e , HIV - 1 p24 antigen release in primary human MDMs infected with HIV - 1 24 h after initiation of daily peptide treatment . Values represent mean ± s . e . m . of triplicate samples . Similar results were observed in MDMs from three independent donors .

O O B-ORGANISM I-ORGANISM I-ORGANISM B-SUBCELLULAR O O O O O B-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O

e, HIV-1 p24 antigen release in primary human MDMs infected with HIV-1 24 h after initiation of daily peptide treatment. Values represent mean ± s.e.m. of triplicate samples. Similar results were observed in MDMs from three independent donors.


f , HIV - 1p24 antigen release in MDMs transduced with nonspecific scrambled shRNA ( shNS ) or ATG5 shRNA ( shATG5 ) and treated daily with peptide ( 5 µM ) . Values represent mean ± s . e . m . of triplicate wells . Similar results were observed in MDMs from two independent donors .

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

f, HIV-1p24 antigen release in MDMs transduced with nonspecific scrambled shRNA (shNS) or ATG5 shRNA (shATG5) and treated daily with peptide (5 µM). Values represent mean ± s.e.m. of triplicate wells. Similar results were observed in MDMs from two independent donors.


g , LC3 immunoblot of MDMs transduced with the indicated shRNA at day 0 and day 10 after HIV - 1 infection . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; t - test .

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

g, LC3 immunoblot of MDMs transduced with the indicated shRNA at day 0 and day 10 after HIV-1 infection. *P  0.05; **P  0.01; ***P  0.001; t-test.


a , GFP - LC3 - positive dots in tissues of 6 - week - old GFP - LC3 mice treated with the indicated peptide ( 20 mg kg − 1 i . p . , 6 h ) . A minimum of ten fields was counted per tissue section . Bars represent mean ± s . e . m . for three mice . Similar results were observed in three independent experiments .

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

a, GFP-LC3-positive dots in tissues of 6-week-old GFP-LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h). A minimum of ten fields was counted per tissue section. Bars represent mean ± s.e.m. for three mice. Similar results were observed in three independent experiments.


b , p62 immunoblot of brains of 5 - day - old GFP - LC3 mice treated with the indicated peptide ( 20 mg kg − 1 i . p . , 6 h ) .

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

b, p62 immunoblot of brains of 5-day-old GFP-LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h).


c , Survival curves of 5 - day - old C57BL / 6J mice infected with CHIKV ( 106 p . f . u . s . c . ) and treated daily with peptide ( 15 mg kg − 1 i . p . beginning 1 day post - infection ) .

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

c, Survival curves of 5-day-old C57BL/6J mice infected with CHIKV (106 p.f.u. s.c.) and treated daily with peptide (15 mg kg−1 i.p. beginning 1 day post-infection).


d - f , Representative images of WNV envelope antigen and TdT - mediated dUTP nick end labelling ( TUNEL ) staining ( d ) ( T , Tat alone ) , quantification of cell death in brain ( e ) and survival curves ( f ) for 5 - day - oldC57BL / 6Jmice infected with WNV ( Egypt strain 101 , 1 p . f . u . intracerebral ( i . c . ) ) and treated daily with peptide ( D - amino acid forms , 20mgkg − 1 i . p . beginning 1day post - infection ) . Images in d are from cerebral cortex day 6 post - infection . Similar results were observed in all regions of the brain for three mice per group . Scale bar , 20μm . Bars in e represent mean ± s . e . m . TUNEL - positive cells per unit area of brain for three mice .

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

d-f, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e) and survival curves (f) for 5-day-oldC57BL/6Jmice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (D-amino acid forms, 20mgkg−1 i.p. beginning 1day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20μm. Bars in e represent mean±s.e.m. TUNEL-positive cells per unit area of brain for three mice. g


d - f , Representative images of WNV envelope antigen and TdT - mediated dUTP nick end labelling ( TUNEL ) staining ( d ) ( T , Tat alone ) , quantification of cell death in brain ( e ) , and survival curves ( f ) for 5 - day - oldC57BL / 6Jmice infected with WNV ( Egypt strain 101 , 1 p . f . u . intracerebral ( i . c . ) ) and treated daily with peptide ( D - amino acid forms , 20mgkg − 1 i . p . beginning 1day post - infection ) . Images in d are from cerebral cortex day 6 post - infection . Similar results were observed in all regions of the brain for three mice per group . Scale bar , 20μm . Bars in e represent mean ± s . e . m . TUNEL - positive cells per unit area of brain for three mice .

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

d-f, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e), and survival curves (f) for 5-day-oldC57BL/6Jmice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (D-amino acid forms, 20mgkg−1 i.p. beginning 1day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20μm. Bars in e represent mean±s.e.m. TUNEL-positive cells per unit area of brain for three mice. g


g , Geometric mean + s . e . m . viral titres of WNV - infected mousebrains day 6 post - infection . Values represent combined data for 12 - 20 mice per treatment group from 10 to 12 litters . Data in c and f represent combined survival probabilities for three and four independent litters , respectively , in each group . Similar results were observed in each independent experiment . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; NS , not significant ; t - test .

O O O O O O O O O O O O O O B-ORGANISM O O B-ORGANISM 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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, Geometric mean + s.e.m. viral titres of WNV-infected mousebrains day 6 post-infection. Values represent combined data for 12-20 mice per treatment group from 10 to 12 litters. Data in c and f represent combined survival probabilities for three and four independent litters, respectively, in each group. Similar results were observed in each independent experiment. *P 0.05; **P 0.01; ***P 0.001; NS, not significant; t-test.


A - B Clearance of a target with a consensus PAM by direct interference . Overlay of fluorescent and phase contrast time - lapse images . Presence of the target plasmid is tracked by its YFP production ( A ) . Reconstructed lineage traces of the imaged population ( A ) from induction of the CRISPR - Cas system over time ( grey ) lineages show some variation in plasmid clearance times ( coloured ) ( B ) .

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

A-B Clearance of a target with a consensus PAM by direct interference. Overlay of fluorescent and phase contrast time-lapse images. Presence of the target plasmid is tracked by its YFP production (A). Reconstructed lineage traces of the imaged population (A) from induction of the CRISPR-Cas system over time (grey) lineages show some variation in plasmid clearance times (coloured) (B).


E - F Clearance of a matching target with a mutated PAM via priming . Overlay of fluorescent and phase contrast time - lapse images as in ( A ) . Reconstructed lineage traces of the imaged population ( E ) ( grey ) . Lineages show large variations in the time taken to clear the plasmid ( coloured ) ( F ) .

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

E-F Clearance of a matching target with a mutated PAM via priming. Overlay of fluorescent and phase contrast time-lapse images as in (A). Reconstructed lineage traces of the imaged population (E) (grey). Lineages show large variations in the time taken to clear the plasmid (coloured) (F).


( C ) Timelapse of ATP sensor FRET efficiency across the wing pouch after addition of 10 μM antimycin A ( ant . A ) .

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

(C) Timelapse of ATP sensor FRET efficiency across the wing pouch after addition of 10 μM antimycin A (ant.A).


( D , F ) Mean FRET efficiency measured over time in the ( D ) AP boundary ( APB ) or ( F ) DV boundary ( DVB ) and non - organizer regions ( NO ) . Shaded regions indicate standard deviation ( SD ) ; dots indicate mean per timepoint , and solid lines indicate the fit to the mean . Black arrows indicate the addition of the drug .

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

(D, F) Mean FRET efficiency measured over time in the (D) AP boundary (APB) or (F) DV boundary (DVB) and non-organizer regions (NO). Shaded regions indicate standard deviation (SD); dots indicate mean per timepoint, and solid lines indicate the fit to the mean. Black arrows indicate the addition of the drug.


( A ) Timelapse of ATP sensor FRET efficiency after combined addition of 10 μM antimycin A ( ant . A ) , 100 μM 3 - Bromopyruvate ( 3BP ) , and 50 mM 2 - deoxy - D - glucose ( 2DG ) . ( B ) Mean FRET efficiency of organizer ( ORG ) regions and non - organizer ( NO ) regions analyzed over time . Shaded regions indicate SD ; dots indicate mean per timepoint , and solid lines indicate the fit to the mean . ( C ) Fit parameters of individual time traces for organizer and non - organizer regions . Each dot represents data from one disc , and lines connect the corresponding regions of the same disc . Box plots summarize the data : boxes encompass the 2nd - 3rd quartiles , with whiskers indicating the 1st and 4th quartiles and the red line indicating the median . * = p - value < 0 . 05 , ns = not significant p - value using a Kruskal - Wallis test ( n = 26 discs ) .

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

(A) Timelapse of ATP sensor FRET efficiency after combined addition of 10 μM antimycin A (ant.A), 100 μM 3-Bromopyruvate (3BP), and 50 mM 2-deoxy-D-glucose (2DG). (B) Mean FRET efficiency of organizer (ORG) regions and non-organizer (NO) regions analyzed over time. Shaded regions indicate SD; dots indicate mean per timepoint, and solid lines indicate the fit to the mean. (C) Fit parameters of individual time traces for organizer and non-organizer regions. Each dot represents data from one disc, and lines connect the corresponding regions of the same disc. Box plots summarize the data: boxes encompass the 2nd-3rd quartiles, with whiskers indicating the 1st and 4th quartiles and the red line indicating the median. * = p-value < 0.05, ns = not significant p-value using a Kruskal-Wallis test (n=26 discs).


( D ) Time lapse montage of ATP sensor FRET efficiency in the wing pouch after 2DG addition . ( E ) Mean FRET efficiency time trace of the entire wing pouch ; shaded region indicates SD ; black arrows indicate the addition of the drugs . Brackets indicate the mean FRET values before and 2h after 2DG addition ( 1 - 10 min and 121 - 130 min , respectively ) . The distribution of mean values within these brackets are shown as violin plots in ( F ) ( solid line indicates median , dotted indicate quartiles ) . These were compared using an unpaired t - test ( ns = not significant p - value , n = 17 discs ) including ( G ) Welch ' s correction and estimation plot ( n = 17 discs for each group ) . In ( G ) , horizontal dotted lines run through the mean values for the two samples ( before 2DG and 2hr after 2DG ) ; on the right , the difference between the two means is plotted on a separate axis ( right ) , where the bars indicate the upper and lower 95 % confidence intervals .

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

(D) Time lapse montage of ATP sensor FRET efficiency in the wing pouch after 2DG addition. (E) Mean FRET efficiency time trace of the entire wing pouch; shaded region indicates SD; black arrows indicate the addition of the drugs. Brackets indicate the mean FRET values before and 2h after 2DG addition (1-10 min and 121-130 min, respectively). The distribution of mean values within these brackets are shown as violin plots in (F) (solid line indicates median, dotted indicate quartiles). These were compared using an unpaired t-test (ns = not significant p-value, n = 17 discs) including (G) Welch's correction and estimation plot (n=17 discs for each group). In (G), horizontal dotted lines run through the mean values for the two samples (before 2DG and 2hr after 2DG); on the right, the difference between the two means is plotted on a separate axis (right), where the bars indicate the upper and lower 95% confidence intervals.


( C ) Timelapse of ATP sensor FRET efficiency in the apGalts > PtcRNAi wing pouch after 10 μM antimycin A ( ant . A ) addition . Red dashed line indicates the position of the AP boundary .

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

(C) Timelapse of ATP sensor FRET efficiency in the apGalts>PtcRNAi wing pouch after 10 μM antimycin A (ant.A) addition. Red dashed line indicates the position of the AP boundary.


( D , F ) Mean FRET efficiency analyzed over time in the ( D ) dorsal anterior ( DA ) and ventral anterior ( VA ) sub - compartments or the ( F ) dorsal anterior ( DA ) and dorsal posterior ( DP ) sub - compartments of apGalts > PtcRNAi wing discs . Shaded regions indicate SD ; dots represent the mean per timepoint ; solid lines illustrate a fit of the mean data .

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 B-GENEPROD O B-GENEPROD B-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

(D, F) Mean FRET efficiency analyzed over time in the (D) dorsal anterior (DA) and ventral anterior (VA) sub-compartments or the (F) dorsal anterior (DA) and dorsal posterior (DP) sub-compartments of apGalts>PtcRNAi wing discs. Shaded regions indicate SD; dots represent the mean per timepoint; solid lines illustrate a fit of the mean data.


( I ) Timelapse of ATP sensor FRET efficiency in apGalts > CiDN wing discs after 10 μM antimycin A addition .

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

(I) Timelapse of ATP sensor FRET efficiency in apGalts > CiDN wing discs after 10 μM antimycin A addition.


( J ) Mean FRET efficiency measured over time in the dorsal and ventral compartments in apGalts > CiDN wings . Shaded regions indicate SD ; dots represent the average per timepoint ; solid lines illustrate a fit of the mean data .

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

(J) Mean FRET efficiency measured over time in the dorsal and ventral compartments in apGalts > CiDN wings. Shaded regions indicate SD; dots represent the average per timepoint; solid lines illustrate a fit of the mean data.


( A ) Timelapse montage of ATP sensor FRET efficiency upon induction of hypoxia in wing disc explants . To highlight the variability in this experiment , we show example discs from two separate experiments .

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

(A) Timelapse montage of ATP sensor FRET efficiency upon induction of hypoxia in wing disc explants. To highlight the variability in this experiment, we show example discs from two separate experiments.


( B , D ) Mean FRET efficiency measured over time in non - organizers compared to ( B ) Anterior - Posterior boundary ( APB ) or ( D ) all organizer regions . Shaded regions indicate SD ; dots are the means per each timepoint , and the solid lines indicate the fit to the mean .

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 O O O O O

(B, D) Mean FRET efficiency measured over time in non-organizers compared to (B) Anterior-Posterior boundary (APB) or (D) all organizer regions. Shaded regions indicate SD; dots are the means per each timepoint, and the solid lines indicate the fit to the mean.


C DNA breaks were measured using alkaline comet assay in BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells treated with 2 µM pyridostatin ( PDS ) for 16 hours and released into fresh medium without pyridostatin . Representative images are shown . Scale bar represents 100 µm . D Quantification of DNA breaks shown in ( C ) . Graph and error bars represent the mean and SEM of n = 3 independent experiments . A minimum of 50 cells were analysed per condition per experiment . P values were calculated using an unpaired two - tailed t - test . * , P ≤ 0 . 05 ; NS , P > 0 . 5 . E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in ( C ) . A minimum of 200 cells were analysed per condition per experiment . Graph and error bars represent the mean and SEM of n = 3 independent experiments . P values were calculated using an unpaired two - tailed t - test . * * * , P ≤ 0 . 001 ; NS , P > 0 . 5 .

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

C DNA breaks were measured using alkaline comet assay in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with 2 µM pyridostatin (PDS) for 16 hours and released into fresh medium without pyridostatin. Representative images are shown. Scale bar represents 100 µm. D Quantification of DNA breaks shown in (C). Graph and error bars represent the mean and SEM of n = 3 independent experiments. A minimum of 50 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; NS, P > 0.5. E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in (C). A minimum of 200 cells were analysed per condition per experiment. Graph and error bars represent the mean and SEM of n = 3 independent experiments. P values were calculated using an unpaired two-tailed t-test. ***, P ≤ 0.001; NS, P > 0.5.


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 .

O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM B-CELL_LINE O 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 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 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 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 .

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 O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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

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 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 .

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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O 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 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 + / + 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 .

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 O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O B-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 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 .

B-CELL_LINE O O 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 B-SMALL_MOLECULE O 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 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 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 .

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

O O O O O O O O B-DISEASE O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY 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 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 test ( * * * * 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 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 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 test (****P <0.0001).


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 .

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 B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-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 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 .

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

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O B-TISSUE 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-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 O O O B-TISSUE O 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 ) 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 .

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

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

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

O 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 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 ) 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 ) .

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-CELL_TYPE O O O B-CELL_TYPE O B-CELL_TYPE O O O B-CELL_TYPE O O B-CELL_TYPE O O O B-CELL_TYPE O B-CELL_TYPE O O O B-CELL_TYPE O 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 ) 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 .

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

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 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 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 ) 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 .

O 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 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

(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 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 .

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

(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 ) 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 .

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

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

O O O O O B-CELL_TYPE B-EXP_ASSAY 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 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 ) 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 .

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 O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O B-GENEPROD O 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 images in coronal plane show abscess formation in the left lower pulmonary lobe ( upper panel ) and in spleen and left axilla ( lower panel ) .

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

O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-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 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 .

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

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

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 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 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 .

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 O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O 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 cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULINWT , OTULINM86I and OTULINW167S or catalytically inactive OTULINC129A as control for 1 hour . Afterwards , samples were subjected to analysis by western blot for the indicated proteins . Images are representative of three independent experiments .

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

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


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

O O O O O B-CELL_LINE B-GENEPROD O O O O O O O O O B-GENEPROD O 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 O B-CELL_TYPE I-CELL_TYPE O O 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.


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

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

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


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

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

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


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

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

O 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 O B-EXP_ASSAY O O O O O B-TISSUE 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 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 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 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 .

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

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O 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 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 . IHC for expression of active β - catenin in colon or wild - type and Angptl2 - / - mice . Scale bar = 50 μm .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O 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 ) 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 .

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 B-ORGANISM O O O B-SMALL_MOLECULE 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 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 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 ) .

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

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

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

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


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

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

O O O O 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 O O O O

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


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

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

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


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

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

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 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 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 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 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 .

O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 IHC in colon from wild - type and Angptl2 - / - mice . The Angptl2 - / - colon image serves as a negative control . Scale bar = 50 μm .

O O B-GENEPROD O O B-TISSUE O O O O O B-GENEPROD O O O B-ORGANISM O 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 levels of indicated genes in wild - type ( n = 4 ) mice based on qRT - PCR analysis .

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

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


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

O O O B-EXP_ASSAY O O O B-TISSUE O O O O B-ORGANISM O O O O O B-GENEPROD O 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 levels of indicated genes in ISEMFs from wild - type ( n = 4 ) and Angptl2 - / - ( n = 3 ) mice based qRT - PCR analysis .

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

O O O O O O O B-GENEPROD 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 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-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 B-GENEPROD O O O O O O O B-TISSUE I-TISSUE 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 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 analysis of isolated colon crypts from wild - type and Angptl2 - / - mice . HSC70 serves as an internal control . Numbers below panels represent normalized protein expression .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O 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 serves as an internal control. Numbers below panels represent normalized protein expression.


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

O O O O O O O O B-TISSUE O O B-CELL_TYPE O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 IHC of integrin α5 and β1 in colon from wild - type mouse . Scale bar = 50 μm .

O O O O O B-GENEPROD I-GENEPROD O 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 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 .

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

O O B-TISSUE I-TISSUE 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 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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O 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 cells ( with antibody complexed ) as indicated . A single experiment representative of 3 independent experiments is shown .

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

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


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

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

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


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

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

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


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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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 / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell viability measured by PrestoBlue

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

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


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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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