( C ) Immunohistochemistry on paraffin sections for cKit ( HSPCs marker ) and F4 / 80 ( macrophage cell marker ) at E13 and E16 stages of mouse fetal liver . ( D ) Distance distribution between cKithigh cells and macrophages ( n = 54 from 3 E13 fetal livers ; n = 64 from 3 E16 fetal livers ) , binned into 5μm intervals . Statistical analysis was completed using t - test . All nuclei marked with DAPI . Scale bar

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(C) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and F4/80 (macrophage cell marker) at E13 and E16 stages of mouse fetal liver. (D) Distance distribution between cKithigh cells and macrophages (n=54 from 3 E13 fetal livers; n=64 from 3 E16 fetal livers), binned into 5μm intervals. Statistical analysis was completed using t-test. All nuclei marked with DAPI. Scale bar 20μm


( E ) Immunohistochemistry on paraffin sections for cKit ( HSPCs marker ) and MBP ( neutrophil cell marker ) at E13 and E16 stages of mouse fetal liver . ( F ) Distance distribution between cKithigh cells and neutrophils ( n = 45 from 3 E13 fetal livers ; n = 53 from 3 E16 fetal livers ) , binned into 20μm intervals . Statistical analysis was completed using t - test * * * P < . 001 . All nuclei marked with DAPI . Scale bar

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(E) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and MBP (neutrophil cell marker) at E13 and E16 stages of mouse fetal liver. (F) Distance distribution between cKithigh cells and neutrophils (n=45 from 3 E13 fetal livers; n=53 from 3 E16 fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test ***P<.001. All nuclei marked with DAPI. Scale bar 20μm


A Anti - SARS - CoV - 2 neutralizing antibodies in plasma of vaccine breakthrough cases ( n = 54 ) and close contacts ( n = 86 ) were measured by surrogate virus neutralization test ( sVNT ) . The percentage inhibition of ACE2 binding to RBD at plasma dilution of 1 : 800 is given . The wild type ( Wuhan ) strain or Delta ( B . 1 . 617 . 2 ) strain RBD was used respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Anti-SARS-CoV-2 neutralizing antibodies in plasma of vaccine breakthrough cases (n=54) and close contacts (n=86) were measured by surrogate virus neutralization test (sVNT). The percentage inhibition of ACE2 binding to RBD at plasma dilution of 1:800 is given. The wild type (Wuhan) strain or Delta (B.1.617.2) strain RBD was used respectively.


B Anti - SARS - CoV - 2 neutralizing antibodies in plasma of vaccine breakthrough cases ( n = 29 ) and close contacts ( n = 86 ) were measured by pseudovirus neutralization 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

B Anti-SARS-CoV-2 neutralizing antibodies in plasma of vaccine breakthrough cases (n=29) and close contacts (n=86) were measured by pseudovirus neutralization assay.


C Anti - SARS - CoV - 2 S protein antibodies in plasma of vaccine breakthrough cases ( n = 48 ) and close contacts ( n = 86 ) was determined by S protein flow cytometry - based assay ( SFB ) . The percentage of plasma antibody - bound cells out of the total population of cells transfected with membrane - anchored SARS - CoV - 2 S protein is given . In all graphs error bars denote median and interquartile range . Red solid circles indicate Delta strain vaccine breakthrough , red open circles indicate non - Delta or unknown strain .

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C Anti-SARS-CoV-2 S protein antibodies in plasma of vaccine breakthrough cases (n=48) and close contacts (n=86) was determined by S protein flow cytometry-based assay (SFB). The percentage of plasma antibody-bound cells out of the total population of cells transfected with membrane-anchored SARS-CoV-2 S protein is given. In all graphs error bars denote median and interquartile range. Red solid circles indicate Delta strain vaccine breakthrough, red open circles indicate non-Delta or unknown strain.


B , C Levels of anti - SARS - CoV - 2 neutralizing antibodies inhibiting ACE2 binding to WT or Delta are determined by surrogate virus neutralization test ( sVNT ) , in supernatant from 5 - day culture of activated memory B cells and in plasma . The difference in sVNT % inhibition between WT and Delta is taken , and this difference is compared between plasma and memory B cell supernatant for vaccine breakthrough cases ( n = 48 pairs ) ( B ) , and for close contacts ( n = 86 pairs ) ( C ) .

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B, C Levels of anti-SARS-CoV-2 neutralizing antibodies inhibiting ACE2 binding to WT or Delta are determined by surrogate virus neutralization test (sVNT), in supernatant from 5-day culture of activated memory B cells and in plasma. The difference in sVNT % inhibition between WT and Delta is taken, and this difference is compared between plasma and memory B cell supernatant for vaccine breakthrough cases (n=48 pairs) (B), and for close contacts (n=86 pairs) (C).


A - D PBMCs from vaccine breakthrough cases ( n = 15 ) and close contacts ( n = 26 ) were examined for T cell responses . PBMCs were left unstimulated ( Unstim ) , were stimulated with pooled SARS - CoV - 2 PepTivator ® S , S1 , M and N peptides for 6 hours ( SARS - CoV - 2 Peptide Stim ) , or non - specifically stimulated with phorbol 12 - myristate 13 - acetate ( PMA ) , then assessed by high - dimensional flow cytometry . CD4 + T cells positive for intracellular staining of IFNγ ( a ) , IL - 2 ( b ) , TNF ( c ) , or CD8 + T cells positive for intracellular staining of granzyme B ( d ) are shown . Error bars indicate median and interquartile range .

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

A-D PBMCs from vaccine breakthrough cases (n=15) and close contacts (n=26) were examined for T cell responses. PBMCs were left unstimulated (Unstim), were stimulated with pooled SARS-CoV-2 PepTivator® S, S1, M and N peptides for 6 hours (SARS-CoV-2 Peptide Stim), or non-specifically stimulated with phorbol 12-myristate 13-acetate (PMA), then assessed by high-dimensional flow cytometry. CD4+ T cells positive for intracellular staining of IFNγ (a), IL-2 (b), TNF (c), or CD8+ T cells positive for intracellular staining of granzyme B (d) are shown. Error bars indicate median and interquartile range.


F The differentiation status of SARS - CoV - 2 peptide - stimulated CD4 + T cells ( left ) and CD8 + T cells ( right ) were compared based on CD27 and CD45RA expression ( Naïve : CD27 + CD45RA + ; T central memory ( TCM ) : CD27 + CD45RA - ; T effector memory ( TEM ) : CD27 - CD45RA - ; TEMRA : CD27 - CD45RA + ) . The average of all vaccine breakthrough cases or close contacts are plotted respectively .

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F The differentiation status of SARS-CoV-2 peptide-stimulated CD4+ T cells (left) and CD8+ T cells (right) were compared based on CD27 and CD45RA expression (Naïve: CD27+ CD45RA+; T central memory (TCM): CD27+ CD45RA-; T effector memory (TEM): CD27- CD45RA-; TEMRA: CD27- CD45RA+). The average of all vaccine breakthrough cases or close contacts are plotted respectively.


B Heatmap of selected immune mediator levels in plasma samples of healthy controls , vaccinated close contacts , COVID - 19 vaccine breakthrough cases , and unvaccinated COVID - 19 patients with primary infection . Each color represents the relative concentration of a particular analyte . Blue and red indicate low and high concentration , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heatmap of selected immune mediator levels in plasma samples of healthy controls, vaccinated close contacts, COVID-19 vaccine breakthrough cases, and unvaccinated COVID-19 patients with primary infection. Each color represents the relative concentration of a particular analyte. Blue and red indicate low and high concentration, respectively.


H , I WT and lincRNA - EPS - / - iBMMs were infected with VSV ( MOI 0 . 1 ) for 6 h or 10 h ( H ) or transfected with polyI : C ( 1 μg / mL ) for 4 h or 8 h ( I ) , phosphorylation and total protein expression were analyzed by Western blot with α - tubulin and β - Actin as loading controls .

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H, I WT and lincRNA-EPS-/- iBMMs were infected with VSV (MOI 0.1) for 6 h or 10 h (H) or transfected with polyI:C (1 μg/mL) for 4 h or 8 h (I), phosphorylation and total protein expression were analyzed by Western blot with α-tubulin and β-Actin as loading controls.


B Western blot for verification of RNA pulldown - MS results with β - Actin as a negative loading control .

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B Western blot for verification of RNA pulldown-MS results with β-Actin as a negative loading control.


D 20 nM siRNA targeting negative control ( NC ) and PKR were transfected into iBMMs for 48 h , the protein and mRNA expression level of PKR were tested by RT - qPCR and Western blot , respectively .

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D 20 nM siRNA targeting negative control (NC) and PKR were transfected into iBMMs for 48 h, the protein and mRNA expression level of PKR were tested by RT-qPCR and Western blot, respectively.


G VSV - N and VSV - G mRNA were transcribed from virus particles for RNA - pulldown , the interactions between VSV RNA with PKR protein was checked by Western blot , β - Actin was shown as a negative control .

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G VSV-N and VSV-G mRNA were transcribed from virus particles for RNA-pulldown, the interactions between VSV RNA with PKR protein was checked by Western blot, β-Actin was shown as a negative control.


H LincRNA - EPS was transcribed in vitro and pulled down proteins from iBMMs infected with VSV for 4 h , PKR protein was detected by Western blot and GAPDH was shown as a loading control .

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H LincRNA-EPS was transcribed in vitro and pulled down proteins from iBMMs infected with VSV for 4 h, PKR protein was detected by Western blot and GAPDH was shown as a loading control.


I RNA - pulldown using biotinylated lincRNA - EPS from cell lysates of polyI : C ( 1 and 2 . 5 μg / mL ) transfected iBMMs for 4 h , PKR protein was detected by Western blot and β - Actin was shown as a loading control .

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I RNA-pulldown using biotinylated lincRNA-EPS from cell lysates of polyI:C (1 and 2.5 μg/mL) transfected iBMMs for 4 h, PKR protein was detected by Western blot and β-Actin was shown as a loading control.


A 20 nM siRNA for negative control ( NC ) and PKR were transfected into iBMMs following infected with VSV ( MOI 0 . 1 ) for 4 h or 8 h , phosphorylation and total levels of STAT1 , eIF2α and PKR were detected by Western blot and β - Actin was shown as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 20 nM siRNA for negative control (NC) and PKR were transfected into iBMMs following infected with VSV (MOI 0.1) for 4 h or 8 h, phosphorylation and total levels of STAT1, eIF2α and PKR were detected by Western blot and β-Actin was shown as a loading control.


B PKR was transfected into A549 cells for 24 h following stimulated with IFN - β ( 50 ng / mL ) for 1 h to test STAT1 phosphorylation by Western blot and β - Actin was shown as a loading control .

B PKR was transfected into A549 cells for 24 h following stimulated with IFN-β (50 ng/mL) for 1 h to test STAT1 phosphorylation by Western blot and β-Actin was shown as a loading control.


( B ) Immunoblot analysis of pro - ( p31 ) and cleaved ( p17 ) IL - 1β , and pro - ( p45 ) and cleaved ( p20 ) caspase - 1 in cytosolic fractions of ASC deficient ( Pycard - / - ) immortalized murine macrophages that were primed with LPS ( 200 ng ml − 1 , for 3h ) . Fractions were incubated with recombinant ASC specks in the presence of 2 , 20 or 200 µg ml − 1 of VHHASC or mutVHHASC . One representative of three independent experiments is shown . ( C ) Quantitative analysis of band densitometry of three independent experiments as shown in B . Data represents the fold change from all conditions ( lanes 1 - 9 ) . vs Pycard − / − lysates incubated with ASC specks alone ( lane 2 ) . ns , p > 0 . 05 ; * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * , p < 0 . 0002 , One - way ANOVA , multiple comparison ( Dunnet test ) . Data is displayed as floating bars with the max / min values and mean ( thicker band ) .

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(B) Immunoblot analysis of pro- (p31) and cleaved (p17) IL-1β, and pro- (p45) and cleaved (p20) caspase-1 in cytosolic fractions of ASC deficient (Pycard-/-) immortalized murine macrophages that were primed with LPS (200 ng ml−1, for 3h). Fractions were incubated with recombinant ASC specks in the presence of 2, 20 or 200 µg ml−1 of VHHASC or mutVHHASC. One representative of three independent experiments is shown. (C) Quantitative analysis of band densitometry of three independent experiments as shown in B. Data represents the fold change from all conditions (lanes 1- 9) .vs Pycard−/− lysates incubated with ASC specks alone (lane 2). ns, p > 0.05; *, p<0.05; **, p<0.005; ***, p<0.0002, One-way ANOVA, multiple comparison (Dunnet test). Data is displayed as floating bars with the max/min values and mean (thicker band).


( A ) Time - lapse confocal imaging of the in vitro nucleation of soluble ASC - mTurquoise ( red ) by ASC - TagGFP ( ASC - GFP ) specks ( green ) , that were left untreated ( None ) , or pre - incubated with VHHASC or mutVHHASC ( 200 µg ml − 1 for 15 min ) . Scale bars : 10 μm . ( B ) Median fluorescence intensity ( MFI ) of mTurquoise graphed over time showing its polymerization seeded by ASC - TagGFP specks . Each line shows the mTurquoise MFI around a seeding ASC - TagGFP speck . Data is one representative of three independent experiments .

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(A) Time-lapse confocal imaging of the in vitro nucleation of soluble ASC-mTurquoise (red) by ASC-TagGFP (ASC-GFP) specks (green), that were left untreated (None), or pre-incubated with VHHASC or mutVHHASC (200 µg ml−1 for 15 min). Scale bars: 10 μm. (B) Median fluorescence intensity (MFI) of mTurquoise graphed over time showing its polymerization seeded by ASC-TagGFP specks. Each line shows the mTurquoise MFI around a seeding ASC-TagGFP speck. Data is one representative of three independent experiments.


( C ) WES capillary electrophoresis and immunoblotting of DSS cross - linked in - vitro - generated human ASC - mTurquoise ( ASC ) specks that were incubated for 1h ( RT ) with 2 , 20 or 200 µg ml − 1 of VHHASC , mutVHHASC or with 20 µg ml − 1 of a polyclonal anti - ASC Ab ( a - ASC pAb ) . Data is from one representative of four independent experiments .

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(C) WES capillary electrophoresis and immunoblotting of DSS cross-linked in-vitro-generated human ASC-mTurquoise (ASC) specks that were incubated for 1h (RT) with 2, 20 or 200 µg ml−1 of VHHASC, mutVHHASC or with 20 µg ml−1 of a polyclonal anti-ASC Ab (a-ASC pAb). Data is from one representative of four independent experiments.


A - D ( A - B ) Human IL - 1β ( hIL - 1β ) concentrations in cell - free supernatants of LPS - primed ( 10 ng ml − 1 , 150 min ) primary human macrophages that were left untreated , or pre - incubated with VHHASC or mutVHHASC ( 100 µg ml − 1 ) , CRID3 ( 50 µM ) or VX - 765 ( 50 µM ) for 30 min before stimulation with ( A ) nigericin ( 10 µM ) , or ( B ) PFO ( 30 ng ml − 1 ) for 2h . ( C - D ) Mouse IL - 1β ( mIL - 1β ) concentrations in cell - free supernatants of LPS - primed mouse BMDMs ( 200 ng ml − 1 , 150 min ) , incubated with VHHs , CRID3 or VX765 , before activation with nigericin ( 10 µM ) , or PFO ( 250 ng ml − 1 ) . Data is combined from two independent experiments , each performed with two donors ( A - B ) or mice ( C - D ) , represented with individual symbols ( 4 donors or mice in total ) . Data is displayed as floating bars with the max / min values and mean ( thicker band ) .

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

A-D (A-B) Human IL-1β (hIL-1β) concentrations in cell-free supernatants of LPS-primed (10 ng ml−1, 150 min) primary human macrophages that were left untreated, or pre-incubated with VHHASC or mutVHHASC (100 µg ml−1), CRID3 (50 µM) or VX-765 (50 µM) for 30 min before stimulation with (A) nigericin (10 µM), or (B) PFO (30 ng ml−1) for 2h. (C-D) Mouse IL-1β (mIL-1β) concentrations in cell-free supernatants of LPS-primed mouse BMDMs (200 ng ml−1, 150 min), incubated with VHHs, CRID3 or VX765, before activation with nigericin (10 µM), or PFO (250 ng ml−1). Data is combined from two independent experiments, each performed with two donors (A-B) or mice (C-D), represented with individual symbols (4 donors or mice in total). Data is displayed as floating bars with the max/min values and mean (thicker band).


E , F ( E ) Epifluorescence microscopy imaging and ( F ) quantification of ASC speck formation in BMDMs from ASC - mCitrine ( Green ) transgenic mice . Cells were primed with LPS ( 200 ng ml − 1 , 150 min ) , pre - treated with VX - 765 ( 50 µM , 30 min ) , then treated with VHHASC , VHHmASC ( 100 µg ml − 1 ) or CRID3 ( 50 µM ) for another 30 min before stimulation with nigericin ( top ) , or PFO ( bottom ) for 2h and finally fixed with 4 % PFA and nuclei stained with DRAQ5 ( Blue ) . Scale bars : 100 μm . Images in ( E ) are representative of one out of three independent experiments that were quantified in F . Data is displayed as floating bars with the max / min values and mean ( thicker band ) .

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

E, F (E) Epifluorescence microscopy imaging and (F) quantification of ASC speck formation in BMDMs from ASC-mCitrine (Green) transgenic mice. Cells were primed with LPS (200 ng ml−1, 150 min), pre-treated with VX-765 (50 µM, 30 min), then treated with VHHASC, VHHmASC (100 µg ml−1) or CRID3 (50 µM) for another 30 min before stimulation with nigericin (top), or PFO (bottom) for 2h and finally fixed with 4% PFA and nuclei stained with DRAQ5 (Blue). Scale bars: 100 μm. Images in (E) are representative of one out of three independent experiments that were quantified in F. Data is displayed as floating bars with the max/min values and mean (thicker band).


THP - 1 cells expressing a Dox - inducible CRISPR - Cas9 cassette targeting GSDMD gene were treated with 1 µg ml - 1 Dox for one or two cycles of 72 h ( 1x , or 2x respectively ) . As control , cells were left untreated and cultured in parallel ( - ) . ( A ) Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA - differentiation , where indicated . Data is from one representative of two independent experiments .

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THP-1 cells expressing a Dox-inducible CRISPR-Cas9 cassette targeting GSDMD gene were treated with 1 µg ml-1 Dox for one or two cycles of 72 h (1x, or 2x respectively). As control, cells were left untreated and cultured in parallel (-). (A) Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA-differentiation, where indicated. Data is from one representative of two independent experiments.


GFP + specks detected in culture supernatants by ( D ) immunoblotting analysis of ASC - GFP oligomeric state ( DSS cross - linked ) of extracellular specks recovered from supernatants of cells treated similarly to ( A - B ) . ( D ) is from one out of at least two independent experiments .

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GFP+ specks detected in culture supernatants by (D) immunoblotting analysis of ASC-GFP oligomeric state (DSS cross-linked) of extracellular specks recovered from supernatants of cells treated similarly to (A-B). (D) is from one out of at least two independent experiments.


A Representative photographs of fly eyes expressing ( CGG ) 90 - EGFP under a GMR - GAL4 driver , with various belle disruptions .

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A Representative photographs of fly eyes expressing (CGG)90-EGFP under a GMR-GAL4 driver, with various belle disruptions.


B Quantitation of GMR - GAL4 , ( CGG ) 90 - EGFP eye phenotypes with belle disruptions ( Mann - Whitney U test with Bonferonni corrections for multiple comparisons ; n = 35 - 77 / genotype ) . Data Information : For all panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All data in all panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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B Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with belle disruptions (Mann-Whitney U test with Bonferonni corrections for multiple comparisons; n=35-77/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


Longevity assays of ( CGG ) 90 - EGFP ; Tub5 - GS ( Log - rank Mantel - Cox test with Bonferroni corrections for multiple comparisons ; n = 110 - 219 / genotype ) flies with belle knockdown . Data Information : For all panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All data in all panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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Longevity assays of (CGG)90-EGFP; Tub5-GS (Log-rank Mantel-Cox test with Bonferroni corrections for multiple comparisons; n=110-219/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


Longevity assays of ( CGG ) 90 - EGFP ; ElaV - GS ( n = 147 - 299 / genotype ) flies with belle knockdown . Data Information : For all panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All data in all panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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Longevity assays of (CGG)90-EGFP; ElaV-GS (n=147-299/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


F Quantitation of FMRpolyG - EGFP band density , normalized to β tubulin band density , from blots in E ( Student ' s t test ; n = 4 - 5 / genotype ) . Data Information : For all panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All data in all panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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F Quantitation of FMRpolyG-EGFP band density, normalized to β tubulin band density, from blots in E (Student's t test; n=4-5/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


G Abundance of ( CGG ) 90 - EGFP mRNA normalized to RPL32 mRNA , following belle knockdown , determined by qRT - PCR ( n = 8 / genotype ) .

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G Abundance of (CGG)90-EGFP mRNA normalized to RPL32 mRNA, following belle knockdown, determined by qRT-PCR (n=8/genotype).


H Western blot of AUG - driven EGFP in EGFP ; Tub5 - GS flies with and without belle knockdown .

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H Western blot of AUG-driven EGFP in EGFP; Tub5-GS flies with and without belle knockdown.


I Quantitation of EGFP band density , normalized to β tubulin band density , from blot in H ( n = 4 / genotype ) .

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I Quantitation of EGFP band density, normalized to β tubulin band density, from blot in H (n=4/genotype).


A Dose - response curves showing the effects of two independent anti - DDX3X siRNAs on the expression of AUG - NL - 3xF ( top ) and ( CGG ) 100 + 1 NL - 3xF ( bottom ) reporters . Plasmid - based reporters were transfected into HeLa cells 24 hours after knockdown , and reporter expression was quantified by luminescence . Nanoluciferase ( NL ) luminescence has been normalized to luminescence from firefly luciferase ( FF ) , which was co - transfected , in order to control for transfection variability . Asterisks refer to comparisons between anti - DDX3X siRNAs and siRNAs against EGFP ( siEGFP ; two - way ANOVA with Dunnett ' s multiple comparisons test ; n = 12 / condition ) . Data Information : For all panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All panels depict data as means ± SD ( compiled from ≥ 3 replicates ) .

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A Dose-response curves showing the effects of two independent anti-DDX3X siRNAs on the expression of AUG-NL-3xF (top) and (CGG)100 +1 NL-3xF (bottom) reporters. Plasmid-based reporters were transfected into HeLa cells 24 hours after knockdown, and reporter expression was quantified by luminescence. Nanoluciferase (NL) luminescence has been normalized to luminescence from firefly luciferase (FF), which was co-transfected, in order to control for transfection variability. Asterisks refer to comparisons between anti-DDX3X siRNAs and siRNAs against EGFP (siEGFP; two-way ANOVA with Dunnett's multiple comparisons test; n=12/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).


( CGG ) n + 1 NL - 3xF expression ( normalized to FF ) with and without DDX3X knockdown across a range of CGG repeat sizes . Black asterisks refer to comparisons between siDDX3X - and siEGFP - treated cells ; orange asterisks refer to comparisons between siDDX3X - treated cells expressing AUG - NL - 3xF and those expressing a different reporter ( two - way ANOVA with Tukey ' s multiple comparisons test ; n = 17 - 30 / condition ) . Data Information : For all panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All panels depict data as means ± SD ( compiled from ≥ 3 replicates ) .

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(CGG)n +1 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).


( CGG ) n + 2 NL - 3xF expression ( normalized to FF ) with and without DDX3X knockdown across a range of CGG repeat sizes . Black asterisks refer to comparisons between siDDX3X - and siEGFP - treated cells ; orange asterisks refer to comparisons between siDDX3X - treated cells expressing AUG - NL - 3xF and those expressing a different reporter ( two - way ANOVA with Tukey ' s multiple comparisons test ; n = 17 - 30 / condition ) . Data Information : For all panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All panels depict data as means ± SD ( compiled from ≥ 3 replicates ) .

(CGG)n +2 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).


Western blots of FMRpolyG - NL - 3xF products with and without DDX3X knockdown across a range of repeat sizes .

Western blots of FMRpolyG-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.


Western blots of FMRpolyA - NL - 3xF products with and without DDX3X knockdown across a range of repeat sizes .

Western blots of FMRpolyA-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.


A Expression of in vitro transcribed AUG , + 1 ( CGG ) 100 , and + 2 ( CGG ) 100 NL - 3xF RNAs following DDX3X knockdown in HeLa cells , expressed as NL luminescence normalized to FF luminescence . ( Student ' s t test with Bonferroni corrections for multiple comparisons ; n = 21 / condition ) . Data Information : For all panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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A Expression of in vitro transcribed AUG, +1 (CGG)100, and +2 (CGG)100 NL-3xF RNAs following DDX3X knockdown in HeLa cells, expressed as NL luminescence normalized to FF luminescence. (Student's t test with Bonferroni corrections for multiple comparisons; n=21/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


B Abundance of reporter mRNAs following DDX3X knockdown and plasmid - reporter transfection , determined by qRT - PCR . ( n = 7 / condition . ) This panel depicts data as means ± SEM .

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B Abundance of reporter mRNAs following DDX3X knockdown and plasmid-reporter transfection, determined by qRT-PCR. (n=7/condition.) This panel depicts data as means ± SEM.


C Expression of AUG - NL - 3xF and + 1 ( CGG ) 100 NL - 3xF in in vitro translation extracts , collected from HeLa cells treated with siRNAs against EGFP or DDX3X ( two - way ANOVA with Tukey ' s multiple comparisons test ; n = 4 / condition ) . Data Information : For all panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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C Expression of AUG-NL-3xF and +1 (CGG)100 NL-3xF in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X (two-way ANOVA with Tukey's multiple comparisons test; n=4/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


D Enrichment of HSPA1A and + 1 ( CGG ) 100 NL - 3xF mRNA following anti - DDX3X RIP , relative to incubation with isotype control IgG . MALAT RNA , in contrast , is not enriched ( Student ' s t test , n = 3 ) .

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D Enrichment of HSPA1A and +1 (CGG)100 NL-3xF mRNA following anti-DDX3X RIP, relative to incubation with isotype control IgG. MALAT RNA, in contrast, is not enriched (Student's t test, n=3).


E Expression of + 1 and + 2 ( CGG ) 100 NL - 3xF plasmid reporters with and without an AUG inserted 5 ' to the CGG repeat , with and without DDX3X knockdown . Black asterisks refer to comparisons between siDDX3X - and siEGFP - treated cells ; orange asterisks refer to comparisons between siDDX3X - treated cells expressing either + 1 or + 2 ( CGG ) 100 NL - 3xF and those expressing the respective AUG - driven variant ( two - way ANOVA with Tukey ' s multiple comparisons test ; n = 11 - 12 / condition ) . Data Information : For all panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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E Expression of +1 and +2 (CGG)100 NL-3xF plasmid reporters with and without an AUG inserted 5' to the CGG repeat, with and without DDX3X knockdown. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing either +1 or +2 (CGG)100 NL-3xF and those expressing the respective AUG-driven variant (two-way ANOVA with Tukey's multiple comparisons test; n=11-12/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


F Expression of NL - 3xF plasmids with initiator AUG codons mutated to near - AUG codons , with and without DDX3X knockdown ( two - way ANOVA with Dunnett ' s multiple comparisons test ; n = 18 - 24 / condition ) . Black asterisks refer to comparisons between siEGFP - treated and siDDX3X - treated cells ; orange asterisks refer to comparisons between siDDX3X - treated cells expressing AUG - NL - 3xF and those expressing a different reporter ; white asterisks refer to comparisons between siDDX3X - treated cells expressing + 1 ( CGG ) 100 NL - 3xF and those expressing a different reporter . Data Information : For all panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . All panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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F Expression of NL-3xF plasmids with initiator AUG codons mutated to near-AUG codons, with and without DDX3X knockdown (two-way ANOVA with Dunnett's multiple comparisons test; n=18-24/condition). Black asterisks refer to comparisons between siEGFP-treated and siDDX3X-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter; white asterisks refer to comparisons between siDDX3X-treated cells expressing +1 (CGG)100 NL-3xF and those expressing a different reporter. Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


G Expression of in vitro transcribed near - AUG reporter RNAs in in vitro translation extracts , collected from HeLa cells treated with siRNAs against EGFP or DDX3X . ( n = 4 / group ) .

G Expression of in vitro transcribed near-AUG reporter RNAs in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X. (n=4/group).


A Representative photographs of GMR - GAL4 ; ( CGG ) 90 - EGFP fly eyes expressing manipulations of eIF4B and eIF4H . ( B ) Quantitation of GMR - GAL4 , ( CGG ) 90 - EGFP eye phenotypes with eIF4B / H manipulations ( Mann - Whitney U test with Bonferroni corrections for multiple comparisons ; n = 26 - 55 / genotype ) . Data Information : For all panels , ns = non - significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 for the specified statistical test . All panels present data as means ± SD ( compiled from ≥ 3 replicates ) .

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A Representative photographs of GMR-GAL4; (CGG)90-EGFP fly eyes expressing manipulations of eIF4B and eIF4H. (B) Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with eIF4B/H manipulations (Mann-Whitney U test with Bonferroni corrections for multiple comparisons; n=26-55/genotype). Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates).


C , D Expression of plasmid - based AUG - NL and + 1 ( CGG ) 100 NL - 3xF reporters ( C ) , or co - transfected AUG - FF reporters ( D ) , following knockdown of EIF4B or EIF4H . Black asterisks refer to comparisons between siEGFP - and siEIF4B / H - treated cells ; pink and blue asterisks refer to comparisons between siEIF4B - ( pink ) or siEIF4H - ( blue ) treated cells expressing AUG - NL - 3xF and those expressing + 1 ( CGG ) 100 ( two - way ANOVA with Tukey ' s multiple comparisons test , n = 9 / condition ) .

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C, D Expression of plasmid-based AUG-NL and +1 (CGG)100 NL-3xF reporters (C), or co-transfected AUG-FF reporters (D), following knockdown of EIF4B or EIF4H. Black asterisks refer to comparisons between siEGFP- and siEIF4B/H-treated cells; pink and blue asterisks refer to comparisons between siEIF4B- (pink) or siEIF4H- (blue) treated cells expressing AUG-NL-3xF and those expressing +1 (CGG)100 (two-way ANOVA with Tukey's multiple comparisons test, n=9/condition).


E Expression of plasmid - based AUG - NL - 3xF and ( CGG ) 100 + 1 NL - 3xF reporters with and without over - expression of EIF4B , EIF4H , or both ( two - way ANOVA with Dunnett ' s multiple comparisons test ; n = 20 / condition ) . Asterisks refer to comparisons between cells over - expressing either EGFP or EIF4B , EIF4H , or EIF4B and EIF4H and expressing the same reporter . Data Information : For all panels , ns = non - significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 for the specified statistical test . All panels present data as means ± SD ( compiled from ≥ 3 replicates ) .

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E Expression of plasmid-based AUG-NL-3xF and (CGG)100 +1 NL-3xF reporters with and without over-expression of EIF4B, EIF4H, or both (two-way ANOVA with Dunnett's multiple comparisons test; n=20/condition). Asterisks refer to comparisons between cells over-expressing either EGFP or EIF4B, EIF4H, or EIF4B and EIF4H and expressing the same reporter. Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates).


A Expression of plasmid - based NL - 3xF reporters in HEK293 cells with and without over - expression of EIF1 ( two - way ANOVA with Sidak ' s multiple comparisons test ; n = 9 - 12 / condition ) . Black asterisks refer to comparisons between Empty Vector - transfected and EIF1 - transfected cells ; green asterisks refer to comparisons between EIF1 - transfected cells expressing AUG - NL - 3xF and those expressing a different reporter . For all panels , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . Bars represent mean ± SEM ( compiled from ≥ 3 replicates ) .

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A Expression of plasmid-based NL-3xF reporters in HEK293 cells with and without over-expression of EIF1 (two-way ANOVA with Sidak's multiple comparisons test; n=9-12/condition). Black asterisks refer to comparisons between Empty Vector-transfected and EIF1-transfected cells; green asterisks refer to comparisons between EIF1-transfected cells expressing AUG-NL-3xF and those expressing a different reporter. For all panels, *** P≤0.001, **** P≤0.0001 for the specified statistical test. Bars represent mean ± SEM (compiled from ≥3 replicates).


B Expression of plasmid - based NL - 3xF reporters in HEK293 cells with and without over - expression of EIF5 ( two - way ANOVA with Sidak ' s multiple comparisons test ; n = 9 - 12 / condition ) . Black asterisks refer to comparisons between Empty Vector - transfected and EIF5 - transfected cells ; pink asterisks refer to comparisons between EIF5 - transfected cells expressing AUG - NL - 3xF and those expressing a different reporter . For all panels , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for the specified statistical test . Bars represent mean ± SEM ( compiled from ≥ 3 replicates ) .

B Expression of plasmid-based NL-3xF reporters in HEK293 cells with and without over-expression of EIF5 (two-way ANOVA with Sidak's multiple comparisons test; n=9-12/condition). Black asterisks refer to comparisons between Empty Vector-transfected and EIF5-transfected cells; pink asterisks refer to comparisons between EIF5-transfected cells expressing AUG-NL-3xF and those expressing a different reporter. For all panels, *** P≤0.001, **** P≤0.0001 for the specified statistical test. Bars represent mean ± SEM (compiled from ≥3 replicates).


A Sample micrographs collected by automated longitudinal fluorescence microscopy , demonstrating the automated determination of cell death .

A Sample micrographs collected by automated longitudinal fluorescence microscopy, demonstrating the automated determination of cell death.


B Anti - DDX3X western blot of B35 cells transfected with either of two independent anti - DDX3X LNAs or a control LNA .

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B Anti-DDX3X western blot of B35 cells transfected with either of two independent anti-DDX3X LNAs or a control LNA.


C Expression of EGFP in primary rat neurons transfected with ( CGG ) 100 ( + 1 ) EGFP and either anti - DDX3X LNAs or a control ( one - way ANOVA with Tukey ' s multiple comparisons test ; n = 2408 - 5689 cells / condition ) . All graphs depict pooled data , normalized first within the replicate . Data Information : For all panels , * * * * P ≤ 0 . 0001 for the specified statistical test ( compiled from ≥ 3 replicates ) .

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C Expression of EGFP in primary rat neurons transfected with (CGG)100 (+1) EGFP and either anti-DDX3X LNAs or a control (one-way ANOVA with Tukey's multiple comparisons test; n=2408-5689 cells/condition). All graphs depict pooled data, normalized first within the replicate. Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).


D , E Transfection of anti - DDX3X LNA # 1 ( D ) or # 2 ( E ) reduced the cumulative risk of death in ( CGG ) 100 ( + 1 ) EGFP - expressing neurons ( Cox proportional hazard analysis ; n = 2408 - 3676 cells / condition ) . Data Information : For all panels , * * * * P ≤ 0 . 0001 for the specified statistical test ( compiled from ≥ 3 replicates ) .

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D, E Transfection of anti-DDX3X LNA #1 (D) or #2 (E) reduced the cumulative risk of death in (CGG)100 (+1) EGFP-expressing neurons (Cox proportional hazard analysis; n=2408-3676 cells/condition). Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).


Survival of age - and sex - matched ( 8 - 10 week old ) Spata2 + / + ( WT ) and Spata2 - / - ( KO ) mice injected intraperitoneally ( i . p . ) with LPS ( 20 mg / kg bodyweight ) . Data pooled from three independent experiments are shown . Statistical analysis was performed using long - rank test .

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Survival of age- and sex-matched (8-10 week old) Spata2+/+ (WT) and Spata2-/- (KO) mice injected intraperitoneally (i.p.) with LPS (20 mg/kg bodyweight). Data pooled from three independent experiments are shown. Statistical analysis was performed using long-rank test.


ELISA of serum IL - 1β , IL - 18 , TNFα and IL - 6 at 12 h after injection of WT or Spata2 - KO mice with 20 mg / kg LPS . Circles and triangles represent individual mice . Horizontal bars indicate mean values . * * P < 0 . 01 ; * * * * P < 0 . 0001 . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

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ELISA of serum IL-1β, IL-18, TNFα and IL-6 at 12 h after injection of WT or Spata2-KO mice with 20 mg/kg LPS. Circles and triangles represent individual mice. Horizontal bars indicate mean values. **P < 0.01; ****P < 0.0001. Statistical analysis was performed using unpaired two-tailed Student's t-test.


Immunoblot analysis of the indicated phosphorylated ( P ) and total proteins in lysates of LPS - stimulated BMDMs derived from WT or Spata2 - KO mice . Molecular weights in kDa are indicated to the right .

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Immunoblot analysis of the indicated phosphorylated (P) and total proteins in lysates of LPS-stimulated BMDMs derived from WT or Spata2-KO mice. Molecular weights in kDa are indicated to the right.


qRT - PCR analysis of the indicated mRNAs in untreated or LPS - stimulated WT or Spata2 - KO BMDMs . Bars and error bars represent the mean ± S . D . of triplicate experiments . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

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qRT-PCR analysis of the indicated mRNAs in untreated or LPS-stimulated WT or Spata2-KO BMDMs. Bars and error bars represent the mean ± S.D. of triplicate experiments. Statistical analysis was performed using unpaired two-tailed Student's t-test.


ELISA of TNFα and IL - 6 in the supernatants from untreated or LPS - stimulated WT or Spata2 - KO BMDMs . Bars and error bars represent the mean ± S . D . of triplicate experiments . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

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

ELISA of TNFα and IL-6 in the supernatants from untreated or LPS-stimulated WT or Spata2-KO BMDMs. Bars and error bars represent the mean ± S.D. of triplicate experiments. Statistical analysis was performed using unpaired two-tailed Student's t-test.


ELISA of IL - 1β secretion by WT or Spata2 - KO BMDMs that were either untreated ( - ) or LPS - primed and then treated with the indicated NLRP3

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

ELISA of IL-1β secretion by WT or Spata2-KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducers


ELISA of IL - 1β secretion by WT or Spata2 - KO BMDMs that were either untreated ( - ) or stimulated with the NLRC4 inducer ( Salmonella infection ) or AIM2 inducer ( Poly ( dA : dT ) transfection ) ( B ) .

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

ELISA of IL-1β secretion by WT or Spata2-KO BMDMs that were either untreated (-) or stimulated with the NLRC4 inducer (Salmonella infection) or AIM2 inducer ( Poly(dA:dT) transfection) (B).


Immunoblot analysis of the mature IL - 1β p17 and the active Caspase - 1 p20 in cell supernatants ( Sup ) and the indicated proteins in the cell extracts ( Cell ext ) of WT or Spata2 - KO BMDMs stimulated as in ( A ) .

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

Immunoblot analysis of the mature IL-1β p17 and the active Caspase-1 p20 in cell supernatants (Sup) and the indicated proteins in the cell extracts (Cell ext) of WT or Spata2-KO BMDMs stimulated as in (A).


WT and Spata2 CRISPR knock out ( KO ) iBMDMs stably expressing GFP - ASC were primed with LPS and stimulated with Nigericin for 1 h . GFP - ASC specks were imaged by confocal microscope , and percentage of ASC speck - positive cells was quantified . Scale bar , 10 µm .

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

WT and Spata2 CRISPR knock out (KO) iBMDMs stably expressing GFP-ASC were primed with LPS and stimulated with Nigericin for 1 h. GFP-ASC specks were imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Scale bar, 10 µm.


BMDMs from Spata2 + / + ( WT ) and Spata2 - / - ( KO ) mice were pretreated with Caspase - 1 inhibitor , Z - YVAD - FMK , for 30 min , primed with LPS , and then stimulated with Nigericin for 1 h . ASC specks were assessed by Immunofluorescence with antibody against ASC and imaged by confocal microscope , and percentage of ASC speck - positive cells was quantified . Scale bar , 10 µm .

BMDMs from Spata2+/+ (WT) and Spata2-/- (KO) mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 1 h. ASC specks were assessed by Immunofluorescence with antibody against ASC and imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Scale bar, 10 µm.


Immunoblot analysis of ASC in soluble cell lysate and DSS cross - linked insoluble fraction of WT or Spata2 - KO BMDMs primed with LPS and stimulated without or with nigericin ( Nig ) for 1 h . Relative levels of ASC in cross - linked samples from LPS + Nig treated cells were quantitated and shown below .

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

Immunoblot analysis of ASC in soluble cell lysate and DSS cross-linked insoluble fraction of WT or Spata2-KO BMDMs primed with LPS and stimulated without or with nigericin (Nig) for 1 h. Relative levels of ASC in cross-linked samples from LPS+ Nig treated cells were quantitated and shown below.


Spata2 + / + ( WT ) and Spata2 - / - ( KO ) mice were injected intraperitoneally ( i . p . ) with alum ( 20 mg / kg bodyweight ) and sacrificed 6 h latter to collect peritoneal lavages . Absolute numbers of neutrophils recruited to the peritoneum were counted by FACS ( G )

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

Spata2+/+ (WT) and Spata2-/- (KO) mice were injected intraperitoneally (i.p.) with alum (20 mg/kg bodyweight) and sacrificed 6 h latter to collect peritoneal lavages. Absolute numbers of neutrophils recruited to the peritoneum were counted by FACS (G)


Spata2 + / + ( WT ) and Spata2 - / - ( KO ) mice were injected intraperitoneally ( i . p . ) with alum ( 20 mg / kg bodyweight ) and sacrificed 6 h latter to collect peritoneal lavages . levels of IL - 1β ( H ) and IL - 6 ( I ) in the peritoneal fluids were measured by ELISA .

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

Spata2+/+ (WT) and Spata2-/- (KO) mice were injected intraperitoneally (i.p.) with alum (20 mg/kg bodyweight) and sacrificed 6 h latter to collect peritoneal lavages. levels of IL-1β (H) and IL-6 (I) in the peritoneal fluids were measured by ELISA.


ELISA of IL - 1β secretion by WT or CYLD KO BMDMs that were either untreated ( - ) or LPS - primed and then treated with the indicated NLRP3 inducers .

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

ELISA of IL-1β secretion by WT or CYLD KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducers.


Immunoblot analysis of IL - 1β and active caspase - 1 p20 in cell supernatants ( Sup ) and the indicated proteins in cell extracts ( Cell ext ) from WT and CYLD KO BMDMs , stimulated as in ( A ) .

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Immunoblot analysis of IL-1β and active caspase-1 p20 in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) from WT and CYLD KO BMDMs, stimulated as in (A).


BMDMs from WT and CYLD KO mice were pretreated with Caspase - 1 inhibitor , Z - YVAD - FMK , for 30 min , primed with LPS , and then stimulated with Nigericin for 30 min . ASC specks were assessed by Immunofluorescence with antibody against ASC and the percentage of ASC speck - positive cells was quantified .

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

BMDMs from WT and CYLD KO mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 30 min. ASC specks were assessed by Immunofluorescence with antibody against ASC and the percentage of ASC speck-positive cells was quantified.


LDH release from WT and Cyld KO BMDMs primed with LPS and stimulated as indicated .

LDH release from WT and Cyld KO BMDMs primed with LPS and stimulated as indicated.


Immunoblotting analysis of active caspase - 1 p20 and IL - 1β in cell supernatants ( Sup ) and the indicated proteins in cell extracts ( Cell ext ) of WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or a Spata2 mutant ( F108A ) defective in CYLD binding that were primed with LPS and stimulated with Nigericin for 1 h .

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

Immunoblotting analysis of active caspase-1 p20 and IL-1β in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or a Spata2 mutant (F108A) defective in CYLD binding that were primed with LPS and stimulated with Nigericin for 1 h.


Confocal immunofluorescence analysis of Spata2 centrosome localization in HEK293 cells stably expressing control shRNA or shRNA against Spata2 , stained with Spata2 and γ - tubulin antibodies and the nuclear dye DAPI . Scale bar , 10 µm .

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Confocal immunofluorescence analysis of Spata2 centrosome localization in HEK293 cells stably expressing control shRNA or shRNA against Spata2, stained with Spata2 and γ-tubulin antibodies and the nuclear dye DAPI. Scale bar, 10 µm.


Confocal immunofluorescence analysis of Spata2 localization in RAW264 . 7 cells stably expressing GFP - Spata2 , stained with γ - tubulin antibody and DAPI . Scale bar , 10 µm .

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Confocal immunofluorescence analysis of Spata2 localization in RAW264.7 cells stably expressing GFP-Spata2, stained with γ-tubulin antibody and DAPI. Scale bar, 10 µm.


Confocal microscopy of HEK293 cells stably expressing GFP - tagged Spata2 or Spata2 mutants ( ΔPUB orΔ51 - 81 ) that were stained with γ - tubulin antibody and DAPI .

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Confocal microscopy of HEK293 cells stably expressing GFP-tagged Spata2 or Spata2 mutants (ΔPUB orΔ51-81) that were stained with γ-tubulin antibody and DAPI.


Confocal microscopy of HEK293 cells stably expressing GFP - tagged CYLD along with an empty vector or vectors encoding Flag - tagged Spata2 or Spata2 mutants ( ΔPUB orΔ51 - 81 ) stained as in ( C ) . Scale bar , 10 µm .

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Confocal microscopy of HEK293 cells stably expressing GFP-tagged CYLD along with an empty vector or vectors encoding Flag-tagged Spata2 or Spata2 mutants (ΔPUB orΔ51-81) stained as in (C). Scale bar, 10 µm.


Co - immunoprecipitation analysis of CYLD physical interaction with Spata2 or Spata2 mutants ( ΔPUB orΔ51 - 81 ) in HEK293 cells transfected with the indicated expression vectors . CYLD and Spata2 proteins were detected by immunoblot using anti - HA and anti - Flag antibodies , respectively .

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Co-immunoprecipitation analysis of CYLD physical interaction with Spata2 or Spata2 mutants (ΔPUB orΔ51-81) in HEK293 cells transfected with the indicated expression vectors. CYLD and Spata2 proteins were detected by immunoblot using anti-HA and anti-Flag antibodies, respectively.


Immunoblotting analysis of the active caspase - 1 p20 in cell supernatants ( Sup ) and pro - caspase - 1 and Flag - Spata2 proteins in cell extracts ( Cell ext ) from WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or Spata2 mutant defective in centrosome localization ( ΔPUB orΔ51 - 81 ) . Cells were primed with LPS and stimulated with Nigericin for 1 h . Data information : For all Immunoblotting data , molecular weights in kDa are indicated to the left .

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

Immunoblotting analysis of the active caspase-1 p20 in cell supernatants (Sup) and pro-caspase-1 and Flag-Spata2 proteins in cell extracts (Cell ext) from WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or Spata2 mutant defective in centrosome localization (ΔPUB orΔ51-81). Cells were primed with LPS and stimulated with Nigericin for 1 h. Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.


WT and Spata2 KO iBMDMs stably expressing an empty vector or a vector encoding Flag - NEK7 were untreated ( NT ) , primed with LPS for 4 h , or primed with LPS for 4 h followed by stimulation with Nigericin for 30 min . Flag - NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblotting . Relative levels of NLRP3 in immunoprecipitates were normalized to Flag - NEK7 and shown below .

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

WT and Spata2 KO iBMDMs stably expressing an empty vector or a vector encoding Flag-NEK7 were untreated (NT), primed with LPS for 4 h, or primed with LPS for 4 h followed by stimulation with Nigericin for 30 min. Flag-NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblotting. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag-NEK7 and shown below.


Co - immunoprecipitation analysis of PLK4 interaction with endogenous Spata2 and CYLD in parental HEK293 cells and HEK293 cells stably expressing GFP - PLK4 .

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Co-immunoprecipitation analysis of PLK4 interaction with endogenous Spata2 and CYLD in parental HEK293 cells and HEK293 cells stably expressing GFP-PLK4.


iBMDMs stably expressing GFP - PLK4 and parental iBMDMs were unstimulated or stimulated with 0 . 5 ug / ml LPS for 4h . The PLK4 interaction with endogenous CYLD was analyzed by co - immunoprecipitation with anti - GFP antibody .

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

iBMDMs stably expressing GFP-PLK4 and parental iBMDMs were unstimulated or stimulated with 0.5 ug/ml LPS for 4h. The PLK4 interaction with endogenous CYLD was analyzed by co-immunoprecipitation with anti-GFP antibody.


Confocal microscopy analysis of ASC specks in iBMDMs stably expressing GFP - ASC , pretreated for 2 days with DMSO and two different PLK4 inhibitors , Centrinone ( 150 nM ) and Centrinone - B ( 500 nM ) , primed with LPS for 4 h , and stimulated with Nigericin for 30 min . GFP - ASC specks were imaged and quantified ( > 500 cells counted ) . Scale bar , 10 µm .

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Confocal microscopy analysis of ASC specks in iBMDMs stably expressing GFP-ASC, pretreated for 2 days with DMSO and two different PLK4 inhibitors, Centrinone (150 nM) and Centrinone-B(500 nM), primed with LPS for 4 h, and stimulated with Nigericin for 30 min. GFP-ASC specks were imaged and quantified (>500 cells counted). Scale bar, 10 µm.


Immunoblot analysis of the indicated proteins in cell supernatants ( Sup ) and cell extracts ( Cell ext ) of LPS - primed and Nigericin - stimulated BMDMs that were pretreated for 2 days with PLK4 inhibitors , Centrinone ( 150 nM ) and Centrinone - B ( 500 nM ) ( E )

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

Immunoblot analysis of the indicated proteins in cell supernatants (Sup) and cell extracts (Cell ext) of LPS-primed and Nigericin-stimulated BMDMs that were pretreated for 2 days with PLK4 inhibitors, Centrinone (150 nM) and Centrinone-B(500 nM) (E)


LPS - primed and Nigericin - stimulated iBMDMs stably expressing a control shRNA or 3 different PLK4 - specific shRNAs ( F ) .

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LPS-primed and Nigericin-stimulated iBMDMs stably expressing a control shRNA or 3 different PLK4-specific shRNAs (F).


Immunoblot analysis of active caspase - 1 ( p20 ) generation in HEK293 cells that were transfected with inflammasome components ( Flag - tagged NLRP3 , NEK7 , ASC , and Caspase - 1 ) along with EGFP , GFP - tagged PLK4 , PLK4 kinase dead mutant ( D154A ) , or PLK1 and stimulated with 10 µM Nigericin for 1h 24h post - transfection .

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Immunoblot analysis of active caspase-1 (p20) generation in HEK293 cells that were transfected with inflammasome components (Flag-tagged NLRP3, NEK7, ASC, and Caspase-1) along with EGFP, GFP-tagged PLK4, PLK4 kinase dead mutant (D154A), or PLK1 and stimulated with 10 µM Nigericin for 1h 24h post-transfection.


WT and Spata2 CRISPR knock out ( KO ) iBMDMs stably expressing GFP - ASC were pretreated with DMSO or PLK4 inhibitor Centrinone at a concentration of 150nM for 2 days , primed with LPS , and stimulated with Nigericin for 1 h . GFP - ASC specks were imaged by confocal microscope , and percentage of ASC speck - positive cells was quantified .

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

WT and Spata2 CRISPR knock out (KO) iBMDMs stably expressing GFP-ASC were pretreated with DMSO or PLK4 inhibitor Centrinone at a concentration of 150nM for 2 days, primed with LPS, and stimulated with Nigericin for 1 h. GFP-ASC specks were imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified.


Co - immunoprecipitation analysis of NEK7 / NLRP3 interaction in iBMDMs stably expressing Flag - NEK7 that were transduced with control or PLK4 - specific shRNA . The cells were untreated , primed with LPS , or primed with LPS and stimulated with Nigericin . Flag - NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblot analysis . Relative levels of NLRP3 in immunoprecipitates were normalized to Flag - NEK7 and shown below .

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

Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in iBMDMs stably expressing Flag-NEK7 that were transduced with control or PLK4-specific shRNA. The cells were untreated, primed with LPS, or primed with LPS and stimulated with Nigericin. Flag-NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblot analysis. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag-NEK7 and shown below.


Co - immunoprecipitation analysis of NEK7 / NLRP3 interaction in HEK293 cells transfected with HA - NEK7 and Flag - NLRP3 along with ( + ) or without ( - ) GFP - PLK4 . The cells were either treated ( + ) or not treated ( - ) with the PLK4 inhibitor Centrinone .

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

Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with HA-NEK7 and Flag-NLRP3 along with (+) or without (-) GFP-PLK4. The cells were either treated (+) or not treated (-) with the PLK4 inhibitor Centrinone.


iBMDMs stably expressing Flag - tagged NEK7 or NEK7 mutant S204A were treated with LPS . Anti - Flag immunoprecipitates were prepared for immunoblot analysis for the interaction between NEK7 and endogenous NLRP3 .

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

iBMDMs stably expressing Flag-tagged NEK7 or NEK7 mutant S204A were treated with LPS. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for the interaction between NEK7 and endogenous NLRP3.


Immunoblot analysis of active caspase - 1 ( p20 ) in supernatants ( Sup ) and the indicated proteins in cell extracts ( Cell ext ) of iBMDMs stably expressing WT NEK7 or NEK7 mutant ( S204A ) , primed with LPS and stimulated with nigericin .

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

Immunoblot analysis of active caspase-1 (p20) in supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of iBMDMs stably expressing WT NEK7 or NEK7 mutant (S204A), primed with LPS and stimulated with nigericin.


iBMDMs stably expressing Flag - tagged WT NEK7 or NEK7 mutant S204A were left untreated or stimulated with LPS for indicated time . Anti - Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204 ( P - NEK7 ) .

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

iBMDMs stably expressing Flag-tagged WT NEK7 or NEK7 mutant S204A were left untreated or stimulated with LPS for indicated time. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204 (P-NEK7).


iBMDMs stably expressing Flag - tagged WT NEK7 were left untreated or pretreated with PLK4 inhibitor Centrinone , and stimulated without or with LPS for 4h . Anti - Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204 .

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

iBMDMs stably expressing Flag-tagged WT NEK7 were left untreated or pretreated with PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4h. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204.


iBMDMs stably expressing GFP - tagged PLK4 and parental iBMDMs were left untreated or stimulated with LPS for 4h . Anti - GFP immunoprecipitates were prepared for immunoblot analysis for the interaction between GFP - PLK4 and endogenous NEK7 .

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

iBMDMs stably expressing GFP-tagged PLK4 and parental iBMDMs were left untreated or stimulated with LPS for 4h. Anti-GFP immunoprecipitates were prepared for immunoblot analysis for the interaction between GFP-PLK4 and endogenous NEK7.


Co - immunoprecipitation analysis of NEK7 / NLRP3 interaction in HEK293 cells transfected with the indicated expression vectors , treated with either DMSO or 150 nM PLK4 inhibitor Centrinone .

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

Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with the indicated expression vectors, treated with either DMSO or 150 nM PLK4 inhibitor Centrinone.


WT and Spata2 KO iBMDMs stably expressing Flag - tagged WT NEK7 were left untreated or pretreated with the PLK4 inhibitor Centrinone , and stimulated without or with LPS for 4h . Anti - Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204 .

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

WT and Spata2 KO iBMDMs stably expressing Flag-tagged WT NEK7 were left untreated or pretreated with the PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4h. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204.


HEK293 cells stably expressing GFP - PLK4Δ24 were transfected with a control siRNA or siRNAs targeting Spata2 or CYLD . GFP - PLK4Δ24 was immunoprecipitated under denaturing conditions and immunoblotted for K63 - , K48 - linked and linear ubiquitination . Relative levels of PLK4 ubiquitination were normalized to GFP - PLK4 and shown below .

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

HEK293 cells stably expressing GFP-PLK4Δ24 were transfected with a control siRNA or siRNAs targeting Spata2 or CYLD. GFP-PLK4Δ24 was immunoprecipitated under denaturing conditions and immunoblotted for K63-, K48-linked and linear ubiquitination. Relative levels of PLK4 ubiquitination were normalized to GFP-PLK4 and shown below.


Co - immunoprecipitation analysis of the PLK4 - NEK7 interaction in HEK293 cells co - transfected with Flag - NEK7 and HA - PLK4 in the presence ( + ) or absence ( - ) of GFP , GFP - Spata2 , GFP - CYLD , or GFP - CYLD C601S , a catalytically inactive mutant .

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

Co-immunoprecipitation analysis of the PLK4-NEK7 interaction in HEK293 cells co-transfected with Flag-NEK7 and HA-PLK4 in the presence (+) or absence (-) of GFP, GFP-Spata2, GFP-CYLD, or GFP-CYLD C601S, a catalytically inactive mutant.


( A Representative images of HUVECs ( A ; n = 6 ) sprouting in a fibrin gel with VEGF ( 25ng / ml ) in the presence or absence of COCO . ( D Quantification of tube surface area of micrographs shown in ( A , ( D : * P = 0 . 0119 ( - vs COCO 30ng / ml ) ; * P = 0 . 0108 ( - vs COCO 60ng / ml )

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(A Representative images of HUVECs (A; n=6) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. (D Quantification of tube surface area of micrographs shown in (A, (D: *P=0.0119 (- vs COCO 30ng/ml); *P=0.0108 (- vs COCO 60ng/ml)


B ) Representative images of HRMECs ( B ; n = 4 ) sprouting in a fibrin gel with VEGF ( 25ng / ml ) in the presence or absence of COCO . E ) Quantification of tube surface area of micrographs shown in B ) . ( E : * * P = 0 . 0013 ( - vs COCO 30ng / ml ) ; * * * P = 0 . 00026 ( - vs COCO 60ng / ml ) ) .

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B) Representative images of HRMECs (B; n=4) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. E) Quantification of tube surface area of micrographs shown in B). (E: **P=0.0013 (- vs COCO 30ng/ml); ***P=0.00026 (- vs COCO 60ng/ml)).


( C ) Representative images of choroidal explants cultured for 5 days in the presence or absence of COCO . Scale bar , 500 μm . ( F ) Quantification of sprouting surface area of micrographs shown in ( C ; n = 6 ) .

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(C) Representative images of choroidal explants cultured for 5 days in the presence or absence of COCO. Scale bar, 500 μm. (F) Quantification of sprouting surface area of micrographs shown in (C; n=6).


( G ) COCO decreases scratch wound migration after 18h . Scale bar , 150 μm . ( H ) Quantification of wound closure .

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(G) COCO decreases scratch wound migration after 18h. Scale bar, 150 μm. (H) Quantification of wound closure.


( I ) COCO decreases EdU incorporation in cultured HUVECs . ( J ) Quantification of EdU incorporation .

(I) COCO decreases EdU incorporation in cultured HUVECs. (J) Quantification of EdU incorporation.


( K ) COCO does not induced apoptosis in HUVECs . ( L ) Quantification of apoptotic ( Annexin V - positive ) and viable ( Annexin V - negative ) cells ( n = 4 ) .

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(K) COCO does not induced apoptosis in HUVECs. (L) Quantification of apoptotic (Annexin V-positive) and viable (Annexin V-negative) cells (n= 4).


( B ) Retinal flat mounts of P5 mice injected with PBS , COCO or Flt1Fc are stained with IB4 ( negative images of the fluorescent signal ) . Scale bar , 100 μm . ( C ) Quantification of vascular length and number of branchpoints ( p - values are versus PBS treatment ) . Vessel length : * * P = 0 . 0059 ( PBS vs COCO ) ; * P = 0 . 0169 ( PBS vs Flt1Fc ) . Vessel junctions : * * P = 0 . 0063 ( PBS vs COCO ) ; * P = 0 . 0159 ( PBS vs Flt1Fc ) ; ( n = 4 mice / group ) .

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(B) Retinal flat mounts of P5 mice injected with PBS, COCO or Flt1Fc are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints (p-values are versus PBS treatment). Vessel length: **P=0.0059 (PBS vs COCO); *P=0.0169 (PBS vs Flt1Fc). Vessel junctions: **P=0.0063 (PBS vs COCO); *P=0.0159 (PBS vs Flt1Fc); (n=4 mice/group).


( D ) COCO does not increase empty collagen IV sleeves . Scale bar , 50 μm . ( E ) Quantification of % of IsoB4 + vessels to Coll IV + vessels ; ( n = 9 ) .

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(D) COCO does not increase empty collagen IV sleeves. Scale bar, 50 μm. (E) Quantification of % of IsoB4+ vessels to Coll IV+ vessels; (n=9).


( F ) Visualization of pericyte coverage ( NG2 : green ; IsoB4 : red ) in PBS - or COCO - injected retinas . Scale bar , 50 μm . ( G ) Quantification of percentage of IsoB4 vascular staining covered by NG2 staining ( % overlay ) ( n = 8 ) .

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(F) Visualization of pericyte coverage (NG2:green; IsoB4:red) in PBS- or COCO-injected retinas. Scale bar, 50 μm. (G) Quantification of percentage of IsoB4 vascular staining covered by NG2 staining (% overlay) (n=8).


( H ) COCO injections ( P1 ) do not result in apoptosis in P5 retinas . Scale bar , 40 μm . ( I ) Quantification of apoptotic cells in control and COCO - injected mice ( n = 3 ) .

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(H) COCO injections (P1) do not result in apoptosis in P5 retinas. Scale bar, 40 μm. (I) Quantification of apoptotic cells in control and COCO-injected mice (n=3).


( B ) Retinal flat mounts of P28 mice injected with PBS , COCO or Flt1Fc are stained with IB4 ( negative images of the fluorescent signal ) . Scale bar , 100 μm . ( C ) Quantification of vascular length and number of branchpoints ( p - values are versus PBS treatment ) . Vessel length : * * P = 0 . 0019 ( PBS vs COCO ) ; * P = 0 . 0128 ( PBS vs Flt1Fc ) . Vessel junctions : * * P = 0 . 0073 ( PBS vs COCO ) ; * P = 0 . 0302 ( PBS vs Flt1Fc ) ; ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Retinal flat mounts of P28 mice injected with PBS, COCO or Flt1Fc are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints (p-values are versus PBS treatment). Vessel length: **P=0.0019 (PBS vs COCO); *P=0.0128 (PBS vs Flt1Fc). Vessel junctions: **P=0.0073 (PBS vs COCO); *P=0.0302 (PBS vs Flt1Fc); (n=4).


( D ) COCO injections ( P1 , P7 and P14 ) do not result in ONL thinning in P28 retinas . Scale bar , 40 μm . ( E ) Quantification of apoptotic cells ( cleaved caspase - 3 ; CC3 ) in control and COCO - injected mice ( p - values are versus PBS treatment ) . ( n = 4 ) .

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(D) COCO injections (P1, P7 and P14) do not result in ONL thinning in P28 retinas. Scale bar, 40 μm. (E) Quantification of apoptotic cells (cleaved caspase-3; CC3) in control and COCO-injected mice (p-values are versus PBS treatment). (n=4).


( B ) Retinal flat mounts of adult mice injected with PBS or COCO for 5 days are stained with IB4 ( negative images of the fluorescent signal ) . Scale bar , 100 μm . ( C ) Quantification of vascular length and number of branchpoints ; ( n = 4 ) .

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(B) Retinal flat mounts of adult mice injected with PBS or COCO for 5 days are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints; (n=4).


( E ) Retinal flat mounts of adult mice injected with PBS or COCO for 28 days are stained with IB4 . Scale bar , 90 μm . ( F ) Quantification of vascular length and number of branchpoints ; ( n = 4 ) .

(E) Retinal flat mounts of adult mice injected with PBS or COCO for 28 days are stained with IB4. Scale bar, 90 μm. (F) Quantification of vascular length and number of branchpoints; (n=4).


( B ) Retinal flat mounts of P17 OIR mice injected with PBS , COCO or Flt1Fc are stained with IB4 . Red areas highlight vascular tufts .

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(B) Retinal flat mounts of P17 OIR mice injected with PBS, COCO or Flt1Fc are stained with IB4. Red areas highlight vascular tufts.


( C ) Quantification of neovascular tuft area in P17 OIR . * * P = 0 . 0021 ( PBS vs COCO ) ; * P = 0 . 0122 ( PBS vs Flt1Fc ) ; ( n = 6 ) . ( D ) Quantification of central avascular area in P17 OIR ; ( n = 6 ) .

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(C) Quantification of neovascular tuft area in P17 OIR. **P=0.0021 (PBS vs COCO); *P=0.0122 (PBS vs Flt1Fc); (n=6). (D) Quantification of central avascular area in P17 OIR; (n=6).


( F ) Choroidal flat mounts of adult mice subjected two weeks prior to Laser - CNV injected with PBS , COCO or Flt1Fc stained with IB4 ( green ) and phalloidin ( red ) . Scale bar , 100 μm . ( G ) Quantification of CNV surface area . * P = 0 . 0210 ( PBS vs COCO ) ; * P = 0 . 0113 ( PBS vs Flt1Fc ) ; ( n = 12 - 14 burns from 5 animals / group ) .

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(F) Choroidal flat mounts of adult mice subjected two weeks prior to Laser-CNV injected with PBS, COCO or Flt1Fc stained with IB4 (green) and phalloidin (red). Scale bar, 100 μm. (G) Quantification of CNV surface area. *P=0.0210 (PBS vs COCO); *P=0.0113(PBS vs Flt1Fc); (n=12-14 burns from 5 animals/group).


( A ) Western blot analysis of HUVECs treated 24 hours prior with COCO and stimulated with VEGF for up to 60 min . Immunoblots are representative of 4 independent experiments . ( B ) Densitometric analysis of western blot experiments . * P = 0 . 0457 ; ( n = 4 ) .

(A) Western blot analysis of HUVECs treated 24 hours prior with COCO and stimulated with VEGF for up to 60 min. Immunoblots are representative of 4 independent experiments. (B) Densitometric analysis of western blot experiments. * P=0.0457; (n=4).


( C ) COCO treatment does not alter VEGF receptor expression in HUVECs . Immunoblots are representative of 3 independent experiments . ( D ) Densitometric analysis of western blot experiments ; ( n = 3 ) .

(C) COCO treatment does not alter VEGF receptor expression in HUVECs. Immunoblots are representative of 3 independent experiments. (D) Densitometric analysis of western blot experiments; (n=3).


( A ) Western blot of whole adult human retina extracts incubated with an anti - human COCO antibody and revealing a unique band at ~ 36 kDa ( arrow ) . Recombinant human COCO was used as positive control .

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(A) Western blot of whole adult human retina extracts incubated with an anti-human COCO antibody and revealing a unique band at ~36 kDa (arrow). Recombinant human COCO was used as positive control.


( B ) Western blot of photoreceptors produced from human embryonic stem cells incubated with an anti - mouse COCO antibody and revealing a unique band at ~ 36 - 38 kDa ( arrow ) . Western blots are representative of 3 independent experiments .

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(B) Western blot of photoreceptors produced from human embryonic stem cells incubated with an anti-mouse COCO antibody and revealing a unique band at ~36-38 kDa (arrow). Western blots are representative of 3 independent experiments.


( C ) Immunofluoresence analysis of adult human retina sections with an anti - mouse COCO antibody . Specific immuno - reactivity was observed in multiple areas ( arrows ) when compared with sections only exposed to the secondary antibody .

(C) Immunofluoresence analysis of adult human retina sections with an anti-mouse COCO antibody. Specific immuno-reactivity was observed in multiple areas (arrows) when compared with sections only exposed to the secondary antibody.


( D ) RNAscope in situ hybridization ( Dand5 , top ; Negative probe ( dapB ) ; down ) and hematoxylin staining of P5 mouse retinas .

(D) RNAscope in situ hybridization (Dand5, top; Negative probe (dapB); down) and hematoxylin staining of P5 mouse retinas.


( E ) Dual RNAscope in situ hybridization and visual - arrestin immunohistochemistry of P5 mouse retinas . Images are representative of 4 animals .

(E) Dual RNAscope in situ hybridization and visual-arrestin immunohistochemistry of P5 mouse retinas. Images are representative of 4 animals.


( F ) Immunofluoresence analysis of HUVECs exposed to COCO for 5 hrs prior to fixation . Exogenously added COCO was detected using an anti - human COCO antibody , showing co - localization with human mitochondria ( MTCO1 antibody ) .

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(F) Immunofluoresence analysis of HUVECs exposed to COCO for 5 hrs prior to fixation. Exogenously added COCO was detected using an anti-human COCO antibody, showing co-localization with human mitochondria (MTCO1 antibody).


( B ) Differential gene expression heatmap generated by DESeq2 of top - altered genes of HUVECs treated with COCO compared to PBS - treated control cells ( n = 4 independent samples / group ) .

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(B) Differential gene expression heatmap generated by DESeq2 of top-altered genes of HUVECs treated with COCO compared to PBS-treated control cells (n = 4 independent samples/group).


( A ) Seahorse analysis of mitochondrial respiration ( OCR ) and glycolysis ( ECAR ) in HUVECs stimulated with COCO for 1 , 2 and 24 hours . * * P = 0 . 0032 for ECAR , 1hour ; * * P = 0 . 0079 for ECAR , 2hours ; * P = 0 . 0255 for OCR , 2hours ; ( n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Seahorse analysis of mitochondrial respiration (OCR) and glycolysis (ECAR) in HUVECs stimulated with COCO for 1, 2 and 24 hours. **P=0.0032 for ECAR, 1hour; **P=0.0079 for ECAR, 2hours; *P=0.0255 for OCR, 2hours; (n=5).


( B ) Media glucose levels ( BioNova analysis ) in HUVECs cultured with COCO for 1 or 6 hours , expressed as % of initial levels * P = 0 . 0459 ; ( n = 4 ) .

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(B) Media glucose levels (BioNova analysis) in HUVECs cultured with COCO for 1 or 6 hours, expressed as % of initial levels *P=0.0459; (n=4).


( C ) Evaluation of ATP content in HUVECs stimulated with COCO for 1 , 6 or 24 hours . * P = 0 . 0120 ( - vs 1hour ) ; * P = 0 . 0109 ( - vs 6hours ) ; * P = 0 . 0219 ( - vs 2 - DG ) ; ( n = 4 ) .

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(C) Evaluation of ATP content in HUVECs stimulated with COCO for 1, 6 or 24 hours. *P=0.0120 (- vs 1hour); *P=0.0109 (- vs 6hours); *P=0.0219 (- vs 2-DG); (n=4).


( D ) Determination of NAD + / NADH ratio in NADH for 6 or 24 hours with COCO . * * P = 0 . 0028 ( 6hours ) ; * P = 0 . 0207 ( 24hours ) ; ( n = 3 ) .

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(D) Determination of NAD+/NADH ratio in NADH for 6 or 24 hours with COCO. **P=0.0028 (6hours); *P=0.0207 (24hours); (n=3).


E ) Evaluation of pyruvate content in HUVECs stimulated with COCO for 1 , 3 , 6 , 12 or 24 hours . * P = 0 . 0244 ; ( n = 4 ) .

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E) Evaluation of pyruvate content in HUVECs stimulated with COCO for 1, 3, 6, 12 or 24 hours. *P=0.0244; (n=4).


( F ) Determination of cellular ROS using DCFDA fluorescence in HUVECs treated with COCO and / or N - acetylcystein . * P = 0 . 0343 ( - vs 48hours - NAC ) ; * P = 0 . 0114 ( - vs H2O2 - NAC ) ; * P = 0 . 0252 ( 48hours - NAC vs 48hours + NAC ) ; * * P = 0 . 0067 ( H2O2 - NAC vs H2O2 + NAC ) ; ( n = 4 ) .

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

(F) Determination of cellular ROS using DCFDA fluorescence in HUVECs treated with COCO and/or N-acetylcystein. *P=0.0343 (- vs 48hours-NAC); *P=0.0114 (- vs H2O2-NAC); *P=0.0252 (48hours-NAC vs 48hours+NAC); **P=0.0067 (H2O2-NAC vs H2O2+NAC); (n=4).


( G ) Determination of mitochondrial superoxide using MitoSox fluorescence in HUVECs treated with COCO . * P = 0 . 0372 ( - vs 24hours ) ; * P = 0 . 0137 ( - vs 3 - NP ) ; ( n = 3 ) .

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(G) Determination of mitochondrial superoxide using MitoSox fluorescence in HUVECs treated with COCO. *P=0.0372 (- vs 24hours); *P=0.0137 (- vs 3-NP); (n=3).


( H ) Representative images of choroidal explants cultured for 4 days in the presence or absence of COCO and / or N - acetylcystein . Scale bar , 500 μm . ( I ) Quantification of sprouting surface area of micrographs shown in ( H ) . * P = 0 . 0126 ( - vs COCO - NAC ) ; * P = 0 . 0200 ( COCO - NAC vs COCO + NAC ) ; ( n = 6 ) .

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

(H) Representative images of choroidal explants cultured for 4 days in the presence or absence of COCO and/or N-acetylcystein. Scale bar, 500 μm. (I) Quantification of sprouting surface area of micrographs shown in (H). *P=0.0126 (- vs COCO-NAC); *P=0.0200 (COCO-NAC vs COCO+NAC); (n=6).


To monitor the progression of the CLL - like population , blood was collected from the animals every four weeks up to an age of 60 weeks and analyzed by flow cytometry . Representative FACSplots show examples of stainingsof B220lowCD5 + B cells ( CLL - like cells ) and the selected gate for B ) .

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To monitor the progression of the CLL-like population, blood was collected from the animals every four weeks up to an age of 60 weeks and analyzed by flow cytometry. Representative FACSplots show examples of stainingsof B220lowCD5+B cells (CLL-like cells) and the selected gate for B).


To monitor the progression of the CLL - like population , blood was collected from the animals every four weeks up to an age of 60 weeks and analyzed by flow cytometry . The diagram displays the percentage of CLL - like cells in relation to all lymphocytesover a period of 60 weeks . Shown are mean values with ± SD . Significant differences between groups were determined by one - way ANOVA with Kruskal - Wallis test ( no normal distribution ) and corrected for multiple comparison with Dunn ' s test , * * p < 0 . 01 , * * * p < 0 . 001 . Summarized data from more than 40 independent experiments for individual time points , comprising n = 4 - 75 animals per time pointand genotype . X = no surviving TCL1 x Siglecg - / - animals at the ageof 60 weeks . + indicates that in these groups only 4 animals were used for the analysis and therefore no calculation of significance was possible .

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

To monitor the progression of the CLL-like population, blood was collected from the animals every four weeks up to an age of 60 weeks and analyzed by flow cytometry. The diagram displays the percentage of CLL-like cells in relation to all lymphocytesover a period of 60 weeks. Shown are mean values with ±SD. Significant differences between groups were determined by one-way ANOVA with Kruskal-Wallis test (no normal distribution) and corrected for multiple comparison with Dunn's test, **p<0.01, ***p<0.001. Summarized data from more than 40 independent experiments for individual time points, comprising n=4-75 animals per time pointand genotype. X = no surviving TCL1 x Siglecg-/-animals at the ageof 60 weeks. + indicates that in these groups only 4 animals were used for the analysis and therefore no calculation of significance was possible.


The absolute cell numbers of B220low CD5 + lymphocytes at different time points in the spleen are shown as mean values with ± SD . Significant differences between groups were tested by one - way ANOVA with Kruskal - Wallis test and corrected for multiple comparison with Dunn ' s test . The different time points were tested separately . Alongside representative dot plots show selection and percentages of B220low CD5 + cells in the spleen pre - gated on single cells , living cells and lymphocytes .

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The absolute cell numbers of B220low CD5+ lymphocytes at different time points in the spleen are shown as mean values with ±SD. Significant differences between groups were tested by one-way ANOVA with Kruskal-Wallis test and corrected for multiple comparison with Dunn's test. The different time points were tested separately. Alongside representative dot plots show selection and percentages of B220low CD5+ cells in the spleen pre-gated on single cells, living cells and lymphocytes.