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 B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD 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 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 )

O O O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY 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. 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 B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 .

B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O

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 .

B-ORGANISM O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O

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 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY 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 .

B-CELL_TYPE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD

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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE

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

B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD

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

B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE

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 )

B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

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 % .

B-ORGANISM O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM 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 .

B-CELL_TYPE O O O O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY 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 .

B-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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-ORGANISM O B-SMALL_MOLECULE 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 .

O O O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

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 B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY 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 B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY 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 B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-EXP_ASSAY 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 .

O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD O O B-TISSUE O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O

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 . ) .

B-CELL_TYPE O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O 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-SMALL_MOLECULE O O O O O O O O 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 ) .

B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O

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 .

B-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

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

B-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY 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 .

B-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

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 ) .

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE O B-CELL_TYPE O O B-GENEPROD O O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O O O B-GENEPROD 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 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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 .

O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O 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 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 B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY 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

B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O

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 B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY 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 .

O B-CELL_TYPE O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

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 ) .

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE O B-CELL_TYPE O O B-GENEPROD O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD 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 .

B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY 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 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY 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 ) .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O 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 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 .

B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD 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 B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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 B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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 B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O 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 B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O 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 B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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 B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O O O O O O

(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 ) .

O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O

(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 ) .

O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-DISEASE O O O O O O O O B-DISEASE O O O O O O O B-TISSUE O O B-GENEPROD O O O O B-DISEASE O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O

(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 ) .

O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O

(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 .

O O O O B-GENEPROD O O O B-DISEASE O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O 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-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O

(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 .

O O O O O B-TISSUE O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O O 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-TISSUE O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O 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) 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 .

O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O B-CELL_LINE O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O

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 .

O O B-CELL_LINE O O O B-ORGANISM B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O

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 .

O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O

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 .

O O B-CELL_LINE O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O

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 .

O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O

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 .

O O B-CELL_LINE O O O 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-SUBCELLULAR O O O O O O O O B-CELL_LINE O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

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

O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O B-CELL_LINE O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR

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

O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR

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

O O B-CELL_LINE O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O O B-SUBCELLULAR

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 .

O O B-CELL_LINE O O O O O O O O O O O O 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-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O

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 .

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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 .

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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 .

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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 .

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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 .

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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

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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


ATG16L1 - / - stably expressing the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag ( green ) and ATG5 ( red ) . Scale bar : 9 μm . Right panels show quantifications of average number of Flag - and ATG5 - positive dots per cell .

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ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and ATG5 (red). Scale bar: 9 μm. Right panels show quantifications of average number of Flag- and ATG5-positive dots per cell.


ATG16L1 - / - stably expressing the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag ( to detect ATG16L1 , red ) and WIPI2 ( green ) . Scale bar : 9 μm . Lower panel shows quantification of average number of WIPI2 positive dots per cell .

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ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag (to detect ATG16L1, red) and WIPI2 (green). Scale bar: 9 μm. Lower panel shows quantification of average number of WIPI2 positive dots per cell.


Average intensities of individual Flag - ATG16L1 dots in ( A ) . Underlying grey circles represent individual data points . Dots were quantified ( n = 1225 for ATG16L1WT ; n = 334 for ATG16L1LD ) .

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Average intensities of individual Flag-ATG16L1 dots in (A). Underlying grey circles represent individual data points. Dots were quantified (n=1225 for ATG16L1WT; n=334 for ATG16L1LD).


WIPI2 - / - cells reconstituted with the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag ( green ) and FIP200 ( red ) . Scale bar : 9 μm . Lower panel shows quantification of average number of Flag positive dots per puncta - positive cell .

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WIPI2-/- cells reconstituted with the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and FIP200 (red). Scale bar: 9 μm. Lower panel shows quantification of average number of Flag positive dots per puncta-positive cell.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Cells were amino acid starved ( AA starved ) to induce mTORC1 - dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Cells were amino acid starved (AA starved) to induce mTORC1-dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Mitophagy was induced by CCCP ( 10 μM ) treatment for 6 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Mitophagy was induced by CCCP (10 μM) treatment for 6 hr prior to lysis.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Glucose starvation ( Glu starved ) for 20 hr was used to induce mTORC1 - independent autophagy . BafA1 was added to all conditions 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Glucose starvation (Glu starved) for 20 hr was used to induce mTORC1-independent autophagy. BafA1 was added to all conditions 2 hr prior to lysis.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . LC3 - associated phagocytosis ( LAP ) - like LC3 lipidation was induced by treating cells with CCCP ( 100 μM ) for 2 hr prior to lysis . Right panel shows quantification of LC3 - II / LC3 - I ratio .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LC3-associated phagocytosis (LAP)-like LC3 lipidation was induced by treating cells with CCCP (100 μM) for 2 hr prior to lysis. Right panel shows quantification of LC3-II/LC3-I ratio.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . LAP - like LC3 lipidation induced by monensin or ammonium chloride ( NH4Cl ) for 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LAP-like LC3 lipidation induced by monensin or ammonium chloride (NH4Cl) for 2 hr prior to lysis.


Coomassie gel of recombinant ATG16L172 - 307 wild type and mutants . * indicates bacterial

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Coomassie gel of recombinant ATG16L172-307 wild type and mutants. * indicates bacterial protein


Liposome co - sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes . Supernatant ( S ) and pellet ( P ) were analysed by immunoblotting against T7 tag to detect ATG16L1 . Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments ( right panel ) .

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Liposome co-sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes. Supernatant (S) and pellet (P) were analysed by immunoblotting against T7 tag to detect ATG16L1. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel).


Liposome co - sedimentation assay , as in ( C ) , using ATG16L172 - 307LE and sonicated liposomes that contain or lack PI3P . Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments ( right panel ) with error bars depicting SEM values .

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Liposome co-sedimentation assay, as in (C), using ATG16L172-307LE and sonicated liposomes that contain or lack PI3P. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel) with error bars depicting SEM values.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LE - expression constructs and autophagy assessed during amino acid starvation ( AA starve ) followed by immunoblotting or immunofluorescence analyses . Immunofluorescence analyses of amino acid starved cells ( 2 hr ) using antibodies against WIPI2 ( red ) or ATG16L1 ( green ) . Scale bar : 9 μm . Cells as in ( A ) with the addition of 3 ' MA ( and pretreatment with the drug for 30 min ) . Scale bar : 9 μm .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LE-expression constructs and autophagy assessed during amino acid starvation (AA starve) followed by immunoblotting or immunofluorescence analyses. Immunofluorescence analyses of amino acid starved cells (2 hr) using antibodies against WIPI2 (red) or ATG16L1 (green). Scale bar: 9 μm. Cells as in (A) with the addition of 3'MA (and pretreatment with the drug for 30 min). Scale bar: 9 μm.


Quantification of percentage of cells positive for ATG16L1 puncta


Cells were left untreated , amino acid starved for 2 hr , or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr . Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1 . Scale bar : 10 μm .

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Cells were left untreated, amino acid starved for 2 hr, or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr. Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1. Scale bar: 10 μm.


Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr . Cell lysates were analysed using the indicated antibodies .

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Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr. Cell lysates were analysed using the indicated antibodies.


WIPI2 - / - cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr . BafA1 treatment was included in all conditions for 2 hr prior to lysis . Cell lysates were analysed by immunoblotting using the indicated antibodies . Quantification of LC3 - II / LC3 - I ratio of three independent experiments is shown on the right panel .

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WIPI2-/- cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr. BafA1 treatment was included in all conditions for 2 hr prior to lysis. Cell lysates were analysed by immunoblotting using the indicated antibodies. Quantification of LC3-II/LC3-I ratio of three independent experiments is shown on the right panel.


Ferroptosis assay in ATG16L1 - / - cells stably expressing ATG16L1WT , ATG16L1LD or ATG16L1LE . Cells were cultured in amino acid free media ( AA starve ) in the presence of 10 % FBS or 10 % dialysed FBS ( diFBS ) for 24 hr . Quantification of percentage of PI - positive cells from at least three independent experiments is shown .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

Ferroptosis assay in ATG16L1-/- cells stably expressing ATG16L1WT, ATG16L1LD or ATG16L1LE. Cells were cultured in amino acid free media (AA starve) in the presence of 10% FBS or 10% dialysed FBS (diFBS) for 24 hr. Quantification of percentage of PI-positive cells from at least three independent experiments is shown.


( a ) Kaplan - Meier survival plot of cohorts of induced Vil Apc , Vil Apc Huwe1het and Vil Apc Huwe1hom mice . Deletion of Huwe1 led to a significant reduction in survival of these animals ( Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom , Log Rank , p < 0 . 001 , n ≥ 10 ) .

O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

(a) Kaplan-Meier survival plot of cohorts of induced Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom, Log Rank, p < 0.001, n ≥ 10).


( b ) Wholemount isolation of small intestines from Vil Apc and Vil Apc Huwe1hom mice culled at clinical endpoint . Note the huge numbers of tiny macroscopic adenomas visible in the Vil Apc Huwe1hom intestine ( red arrows ) .

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(b) Wholemount isolation of small intestines from Vil Apc and Vil Apc Huwe1hom mice culled at clinical endpoint. Note the huge numbers of tiny macroscopic adenomas visible in the Vil Apc Huwe1hom intestine (red arrows).


( c ) H & amp ; amp ; E staining of intestinal cross sections from Vil Apc and Vil Apc Huwe1hom mice demonstrating the increased adenoma burden following Huwe1 deletion . Black arrows indicate individual adenomas . Scale bars = 200 µm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O B-TISSUE O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O

(c) H&amp;E staining of intestinal cross sections from Vil Apc and Vil Apc Huwe1hom mice demonstrating the increased adenoma burden following Huwe1 deletion. Black arrows indicate individual adenomas. Scale bars = 200 µm.


( d ) Quantification of total tumour numbers per gut in sacrificed Vil Apc , Vil Apc Huwe1het and Vil Apc Huwe1hom mice . Deletion of Huwe1 led to a significant increase in the number of tumours per gut ( Mann Whitney , n ≥ 5 ) .

O O O O O O B-TISSUE O O B-TISSUE O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O

(d) Quantification of total tumour numbers per gut in sacrificed Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant increase in the number of tumours per gut (Mann Whitney, n ≥ 5).


( a ) H & amp ; amp ; E staining of control and Huwe1 deleted intestinal epithelium . Shortened villi in Huwe1 deficient tissue are indicated . Scale bars = 100 µm . ( b ) BrdU IHC of control and Huwe1 deleted intestinal epithelium . Scale bars = 100 µm . ( c ) PAS staining identifying Goblet cells . No gross changes were observed . Scale bars = 100 µm . ( d ) Lysozyme staining ( Paneth cell marker ) of control and Huwe1 deleted small intestine . Note the occurrence of lysozyme positive cells away from the crypt base ( black arrows ) . Scale bars = 100 µm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-TISSUE I-TISSUE O O B-TISSUE O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O

(a) H&amp;E staining of control and Huwe1 deleted intestinal epithelium. Shortened villi in Huwe1 deficient tissue are indicated. Scale bars = 100 µm. (b) BrdU IHC of control and Huwe1 deleted intestinal epithelium. Scale bars = 100 µm. (c) PAS staining identifying Goblet cells. No gross changes were observed. Scale bars = 100 µm. (d) Lysozyme staining (Paneth cell marker) of control and Huwe1 deleted small intestine. Note the occurrence of lysozyme positive cells away from the crypt base (black arrows). Scale bars = 100 µm.


( e ) Dual Periodic acid - Schiff / Alcian blue staining to identify Paneth cell secretory vesicles ( light blue / pink - marked with black arrows ) and goblet cells ( dark blue / purple - marked with red arrows ) . Note Paneth cell secretory vesicles are restricted to crypt base in both control and Huwe1 deficient small intestines ( inset - black arrows ) . Scale bars = 100µm .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O O O

(e) Dual Periodic acid-Schiff / Alcian blue staining to identify Paneth cell secretory vesicles (light blue / pink - marked with black arrows) and goblet cells (dark blue / purple - marked with red arrows). Note Paneth cell secretory vesicles are restricted to crypt base in both control and Huwe1 deficient small intestines (inset - black arrows). Scale bars = 100µm.


( f ) MMP7 staining of control and Huwe1 deleted small intestine . Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines . Scale bars = 100µm .

O O O B-GENEPROD O O O O B-GENEPROD O B-TISSUE I-TISSUE O O B-GENEPROD O O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O O O O O

(f) MMP7 staining of control and Huwe1 deleted small intestine. Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines. Scale bars = 100µm.


( a ) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines . Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-TISSUE O O O B-GENEPROD O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O

(a) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines. Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( b ) QRT - PCR analysis of Myc expression in control and Huwe1 deficient intestines .

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(b) QRT-PCR analysis of Myc expression in control and Huwe1 deficient intestines.