F BRCA2 - deficient ( - BRCA2 ) human DLD1 were treated with 2 µM pyridostatin ( PDS ) for 24 hours . Next , pyridostatin was removed were released in a medium containing 250 nM NU - 7441 ( NU ) . Whole - extracts were prepared at indicated timepoints after release immunoblotted as shown . SMC1 was used as a loading control . KAP1 phosphorylation site is indicated in red .

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

F BRCA2-deficient (-BRCA2) human DLD1 cells were treated with 2 µM pyridostatin (PDS) for 24 hours. Next, pyridostatin was removed and cells were released in a medium containing 250 nM NU-7441 (NU). Whole-cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown. SMC1 was used as a loading control. KAP1 phosphorylation site is indicated in red.


G Dose - dependent viability assays of BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 treated with pyridostatin ( PDS ) NU - 7441 at indicated concentrations for six days . Graphs represent average values obtained from of n = 3 independent experiments , each performed in technical triplicates .

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

G Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with pyridostatin (PDS) and NU-7441 at the indicated concentrations for six days. Graphs represent average values obtained from of n = 3 independent experiments, each performed in technical triplicates.


Quantitative RT - PCR of grown as in ( A ) treated with 10 µM pyridostatin ( PDS ) for 3 days was performed using primers specific for indicated genes . mRNA are expressed relative to GAPDH to - DOX ( + BRCA2 ) . Error bars represent of n = 3 independent experiments , each performed in technical triplicate . P values were calculated using an unpaired two - t - . * , P ≤ 0 . 05 ; * * * , P ≤ 0 . 001 .

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

Quantitative RT-PCR of cells grown as in (A) and treated with 10 µM pyridostatin (PDS) for 3 days was performed using primers specific for the indicated genes. mRNA levels are expressed relative to GAPDH and to -DOX (+BRCA2) cells. Error bars represent the SEM of n = 3 independent experiments, each performed in technical triplicate. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; ***, P ≤ 0.001.


BRCA2 + / + BRCA2 - / - RPE - 1 were treated with 10 μM pyridostatin ( PDS ) for 2 days . Whole - extracts were immunoblotted as indicated . KAP1 , IRF3 STAT1 phosphorylation sites are shown in red . Tubulin GAPDH were used as loading controls .

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

BRCA2+/+ and BRCA2-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. Tubulin and GAPDH were used as loading controls.


H1299 + shBRCA2DOX treated as in ( B ) were fixed prepared for immunofluorescence with antibody against cGAS . was counterstained with DAPI . Scale bar represents 20 µm . of cGAS - positive micronuclei per shown in ( E ) . Graph error bars represent mean of of n = 3 independent experiments . A minimum of 250 were analysed per condition per experiment . P values were calculated using an unpaired two - t - . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; NS , P > 0 . 5 .

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

H1299+shBRCA2DOX cells treated as in (B) were fixed and prepared for immunofluorescence with antibody against cGAS. DNA was counterstained with DAPI. Scale bar represents 20 µm. Quantification of cGAS-positive micronuclei per cells shown in (E). Graph and error bars represent the mean and SEM of of n = 3 independent experiments. A minimum of 250 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; **, P ≤ 0.01; NS, P > 0.5.


FVB mice were injected intramuscularly with ( A ) BRCA1 - proficient KP3 . 33 ( Brca1 + / + ) or ( B ) BRCA1 / 53BP1 - deficient KB1PM5 ( Brca1 - / - , Tp53bp1 - / - ) mouse mammary tumour . Pyridostatin ( PDS ) was administered intravenously ( i . v . ; 7 . 5 mg / kg / day ) talazoparib was administered orally ( p . o . ; 0 . 33 mg / kg / day ) , over indicated periods of time . Vertical dotted indicates end of treatment . Tumour volume was measured at timepoints shown on graph expressed relative to tumour volume at beginning of treatment . Each experimental group included n = 5 mice . Error bars represent . P values were calculated between treated untreated tumours at day 16 , using an unpaired two - t - . * * * * , P ≤ 0 . 0001 ; NS , P > 0 . 5 .

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

FVB female mice were injected intramuscularly with (A) BRCA1-proficient KP3.33 (Brca1+/+) or (B) BRCA1/53BP1-deficient KB1PM5 (Brca1-/-, Tp53bp1-/-) mouse mammary tumour cells. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) and talazoparib was administered orally (p.o.; 0.33 mg/kg/day), over the indicated periods of time. Vertical dotted line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 5 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 16, using an unpaired two-tailed t-test. ****, P ≤ 0.0001; NS, P > 0.5.


BRCA1 + / + , BRCA1 - / - BRCA1 - / - / TP53BP1 - / - RPE - 1 were treated with 10 μM pyridostatin ( PDS ) or 2 μM olaparib for 2 days . Whole - extracts were immunoblotted as indicated . KAP1 , IRF3 STAT1 phosphorylation sites are shown in red . SMC1 GAPDH were used as loading controls .

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

BRCA1+/+, BRCA1-/- and BRCA1-/-/TP53BP1-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) or 2 μM olaparib for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. SMC1 and GAPDH were used as loading controls.


B VHIO179 PDTXs were grafted into CB17 - SCID mice . Pyridostatin was administered intravenously ( 7 . 5 mg / kg / day ) over indicated periods of time . Vertical dotted indicates end of treatment . Tumour volume was measured at timepoints shown on graph expressed relative to tumour volume at beginning of treatment . Each experimental group included n = 7 mice . Error bars represent . P values were calculated between treated untreated tumours at day 24 , using an unpaired two - t - . * * * * , P ≤ 0 . 0001 .

O O O O O O O O B-DISEASE 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 O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 O O O O O O O O

B VHIO179 PDTXs were grafted into CB17-SCID female mice. Pyridostatin was administered intravenously (7.5 mg/kg/day) over the indicated periods of time. Vertical dotted line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 7 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 24, using an unpaired two-tailed t-test. ****, P ≤ 0.0001.


Kaplan - Meier survival curve for mice treated as in ( C ) , with each treatment group containing n = 5 mice . Statistical significance was assessed by Log - rank ( * * * * P < 0 . 0001 ) .

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

Kaplan-Meier survival curve for mice treated as in (C), with each treatment group containing n = 5 mice. Statistical significance was assessed by Log-rank test (****P <0.0001).


Kaplan - Meier survival curve for mice treated as in ( C ) , with each treatment group containing n = 6 mice . Statistical significance was assessed by Log - rank ( * * * * P < 0 . 0001 ) .

Kaplan-Meier survival curve for mice treated as in (C), with each treatment group containing n = 6 mice. Statistical significance was assessed by Log-rank test (****P <0.0001).


BN - PAGE of 20 µg solubilized BHM with several of distinct complexes annotated . dashed box indicates monomeric ATP synthase ( V ) band subjected to IGX - MS .

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

BN-PAGE of 20 µg solubilized BHM with several of the distinct complexes annotated. The dashed box indicates the monomeric ATP synthase (complex V) band subjected to IGX-MS.


( a ) Manually segmented rosettes on 3D reconstructions from Z - stacks of 25 μm of an E5 . 75 imaged using lightsheet microscopy . AVE are in green , membranes are labelled in grey . Left : anterior view ; Middle : lateral view ( anterior to left ) ; Right : posterior view . Scale bars : 20 μm . ( a ' ) Mouse strategy used to perform live imaging . ( a " ) Left : Number of rosettes normalized by total number of in epiblast region in focus expressed in . Right : Frequency of rosettes , normalized by in mm2 of epiblast region in focus time of observation in minutes . Data information : Mean ± , n = 41 frames from 3 . ( b , c ) Rosettes outlines on optical slices from E5 . 75 ( b ) E6 . 25 ( c ) imaged by confocal microscopy . AVE are in green , membranes are labelled in grey . Left : anterior view ; Middle : lateral view ( anterior to left ) ; Right : posterior view . Scale bars : 20 μm . ( b ' ) Mouse lines strategy used to perform live imaging . ( b ' ' , c ' ) Left : Number of rosettes normalized by total number of expressed in . Right : Density of rosettes , normalized by in mm2 of epiblast region in focus . posterior region includes PS region , as it could not be precisely discriminated . Data information : Mean ± . E5 . 75 : n anterior = 19 frames from 3 , n lateral = 23 frames from 5 , n posterior = 20 frames from 3 . E6 . 25 : n anterior = 16 frames from 2 , n lateral = 12 frames from 1 n posterior = 16 frames from 2 .

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 B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O O O O 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 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 B-TISSUE 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 O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O O O O 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 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 B-EXP_ASSAY O O O O O O O O B-TISSUE 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 O O O O O 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) Manually segmented rosettes on 3D reconstructions from Z-stacks of 25 μm of an E5.75 embryo imaged using lightsheet microscopy. AVE cells are in green, and all cell membranes are labelled in grey. Left: anterior view; Middle: lateral view (anterior to the left); Right: posterior view. Scale bars: 20 μm. (a') Mouse line strategy used to perform live imaging. (a") Left: Number of rosettes normalized by the total number of cells in the epiblast region in focus expressed in percentage. Right: Frequency of rosettes, normalized by the area in mm2 of the epiblast region in focus and the time of observation in minutes. Data information: Mean ± SEM, n=41 frames from 3 embryos. (b, c) Rosettes outlines on optical slices from E5.75 (b) and E6.25 (c) embryos imaged by confocal microscopy. AVE cells are in green, and all cell membranes are labelled in grey. Left: anterior view; Middle: lateral view (anterior to the left); Right: posterior view. Scale bars: 20 μm. (b') Mouse lines strategy used to perform live imaging. (b'', c') Left: Number of rosettes normalized by the total number of cells expressed in percentage. Right: Density of rosettes, normalized by the area in mm2 of the epiblast region in focus. The posterior region includes the PS region, as it could not be precisely discriminated. Data information: Mean ± SEM. E5.75: n anterior=19 frames from 3 embryos, n lateral=23 frames from 5 embryos, n posterior=20 frames from 3 embryos. E6.25: n anterior=16 frames from 2 embryos, n lateral=12 frames from 1 embryo and n posterior=16 frames from 2 embryos.


( a ) Z - projection of an E7 . 25 mosaically labelled through OH - tamoxifen injection , imaged by two - photon microscopy . Scale bar : 50 μm . Data information : n = 54 . ( a ' ) Mouse strategy used to produce ubiquitous for tdTomato ( grey ) mosaic for GFP ( mGFP , green ) in epiblast . ( b ) Z - projections of stills from live imaging recording of a bottle - shaped ( indicated by a arrow ) that delaminates through basal at time 0min , 25 min , 50 min , 1h15min 1h40min . ( b ' ) Top : Bottle shape events : GFP + bottle shape over total GFP + , expressed in , in anterior versus posterior region of epiblast . posterior region includes PS region , as it could not be precisely discriminated . of bottle shape exiting epiblast among total number of GFP + bottle shape , in anterior versus posterior epiblast . dotted lines outline basal ( top ) apical ( bottom ) borders of epiblast .

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

(a) Z-projection of an E7.25 embryo mosaically labelled through OH-tamoxifen injection, imaged by two-photon microscopy. Scale bar: 50 μm. Data information: n embryos=54. (a') Mouse line strategy used to produce embryos ubiquitous for membrane tdTomato (grey) and mosaic for membrane GFP (mGFP, green) in the epiblast. (b) Z-projections of stills from live imaging recording of a bottle-shaped cell (indicated by a white arrow) that delaminates through the basal membrane at time 0min, 25 min, 50 min, 1h15min and 1h40min. (b') Top: Bottle shape events: GFP+ bottle shape cells over total GFP+ cells, expressed in percentage, in anterior versus posterior region of the epiblast. The posterior region includes the PS region, as it could not be precisely discriminated. Percentage of bottle shape cells exiting the epiblast among total number of GFP+ bottle shape cells, in anterior versus posterior epiblast. white dotted lines outline basal (top) and apical (bottom) borders of the epiblast.


( c ) Z projection ( c ) 3D reconstruction ( c ' ) of stills from live imaging recording showing an apical division without basal contact , followed by daughter dispersion at 0min , 25 min , 50 min , 1h15min 1h40min . dotted lines outline basal ( top ) apical ( bottom ) borders of epiblast . ( c " ) Left : Frequency of apical division ( GFP + apical division over total GFP + , expressed in ) in anterior posterior epiblast . Middle : Apical division with or without persistence of contact with epiblast basal pole , normalized by total number of GFP + apical division , in anterior versus posterior epiblast . Right : dispersion ( defined as loss of basolateral contact between daughter after reinsertion in epiblast layer ) after apical division in anterior posterior epiblast .

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

(c) Z projection (c) and 3D reconstruction (c') of stills from live imaging recording showing an apical division without basal contact, followed by daughter cells dispersion at 0min, 25 min, 50 min, 1h15min and 1h40min. white dotted lines outline basal (top) and apical (bottom) borders of the epiblast. (c") Left: Frequency of apical division (GFP+ apical division over total GFP+ cells, expressed in percentage) in anterior and posterior epiblast. Middle: Apical division with or without persistence of contact with the epiblast basal pole, normalized by the total number of GFP+ apical division, in anterior versus posterior epiblast. Right: Cell dispersion (defined as loss of basolateral contact between daughter cells after reinsertion in the epiblast layer) after apical division in anterior and posterior epiblast.


( a ) Z - projection ( a ) 3D reconstruction ( a ' ) of stills from two - photon live imaging of an E7 . 25 mosaically labelled for epiblast showing a division giving rise to an epiblast a mesoderm that will exit from epiblast layer . dotted lines outline basal ( top ) apical ( bottom ) borders of epiblast at 0min , 25 min , 50min , 1h15min 1h40min . ( b ) ( c ) Z projection of stills from two - photon live imaging of an E7 . 25 mosaically labelled for epiblast . Non - apical division can be observed in posterior epiblast only , without ( b ) or with ( c ) attachment to apical side . Stills were taken at 0min , 25 min , 50min , 1h15min 1h40min for ( b ) 50min , 1h15min , 1h40min , 2h05min 2h30min for ( c ) . dotted lines outline basal ( top ) apical ( bottom ) borders of epiblast .

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

(a) Z-projection (a) and 3D cell reconstruction (a') of stills from two-photon live imaging of an E7.25 embryo mosaically labelled for epiblast cells showing a division giving rise to an epiblast and a mesoderm cell that will exit from the epiblast layer. White dotted lines outline basal (top) and apical (bottom) borders of the epiblast at 0min, 25 min, 50min, 1h15min and 1h40min. (b) (c) Z projection of stills from two-photon live imaging of an E7.25 embryo mosaically labelled for epiblast cells. Non-apical division can be observed in the posterior epiblast only, without (b) or with (c) attachment to the apical side. Stills were taken at 0min, 25 min, 50min, 1h15min and 1h40min for (b) and 50min, 1h15min, 1h40min, 2h05min and 2h30min for (c). White dotted lines outline basal (top) and apical (bottom) borders of the epiblast.


( d ) Mitotic Index ( GFP + dividing over total number of GFP + ) in anterior posterior region , as well as among exiting epiblast at PS . posterior region includes PS region , as it could not be precisely discriminated .

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

(d) Mitotic Index (GFP+ dividing cells over total number of GFP+ cells) in the anterior and posterior region, as well as among cells exiting the epiblast at the PS. The posterior region includes the PS region, as it could not be precisely discriminated.


( e ) of non - apical rounding ( non - apical GFP + rounding divided by total GFP + rounding ) expressed in for anterior posterior regions .

O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O

(e) Ratio of non-apical rounding (non-apical GFP+ rounding divided by the total GFP+ rounding) and expressed in percentage for the anterior and posterior regions.


( f ) of non - apical division ( non - apical GFP + division over total GFP + division ) expressed in for anterior posterior region of .

O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O

(f) Ratio of non-apical division (non-apical GFP+ division over total GFP+ division) and expressed in percentage for the anterior and posterior region of the embryo.


( g ) of posterior division outcome for apical non - apical divisions . Outcomes are 2 epiblast daughter ( epi / epi ) , one epiblast one mesoderm daughter ( epi / meso ) or 2 mesoderm daughter ( meso / meso ) .

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

(g) Quantification of posterior cell division outcome for apical and non-apical divisions. Outcomes are 2 epiblast daughter cells (epi/epi), one epiblast and one mesoderm daughter cells (epi/meso) or 2 mesoderm daughter cells (meso/meso).


( c ) Z - projections of transverse sections from E6 . 5 ( top ) , E6 . 75 ( mid ) E7 ( bottom ) . Samples were stained for F - actin ( Phalloidin , grey ) mitosis ( Phh3 , magenta ) on left nuclei ( DAPI , cyan ) basement ( collagen IV , yellow ) on right . Yellow arrowheads show non - apical mitosis . arrows delimitate borders of basement degradation region . A : anterior , P : posterior .

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

(c) Z-projections of transverse sections from E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. Samples were stained for F-actin (Phalloidin, grey) and mitosis (Phh3, magenta) on the left and nuclei (DAPI, cyan) and basement membrane (collagen IV, yellow) on the right. Yellow arrowheads show non-apical mitosis. White arrows delimitate borders of the basement membrane degradation region. A: anterior, P: posterior.


( d ) Mitotic Index ( Phh3 / DAPI ) in at anterior , posterior , PS regions of E6 . 5 ( top ) , E6 . 75 ( mid ) E7 ( bottom ) . posterior region excludes from PS region .

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

(d) Mitotic Index (Phh3/DAPI) in percentage at the anterior, posterior, and PS regions of E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. The posterior region quantification excludes counts from the PS region.


( e ) of non - apical mitosis ( non - apical Phh3 / Phh3 , normalized by number of nuclei ) in at anterior , posterior , PS regions of E6 . 5 ( top ) , E6 . 75 ( mid ) E7 ( bottom ) .

O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR 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

(e) Ratio of non-apical mitosis (non-apical Phh3/ Phh3, normalized by number of nuclei) in percentage at the anterior, posterior, and PS regions of E6.5 (top), E6.75 (mid) and E7 (bottom) embryos.


( f ) Violin plots representing distribution of in G2 / M phase in with high , compared to low , Brachyury expression , among annotated as primitive streak mesoderm harvested from E6 . 5 , E7 , E7 . 25 . Violin plots are overlaid with box plots marking 25th percentile , median 75th percentile .

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

(f) Violin plots representing the distribution of cells in G2/M phase in cells with high, compared to low, Brachyury expression, among cells annotated as primitive streak and mesoderm harvested from E6.5, E7, and E7.25 embryos. Violin plots are overlaid with white box plots marking 25th percentile, median and 75th percentile.


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O O O O B-EXP_ASSAY O O O O B-DISEASE O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O O B-TISSUE O O O O B-GENEPROD O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

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


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

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

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


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

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

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


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

O O O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


E . Organoid growth efficiency 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 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 were stimulated with ( 50 000 particles / ) AdV - GFP 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 B-ORGANISM O B-GENEPROD 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.


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

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


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

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 B-GENEPROD I-GENEPROD I-GENEPROD 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 + / - antibody . Cytokines in 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 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 ( with antibody complexed ) as indicated . A single experiment representative of 3 independent experiments is shown .

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


THP - 1s were stimulated with a constant dose of AdV - GFP ( 50 000 pp / ) , 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 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 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

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 / ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h 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 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 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 / ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h 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 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 B-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 , stimulated with AdV ( 50 000 pp / ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h was measured by LDH

B-CELL_TYPE 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 O O O 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 , stimulated with AdV ( 50 000 pp / ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h viability measured by PrestoBlue

B-CELL_TYPE 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 O O O B-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 / ) complexed with antibody at indicated doses for 2h 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 B-EXP_ASSAY 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 of 1 : 200 50 000 pp / of this mix was complexed with h9C12 IVIg indicated doses . HMDM were stimulated with these complexes for 16h infection measured as mCherry by flow cytometry . In alone of mCherry positive was ~ 20 % , relative infection with antibodies was normalised to 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

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


HMDM were stimulated for 3 h with AdV - Ab complexes as in ( A , B ) or with LeuLeu at 10μM , lysosomal damage measured by loss of acridine orange red 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 B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 ) was assessed by an increase in e780 . mean of 3 independent donors is shown .

B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O

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


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

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 B-ORGANISM O B-SMALL_MOLECULE 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 / ) + / - 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 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 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 I-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 / ) 20 μg / ml h9C12 or 20 mg / ml IVIg or with 200 ng / well HT - or 10 μM Nigericin for 16h , IL - 1β measured in 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 B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 / ) 20 μg / ml h9C12 or 20 mg / ml IVIg or with 200 ng / well HT - or 10 μM Nigericin for 16h , TNF measured in 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 B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD 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 next day injected i . v . with 2 . 5 x 1011 pp AdV - GFP . 4h later spleens were harvested 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 B-ORGANISM O B-GENEPROD I-GENEPROD O O B-TISSUE 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 / ) in presence of h9C12 antibodies at indicated doses . Infection was measured after 24h by flow cytometry . Infection relative to AdV alone is shown graphed on left ( n = 4 mean ± s . e . m ) . Representative plots a comparison of different h9C12 mutants at 0 . 2 ug / ml are also shown in graph on 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 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 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

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 / ) antibody ( h9C12 ; 20 ug / ml , IVIg : 20 mg / ml ) or 10 μg / ml pI : C for 3h 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 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 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 pro - IL - 1β in 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 B-GENEPROD I-GENEPROD I-GENEPROD 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 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 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 ) NLRP3 in cytosol measured by western blot . Blot is representative of two independent donors .

B-CELL_TYPE O O O O O O O B-GENEPROD 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 from PBMCs , CD19 + ve B , CD14 + ve monocytes or HMDM derived from CD14 + ve monocytes were assessed by qPCR copy number was determined relative to actin copy number ( n = 5 mean ± s . e . m ) .

B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O B-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 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 antibody or 200 ng / well HT - or 10 μM Nigericin , in presence of pan - caspase inhibitor zVAD - fmk . A representative image ( scale bar 100 μm ) of number of with ASC specks from three independent experiments ( mean ± s . e . m , * P - value ≤ 0 . 05 , paired , two - t - ) is shown .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD 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 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 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

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 / ) 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 supernatants . Data shows average ± s . d . of two independent donors 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 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

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 ) IL - 1β in 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 B-GENEPROD I-GENEPROD I-GENEPROD 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 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

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 TNF were measured in 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 I-GENEPROD 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 20 μg / ml h9C12 or 10 μM Nigericin for 3h , 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 B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 from PBMCs , CD19 + ve B , CD14 + ve Monocytes or HMDM derived from CD14 + ve monocytes . were assessed by qPCR copy number was determined relative to Actin copy number ( n = 5 mean ± s . e . m ) .

B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O B-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 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 / ) 20 μg / ml h9C12 for 1h , washed 2x with SFM , then whole lysates harvested after a further 5h . Viral hexon transgene ( GFP ) expression in 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 B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O B-GENEPROD 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 or targeting cGAS STING were generated stimulated with 1000 U / ml IFNα for 4 h assessed by western blot .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O 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 STING were stimulated with AdV ( 50 000 pp / ) 20 μg / ml h9C12 , 200 ng / well HT - , 10 μM Nigericin or 10 ng / ml LPS for 16 h . Data shows combined data ( mean ± s . e . m ) of 3 experiments with absolute values ( upper panel ) or as % cytokine output of KO relative to Ctrl treated ( lower panel ) , * P - value ≤ 0 . 05 , * * * P - value ≤ 0 . 001 unpaired , two - t - ) .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD 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 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 O O O O O O O O O O O O O O O O O O O O O O O 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 relative to media treated ( * P - value ≤ 0 . 05 , * * * P - value ≤ 0 . 001 unpaired , two - t - ) .

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

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 / ) + 20 μg / ml h9C12 or 20 mg / ml IVIg or 200 ng / well HT - for 8h . A representative image ( scale bar 50 μm ) of number of 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 B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY 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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A ) ( B , C ) composition . Compounds were administered by daily subcutaneous injections . Data represent means ± ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O B-ORGANISM I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A , B ) energy expenditure . Compounds were administered by daily subcutaneous injections . Data represent means ± ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O B-ORGANISM I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( C ) respiratory exchange ( RER ) . Compounds were administered by daily subcutaneous injections . Data represent means ± ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O B-ORGANISM I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( D ) locomotor activity . Compounds were administered by daily subcutaneous injections . Data represent means ± ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O B-ORGANISM I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( E ) cumulative food intake , ( F ) meal number ( G ) meal . Compounds were administered by daily subcutaneous injections . Data represent means ± ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O B-ORGANISM I-ORGANISM 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 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 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-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 ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Compounds were administered by daily subcutaneous injections , last injection was provided 2 h prior to tissues sampling . Data represent means ± ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , § P < 0 , 001 , comparison between treatment 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 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 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 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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( A ) Fasted blood glucose . Data represent means ± ; 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 I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( B , C ) glucose tolerance . Data represent means ± ; 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 I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( D ) fasted insulin , . Data represent means ± ; 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 I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( E , F ) glucose - induced insulin secretion . Data represent means ± ; 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 I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( G ) HOMA - IR . Data represent means ± ; 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 I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( H ) insulin sensitivity . Data represent means ± ; 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 I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) 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 ± ; 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 I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( A ) Plasma lipoprotein fractions . Data represent means ± ; 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 I-ORGANISM 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 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

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 mice with vehicle ( ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( B ) liver cholesterol . Data represent means ± ; 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 I-ORGANISM 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 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

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.