (B) SPR measurements and steady-state binding curves with calculated dissociation constants (Kd) after injection of a concentration series of OTULINC129A, OTULINC129A/M86I or OTULINC129A/W167S to CM5-immobilized di-ubiquitin chains.


( C ) Linear ubiquitin linkages isolated from A549 OTULIN KO cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULINWT , OTULINM86I and OTULINW167S or catalytically inactive OTULINC129A as control for 1 hour . Afterwards , samples were subjected to analysis by western blot for the indicated proteins . Images are representative of three independent experiments .

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

(C) Linear ubiquitin linkages isolated from A549 OTULIN KO cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULINWT, OTULINM86I and OTULINW167S or catalytically inactive OTULINC129A as control for 1 hour. Afterwards, samples were subjected to analysis by western blot for the indicated proteins. Images are representative of three independent experiments.


( A - C ) A549 OTULIN KO cells were transfected with empty vector or different OTULIN constructs as indicated and analyzed by western blot . ( D ) FLAG - tagged tandem ubiquitin binding entity ( TUBE ) assay was performed in B cells from control , patient , mother and father to pull down linear ubiquitin linkages . One representative ( A - D ) of three independent experiments is shown .

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

(A - C) A549 OTULIN KO cells were transfected with empty vector or different OTULIN constructs as indicated and analyzed by western blot. (D) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed in B cells from control, patient, mother and father to pull down linear ubiquitin linkages. One representative (A - D) of three independent experiments is shown.


Patient - derived and control fibroblasts were used . ( A ) FLAG - tagged tandem ubiquitin binding entity ( TUBE ) assay was performed to pull down linear ubiquitin linkages upon stimulation with 500ng / ml TNF . ( B ) The TNFR1 - SC was isolated using 500ng / ml TAP - TNF . ( C ) Cells were stimulated with 100ng / ml TNF for the indicated times and analyzed by western blot . One representative ( A - C ) of three independent experiments is shown .

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

Patient-derived and control fibroblasts were used. (A) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed to pull down linear ubiquitin linkages upon stimulation with 500ng/ml TNF. (B) The TNFR1-SC was isolated using 500ng/ml TAP-TNF. (C) Cells were stimulated with 100ng/ml TNF for the indicated times and analyzed by western blot. One representative (A - C) of three independent experiments is shown.


( E ) B cells were incubated with 50µg / ml cycloheximide ( CHX ) for the indicated times and subjected to analysis by western blot . One representative ( left panel ) out of 4 individual experiments analyzed by densitometry ( right panel ) is shown . Data are presented as mean ± SD . * , P = 0 . 02 , unpaired t - test .

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(E) B cells were incubated with 50µg/ml cycloheximide (CHX) for the indicated times and subjected to analysis by western blot. One representative (left panel) out of 4 individual experiments analyzed by densitometry (right panel) is shown. Data are presented as mean ± SD. *, P = 0.02, unpaired t-test.


( G ) Fibroblasts were incubated with 50µg / ml cycloheximide ( CHX ) or DMSO and stimulated with TNF as indicated . Viability was measured after 24 hours . Data are presented as mean ± SD of 5 individual experiments performed in three technical replicates ; * * * , P = 0 . 0008 , mixed - effects analysis with Tukey ' s multiple comparisons test .

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(G) Fibroblasts were incubated with 50µg/ml cycloheximide (CHX) or DMSO and stimulated with TNF as indicated. Viability was measured after 24 hours. Data are presented as mean ± SD of 5 individual experiments performed in three technical replicates; ***, P = 0.0008, mixed-effects analysis with Tukey's multiple comparisons test.


A , B . Representative images of H & amp ; amp ; E - ( A ) and PAS - ( B ) stained colon sections from wild - type and Angptl2 - / - mice . Scale bar = 100μm . C , D . Representative images of colon crypts as assessed by Ki - 67 ( C ) and pH3 ( D ) staining . Scale bar = 100 μm .

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A, B. Representative images of H&amp;E- (A) and PAS- (B) stained colon sections from wild-type and Angptl2-/- mice. Scale bar = 100μm.C, D. Representative images of colon crypts as assessed by Ki-67 (C) and pH3 (D) staining. Scale bar = 100 μm.


E . mRNA levels of indicated proliferation and ISC markers in isolated crypts from wild - type ( n = 4 ) and Angptl2 - / - ( n = 4 ) mice based on qRT - PCR analysis .

E. mRNA levels of indicated proliferation and ISC markers in isolated crypts from wild-type (n=4) and Angptl2-/- (n=4) mice based on qRT-PCR analysis.


F . Western blotting analysis of isolated colon crypts from wild - type and Angptl2 - / - mice . HSC70 served as an internal control . Numbers below panels represent normalized expression of proteins .

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F. Western blotting analysis of isolated colon crypts from wild-type and Angptl2-/- mice. HSC70 served as an internal control. Numbers below panels represent normalized expression of proteins.


G . IHC for expression of active β - catenin in colon or wild - type and Angptl2 - / - mice . Scale bar = 50 μm .

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G. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice. Scale bar = 50 μm.


A . Survival rates of wild - type ( n = 33 ) and Angptl2 - / - ( n = 33 ) mice supplied with drinking water containing 2 . 5 % DSS for 6 days and then untreated water for 6 more days , from two different experiments . * * * p < 0 . 001 , by log - rank test .

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A. Survival rates of wild-type (n=33) and Angptl2-/- (n=33) mice supplied with drinking water containing 2.5 % DSS for 6 days and then untreated water for 6 more days, from two different experiments. ***p < 0.001, by log-rank test.


B . Representative images of colons from wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) . C . Colon length of wild - type ( n = 7 ) and Angptl2 - / - ( n = 6 ) mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) .

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B. Representative images of colons from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).C. Colon length of wild-type (n=7) and Angptl2-/- (n=6) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).


D , E , F . Representative images of colon crypts as assessed by H & amp ; amp ; E ( D ) , Ki - 67 ( E ) , and pH3 ( F ) staining in wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) . Scale bar = 100 μm . G , H . The number of regenerating crypts as quantified using Ki - 67 ( G ) and pH3 ( H ) staining . Each data point represents the number of Ki - 67 - positive viable crypts or the number of pH3 - positive cells in a single field of view . Multiple areas in at least 6 mice per genotype were quantified . Numbers below panels represent the average .

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D, E, F. Representative images of colon crypts as assessed by H&amp;E (D), Ki-67 (E), and pH3 (F) staining in wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 100 μm.G, H. The number of regenerating crypts as quantified using Ki-67 (G) and pH3 (H) staining. Each data point represents the number of Ki-67-positive viable crypts or the number of pH3-positive cells in a single field of view. Multiple areas in at least 6 mice per genotype were quantified. Numbers below panels represent the average.


I . IHC for expression of active β - catenin in colon or wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) . Scale bar = 50 μm .

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

I. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 50 μm.


J . mRNA levels of indicated genes in isolated crypts from wild - type ( n = 4 ) and Angptl2 - / - ( n = 4 ) mice following DSS treatment for 6 days and then untreated water for 6 more days ( at days 12 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. mRNA levels of indicated genes in isolated crypts from wild-type (n=4) and Angptl2-/-(n=4) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).


A . Weight loss of wild - type ( n = 4 ) and Angptl2 - / - ( n = 5 ) mice in days following 12 Gy irradiation .

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A. Weight loss of wild-type (n=4) and Angptl2-/- (n=5) mice in days following 12 Gy irradiation.


B . Small intestine and colon length from wild - type ( n = 4 ) and Angptl2 - / - ( n = 5 ) mice 5 days after 12 Gy irradiation .

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B. Small intestine and colon length from wild-type (n=4) and Angptl2-/- (n=5) mice 5 days after 12 Gy irradiation.


C , D . Representative images of crypts of small intestine as assessed by H & amp ; amp ; E ( C ) and Ki - 67 ( D ) staining in wild - type and Angptl2 - / - mice 5 days after 12 Gy irradiation . Scale bar = 100 μm . E . The number of regenerating crypts in small intestine as quantified by Ki - 67 ( D ) staining at days 3 and 5 . Each data point represents the number of Ki - 67 - positive viable crypts in a single field of view . Multiple areas in at least 3 mice per genotype were quantified . Numbers below panels represent average .

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C, D. Representative images of crypts of small intestine as assessed by H&amp;E (C) and Ki-67 (D) staining in wild-type and Angptl2-/- mice 5 days after 12 Gy irradiation. Scale bar = 100 μm.E. The number of regenerating crypts in small intestine as quantified by Ki-67 (D) staining at days 3 and 5. Each data point represents the number of Ki-67-positive viable crypts in a single field of view. Multiple areas in at least 3 mice per genotype were quantified. Numbers below panels represent average.


F , G . Representative images of colon crypts as assessed by H & amp ; amp ; E ( F ) or Ki - 67 ( G ) staining in wild - type and Angptl2 - / - mice 5 days after 12 Gy irradiation . Scale bar = 100 μm . H . The number of regenerating colon crypts as quantified by using Ki - 67 ( G ) staining at days 3 and 5 . Each data point represents the number of Ki - 67 - positive viable crypts in a single field of view . Multiple areas in at least 3 mice per genotype were quantified . Numbers below panels represent average .

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F, G. Representative images of colon crypts as assessed by H&amp;E (F) or Ki-67 (G) staining in wild-type and Angptl2-/- mice 5 days after 12 Gy irradiation. Scale bar = 100 μm.H. The number of regenerating colon crypts as quantified by using Ki-67 (G) staining at days 3 and 5. Each data point represents the number of Ki-67-positive viable crypts in a single field of view. Multiple areas in at least 3 mice per genotype were quantified. Numbers below panels represent average.


A . Angptl2 mRNA levels in small intestine ( proximal , mid and distal ) , cecum and colon ( proximal and distal ) of wild - type mice based on qRT - PCR analysis . n = 5 .

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A. Angptl2 mRNA levels in small intestine (proximal, mid and distal), cecum and colon (proximal and distal) of wild-type mice based on qRT-PCR analysis. n=5.


B . ANGPTL2 IHC in colon from wild - type and Angptl2 - / - mice . The Angptl2 - / - colon image serves as a negative control . Scale bar = 50 μm .

B. ANGPTL2 IHC in colon from wild-type and Angptl2-/- mice. The Angptl2-/- colon image serves as a negative control. Scale bar = 50 μm.


C . mRNA levels of indicated genes in wild - type ( n = 4 ) mice based on qRT - PCR analysis .

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C. mRNA levels of indicated genes in wild-type (n=4) mice based on qRT-PCR analysis.


D . Representative IF images of distal colon from wild - type mice as assessed with anti - ANGPTL2 ( red ) and anti - α - SMA ( green ) . Nuclei are counterstained with DAPI ( blue ) . Scale bar = 100 μm .

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D. Representative IF images of distal colon from wild-type mice as assessed with anti-ANGPTL2 (red) and anti-α-SMA (green). Nuclei are counterstained with DAPI (blue). Scale bar = 100 μm.


A , B , C . mRNA levels of indicated genes in ISEMFs from wild - type ( n = 4 ) and Angptl2 - / - ( n = 3 ) mice based qRT - PCR analysis .

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A, B, C. mRNA levels of indicated genes in ISEMFs from wild-type (n=4) and Angptl2-/- (n=3) mice based qRT-PCR analysis.


D . Representative staining for P - Smad1 / 5 in crypts from wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( DSS ) or untreated water for 12 days ( Untreated ) . Dashed lines indicate half of the crypt and arrowheads show P - Smad1 / 5 - positive cells in lower halves of crypts . Scale bar = 50 μm . E . The number of P - Smad1 / 5 - positive cells in the crypt base ( n = 60 ) from wild - type and Angptl2 - / - mice following DSS treatment for 6 days and then untreated water for 6 more days ( DSS ) or untreated water for 12 days ( Untreated ) . Multiple areas in 3 mice per genotype were quantified .

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D. Representative staining for P-Smad1/5 in crypts from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Dashed lines indicate half of the crypt and arrowheads show P-Smad1/5-positive cells in lower halves of crypts. Scale bar = 50 μm.E. The number of P-Smad1/5-positive cells in the crypt base (n=60) from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Multiple areas in 3 mice per genotype were quantified.


F . Western blotting analysis of isolated colon crypts from wild - type and Angptl2 - / - mice . HSC70 serves as an internal control . Numbers below panels represent normalized protein expression .

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F. Western blotting analysis of isolated colon crypts from wild-type and Angptl2-/- mice. HSC70 serves as an internal control. Numbers below panels represent normalized protein expression.


A . mRNA levels of indicated genes in colon and cultured ISEMFs from wild - type mice ( n = 4 ) based on qRT - PCR analysis . Inta5 , Integrin α5 ; Intb1 , Integrin β1 .

A. mRNA levels of indicated genes in colon and cultured ISEMFs from wild-type mice (n=4) based on qRT-PCR analysis. Inta5, Integrin α5; Intb1, Integrin β1.


B . Representative IHC of integrin α5 and β1 in colon from wild - type mouse . Scale bar = 50 μm .

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B. Representative IHC of integrin α5 and β1 in colon from wild-type mouse. Scale bar = 50 μm.


C , D . mRNA levels of Bmp2 and Bmp7 in ISEMFs from wild - type mice 24 h after treatment with integrinα5β1 antibody or various signaling pathway inhibitors . n = 3 . U0126 , ERK inhibitor ; LY294002 , PI3K inhibitor ; BAY11 - 7085 , NF - κB inhibitor .

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C, D. mRNA levels of Bmp2 and Bmp7 in ISEMFs from wild-type mice 24 h after treatment with integrinα5β1 antibody or various signaling pathway inhibitors. n=3. U0126, ERK inhibitor; LY294002, PI3K inhibitor; BAY11-7085, NF-κB inhibitor.


A . Colon organoid cultures treated with vehicle or rANGPTL2 protein . Scale bar = 200 μm . B . Organoid growth efficiency and size following treatment with vehicle or rANGPTL2 . rA2 , recombinant ANGPTL2 . n = 4 . Data from vehicle / Wnt ( + ) was set at 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Colon organoid cultures treated with vehicle or rANGPTL2 protein. Scale bar = 200 μm.B. Organoid growth efficiency and size following treatment with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. n=4. Data from vehicle/Wnt (+) was set at 1.


D . Colon organoid cultures in presence of ISEMFs with or without Noggin ( Nog ) . Scale bar = 50

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

D. Colon organoid cultures in presence of ISEMFs with or without Noggin (Nog). Scale bar = 50 μm


E . Organoid growth efficiency and size in presence of ISEMFs with or without Noggin ( Nog ) . n = 4 . Data from Wild - type organoids / Wild - type ISEMFs / Nog ( + ) was set at 1 .

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

E. Organoid growth efficiency and size in presence of ISEMFs with or without Noggin (Nog). n=4. Data from Wild-type organoids/Wild-type ISEMFs/Nog (+) was set at 1.


THP - 1 cells were stimulated with ( 50 000 particles / cell ) AdV - GFP and infection after 24 h measured by flow cytometry .

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

THP-1 cells were stimulated with (50 000 particles/cell) AdV-GFP and infection after 24 h measured by flow cytometry.


Cells were stimulated as in ( A ) with AdV - GFP or AdV - GFP complexed with 20 μg / ml h9C12 or 20 mg / ml human IVIg . Cell supernatants were harvested at indicated time - points and cytokines measured by ELISA ( B )

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Cells were stimulated as in (A) with AdV-GFP or AdV-GFP complexed with 20 μg/ml h9C12 or 20 mg/ml human IVIg. Cell supernatants were harvested at indicated time-points and cytokines measured by ELISA (B)


Cells were stimulated as in ( A ) with AdV - GFP or AdV - GFP complexed with 20 μg / ml h9C12 or 20 mg / ml human IVIg . pro - and cleaved IL - 1β was measured in whole cell lysates or in supernatants by western blot ( C ) .

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 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

Cells were stimulated as in (A) with AdV-GFP or AdV-GFP complexed with 20 μg/ml h9C12 or 20 mg/ml human IVIg. pro- and cleaved IL-1β was measured in whole cell lysates or in supernatants by western blot (C).


HMDM were primed with 10 μg / ml pI : C for 2h , then stimulated for 16h with virus + / - antibody . Cytokines in the supernatants were measured by ELISA .

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HMDM were primed with 10 μg/ml pI:C for 2h, then stimulated for 16h with virus +/- antibody. Cytokines in the supernatants were measured by ELISA.


AdV - GFP was incubated with 20 ug / ml h9C12 antibody or 20 mg / ml IVIg in PBS . These AdV - Ab complexes were then diluted 1 : 2 to give final doses of viral particles being added to WT THP - 1 cells ( with antibody complexed ) as indicated . A single experiment representative of 3 independent experiments is shown .

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AdV-GFP was incubated with 20 ug/ml h9C12 antibody or 20 mg/ml IVIg in PBS. These AdV-Ab complexes were then diluted 1:2 to give final doses of viral particles being added to WT THP-1 cells (with antibody complexed) as indicated. A single experiment representative of 3 independent experiments is shown.


THP - 1s were stimulated with a constant dose of AdV - GFP ( 50 000 pp / cell ) , and complexed with decreasing antibody concentrations as indicated . A single experiment representative of 2 independent experiments is shown .

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

THP-1s were stimulated with a constant dose of AdV-GFP (50 000 pp/cell), and complexed with decreasing antibody concentrations as indicated. A single experiment representative of 2 independent experiments is shown.


HMDM were stimulated directly ( TNF ) or primed ( IL - 1β , LDH ) with AdV - Ab complexes as in ( B ) . Data for each individual donor ( 2 total ) is shown .

HMDM were stimulated directly (TNF) or primed (IL-1β, LDH) with AdV-Ab complexes as in (B). Data for each individual donor (2 total) is shown.


THP - 1 were stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell death was measured by LDH

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THP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH release


THP - 1 were stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell viability measured by PrestoBlue

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

THP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell viability measured by PrestoBlue assay


HMDM were primed , or not , and stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell death was measured by LDH

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

HMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH release


HMDM were primed , or not , and stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell viability measured by PrestoBlue

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

HMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell viability measured by PrestoBlue assay


HMDM were stimulated with AdV ( 50 000 pp / cell ) complexed with antibody at indicated doses for 2h and viral genomes were measured by qPCR . ( n = 4 , mean ± s . e . m )

HMDM were stimulated with AdV (50 000 pp/cell) complexed with antibody at indicated doses for 2h and viral genomes were measured by qPCR. (n = 4, mean ± s.e.m)


AdV - mCherry was added to AdV - GFP at a ratio of 1 : 200 and 50 000 pp / cell of this mix was complexed with h9C12 and IVIg and indicated doses . HMDM were stimulated with these complexes for 16h and infection measured as mCherry fluorescence by flow cytometry . In virus alone the percentage of mCherry positive cells was ~ 20 % , the relative infection with antibodies was normalised to virus alone as 100 % .

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

AdV-mCherry was added to AdV-GFP at a ratio of 1:200 and 50 000 pp/cell of this mix was complexed with h9C12 and IVIg and indicated doses. HMDM were stimulated with these complexes for 16h and infection measured as mCherry fluorescence by flow cytometry. In virus alone the percentage of mCherry positive cells was ~ 20%, the relative infection with antibodies was normalised to virus alone as 100%.


HMDM were stimulated for 3 h with AdV - Ab complexes as in ( A , B ) or with LeuLeu at 10μM , and lysosomal damage measured by loss of acridine orange red fluorescence using flow cytometry .

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

HMDM were stimulated for 3 h with AdV-Ab complexes as in (A,B) or with LeuLeu at 10μM, and lysosomal damage measured by loss of acridine orange red fluorescence using flow cytometry.


HMDM were stimulated as in ( C ) and cell death was assessed by an increase in e780 fluorescence . The mean of 3 independent donors is shown .

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

HMDM were stimulated as in (C) and cell death was assessed by an increase in e780 fluorescence. The mean of 3 independent donors is shown.


( E ) The MFI of the AO - red channel ( 685nM ) for the populations determined as e780 positive ( dying ) or e780 negative ( live ) for the condition of AdV + IVIg 20 was measured and plotted as mean + SEM of the three donors shown in ( D ) .

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

(E)The MFI of the AO- red channel (685nM) for the populations determined as e780 positive (dying) or e780 negative (live) for the condition of AdV + IVIg 20 was measured and plotted as mean + SEM of the three donors shown in (D).


Primed or ( C ) un - primed HMDM were stimulated with AdV ( 50 000 pp / cell ) + / - h9C12 ( 20 μg / ml ) for 16h .

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Primed or (C) un- primed HMDM were stimulated with AdV (50 000 pp/cell) +/- h9C12 (20 μg/ml) for 16h.


Ctrl or Trim21 - deficient THP - 1s were stimulated for 4 h with 1000 U / ml IFNα and TRIM21 expression measured by immunoblot .

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

Ctrl or Trim21- deficient THP-1s were stimulated for 4 h with 1000 U/ml IFNα and TRIM21 expression measured by immunoblot.


WT or TRIM21 - deficient THP - 1s were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 or 20 mg / ml IVIg or with 200 ng / well HT - DNA or 10 μM Nigericin for 16h , and IL - 1β measured in the supernatant by ELISA .

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

WT or TRIM21- deficient THP-1s were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT-DNA or 10 μM Nigericin for 16h, and IL-1β measured in the supernatant by ELISA.


WT or TRIM21 - deficient THP - 1s were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 or 20 mg / ml IVIg or with 200 ng / well HT - DNA or 10 μM Nigericin for 16h , and TNF measured in the supernatant by ELISA .

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

WT or TRIM21- deficient THP-1s were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT-DNA or 10 μM Nigericin for 16h, and TNF measured in the supernatant by ELISA.


WT mice were injected i . v . with 2 . 5ug WT or H433A h9C12 antibody , then the next day injected i . v . with 2 . 5 x 1011 pp AdV - GFP . 4h later spleens were harvested and neutrophil influx measured by flow cytometry .

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WT mice were injected i.v. with 2.5ug WT or H433A h9C12 antibody, then the next day injected i.v. with 2.5 x 1011 pp AdV-GFP. 4h later spleens were harvested and neutrophil influx measured by flow cytometry.


HMDM were stimulated with AdV - GFP ( 250 pp / cell ) in the presence of h9C12 antibodies at indicated doses . Infection was measured after 24h by flow cytometry . Infection relative to AdV alone is shown graphed on the left ( n = 4 mean ± s . e . m ) . Representative plots and a comparison of the different h9C12 mutants at 0 . 2 ug / ml are also shown in the graph on the right ( n = 4 , mean ± s . e . m . ) .

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 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

HMDM were stimulated with AdV-GFP (250 pp/cell) in the presence of h9C12 antibodies at indicated doses. Infection was measured after 24h by flow cytometry. Infection relative to AdV alone is shown graphed on the left (n = 4 mean ± s.e.m). Representative plots and a comparison of the different h9C12 mutants at 0.2 ug/ml are also shown in the graph on the right(n = 4,mean ± s.e.m.).


HMDM were stimulated with AdV ( 50 000 pp / cell ) and antibody ( h9C12 ; 20 ug / ml , IVIg : 20 mg / ml ) or 10 μg / ml pI : C for 3h and gene expression was measured by qPCR ( n = 4 mean ± s . e . m ) .

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HMDM were stimulated with AdV (50 000 pp/cell) and antibody (h9C12; 20 ug/ml, IVIg: 20 mg/ml) or 10 μg/ml pI:C for 3h and gene expression was measured by qPCR (n = 4 mean ± s.e.m).


HMDM were stimulated as in ( A ) , or with 10 ng / ml LPS for 6 h and pro - IL - 1β levels in the cytosol measured by western blot . Blot is representative of three independent donors .

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HMDM were stimulated as in (A), or with 10 ng/ml LPS for 6 h and pro-IL-1β levels in the cytosol measured by western blot. Blot is representative of three independent donors.


HMDM were stimulated as in ( A ) , or with 10 ng / ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR . Data shows average ± s . d . of 2 independent

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

HMDM were stimulated as in (A), or with 10 ng/ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR. Data shows average ± s.d. of 2 independent donors


HMDM were stimulated as in ( B ) and NLRP3 levels in the cytosol measured by western blot . Blot is representative of two independent donors .

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HMDM were stimulated as in (B) and NLRP3 levels in the cytosol measured by western blot. Blot is representative of two independent donors.


TLR9 mRNA levels from PBMCs , CD19 + ve B cells , CD14 + ve monocytes or HMDM derived from CD14 + ve monocytes were assessed by qPCR and copy number was determined relative to actin copy number ( n = 5 mean ± s . e . m ) .

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

TLR9 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve monocytes or HMDM derived from CD14+ve monocytes were assessed by qPCR and copy number was determined relative to actin copy number (n = 5 mean ± s.e.m).


THP - 1s expressing ASC - GFP were stimulated for 6 h with virus and antibody or 200 ng / well HT - DNA or 10 μM Nigericin , in the presence of the pan - caspase inhibitor zVAD - fmk . A representative image ( scale bar 100 μm ) and quantification of number of cells with ASC specks from three independent experiments ( mean ± s . e . m , * P - value ≤ 0 . 05 , paired , two - tailed t - test ) is shown .

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

THP-1s expressing ASC-GFP were stimulated for 6 h with virus and antibody or 200 ng/well HT-DNA or 10 μM Nigericin, in the presence of the pan-caspase inhibitor zVAD-fmk. A representative image (scale bar 100 μm) and quantification of number of cells with ASC specks from three independent experiments (mean ± s.e.m, *P-value ≤ 0.05, paired, two-tailed t-test) is shown.


Primed HMDM were treated with MCC950 ( 1 μM ) for 30 mins before stimulation overnight with AdV ( 50 000 pp / cell ) and h9C12 ( 20 μg / ml ) or IVIg ( 20 mg / ml ) or 10 μM Nigericin . IL - 1β ( A ) or TNF ( B ) or LDH release ( C ) were measured in cell supernatants . Data shows average ± s . d . of two independent donors and is representative of three independent experiments .

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Primed HMDM were treated with MCC950 (1 μM) for 30 mins before stimulation overnight with AdV (50 000 pp/cell) and h9C12 (20 μg/ml) or IVIg (20 mg/ml) or 10 μM Nigericin. IL-1β (A) or TNF (B) or LDH release (C) were measured in cell supernatants. Data shows average ± s.d. of two independent donors and is representative of three independent experiments.


WT , NLRP3 , ASC or Caspase - 1 - deficient THP - 1s were stimulated as in ( A ) and IL - 1β in the supernatant measured by ELISA . Data is representative of two independent experiments .

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

WT, NLRP3, ASC or Caspase-1 - deficient THP-1s were stimulated as in (A) and IL-1β in the supernatant measured by ELISA. Data is representative of two independent experiments.


HMDM were primed with 10 ug / ml pI : C for 2h , treated with 100 μM VX - 765 for 30 mins before stimulation for 16 h as in ( A ) . IL - 1β or LDH release was measured in cell supernatants . Data shows average ± s . d . of two independent

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HMDM were primed with 10 ug/ml pI:C for 2h, treated with 100 μM VX-765 for 30 mins before stimulation for 16 h as in (A). IL-1β or LDH release was measured in cell supernatants. Data shows average ± s.d. of two independent donors


Primed HMDM were treated with KCl as indicated for 1h , before being stimulated for a further 6h as in ( A ) . IL - 1β and TNF were measured in cell supernatants by ELISA . Data is representative of three independent donors .

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

Primed HMDM were treated with KCl as indicated for 1h, before being stimulated for a further 6h as in (A). IL-1β and TNF were measured in cell supernatants by ELISA. Data is representative of three independent donors.


Primed HMDM were stimulated with AdV and 20 μg / ml h9C12 or 10 μM Nigericin for 3h , and then immunostained for ASC or intracellular antibody . Co - localisation was assessed by confocal microscopy . Data is representative of two independent donors .

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Primed HMDM were stimulated with AdV and 20 μg/ml h9C12 or 10 μM Nigericin for 3h, and then immunostained for ASC or intracellular antibody. Co-localisation was assessed by confocal microscopy. Data is representative of two independent donors.


AIM2 mRNA levels from PBMCs , CD19 + ve B cells , CD14 + ve Monocytes or HMDM derived from CD14 + ve monocytes . were assessed by qPCR and copy number was determined relative to Actin copy number ( n = 5 mean ± s . e . m ) .

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

AIM2 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve Monocytes or HMDM derived from CD14+ve monocytes. were assessed by qPCR and copy number was determined relative to Actin copy number (n = 5 mean ± s.e.m).


HMDM were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 for 1h , washed 2x with SFM , then whole cell lysates harvested after a further 5h . Viral hexon and transgene ( GFP ) expression in the cytosol was assessed by western blot . Data is representative of two independent experiments .

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

HMDM were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 for 1h, washed 2x with SFM, then whole cell lysates harvested after a further 5h. Viral hexon and transgene (GFP) expression in the cytosol was assessed by western blot. Data is representative of two independent experiments.


THP - 1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1000 U / ml IFNα for 4 h and protein levels assessed by western blot .

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

THP-1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1000 U/ml IFNα for 4 h and protein levels assessed by western blot.


THP - 1s deficient in cGAS and STING were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 , 200 ng / well HT - DNA , 10 μM Nigericin or 10 ng / ml LPS for 16 h . Data shows combined data ( mean ± s . e . m ) of 3 experiments with absolute protein values ( upper panel ) or as % cytokine output of KO cells relative to Ctrl treated cells ( lower panel ) , * P - value ≤ 0 . 05 , * * * P - value ≤ 0 . 001 unpaired , two - tailed t - test ) .

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THP-1s deficient in cGAS and STING were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12, 200 ng/ well HT-DNA, 10 μM Nigericin or 10 ng/ml LPS for 16 h. Data shows combined data (mean ± s.e.m) of 3 experiments with absolute protein values (upper panel) or as % cytokine output of KO cells relative to Ctrl treated cells (lower panel), *P-value ≤ 0.05, ***P-value ≤ 0.001 unpaired, two-tailed t-test).


WT THP - 1s were treated with 5 μM H151 for 30 mins before stimulation as in ( D ) . Data in upper panel is representative of 3 independent experiments . Data in lower panel shows combined data of these 3 experiments ( mean ± s . e . m ) showing H151 treated cells relative to media treated cells ( * P - value ≤ 0 . 05 , * * * P - value ≤ 0 . 001 unpaired , two - tailed t - test ) .

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WT THP-1s were treated with 5 μM H151 for 30 mins before stimulation as in (D). Data in upper panel is representative of 3 independent experiments. Data in lower panel shows combined data of these 3 experiments (mean ± s.e.m) showing H151 treated cells relative to media treated cells (*P-value ≤ 0.05, ***P-value ≤ 0.001 unpaired, two-tailed t-test).


ASC - GFP THP - 1s were treated with 5 μM H151 for 30 mins before stimulation with AdV - mCherry ( 50 000 pp / cell ) + 20 μg / ml h9C12 or 20 mg / ml IVIg or 200 ng / well HT - DNA for 8h . A representative image ( scale bar 50 μm ) and quantification of number of cells with ASC specks from one representative experiment of three is shown .

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

ASC-GFP THP-1s were treated with 5 μM H151 for 30 mins before stimulation with AdV-mCherry (50 000 pp/cell) + 20 μg/ml h9C12 or 20 mg/ml IVIg or 200 ng/ well HT-DNA for 8h. A representative image (scale bar 50 μm) and quantification of number of cells with ASC specks from one representative experiment of three is shown.


A - C Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A ) body weight and ( B , C ) body composition . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * 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 B-CONTROLLED_VAR O O 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 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 O O O O O O O

A-C Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A) body weight and (B, C) body composition. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A , B ) energy expenditure . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * 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 B-CONTROLLED_VAR O O 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 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 O

A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A, B) energy expenditure. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( C ) respiratory exchange ratio ( RER ) . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * 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 B-CONTROLLED_VAR O O 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 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 O O O

A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (C) respiratory exchange ratio (RER). Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( D ) locomotor activity . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * 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 B-CONTROLLED_VAR O O 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 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

A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (D) locomotor activity. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( E ) cumulative food intake , ( F ) meal number and ( G ) meal size . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * 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 B-CONTROLLED_VAR O O 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 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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (E) cumulative food intake, (F) meal number and (G) meal size. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A , B Treatment - induced changes in hypothalamic gene expression in DIO mice treated for 2 ( A ) or 5 ( B ) days with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Compounds were administered by daily subcutaneous injections , and the last injection was provided 2 h prior to tissues sampling . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , § P < 0 , 001 , comparison between treatment and vehicle control . # P < 0 . 05 , # # P < 0 . 01 , # # # P < 0 . 001 , comparison to liraglutide . + P < 0 . 05 , comparison to RM - 493 .

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

A, B Treatment-induced changes in hypothalamic gene expression in DIO mice treated for 2 (A) or 5 (B) days with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Compounds were administered by daily subcutaneous injections, and the last injection was provided 2 h prior to tissues sampling. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, §P < 0,001, comparison between treatment and vehicle control. #P < 0.05, ##P < 0.01, ###P < 0.001, comparison to liraglutide. +P < 0.05, comparison to RM-493.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( A ) Fasted blood glucose levels . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 B-CONTROLLED_VAR O O 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 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 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

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Fasted blood glucose levels. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( B , C ) glucose tolerance . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 B-CONTROLLED_VAR O O 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 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 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

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered).(B, C) glucose tolerance. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( D ) fasted insulin levels , . Data represent means ± SEM ; n = 8 in ( A - H ) ; * 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 B-CONTROLLED_VAR O O 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 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 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

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (D) fasted insulin levels,. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( E , F ) glucose - induced insulin secretion . Data represent means ± SEM ; n = 8 in ( A - H ) ; * 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 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 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 B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (E, F) glucose-induced insulin secretion. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( G ) HOMA - IR . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 B-CONTROLLED_VAR O O 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 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

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (G) HOMA-IR. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( H ) insulin sensitivity . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 B-CONTROLLED_VAR O O 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 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 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

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (H) insulin sensitivity. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( I ) hepatic phosphorylation of AKT ( p - AKTSer473 ) were analyzed . Compounds were administered by daily subcutaneous injections . To assess insulin - stimulated p - AKTSer473 , insulin ( n = 5 ) or saline ( n = 3 ) was injected 10 min prior to liver sampling . Data represent means ± SEM ; n = 8 in ( A - H ) ; * 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 B-CONTROLLED_VAR O O 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 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 B-CONTROLLED_VAR 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-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (I) hepatic phosphorylation of AKT (p-AKTSer473) were analyzed. Compounds were administered by daily subcutaneous injections. To assess insulin-stimulated p-AKTSer473, insulin (n = 5) or saline (n = 3) was injected 10 min prior to liver sampling. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( A ) Plasma lipoprotein fractions . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 B-CONTROLLED_VAR O O 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 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 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

A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Plasma lipoprotein fractions. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( B ) liver cholesterol levels . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (B) liver cholesterol levels. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( C ) expression of genes implicated in hepatic cholesterol and lipoprotein uptake . Data represent means ± SEM ; n = 8 ; * 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 B-CONTROLLED_VAR O O 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 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

A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (C) expression of genes implicated in hepatic cholesterol and lipoprotein uptake. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( D ) expression of genes implicated in cholesterol and bile acid metabolism and excretion were analyzed . Data represent means ± SEM ; n = 8 ; * 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 B-CONTROLLED_VAR O O 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 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 O

A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (D) expression of genes implicated in cholesterol and bile acid metabolism and excretion were analyzed. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A ) body weight . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A) body weight. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( B ) cumulative food intake . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * 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 B-CONTROLLED_VAR O O 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 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 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

A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (B) cumulative food intake. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( C , D ) body composition . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (C, D) body composition. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( E , F ) glucose tolerance . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * 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 B-CONTROLLED_VAR O O 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 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 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

A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (E, F) glucose tolerance. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


( A ) Bar chart showing the relative abundance ( signal intensity ) of the phosphorylated tau species identified using an exploratory IP - MS approach using HT7 antibody on TBS - soluble control and Alzheimer ' s disease ( AD ) pools . Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP - MS : mono - phosphorylated p - tau231 and double phosphorylated p - tau ( 231 + 235 ) .

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(A) Bar chart showing the relative abundance (signal intensity) of the phosphorylated tau species identified using an exploratory IP-MS approach using HT7 antibody on TBS-soluble control and Alzheimer's disease (AD) pools. Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP-MS: mono-phosphorylated p-tau231 and double phosphorylated p-tau(231+235).


( A ) Box - and - whiskers plot showing the levels of CSF p - tau235 in neurological controls ( n = 21 ) and biologically defined Alzheimer ' s disease ( AD ) cases ( n = 19 ) .

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(A) Box-and-whiskers plot showing the levels of CSF p-tau235 in neurological controls (n=21) and biologically defined Alzheimer's disease (AD) cases (n=19).


( A ) Box - and - whiskers plot showing CSF p - tau235 concentrations in the different groups : amyloid - negative cognitively unimpaired ( CU - ) participants ( n = 50 ) , participants across the Alzheimer ' s disease ( AD ) continuum ( amyloid - positive cognitively unimpaired ( CU + , n = 32 ) ; amyloid - positive mild cognitive impairment ( MCI + , n = 20 ) ; AD ( n = 20 ) and non - AD cases ( n = 19 ) . CSF p - tau235 is highly specific for AD pathology . Cut - off value for CSF p - tau235 positivity is displayed with black dashed line ( 19 . 92 pg / mL ) .

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(A) Box-and-whiskers plot showing CSF p-tau235 concentrations in the different groups: amyloid-negative cognitively unimpaired (CU-) participants (n=50), participants across the Alzheimer's disease (AD) continuum (amyloid-positive cognitively unimpaired (CU+, n=32); amyloid-positive mild cognitive impairment (MCI+, n=20); AD (n=20) and non-AD cases (n=19). CSF p-tau235 is highly specific for AD pathology. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL).


( A ) Box - and - whiskers plot showing CSF p - tau235 concentrations in A - T - participants and across preclinical Alzheimer ' s disease ( AD , dichotomised as A + T - and A + T + ) . CSF p - tau235 was increased already in A + T - , which represents the early cases within preclinical AD . A prominent increase was observed from A + T - to A + T + cases , the latter representing late preclinical AD cases . Cut - off value for CSF p - tau235 positivity is displayed with black dashed line ( 19 . 92 pg / mL ) .

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(A) Box-and-whiskers plot showing CSF p-tau235 concentrations in A-T- participants and across preclinical Alzheimer's disease (AD, dichotomised as A+T- and A+T+). CSF p-tau235 was increased already in A+T-, which represents the early cases within preclinical AD. A prominent increase was observed from A+T- to A+T+ cases, the latter representing late preclinical AD cases. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL).


Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method ( Loess curve ) . Changes on biomarkers levels in CSF are represented as z - scores using Aβ PET in centiloid scale ( CL ) as a proxy of disease progression . Abnormal biomarker levels were determined as two standard deviations ( SD ) above the mean . Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 ( Mila - Aloma et al . , 2020 ) . All samples were run in singlicates .

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Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method (Loess curve). Changes on biomarkers levels in CSF are represented as z-scores using Aβ PET in centiloid scale (CL) as a proxy of disease progression. Abnormal biomarker levels were determined as two standard deviations (SD) above the mean. Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 (Mila-Aloma et al., 2020). All samples were run in singlicates.


( A ) Stacked bar chart depicting the percentages of [ 231 + / 235 + ] , [ 231 + / 235 - ] , [ 231 - / 235 - ] and [ 231 - / 235 + ] cases in each preclinical group . Table below indicates the exact percentages of participants in each group ( exact number of participants in parenthesis ) . Discordant cases with the sequential phosphorylation hypothesis [ 231 - / + 235 ] shown in grey .

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(A) Stacked bar chart depicting the percentages of [231+/235+], [231+/235-], [231-/235-] and [231-/235+] cases in each preclinical group. Table below indicates the exact percentages of participants in each group (exact number of participants in parenthesis). Discordant cases with the sequential phosphorylation hypothesis [231-/+235] shown in grey.


( B ) Correlation between CSF p - tau235 and p - tau231 in the whole ALFA + cohort ( Spearman ' s rank correlation : rS = 0 . 80 , P ˂ 0 . 0001 ) . Assay cut - offs were determined as the mean + 2 SD of the A - T - group ( defining p - tau231 and p - tau235 positivity or negativity in each participant ) . Cut - off values are indicated in red ( 19 . 92 and 9 . 59 pg / mL for CSF p - tau235 and p - tau231 respectively ) and displayed with black dashed lines , resulting in four quadrants , each of them representing the four different positive or negatively status for each biomarker . Discordant cases with the sequential phosphorylation hypothesis ( [ 231 - / + 235 ] ) showed on the lower right quadrant ( 2 . 35 % of the total , 9 participants out 383 ) . All samples were run in singlicates .

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(B) Correlation between CSF p-tau235 and p-tau231 in the whole ALFA+ cohort (Spearman's rank correlation: rS = 0.80, P˂0.0001). Assay cut-offs were determined as the mean + 2 SD of the A-T- group (defining p-tau231 and p-tau235 positivity or negativity in each participant). Cut-off values are indicated in red (19.92 and 9.59 pg/mL for CSF p-tau235 and p-tau231 respectively) and displayed with black dashed lines, resulting in four quadrants, each of them representing the four different positive or negatively status for each biomarker. Discordant cases with the sequential phosphorylation hypothesis ([231-/+235]) showed on the lower right quadrant (2.35% of the total, 9 participants out 383). All samples were run in singlicates.


A . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation . Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated . Note that the PEG pellets did not contain mitochondrial protein TOMM20 , suggesting that the PEG pellets were not contaminated by intracellular organelles .

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A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. Note that the PEG pellets did not contain mitochondrial protein TOMM20, suggesting that the PEG pellets were not contaminated by intracellular organelles.


B . PEG pellets were subjected to OptiPrep flotation analysis .

B. PEG pellets were subjected to OptiPrep flotation analysis.


C . MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

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C. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


D . sEVs were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis ( NTA ) . Representative NTA traces were shown . E . Quantification of the NTA data obtained in five independent experiments .

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D. sEVs were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis (NTA). Representative NTA traces were shown. E. Quantification of the NTA data obtained in five independent experiments.


F . MDCK cells were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody .

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F. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody.


G . sEVs were eluted from the beads with a glycine buffer and analyzed by NTA . Representative NTA traces were shown . H . Quantification of the NTA data obtained in five independent experiments .

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G. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. H. Quantification of the NTA data obtained in five independent experiments.


A . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with serum - free medium . sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation . Apical and basolateral sEVs ( P100 ) were analyzed by silver staining .

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A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with serum-free medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation. Apical and basolateral sEVs (P100) were analyzed by silver staining.


C . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation . Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated . GPRC5C was detected only in the apical sEV

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C. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. GPRC5C was detected only in the apical sEV samples


D . PEG pellets were subjected to OptiPrep flotation analysis . GPRC5C was detected only in the apical sEV

D. PEG pellets were subjected to OptiPrep flotation analysis. GPRC5C was detected only in the apical sEV samples


E . MDCK cells were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . GPRC5C was detected only in the apical sEV

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E. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. GPRC5C was detected only in the apical sEV samples


F . MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

F. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


A . MDCK cells were transfected with siControl or the siRNAs indicated , and the cells were transferred to cell culture inserts and cultured for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated .

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A. MDCK cells were transfected with siControl or the siRNAs indicated, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.


B . The intensity of the bands was measured in three independent experiments .

B. The intensity of the bands was measured in three independent experiments.


C . sEVs were eluted from the beads with a glycine buffer and analyzed by NTA . Representative NTA traces were shown .

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C. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown.


D . Quantification of the NTA data obtained in five independent experiments .

D. Quantification of the NTA data obtained in five independent experiments.


A . MDCK cells were transfected with siControl or siALIX , and the cells were transferred to cell culture inserts and cultured for 4 days . On the last day , the culture medium was replaced with EV - depleted medium with or without 10 nM GW4869 . sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated .

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A. MDCK cells were transfected with siControl or siALIX, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium with or without 10 nM GW4869. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.


Analyses of protein expression in lysates of various cell lines by western blotting against the indicated antibodies .

Analyses of protein expression in lysates of various cell lines by western blotting against the indicated antibodies.


Fluorescence analyses of GFP - ATG5 or ATG16L1 - GFP stably expressed in ATG5 - / - or ATG16L1 - / - . Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence . Scale bar : 10 μm .

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Fluorescence analyses of GFP-ATG5 or ATG16L1-GFP stably expressed in ATG5-/- or ATG16L1-/-. Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence. Scale bar: 10 μm.


Assessment of ATG16L1 levels in the cytosolic ( C ) and membrane ( M ) fractions in lysates of the indicated cell lines using western blot analyses and antibodies against ATG16L1 . ATG16L1 - / - and ATG5 - / - stably expressed ATG16L1 - GFP while ATG3 - / - stably expressed Flag - S - ATG16L1 . Antibodies against α - tubulin and integrin β1 were used as controls for fractionation . Exogenous ( exo ) ATG16L1 was detected using antibodies against ATG16L1 .

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Assessment of ATG16L1 levels in the cytosolic (C) and membrane (M) fractions in lysates of the indicated cell lines using western blot analyses and antibodies against ATG16L1. ATG16L1-/- and ATG5-/- stably expressed ATG16L1-GFP while ATG3-/- stably expressed Flag-S-ATG16L1. Antibodies against α-tubulin and integrin β1 were used as controls for fractionation. Exogenous (exo) ATG16L1 was detected using antibodies against ATG16L1.


Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs ( WIPI2 + / + ) and WIPI2 - / - cells .

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Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs (WIPI2+/+) and WIPI2-/- cells.


Immunofluorescence analyses of ATG16L1 - / - and WIPI2 - / - stably expressing Flag - S - ATG16L1 . Cells were amino acid starved ( AA starve ) for 2 hr in the presence or absence of 3 ' MA ( and additional pretreatment for 30 min ) followed by fixation and immuno - staining using antibodies against Flag tag to detect ATG16L1 . Scale bar : 9 μm .

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Immunofluorescence analyses of ATG16L1-/- and WIPI2-/- stably expressing Flag-S-ATG16L1. Cells were amino acid starved (AA starve) for 2 hr in the presence or absence of 3'MA (and additional pretreatment for 30 min) followed by fixation and immuno-staining using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.


Fragments depicted in ( A ) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S - ATG16L1 . Scale bar : 10 μm .

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Fragments depicted in (A) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S-ATG16L1. Scale bar: 10 μm.


U2OS cells expressing Flag - ATG16L1 ∆ 2 or Flag - ATG16L1 ∆ 2 ∆ 182 - 205 were treated as in ( B ) and stained using antibodies against Flag tag ( to detect ATG16L1 , green ) and FIP200 ( red ) . Scale bar : 10 μm .

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U2OS cells expressing Flag-ATG16L1∆2 or Flag-ATG16L1∆2∆182-205 were treated as in (B) and stained using antibodies against Flag tag (to detect ATG16L1, green) and FIP200 (red). Scale bar: 10 μm.


Protein - protein interaction assay in 293T cells transiently transfected with the indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies .

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Protein-protein interaction assay in 293T cells transiently transfected with the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


Homodimerisation assay in 293T cells transiently transfected with ATG16L1 - GFP and the indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies .

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Homodimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


Flag - ATG16L1WT or Flag - ATG16L1 ∆ 182 - 205 were stably expressed in ATG5 - / - cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1 . Scale bar : 9 μm .

Flag-ATG16L1WT or Flag-ATG16L1∆182-205 were stably expressed in ATG5-/- cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.


Microscopy - based protein - liposome binding assay . ATG16L1 - GFP immobilised on beads incubated in the presence of rhodamine - labelled liposome preparations containing the indicated phosphoinositides . Scale bar : 50 μm .

Microscopy-based protein-liposome binding assay. ATG16L1-GFP immobilised on beads incubated in the presence of rhodamine-labelled liposome preparations containing the indicated phosphoinositides. Scale bar: 50 μm.


Quantification of relative liposome binding


Microscopy - based protein - liposome binding assay as in ( B ) . ATG16L1WT - and ATG16L1LD - GFP immobilised on beads were incubated with rhodamine - labelled , PI3P - positive liposome preparations . Scale bar : 50 μm . Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values .

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

Microscopy-based protein-liposome binding assay as in (B). ATG16L1WT- and ATG16L1LD-GFP immobilised on beads were incubated with rhodamine-labelled, PI3P-positive liposome preparations. Scale bar: 50 μm. Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values.


Protein - protein interaction assay in 293T cells transiently transfected with the indicated S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies .

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

Protein-protein interaction assay in 293T cells transiently transfected with the indicated S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


by immunoblotting using the indicated antibodies . Dimerisation assay in 293T cells transiently transfected with ATG16L1 - GFP and the indicated S - tagged ATG16L1 constructs and

by immunoblotting using the indicated antibodies. Dimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated S-tagged ATG16L1 constructs and analysed