C , D of mitophagy in worms transgenic for mito - Rosella but not tau ( WT ) , that were treated with NaN3 ( 8 mM , 1 h ) or CCCP ( 15 µM , 2 h ) . Data were analysed by unpaired t , NaN3 t = 5 . 56 , p < 0 . 0001 ; CCCP t = 3 . 92 p = 0 . 0002 . Data information : n = 16 - 25 worms / group for NaN3 23 - 39 worms / groups for CCCP experiments . Data are given as mean . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . are high - magnification of a small number of mitochondria in ventral

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

C, D Analysis of mitophagy in worms transgenic for mito-Rosella but not tau (WT), that were treated with NaN3 (8 mM, 1 h) or CCCP (15 µM, 2 h). Data were analysed by unpaired t test, NaN3 t = 5.56, p < 0.0001; CCCP t = 3.92 p = 0.0002. Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cord


E , F of mitophagy in worms expressing hTau in response to NaN3 treatment ( unpaired t , t = 1 . 62 , p = 0 . 1126 ) CCCP treatment ( Mann - Whitney , U = 113 , p = 0 . 002 ) . Data information : n = 16 - 25 worms / group for NaN3 23 - 39 worms / groups for CCCP experiments . Data are given as mean . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . are high - magnification of a small number of mitochondria in ventral

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

E, F Analysis of mitophagy in worms expressing hTau in response to NaN3 treatment (unpaired t test, t = 1.62, p = 0.1126) and CCCP treatment (Mann-Whitney test, U = 113, p = 0.002). Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cord


G , H of mitophagy in worms expressing hP301L in response to either NaN3 ( Mann - Whitney , U = 126 , p = 0 . 2711 ) or CCCP ( Mann - Whitney , U = 375 , p = 0 . 3697 ) treatment . Data information : n = 16 - 25 worms / group for NaN3 23 - 39 worms / groups for CCCP experiments . Data are given as mean . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . are high - magnification of a small number of mitochondria in ventral

O O O O B-EXP_ASSAY O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O B-ORGANISM 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 B-SUBCELLULAR O B-TISSUE

G, H Analysis of mitophagy in worms expressing hP301L in response to either NaN3 (Mann-Whitney test, U = 126, p = 0.2711) or CCCP (Mann-Whitney test, U = 375, p = 0.3697) treatment. Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cord


I Tau expression in worm lysates , as detected using human tau - specific antibody HT7 .

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I Tau expression in worm lysates, as detected using the human tau-specific antibody HT7.


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

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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 IL - 6 at 12 h after injection of WT or Spata2 - KO mice with 20 mg / kg LPS . Circles triangles represent mice . Horizontal bars indicate mean values . * * P < 0 . 01 ; * * * * P < 0 . 0001 . Statistical was performed using unpaired two - Student ' s t - .

B-EXP_ASSAY O B-TISSUE B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O B-ORGANISM 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 O O 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 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 of indicated phosphorylated ( P ) total in lysates of LPS - stimulated BMDMs derived from WT or Spata2 - KO mice . Molecular weights in kDa are indicated to 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 of indicated mRNAs in untreated or LPS - stimulated WT or Spata2 - KO BMDMs . Bars error bars represent mean ± S . D . of triplicate experiments . Statistical was performed using unpaired two - Student ' s t - .

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

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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 then treated with indicated NLRP3

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

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 NLRC4 inducer ( Salmonella infection ) or AIM2 inducer ( Poly ( dA : dT ) transfection ) ( B ) .

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 of mature IL - 1β p17 active Caspase - 1 p20 in supernatants ( Sup ) indicated in extracts ( ext ) of WT or Spata2 - KO BMDMs stimulated as in ( A ) .

B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-CELL_TYPE 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 Spata2 CRISPR knock out ( KO ) iBMDMs stably expressing GFP - ASC were primed with LPS stimulated with Nigericin for 1 h . GFP - ASC specks were imaged by confocal microscope , of ASC speck - positive was quantified . Scale bar , 10 µm .

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

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

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

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

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

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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 then treated with indicated NLRP3 inducers .

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD 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 of IL - 1β active caspase - 1 p20 in supernatants ( Sup ) indicated in extracts ( ext ) from WT CYLD KO BMDMs , stimulated as in ( A ) .

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

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

B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD 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 Cyld KO BMDMs primed with LPS stimulated as indicated .

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


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

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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 of Spata2 centrosome localization in HEK293 stably expressing control shRNA or shRNA against Spata2 , stained with Spata2 γ - tubulin antibodies dye DAPI . Scale bar , 10 µm .

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

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

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

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 stably expressing GFP - tagged Spata2 or Spata2 mutants ( ΔPUB orΔ51 - 81 ) that were stained with γ - tubulin antibody 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 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 of CYLD physical interaction with Spata2 or Spata2 mutants ( ΔPUB orΔ51 - 81 ) in HEK293 transfected with indicated expression vectors . CYLD Spata2 were detected by immunoblot using anti - HA 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 of active caspase - 1 p20 in supernatants ( Sup ) pro - caspase - 1 Flag - Spata2 in extracts ( ext ) from WT iBMDMs Spata2 KO iBMDMs reconstituted with WT Spata2 or Spata2 mutant defective in centrosome localization ( ΔPUB orΔ51 - 81 ) . were primed with LPS stimulated with Nigericin for 1 h . Data information : For Immunoblotting data , molecular weights in kDa are indicated to left .

B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY 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 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 association of endogenous NLRP3 was assessed by immunoblotting . Relative of NLRP3 in immunoprecipitates were normalized to Flag - NEK7 shown below .

O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-GENEPROD 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 B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD 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 of PLK4 interaction with endogenous Spata2 CYLD in parental HEK293 HEK293 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 parental iBMDMs were unstimulated or stimulated with 0 . 5 ug / ml LPS for 4h . PLK4 interaction with endogenous CYLD was analyzed by co - immunoprecipitation with anti - GFP antibody .

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

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

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

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

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

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


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

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

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

O B-GENEPROD O O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD 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 of NEK7 / NLRP3 interaction in iBMDMs stably expressing Flag - NEK7 that were transduced with control or PLK4 - specific shRNA . were untreated , primed with LPS , or primed with LPS stimulated with Nigericin . Flag - NEK7 was immunoprecipitated association of endogenous NLRP3 was assessed by immunoblot . Relative of NLRP3 in immunoprecipitates were normalized to Flag - NEK7 shown below .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD 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 of NEK7 / NLRP3 interaction in HEK293 transfected with HA - NEK7 Flag - NLRP3 along with ( + ) or without ( - ) GFP - PLK4 . were either treated ( + ) or not treated ( - ) with PLK4 inhibitor Centrinone .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD 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 B-GENEPROD O B-SMALL_MOLECULE 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 for interaction between NEK7 endogenous NLRP3 .

B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD 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 of active caspase - 1 ( p20 ) in supernatants ( Sup ) indicated in extracts ( ext ) of iBMDMs stably expressing WT NEK7 or NEK7 mutant ( S204A ) , primed with LPS stimulated with nigericin .

B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 for NEK7 phosphorylation at Ser 204 ( P - NEK7 ) .

B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD 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 , stimulated without or with LPS for 4h . Anti - Flag immunoprecipitates were prepared for immunoblot for NEK7 phosphorylation at Ser 204 .

B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE 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 parental iBMDMs were left untreated or stimulated with LPS for 4h . Anti - GFP immunoprecipitates were prepared for immunoblot for interaction between GFP - PLK4 endogenous NEK7 .

B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 of NEK7 / NLRP3 interaction in HEK293 transfected with indicated expression vectors , treated with either DMSO or 150 nM PLK4 inhibitor Centrinone .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE 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 Spata2 KO iBMDMs stably expressing Flag - tagged WT NEK7 were left untreated or pretreated with PLK4 inhibitor Centrinone , stimulated without or with LPS for 4h . Anti - Flag immunoprecipitates were prepared for immunoblot for NEK7 phosphorylation at Ser 204 .

O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE 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 stably expressing GFP - PLK4Δ24 were transfected with a control siRNA or siRNAs targeting Spata2 or CYLD . GFP - PLK4Δ24 was immunoprecipitated under denaturing conditions immunoblotted for K63 - , K48 - linked linear ubiquitination . Relative of PLK4 ubiquitination were normalized to GFP - PLK4 shown below .

B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD 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 of PLK4 - NEK7 interaction in HEK293 co - transfected with Flag - NEK7 HA - PLK4 in presence ( + ) or absence ( - ) of GFP , GFP - Spata2 , GFP - CYLD , or GFP - CYLD C601S , a catalytically inactive mutant .

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


( C ) NAD + / NADH was measured in mBA lines 17 . 5 days liver . Data represented as mean ± SEMs , obtained from three different liver . Significance determined by Student ' s t , two - , * P < 0 . 05 , * * P < 0 . 01 .

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

(C) NAD+/NADH ratio was measured in mBA cell lines and 17.5 days embryo liver. Data represented as mean ±SEMs, and obtained from three different cell line and embryo liver. Significance determined by Student's t test, two-tailed, *P < 0.05, **P < 0.01.


( D ) Immunostaining of 17 . 5 days tissues ( mid - brain , heart , liver , skin ) with anti - PAR antibody . Insets show higher magnification . Scale bar indicates 100 μm ( insert ) .

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

(D) Immunostaining of 17.5 days embryo tissues (mid-brain, heart, liver, and skin) with anti-PAR antibody. Insets show higher magnification. Scale bar indicates 100 μm (insert).


( E ) Wild - type ( WT ) Ews - / - mid - brain at E17 . 5 days were subjected to TUNEL . Scale bar indicates 100 μm ( lnsert ) .

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

(E) Wild-type (WT) and Ews-/- embryos mid-brain at E17.5 days were subjected to TUNEL assay. Scale bar indicates 100 μm (lnsert).


( A ) Western blot of Parp1 damage markers in Wild - type ( WT ) Ews - / - mBA . were treated with MMS ( 0 . 02 % ) in a time dependent manner . were fractionated into two groups , - bound ( Chro ) soluble ( Solu ) . ( B ) After treatment with MMS ( 0 . 02 % , 1hour ) , media was replaced to release damage . were fractionated specific kinetics at was analyzed by western blot .

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

(A) Western blot analysis of Parp1 and DNA damage markers in Wild-type (WT) and Ews-/- mBA cells. Cells were treated with MMS (0.02%) in a time dependent manner. Proteins were fractionated into two groups, chromatin-bound (Chro) proteins and soluble (Solu) proteins. (B) After treatment with MMS (0.02%, 1hour), the media was replaced to release the DNA damage. The proteins were fractionated and the specific protein kinetics at chromatin was analyzed by western blot.


( C ) GFP - PARP1 U2OS were transfected with siControl siEWS . Local damage was induced by micro - irradiation using a 405nm . Data represented as mean ± SEMs more than six were analyzed from six independent experiments . statistical significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicates 5 μm .

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

(C) GFP-PARP1 U2OS cells were transfected with siControl and siEWS. Local DNA damage was induced by micro-irradiation using a 405nm laser. Data represented as mean ±SEMs and more than six cells were analyzed from six independent experiments. The statistical significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicates 5 μm.


( D ) correlation spectroscopy ( FCS ) was measured after 15 minutes later following micro - irradiation ( Upper ) or under normal condition in GFP - PARP1 U2OS with either siControl or siEWS . Data represented as mean ± SEMs , more than 50 were analyzed , significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicate 5 μm .

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

(D) Fluorescence correlation spectroscopy (FCS) was measured after 15 minutes later following micro-irradiation (Upper) or under normal condition in GFP-PARP1 U2OS cells with either siControl or siEWS. Data represented as mean ±SEMs, more than 50 cells were analyzed, and significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicate 5 μm.


( E ) AsiSI endonuclease - integrated U2OS were transfected with siControl siEWS . were incubated with doxycycline for 4 hours to induce DSBs . With or without changing Dox - added media to fresh media for 2 hours ( for release samples ) , amount of - associated PARP1 was measured using ChIP . ( N . T : Non - treat , T : AsiSI treat , Release : Damage released samples ) . Data represented as mean ± SEMs , technical repeats ( n = 3 ) , significance determined by two - way ANOVA , * * * P < 0 . 001 .

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

(E) AsiSI endonuclease-integrated U2OS cells were transfected with siControl and siEWS. Cells were incubated with doxycycline for 4 hours to induce DSBs. With or without changing the Dox-added media to fresh media for 2 hours (for release samples), the amount of chromatin-associated PARP1 was measured using ChIP assay. (N.T: Non-treat, T: AsiSI treat, Release: Damage released samples). Data represented as mean ±SEMs, and technical repeats (n=3), significance determined by two-way ANOVA, ***P< 0.001.


( A ) Total of PAR in whole lysate were measured using western blot . Wild - type ( WT ) Ews - / - mBA were treated with H2O2 , ( 1 mM , 20 minutes ) with or without Olaparib ( 5 μM , 7 hours ) .

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

(A) Total levels of PAR in whole cell lysate were measured using western blot analysis. Wild-type (WT) and Ews-/- mBA cells were treated with H2O2, (1 mM, 20 minutes) with or without Olaparib (5 μM, 7 hours).


( B ) kinetics of PAR accumulation was analyzed by western blotting . WT Ews - / - were treated with H2O2 ( 1 mM , 20 minutes ) , followed by incubation in fresh media for indicated times to allow release of

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

(B) The kinetics of PAR accumulation was analyzed by western blotting. WT and Ews-/- cells were treated with H2O2 (1 mM, 20 minutes), followed by incubation in fresh media for the indicated times to allow release of DNA damage


( C ) Immunohistochemistry detection of PAR by ADP - ribose antibody in WT Ews - / - after H2O2 treatment ( 1 mM , 20 minutes ) recovery from damage . Right graph displays mean of intensities measured from 500 . Data represented as mean ± SEMs , significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicate 20 μm .

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

(C) Immunohistochemistry detection of PAR by ADP-ribose antibody in WT and Ews-/- cells after H2O2 treatment (1 mM, 20 minutes) and recovery from DNA damage. Right graph displays mean of intensities measured from 500 cells. Data represented as mean ±SEMs, significance determined by One-way ANOVA, ***P < 0.001. Scale bar indicate 20 μm.


( D , E ) Whole level of PAR was measured in WT Ews - / - mBA upon treatment with ( D ) MMS ( 0 . 02 % , 60 minutes ) ( E ) with or without recovery from damage .

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

(D, E) Whole cell level of PAR was measured in WT and Ews-/- mBA cells upon treatment with (D) MMS (0.02%, 60 minutes) (E) with or without recovery from DNA damage.


( F ) NAD + / NADH was measured in mBA lines following treatment of H2O2 ( 1 mM , 10 minutes ) . Data represented as mean ± SEMs , technical repeats ( n = 3 ) . Significance determined by Student ' s t , two - , * * P < 0 . 01 , * * * P < 0 . 001 .

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

(F) NAD+/NADH ratio was measured in mBA cell lines following treatment of H2O2 (1 mM, 10 minutes). Data represented as mean ±SEMs, and technical repeats (n=3). Significance determined by Student's t test, two-tailed, **P < 0.01, ***P< 0.001.


( A ) PARP1 EWS interaction was visualized in using CUPID system . After transfection of mRCD - EWS GC3 - PARP1 ( EGFP - PARP1 ) plasmid , were treated with either H2O2 ( 1 mM , 10 minutes ) or PMA . Scale bar indicates 5 μm .

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

(A) PARP1 and EWS interaction was visualized in cells using the CUPID system. After transfection of mRCD-EWS and GC3-PARP1 (EGFP-PARP1) plasmid, the cells were treated with either H2O2 (1 mM, 10 minutes) or PMA. Scale bar indicates 5 μm.


( B ) Endogenous interaction between EWS PARP1 was measured using immunoprecipitation western blot . were treated with H2O2 ( 1 mM , 10 minutes ) with or without Olaparib ( 5 μM , 7 hours ) .

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

(B) Endogenous interaction between EWS and PARP1 was measured using immunoprecipitation western blot analysis. Cells were treated with H2O2 (1 mM, 10 minutes) with or without Olaparib (5 μM, 7 hours).


( C ) MMS - treated , with or without Olaparib , were immunoprecipitated using an anti - EWS antibody immunoblotted by anti - PAR antibody ( upper ) . Pre - Olaparib group was treated with Olaparib for 3 hours before MMS treatment ( 0 . 02 % 30 minutes ) after - Olaparib group treated Olaparib after 10 minutes following treatment of MMS . Lower blot indicates input of anti - EWS antibody .

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

(C) MMS-treated cells, with or without Olaparib, were immunoprecipitated using an anti-EWS antibody and immunoblotted by anti-PAR antibody (upper). Pre-Olaparib group was treated with Olaparib for 3 hours before MMS treatment (0.02% 30 minutes) and after-Olaparib group treated Olaparib after 10 minutes following treatment of MMS. Lower blot indicates input of anti-EWS antibody.


( D ) In vitro PAR binding . THRAP3 binding to PAR was used as a positive control BSA binding to PAR was used as a negative control .

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(D) In vitro PAR binding assay. THRAP3 binding to PAR was used as a positive control and BSA binding to PAR was used as a negative control.


( E ) Schematic map of nine EWS mutants ( left ) IP - western blot ( right ) . GFP - PARP1 siPARG were transfected into HEK - 293 with each EWS mutants plasmid . After H2O2 treatment , were conducted to IP with anti - Flag antibody .

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(E) Schematic map of nine EWS mutants (left) and IP-western blot analysis (right). GFP-PARP1 and siPARG were transfected into HEK-293 cells with each EWS mutants plasmid. After H2O2 treatment, the cells were conducted to IP with anti-Flag antibody.


( F ) Western blot of PARP1 accumulation on . EWS - WT M4 mutants were transfected into EWS depleted HEK - 293 . After treatment of MMS ( 0 . 02 % , 1 hour ) , were fractionated to either - bound or soluble fraction . ( G ) of relative amount of PARP1 γH2AX on , divided by H3 , from three independent western experiments are presented . Data represented as mean ± SEMs , were measured from 3 independent experiments . Significance determined by t - . * P < 0 . 05 , * * P < 0 . 01 . n . s indicate non -

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(F) Western blot analysis of PARP1 accumulation on chromatin. EWS-WT and M4 mutants were transfected into EWS depleted HEK-293 cells. After treatment of MMS (0.02%, 1 hour), the proteins were fractionated to either chromatin-bound or soluble fraction. (G) Quantification of relative amount of PARP1 and γH2AX on chromatin, divided by chromatin H3, from three independent western experiments are presented. Data represented as mean ±SEMs, were measured from 3 independent experiments. Significance determined by t-test. * P < 0.05, **P < 0.01. n.s indicate non-significance


( H ) WT Ews - / - were transfected with EWS - WT - M4 mutant viability was measured 24 hours after MMS treatment ( indicated ) . Error bars represent as mean ± SEMs , technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * P < 0 . 01 . n . s indicate non - significance ,

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(H) WT and Ews-/- cells were transfected with EWS-WT and -M4 mutant and cell viability was measured 24 hours after MMS treatment (indicated concentration). Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, **P < 0.01. n.s indicate non-significance,


( A ) Relative viability of transfected with siCon ( WT ) , siEWS ( EWS KD ) , PARP1 ( PARP - 1 KD ) , double siRNA ( DKD ) after MMS treatment . Error bars represent as mean ± SEMs , technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . ( * * * ) indicates differences between DKD with EWS KD .

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(A) Relative cell viability of cells transfected with siCon (WT), siEWS (EWS KD), PARP1 (PARP-1 KD), and double siRNA (DKD) after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. Analysis (***) indicates differences between DKD with EWS KD.


( B ) Additional Parp1 knockout in Ews knockout ( DKO ) were subjected to viability after MMS treatment . Error bars represent as mean ± SEMs , technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . * * * indicated differences between DKO with EWS - KO .

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(B) Additional Parp1 knockout in Ews knockout cells (DKO) were subjected to viability test after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. *** indicated differences between DKO with EWS-KO.


C ) After transfection of siCon , siEWS , PARP1 , double siRNA , effect of co - depletion of PARP1 EWS on γH2AX phosphorylated CHK1 upon MMS treatment was analyzed by western blot .

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C) After transfection of siCon, siEWS, PARP1, and double siRNA, the effect of co-depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by western blot.


D ) After treatment of MMS , effect of additional Parp1 knockout in Ews knockout ( Double KO ) to γH2AX phosphorylated CHK1 were analyzed using western blot .

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D) After treatment of MMS, the effect of additional Parp1 knockout in Ews knockout cells (Double KO) to the γH2AX and phosphorylated CHK1 were analyzed using western blot.


E ) Whole expression of PAR in mBA ( WT : Ews - WT , KO : Ews - KO , DKO : double KO ) .

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E) Whole cell expression of PAR in mBA (WT: Ews-WT, KO: Ews-KO, DKO: double KO).


F ) Relative NAD + / NADH ratios were measured in WT , Ews - / - DKO . Error bars represent as mean ± SEMs , technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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F) Relative NAD+/NADH ratios were measured in WT, Ews-/- and DKO cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.


G ) PARGi ( 5 μM , 24 hours ) treated WT Ews - / - were subjected to viability . Error bars represent as mean ± SEMs , technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA .

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G) PARGi (5 μM, 24 hours) treated WT and Ews-/- cells were subjected to cellular viability assay. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA.


H ) Relative viability was measured after NMN treatment , with or without MMS pretreatment , in WT Ews - / - . Error bars represent as mean ± SEMs , technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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H) Relative cellular viability was measured after NMN treatment, with or without MMS pretreatment, in WT and Ews-/- cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.


I ) - bound PARP1 was quantified by western blot in WT Ews - / - treated with Olaparib ( 5 μM , 24 hours ) treatment .

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I) Chromatin-bound PARP1 was quantified by western blot in WT and Ews-/- cells treated with Olaparib (5 μM, 24 hours) treatment.


J ) Relative viability was measured in WT Ews - / - upon treatment of Olaparib for 24 hours . Error bars represent as mean ± SEMs , technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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J) Relative viability was measured in WT and Ews-/- cells upon treatment of Olaparib for 24 hours. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.


( A ) Western blot of PARP1 in . Two Ewing Sarcoma ( CHP100 A4573 ) were treated with MMS ( 0 . 02 % , 1 hour ) with or without release ( 1 hour ) from MMS ( N . T : non treat , T : treat , R : release sample ) .

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(A) Western blot analysis of PARP1 in chromatin. Two Ewing Sarcoma cells (CHP100 and A4573) were treated with MMS (0.02%, 1 hour) with or without release (1 hour) from MMS (N.T: non treat, T: treat, R: release sample).


( B ) U2OS two Ewing Sarcoma were treated MMS ( 0 . 02 % , 30 minutes ) subjected to immunofluorescence by PARP1 antibody after bound fraction . Scale bar indicates 20 μm ( left ) . Red indicates mean more than 300 were analyzed . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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(B) U2OS and two Ewing Sarcoma cells were treated MMS (0.02%, 30 minutes) and subjected to immunofluorescence by PARP1 antibody after chromatin bound fraction. Scale bar indicates 20 μm (left). Red line indicates mean and more than 300 cells were analyzed. Significance determined by Two-way ANOVA, ***P < 0.001.


( C ) Human Ewing Sarcoma histology samples were analyzed by immuno - histochemistry using PAR antibody . Scale bar represents 100 mm .

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(C) Human Ewing Sarcoma histology samples were analyzed by immuno-histochemistry using PAR antibody. Scale bar represents 100 mm.


( C ) Reaction progress was measured for Twist COVID - 19 synthetic ( MT007544 . 1 ) from 1 million molecules of ( 106 ) , then titrated down by log10 dilutions . colorimetric findings of LAMP are on a yellow to pink gradient with higher copies of SARS - CoV - 2 corresponding to a yellow color . limit of detection ( LoD ) range is shown with a gradient after 30 minutes between 10 100 viral copies ( lower right ) .

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(C) Reaction progress was measured for the Twist COVID-19 synthetic RNA (MT007544.1) from 1 million molecules of virus (106), then titrated down by log10 dilutions. The colorimetric findings of the LAMP assay are based on a yellow to pink gradient with higher copies of SARS-CoV-2 RNA corresponding to a yellow color. The limit of detection (LoD) range is shown with a gradient after 30 minutes between 10 and 100 viral copies (lower right).


( D ) Replicates of titrated viral copies using LAMP , as measured by Quantifluor .

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(D) Replicates of the titrated viral copies using LAMP, as measured by Quantifluor fluorescence.


( E ) sensitivity specificity of LAMP from 201 ( 132 negative 69 positive for SARS - CoV - 2 , as measured by qRT - PCR ) . Thresholds are quantified by Quantifluor .

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(E) The sensitivity and specificity of the LAMP assay from 201 patients (132 negative and 69 positive for SARS-CoV-2, as measured by qRT-PCR). Thresholds are DNA quantified by the Quantifluor.


LAMP . ( a ) Clinical samples tested by qRT - PCR ( Positive , red or Negative , grey ) were sequenced run through LAMP . These results were compared to buffer blanks ( Negative Control ) , grey , Synthetic RNAs or Vero 6 extracts with SARS - CoV - 2 infection ( Positive Controls , red ) , Subway Samples ( Environmental , blue ) . Read proportions are shown on y - axis .

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LAMP.(a) Clinical samples tested by qRT-PCR (Positive, dark red or Negative, light grey) were sequenced and run through the LAMP assay. These results were compared to the buffer blanks (Negative Control), dark grey, Synthetic RNAs or Vero 6 cell extracts with SARS-CoV-2 infection (Positive Controls, light red), and Subway Samples (Environmental, blue). Read proportions are shown on the y-axis.


( b ) SARS - CoV - 2 abundance , as measured with NGS of reads ( y - axis ) is compared to Ct Threshold for qRT - PCR ( x - axis ) , with lower Ct values representing higher viral abundance , LAMP reaction output ( Fluorimeter values , black to yellow scale ) .

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(b) SARS-CoV-2 abundance, as measured with NGS and percentage of reads (y-axis) is compared to the Ct Threshold for qRT-PCR (x-axis), with lower Ct values representing higher viral abundance, and the LAMP reaction output (Fluorimeter values, black to yellow scale).


( c , d ) Read mapping to archaea ( red ) , ( green ) , fungi ( yellow ) , human ( blue ) , SARS - CoV - 2 ( orange ) , other ( grey ) , across clinical controls ( CN , CP ) , environmental samples , qRT - PCR negative , qRT - PCR positive samples .

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(c,d) Read mapping to archaea (red), bacteria (green), fungi (yellow), human (blue), and SARS-CoV-2 (orange), and other viruses (grey), across the clinical controls (CN, CP), environmental samples, qRT-PCR negative, and qRT-PCR positive samples.


coverage ( a ) coverage plot across SARS - CoV - 2 genomes ( viral coordinate on bottom , colored by sample ) from a representative set of clinical positive samples . Sample names with suffixes CN N are clinical negative ( buffer ) , P are qRT - PCR positive , CP are Vero 6 with .

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coverage(a) The coverage plot across the SARS-CoV-2 genomes (viral coordinate on bottom, colored by sample) from a representative set of clinical positive samples. Sample names with the suffixes CN and N are clinical negative (buffer), P are qRT-PCR positive, and CP are Vero 6 cells with virus.


phylogenetic placement of these COVID samples is shown on tree ( left ) global map of known COVID genomes ( right ) . Genetic variants called from - seq data show a range of variants that are distinct from Wuhan reference , samples from this study , highlighted in blue , show enrichment for European Asian alleles .

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The phylogenetic placement of these COVID samples is shown on the tree (left) and the global map of known COVID genomes (right). Genetic variants called from the RNA-seq data show a range of variants that are distinct from the Wuhan reference strain, and the samples from this study, highlighted in blue, show enrichment for European and Asian alleles.


( b ) Proportion of L ( green ) S ( yellow ) are shown for NYC . Phylogeny of samples from this study on left total GISAID samples on right , with a map of variants in this study ' s samples in middle , colored by event type sized by number of nucleotides impacted . Annotation track on bottom shows frequency of alternate alleles in this study in GISAID database .

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(b) Proportion of the L (green) and S strain (yellow) are shown for the NYC viruses. Phylogeny of samples from this study on the left and total GISAID samples on the right, with a map of variants in this study's samples in the middle, colored by event type and sized by number of nucleotides impacted. Annotation track on the bottom shows frequency of alternate alleles in this study and in the GISAID database.


( c ) 9 - bp deletion in ORRF1b ( NSP1 ) that was detected in samples from two was confirmed in GISAID database ( below ) .

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(c) The 9-bp deletion in ORRF1b (NSP1) that was detected in samples from two patients was confirmed in the GISAID database (below).


( d ) Comparison of ORF1b of SARS - CoV - 2 SARS - CoV genomes with 9 - bp deletion region shown enlarged .

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(d) Comparison of the ORF1b of the SARS-CoV-2 and SARS-CoV genomes with the 9-bp deletion region shown enlarged.


( a ) Six variant alleles were found to be significantly enriched by population allelic fraction within this set of 93 WCM / NYP cases as compared with non - NYC strains of Nextstrain clade A2a ( 1059 : C > T P = 6 . 26e - 22 , 11916 : C > T P = 3 . 09e - 9 , 18998 : C > T P = 2 . 51e - 7 , 20755 : A > C P = 1 . 32e - 4 , 25563 : G > T P = 9 . 6e - 23 , 29540 : G > A P = 2 . 51e - 7 ) . These variants demonstrated similar PAF enrichment within other NYC strains as compared to non - NYC clade A2a ( 1059 : C > T P = 4 . 35e - 84 , 11916 : C > T P = 2 . 16e - 43 , 18998 : C > T P = 1 . 34e - 25 , 20755 : A > C P = 1 . 67e - 4 , 25563 : G > T P = 2 . 60e - 83 , 29540 : G > A P = 1 . 34e - 46 ) .

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(a) Six variant alleles were found to be significantly enriched by population allelic fraction within this set of 93 WCM/NYP cases as compared with non-NYC strains of Nextstrain clade A2a (1059:C>T P = 6.26e-22, 11916:C>T P = 3.09e-9, 18998:C>T P= 2.51e-7, 20755:A>C P = 1.32e-4, 25563:G>T P = 9.6e-23, 29540:G>A P = 2.51e-7). These variants demonstrated similar PAF enrichment within all other NYC strains as compared to non-NYC clade A2a (1059:C>T P = 4.35e-84, 11916:C>T P = 2.16e-43, 18998:C>T P= 1.34e-25, 20755:A>C P = 1.67e-4, 25563:G>T P = 2.60e-83, 29540:G>A P = 1.34e-46).


( b ) ( left ) Phylogenetic tree produced by Nextstrain with clade affiliations nodes corresponding to WCM / NYP in red other NYC cases in green . ( right ) occurrence of six NYC - enriched alleles 9 nucleotide deletion across genomes .

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(b) (left) Phylogenetic tree produced by the Nextstrain analysis with clade affiliations and nodes corresponding to WCM/NYP in red and other NYC cases in green. (right) occurrence of the six NYC-enriched alleles and the 9 nucleotide deletion across genomes.


Samples were quantified by a range of viral detection methods , including LAMP ( Quantifluor ) , - seq ( log10 SARS - CoV - 2 % of reads ) , qRT - PCR ( Ct values ) to create a three - tier range of viral load for positive samples ( right ) compared to clinically - annotated negative samples ( class , red or grey ) . ( bottom ) differentially expressed genes of COVID + compared to COVID - showed up - regulated ( orange ) genes as well as down - regulated ( purple ) genes .

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Samples were quantified by a range of viral detection methods, including LAMP (Quantifluor), RNA-seq (log10 SARS-CoV-2 % of reads), and qRT-PCR (Ct values) to create a three-tier range of viral load for the positive samples (right) compared to the clinically-annotated negative samples (class, red or grey). (bottom) The differentially expressed genes of COVID+ patients compared to COVID-patients showed up-regulated (orange) genes as well as down-regulated (purple) genes.


( b ) Up - regulated genes , with boxplots across samples , include IFI6 , ACE2 , SHFL , HERC6 , IFI27 , IFIT1 , on data from ( c ) , which is total set of DEGs . full set is shown in an intersecting heat map , with a set of up - regulated genes ( orange ) distinct form set of down - regulated genes ( purple ) , compared to genes that are not significantly differently expressed ( grey ) in any comparison ( DESeq2 , q - value < 0 . 01 , | logFC | > 0 . 58 ) .

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(b) Up-regulated genes, with boxplots across all samples, include IFI6, ACE2, SHFL, HERC6, IFI27, and IFIT1, based on data from (c), which is the total set of DEGs. The full set is shown in an intersecting heat map, with a core set of up-regulated genes (orange) distinct form the set of down-regulated genes (purple), compared to genes that are not significantly differently expressed (grey) in any comparison (DESeq2, q-value <0.01, |logFC| >0.58).


( e ) Survival curves for requiring mechanical respiration ( identified by intubation procedure notes ) ( e ) mortality ( f ) . with a history of ACE inhibitor exposure were more likely to require intubation ( HR = 2 . 63 95 % CI 2 . 01 - 3 . 43 , p = 1 . 22E - 12 ; ( e ) less likely to survive ( HR = 1 . 68 95 % CI : 1 . 22 - 2 . 31 , p = 1 . 42E - 03 ; ( f ) . Because several individuals were intubated shortly before they first tested positive for SARS - CoV - 2 infection , each first positive was set back by seven days to account for testing delays .

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(e)Survival curves for requiring mechanical respiration (identified by intubation procedure notes) (e) and mortality (f). Patients with a history of ACE inhibitor exposure were more likely to require intubation (HR=2.63 95%CI 2.01-3.43, p=1.22E-12; (e) and less likely to survive (HR=1.68 95%CI: 1.22-2.31, p=1.42E-03; (f). Because several individuals were intubated shortly before they first tested positive for SARS-CoV-2 infection, each first positive test was set back by seven days to account for testing delays.


A - C . Enzyme - linked immunosorbent ( ELISA ) of IL - 6 , TNF - α IFN - β real - time PCR of IL - 6 , TNF - α IFN - β mRNA in peritoneal macrophage isolated from wild type RKIP - deficient mice treated with VSV ( MOI , 0 . 1 ) ( A ) .

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

A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice treated with VSV (MOI,0.1) (A).


A - C . Enzyme - linked immunosorbent ( ELISA ) of IL - 6 , TNF - α IFN - β real - time PCR of IL - 6 , TNF - α IFN - β mRNA in peritoneal macrophage isolated from wild type RKIP - deficient mice treated with SEV ( MOI , 10 ) ( B ) .

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

A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice treated with SEV (MOI,10) (B).


A - C . Enzyme - linked immunosorbent ( ELISA ) of IL - 6 , TNF - α IFN - β real - time PCR of IL - 6 , TNF - α IFN - β mRNA in peritoneal macrophage isolated from wild type RKIP - deficient mice transfected with poly ( I : C ) ( 1 μg / ml ) ( C ) .

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

A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice transfected with poly(I:C) ( 1 μg/ml) (C).


D . - activated sorting ( FACS ) TCID50 ( 50 % tissue culture infective dose ) assessing proliferation level of VSV . RAW264 . 7 stably overexpressed flag - RKIP was infected with VSV - eGFP at MOI of 1 for indicated time . fusion fluorophore were analyzed by FACS . Then fresh medium with VSV - eGFP infection was serially diluted on monolayer of HEK293 for 3 - 7 days TCID50 was measured .

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D. Fluorescence-activated cell sorting (FACS) and TCID50 (50% tissue culture infective dose) assessing the proliferation level of VSV. RAW264.7 cells line stably overexpressed flag-RKIP was infected with VSV-eGFP at MOI of 1 for the indicated time. The fusion protein fluorophore were analyzed by FACS. Then fresh medium with the VSV-eGFP infection was serially diluted on the monolayer of HEK293 cells for 3-7 days and TCID50 was measured.


E . FACS infection of wild type RKIP - deficient peritoneal macrophage infected with VSV - eGFP at MOI of 1 . fresh medium with VSV - eGFP infection 9 hr were collected for TCID50 assessing .

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

E. FACS analysis the infection of wild type and RKIP-deficient peritoneal macrophage infected with VSV-eGFP at MOI of 1. The fresh medium with the VSV-eGFP infection 9 hr were collected for TCID50 assessing.


A . Wide - type ( RKIP + / + ) RKIP deficiency ( RKIP - / - ) chimera were intraperitoneally injected with VSV ( 1x107 pfu / g ) , 12 hours later , ELISA of IFN - β IL - 6 in serum ( n = 6 per group ) .

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

A. Wide-type (RKIP+/+) and RKIP deficiency (RKIP-/-) chimera were intraperitoneally injected with VSV (1x107 pfu/g), 12 hours later, ELISA of IFN-β and IL-6 in serum (n=6 per group).


B . Determination of VSV loads in organs by TCID50 12 hours later after RKIP + / + RKIP - / - chimera mice ( n = 6 per group ) intraperitoneally injected with VSV ( 1x107 pfu / g ) .

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B. Determination of VSV loads in organs by TCID50 assay 12 hours later after RKIP+/+ and RKIP-/- chimera mice (n = 6 per group) intraperitoneally injected with VSV (1x107 pfu/g).


C . Real - time PCR of IFN - β mRNA VSV expression in liver from RKIP + / + RKIP - / - chimera mice 12 hours later intraperitoneally injected with VSV ( 1x107 pfu / g ) ( n = 6 per group ) .

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C. Real-time PCR analysis of IFN-β mRNA and VSV expression in liver from RKIP+/+ and RKIP-/- chimera mice 12 hours later intraperitoneally injected with VSV (1x107 pfu/g) (n = 6 per group).


D . Pathology of wild type RKIP deficient chimeric mice in response to VSV infection . Hematoxylin eosin of liver lung sections from mice in ( A ) . Scale bar , 100 μm ( left ) or 50 μm ( right ) .

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D. Pathology of wild type and RKIP deficient chimeric mice in response to VSV infection. Hematoxylin and eosin staining of liver and lung sections from mice in (A). Scale bar, 100 μm (left) or 50 μm (right).


E . Real - time PCR of IFN - β mRNA expression in peritoneal macrophages from RKIP + / + RKIP - / - chimera mice 12 hours after intraperitoneally injected with VSV ( 1x107 pfu / g ) ( n = 6 per group ) .

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E. Real-time PCR analysis of IFN-β mRNA expression in peritoneal macrophages from RKIP+/+ and RKIP-/- chimera mice 12 hours after intraperitoneally injected with VSV (1x107 pfu/g) (n = 6 per group).


F . Survival curve for 8 - old wild type RKIP deficient chimeras challenged with VSV ( 1x108 pfu / g , i . v . ) ( n = 10 per group ) . * p < 0 . 05 Wilcoxon .

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F. Survival curve for 8 weeks-old wild type and RKIP deficient chimeras challenged with VSV (1x108 pfu/g, i.v.) (n=10 per group). *p<0.05 Wilcoxon Test .


A . Immunoblot of p - TBK1 , - IKKi , - IRF3 , - P38 , - JNK1 / 2 , - ERK1 / 2 - IκBα in lysates of RKIP + / + , RKIP + / - RKIP - / - peritoneal macrophages stimulated with VSV ( MOI , 0 . 1 ) for indicated time .

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A. Immunoblot analysis of p-TBK1, -IKKi, -IRF3, -P38, -JNK1/2, -ERK1/2 and -IκBα in lysates of RKIP+/+, RKIP+/- and RKIP-/- peritoneal macrophages stimulated with VSV (MOI, 0.1) for the indicated time.


B . Immunoblot of RKIP + / + , RKIP + / - RKIP - / - peritoneal macrophage treated with intracellular poly ( I : C ) ( 1 μg / ml ) .

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B. Immunoblot assay of RKIP+/+, RKIP+/- and RKIP-/- peritoneal macrophage cells treated with intracellular poly(I:C) (1 μg/ml).


C . Immunoblot of phosphorylated ( p - ) TBK1 , IKKi , IRF3 , P38 , JNK1 / 2 , ERK1 / 2 in peritoneal macrophage transfected with RKIP - specific or scrambled siRNA , followed by infection with VSV ( MOI , 0 . 1 ) . β - actin was used as a loading control .

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C. Immunoblot analysis of phosphorylated (p-) TBK1, IKKi, IRF3, P38, JNK1/2, ERK1/2 in peritoneal macrophage transfected with RKIP-specific or scrambled siRNA, followed by infection with VSV (MOI, 0.1). β-actin was used as a loading control.


D . Immunoblot of p - TBK1 , - IKKi , - IRF3 , - P38 , - JNK1 / 2 , - ERK1 / 2 - IκBα in lysates of VSV - treated RAW264 . 7 stably over - expressing RKIP .

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D. Immunoblot analysis of p- TBK1, -IKKi, -IRF3, -P38, -JNK1/2, -ERK1/2 and -IκBα in lysates of VSV-treated RAW264.7 cells stably over-expressing RKIP.


E . Immunoblot in lysates of RAW264 . 7 stably overexpressing RKIP treated with intracellular poly ( I : C ) ( 1 μg / ml ) .

E. Immunoblot analysis in lysates of RAW264.7 cells stably overexpressing RKIP treated with intracellular poly(I:C) (1 μg/ml).


F . Luciferase activity in 293T transfected with plasmid encoding a luciferase reporter for IFN - β ( IFN - β - luc ) or ISRE ( ISRE - luc ) , an expression vector for RKIP ( 0 , 50 , 100 , 200 ng each ) , together with plasmids encoding RIG - I CARD , TBK1 , IKKi IRF3 5D .

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F. Luciferase activity in 293T cells transfected with plasmid encoding a luciferase reporter for IFN-β (IFN-β-luc) or ISRE (ISRE-luc), and an expression vector for RKIP (0, 50, 100, 200 ng each), together with plasmids encoding RIG-I CARD, TBK1, IKKi and IRF3 5D.


A . Immunoblot of extracts of RAW264 . 7 stably overexpressing flag - RKIP infected with VSV ( MOI , 0 . 1 ) for indicated times followed by immunoprecipitation ( IP ) . WCL , whole lysates .

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A. Immunoblot of extracts of RAW264.7 cells stably overexpressing flag-RKIP infected with VSV (MOI, 0.1) for the indicated times followed by immunoprecipitation (IP). WCL, whole cell lysates.


B . Coimmunoprecipitation immunoblot of 293T transfected with vector for myc - RKIP alone or with deletion mutants of TBK1 . Numbers in parentheses indicate included in construct . FL , full ; KD , kinase domain ; ULD , ubiquitin - like domain ; SDD , α - helical scaffold domain ; NBD , C - terminal domain . * indicates targeted band .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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. Coimmunoprecipitation and immunoblot analysis of 293T cells transfected with vector for myc-RKIP alone or with the deletion mutants of TBK1. Numbers in parentheses indicate amino acids included in construct. FL, full length; KD, kinase domain; ULD, ubiquitin-like domain; SDD, α-helical scaffold domain; NBD, C-terminal domain.* indicates targeted band.


C . Immunoblot of 293T transfected with vector for myc - TBK1 together with plasmid for HA - GSK3β or HA - RKIP , followed by immunoprecipitation . * indicates targeted band .

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

C. Immunoblot of 293T cells transfected with vector for myc-TBK1 together with plasmid for HA-GSK3β or HA-RKIP, followed by immunoprecipitation. * indicates targeted band.


D . In vitro kinase was carried out in reaction mixture containing 2 mM ATP , 100 μM GST - RKIP or 100 μM GST - GFP 5 μM flag - TBK1 at 30 ° C for 30 min then followed by immunoblotting with indicated Antibody .

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

D. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP and 5 μM flag-TBK1 at 30 °C for 30 min and then followed by immunoblotting with the indicated Antibody.


A . Real - time PCR of IL - 6 , TNF - α IFN - β mRNA in RAW264 . 7 stably overexpressing flag - RKIP , flag - S109A flag - S109D , followed by infection with VSV ( MOI , 0 . 1 ) .

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

A. Real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, followed by infection with VSV (MOI, 0.1).


B . Immunoblot of p - TBK1 , - IRF3 , - P38 of as in ( A ) .

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B. Immunoblot analysis of p- TBK1, -IRF3, -P38 of cells as in (A).


C . FACS of RAW264 . 7 stably overexpressing flag - RKIP , flag - S109A flag - S109D infected with VSV - eGFP at MOI of 1 .

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C. FACS analysis of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D infected with VSV-eGFP at MOI of 1.


D . Immunoblot of phosphorylated - TBK1 , - IRF3 , - P38 in lysates of RKIP + / + or RKIP - / - bone marrow derived macrophages ( BMDM ) transfected with flag - RKIP , flag - S109A flag - S109D , then stimulated with VSV ( MOI , 0 . 1 ) .

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D. Immunoblot analysis of phosphorylated-TBK1, -IRF3, -P38 in lysates of RKIP+/+ or RKIP-/- bone marrow derived macrophages (BMDM) transfected with flag-RKIP, flag-S109A and flag-S109D, and then stimulated with VSV (MOI, 0.1).


E . ELISA of IFN - β TNF - α of in ( D ) .

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E. ELISA of IFN-β and TNF-α of cells in (D).


A . BMDM were infected with VSV ( MOI , 0 . 1 ) or HSV ( MOI , 5 ) for indicated times . lysates were analyzed by immunoblotting with indicated antibodies .

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A. BMDM cells were infected with VSV (MOI, 0.1) or HSV (MOI, 5) for the indicated times. Cell lysates were analyzed by immunoblotting with the indicated antibodies.


B . BMDM were transfected with control or TBK1 siRNA . 72 hours after transfection , were analyzed by immunoblotting with antibody for phospho - RKIP or TBK1 .

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B. BMDM cells were transfected with control or TBK1 siRNA. 72 hours after transfection, cells were analyzed by immunoblotting with antibody for phospho-RKIP or TBK1.


C . Immunoblot of RKIP phosphorylation in control or TBK1 - / - mouse embryonic fibroblasts after VSV ( MOI , 0 . 1 ) infection for indicated times .

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C. Immunoblot analysis of RKIP phosphorylation in control or TBK1-/- mouse embryonic fibroblasts after VSV (MOI, 0.1) infection for the indicated times.


D . Recombinant flag - tagged TBK1 was immunoprecipitated with anti - flag M2 beads in flag - TBK1 transfected 293T . Followed by in vitro kinase , which was carried out in reaction mixture containing 2 mM ATP , 100 μM GST - RKIP or 100 μM GST - GFP together with 5 μM flag - TBK1 at 30 ° C for 30 min . mixtures were immunoblotted with indicated antibody for phospho - RKIP , flag , GST or his .

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D. Recombinant flag-tagged TBK1 was immunoprecipitated with anti-flag M2 beads in flag-TBK1 transfected 293T cells. Followed by in vitro kinase assay, which was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP together with 5 μM flag-TBK1 at 30 °C for 30 min. Assay mixtures were immunoblotted with the indicated antibody for phospho-RKIP, flag, GST or his.


E . Immunoblot of extracts of RAW264 . 7 stably overexpressing flag - RKIP , flag - S109A flag - S109D , infected with VSV ( MOI , 0 . 1 ) for indicated times , followed by immunoprecipitation with anti - flag beads .

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E. Immunoblot analysis of extracts of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, infected with VSV (MOI, 0.1) for the indicated times, followed by immunoprecipitation with anti-flag beads.


F . BMDM were infected with VSV ( MOI , 0 . 1 ) for indicated times . lysates were immunoprecipitated with anti - TBK1 ( left ) or anti - RKIP ( right ) . immunoprecipitates were analyzed by immunoblot with anti - RKIP or anti - TBK1 antibody . of endogenous RKIP , TBK1 , p - RKIP were detected by immunoblot .

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F. BMDM cells were infected with VSV (MOI, 0.1) for the indicated times. The cell lysates were immunoprecipitated with anti-TBK1 (left) or anti-RKIP (right). The immunoprecipitates were analyzed by immunoblot with anti-RKIP or anti-TBK1 antibody. The levels of the endogenous RKIP, TBK1, and p-RKIP were detected by immunoblot analysis.


G . Co - immunoprecipitation immunoblot of 293T transfected with myc - TBK1 alone or with flag - RKIP , flag - S109A or flag - S109D .

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G. Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with myc-TBK1 alone or with flag-RKIP, flag-S109A or flag-S109D.


H . GST - pull down experiment was performed with 1 μg fusion GST - GFP , GST - his - RKIP or GST - his - S109D mixed with 20 μl precleared agarose beads in 800 μl reaction medium , followed by adding 1 μg flag - TBK1 incubating at 4 ° C for 3 hours with gentle rotation . Pull down ( 1st - 3th lane ) input samples ( 4th lane ) were separated by SDS - PAGE followed by immunoblotting with anti - GST or anti - flag antibody .

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H. GST-pull down experiment was performed with 1 μg fusion protein GST-GFP, GST-his-RKIP or GST-his-S109D mixed with 20 μl precleared agarose beads in 800 μl reaction medium, followed by adding 1 μg flag-TBK1 and incubating at 4°C for 3 hours with gentle rotation. Pull down (1st-3th lane) and input samples (4th lane) were separated by SDS-PAGE followed by immunoblotting with anti-GST or anti-flag antibody.


I . Immunoprecipitation immunoblot of 293T transfected with indicated combinations of vector for myc - TBK1 , flag - RKIP , flag - S109A , flag - S109D HA - GSK3β or flag - GFP .

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I. Immunoprecipitation and immunoblot analysis of 293T cells transfected with indicated combinations of vector for myc-TBK1, flag-RKIP, flag-S109A, flag-S109D and HA-GSK3β or flag-GFP.


J . In vitro kinase was carried out in reaction mixture containing 2 mM ATP , 50 μM GST - TBK1 together with 100 μM GST - his - RKIP , 100 μM GST - his - S109D , 100 μM GST - his - S109A or 100 μM GST , respectively . React at 30 ° C for 30 min . Then followed by immunoblotting with anti - p - TBK1or anti - GST antibody .

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J. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 50 μM GST-TBK1 together with 100 μM GST-his-RKIP, 100 μM GST-his-S109D, 100 μM GST-his-S109A or 100 μM GST, respectively. React at 30 °C for 30 min. Then followed by immunoblotting with anti-p-TBK1or anti-GST antibody.


( D ) Immunostaining of PGCLCs d5 cryosections for SOX2 ( magenta ) TFAP2C ( cyan ) . Barplot indicates of SOX2 + , TFAP2C + , SOX2 + / TFAP2C + . n = 1150 , from n = 3 separate inductions , using two biological clones . squares represent position of magnified region at bottom . Scale bar , 50 µm 10 µm for magnified region .

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(D) Immunostaining of PGCLCs d5 cryosections for SOX2 (magenta) and TFAP2C (cyan). Barplot indicates the quantification of SOX2+ cells, TFAP2C+ cells, and SOX2+/TFAP2C+. n=1150 cells, from n=3 separate inductions, using two biological clones. The white squares represent the position of the magnified region at the bottom. Scale bar, 50 µm and 10 µm for the magnified region.


( C ) MA plot of differential gene expression changes between XGFP - XGFP + PGCLCs as determined by - seq . Log2 - mean expression ( log2 - normalised from DESeq2 ) on x - axis log2 - fold change on y - axis are shown . Significantly upregulated downregulated genes are highlighted in red green respectively . False Discovery Rate ( FDR ) < 0 . 001 . Non - significant genes with log2 - mean expression between 0 0 . 2 were removed for easier plot visualisation .

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(C) MA plot of differential gene expression changes between XGFP- and XGFP+ PGCLCs as determined by RNA-seq. Log2-mean expression (log2-normalised counts from DESeq2) on the x-axis and the log2-fold change on the y-axis are shown. Significantly upregulated and downregulated genes are highlighted in red and green respectively. False Discovery Rate (FDR) < 0.001. Non-significant genes with log2-mean expression between 0 and 0.2 were removed for easier plot visualisation.


( D ) Expression ( normalised DEseq2 ) of selected differentially expressed genes between XGFP - XGFP + PGCLCs during differentiation time course . Genes with FDR < 0 . 001 were considered significantly differentially expressed . Points indicate expression of biological replicates .

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(D) Expression levels (normalised DEseq2 counts) of selected differentially expressed genes between XGFP- and XGFP+ PGCLCs during the differentiation time course. Genes with FDR < 0.001 were considered significantly differentially expressed. Points indicate expression of individual biological replicates.


( E ) of - seq normalised showing Zscore across PGCLC induction timepoints of 31 manually selected manually ordered marker genes belonging to categories reported on side .

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(E) Heatmap of RNA-seq normalised counts showing the Zscore across PGCLC induction timepoints of 31 manually selected and manually ordered marker genes belonging to the categories reported on the side.


( G ) FACS of cycle using DAPI . Numbers indicate of in G1 , S G2 / M respectively .

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(G) FACS analysis of cell cycle using DAPI. Numbers indicate the percentage of cells in G1, S and G2/M respectively.


( H ) Alkaline phosphatase for ESCs , XGFP + PGCLCs XGFP - PGCLCs grown for 7 days in 2i / LIF medium on immortalised mouse embryonic fibroblasts . ( I ) Barplot indicating absolute numbers of Alkaline Phosphatase ( AP ) positive colonies in each type after 7 days of culture in 2i / LIF medium on immortalised mouse embryonic fibroblasts . Y - axis is in square root scale ( sqrt ) for better plot visualisation . Each dot represents one technical replicate .

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(H) Alkaline phosphatase staining for ESCs, XGFP+ PGCLCs and XGFP- PGCLCs grown for 7 days in 2i/LIF medium on immortalised mouse embryonic fibroblasts. (I) Barplot indicating the absolute numbers of Alkaline Phosphatase (AP) positive colonies in each cell type after 7 days of culture in 2i/LIF medium on immortalised mouse embryonic fibroblasts. Y-axis is in square root scale (sqrt) for better plot visualisation. Each white dot represents one technical replicate.