( k , l ) . PINK1 regulates S616 S637 phosphorylation of Drp1 via independent mechanisms . DRP1KO HEK293 co - transfected FRB - MTS / Δ110 - PINK1 - GFP - FKBP ( PINK1WT ) with either myc - Drp1WT or myc - Drp1S616A were treated with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase . treated with solvent ( DMSO ) were included as a control . lysates were immunoblotting with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho - ( Ser637 ) - Drp1 ( pS637 ) , Drp1 GFP ( to detect PINK1 - FKBP ) ( k ) . β - actin was detected as a loading control . Quantitation of pS637 / Drp1 in various conditions was shown ( l ) . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments .

O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_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 B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_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

(k, l). PINK1 regulates S616 and S637 phosphorylation of Drp1 via independent mechanisms. DRP1KO HEK293 cells co-transfected FRB-MTS/Δ110-PINK1-GFP-FKBP (PINK1WT) with either myc-Drp1WT or myc-Drp1S616A were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cell lysates were immunoblotting with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho-(Ser637)-Drp1 (pS637), Drp1 and GFP (to detect PINK1-FKBP) (k). β-actin was detected as a loading control. Quantitation of pS637/Drp1 in various conditions was shown (l). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( a - e ) . ATG5 is dispensable for PINK1 - mediated mitochondrial fission . MEF derived from ATG5 - null ( ATG5KO ) their wildtype control mice ( ATG5WT ) were co - transfected Δ110 - PINK1 - GFP - FKBP / FRB - MTS with either PINK1WT or a kinase - dead mutant PINK1D384N . were induced with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , red ) Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( PINK1 - FKBP , green ) . treated with solvent ( DMSO ) were used as a treatment control . Representative are shown , scale bar = 25 μm ( a ) . Higher magnification are also included ( panels beneath large , scale bar = 10 μm ) . Mitochondrial in different transfections was quantified ( b - e ) . Student ' s . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments . For each condition , > 100 were analyzed .

O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_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 O O O O O O O O O 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-e). ATG5 is dispensable for PINK1-mediated mitochondrial fission. MEF cells derived from ATG5-null (ATG5KO) and their wildtype control mice (ATG5WT) were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either PINK1WT or a kinase-dead mutant PINK1D384N. Cells were induced with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images are shown, scale bar=25 μm (a). Higher magnification images are also included (panels beneath the large cell images, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified (b-e). Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( f - i ) . Drp1 is dispensable for recruitment of parkin to mitochondria . Drp1WT Drp1KO HeLa were transfected with GFP - parkin . were treated with 20 μM CCCP for 2 hours ( f ) followed by immunodetecting mitochondria ( TOM20 , red ) parkin ( GFP - parkin , green ) . Nuclei were labeled with DAPI ( blue ) . treated with solvent ( DMSO ) were used as a treatment control . Representative were shown , scale bar = 25 μm . with mitochondrial parkin ( % ) in different experimental group was quantified ( g , i ) . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments . For each condition , > 100 were analyzed .

O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O B-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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_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 B-MEASURED_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

(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 20 μM CCCP for 2 hours (f) followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( f - i ) . Drp1 is dispensable for recruitment of parkin to mitochondria . Drp1WT Drp1KO HeLa were transfected with GFP - parkin . were treated with 150 μM actinonin for 6 hours ( h ) , followed by immunodetecting mitochondria ( TOM20 , red ) parkin ( GFP - parkin , green ) . Nuclei were labeled with DAPI ( blue ) . treated with solvent ( DMSO ) were used as a treatment control . Representative were shown , scale bar = 25 μm . with mitochondrial parkin ( % ) in different experimental group was quantified ( g , i ) . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments . For each condition , > 100 were analyzed .

O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O B-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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_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 B-MEASURED_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

(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 150 μM actinonin for 6 hours (h), followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( a , b ) . Drp1WT Drp1S616D , but not Drp1S616A , rescue PINK1 - null induced crushed thorax in Drosophila . Representative thorax of PINK1WT PINK1 - deficiency ( PINK1KO ) Drosophila expressing either mhc - gal4 ( Mhc / + ) , mhc - gal4 driven human Drp1wt ( Mhc > hDrp1WT ) or mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . were taken either under microscopy ( a , upper panels , yellow arrowhead indicates collapsed throax ) or ( b , lower panels ) . Quantitation of crushed thorax is shown ( b ) . > 200 flies for each experiment are analyzed . Scale bar = 200µm . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 . Data was presented as mean ± of three independent experiments . For each condition , > 200 flies were quantified .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-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 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 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 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, b). Drp1WT and Drp1S616D, but not Drp1S616A, rescue PINK1-null induced crushed thorax in Drosophila. Representative thorax images of PINK1WT and PINK1-deficiency (PINK1KO) Drosophila expressing either mhc-gal4 (Mhc/+), mhc-gal4 driven human Drp1wt (Mhc>hDrp1WT) or mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Images were taken either under light microscopy (a, upper panels, yellow arrowhead indicates the collapsed throax) or SEM (b, lower panels). Quantitation of crushed thorax is shown (b). > 200 flies for each experiment are analyzed. Scale bar=200µm. One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments. For each condition, >200 flies were quantified.


( c ) Drp1WT Drp1S616D , but not Drp1S616A , restore abnormal ATP production in PINK1 - null flies . ATP production was measured using muscle lysates generated from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments .

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

(c) Drp1WT and Drp1S616D, but not Drp1S616A, restore abnormal ATP production in PINK1-null flies. ATP production was measured using muscle lysates generated from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Drp1WT Drp1S616D , but not Drp1S616A , rescue mitochondrial structures in PINK1KO flies . Representative of Mito - GFP ( green ) labeled mitochondria ( d ) from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Scale bar = 10 μm ( d ) Average mitochondrial was quantified , > 3 flies / group 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . One - way ANOVA followed with Tukey ' s . * p < 0 . 05 , * * * p < 0 . 001 . Data was presented as mean ± of three independent experiments ( e ) .

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

Drp1WT and Drp1S616D, but not Drp1S616A, rescue mitochondrial structures in PINK1KO flies. Representative images of Mito-GFP (green) labeled mitochondria (d) from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Scale bar=10 μm (d) Average mitochondrial size was quantified, >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001. Data was presented as mean ± SEM of three independent experiments (e).


Drp1WT Drp1S616D , but not Drp1S616A , rescue mitochondrial structures in PINK1KO flies . Representative of ( f ) of IFMs from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Scale bar = 1 μm ( f ) . Average mitochondrial was quantified , > 3 flies / group 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Normal abnormal mitochondrial number per mm2 were quantified . > 3 flies / group 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Bars represent mean ± of three independent experiments ( g ) .

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

Drp1WT and Drp1S616D, but not Drp1S616A, rescue mitochondrial structures in PINK1KO flies. Representative images of TEM analysis (f) of IFMs from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Scale bar= 1 μm (f). Average mitochondrial size was quantified, >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Normal and abnormal mitochondrial number per mm2 were quantified. >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (g).


( h , i ) . Drp1WT Drp1S616D , but not Drp1S616A , suppress in PINK1KO flies . Representative TUNEL ( Red ) of IFM sections from PINK1WT PINK1KO flies expressing either Mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1WT ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Nuclei were counter - stained with DAPI ( blue ) , scale bar = 10 μm . Apoptotic ( % ) in different genotype flies were quantified ( i ) . > 5 flies / group 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 . Data was presented as mean ± of three independent experiments .

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

(h, i). Drp1WT and Drp1S616D, but not Drp1S616A, suppress cell death in PINK1KO flies. Representative TUNEL staining (Red) images of IFM sections from PINK1WT and PINK1KO flies expressing either Mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1WT) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Nuclei were counter-stained with DAPI (blue), scale bar=10 μm. Apoptotic cells (%) in different genotype flies were quantified (i). >5 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat.One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( j ) . Expression phosphorylation of hDrp1 in Drosophila . Fly muscle lysates generated from PINK1WT or PINK1KO flies expressing mhc - gal4 driven hDrp1WT ( WT ) hDrp1S616A ( S616A ) were immunoblotted with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) ( to detect phosphorylated hDrp1S616 ) , Drp1 ( to detect both endogenous exogenous Drp1 ) , myc - tag ( to detect exogenous Drp1 ) . α - tubulin was detected as a loading control . Flies expressing mhc - gal4 ( - ) were included as an expression control .

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 O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_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

(j). Expression and phosphorylation of hDrp1 in Drosophila. Fly muscle lysates generated from PINK1WT or PINK1KO flies expressing mhc-gal4 driven hDrp1WT (WT) and hDrp1S616A (S616A) were immunoblotted with antibodies against either phospho(Ser616)-Drp1(pS616) (to detect phosphorylated hDrp1S616), Drp1 (to detect both endogenous and exogenous Drp1), myc-tag (to detect exogenous Drp1). α-tubulin was detected as a loading control. Flies expressing mhc-gal4 (-) were included as an expression control.


( a , b ) . Drp1 rescues PINK1KO induced collapsed thorax phenotype in dATG7 - deficient Drosophila . Representative of thorax from PINK1WT , ( upper panels ) PINK1KO ( lower panels ) flies combined with dATP7 deficiency ( dATG7KO ) or their controls ( dATG7WT ) , followed by expressing either mhc - gal4 ( Mhc / + ) alone , or Mhc - gal4 driven expression of human Drp1 ( Mhc > hDrp1WT ) , mhc - gal4 driven expression of human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven expression of human Drp1S616D ( Mhc > hDrp1S616D ) are shown ( a ) . Quantitation of crushed thorax in flies with indicated genotypes is presented ( b ) . > 100 flies for each experiment are analyzed . Scale bar = 200µm . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 . Data was presented as mean ± of three independent experiments .

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 B-CONTROLLED_VAR 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 B-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 O O O O O 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, b). Drp1 rescues PINK1KO induced collapsed thorax phenotype in dATG7-deficient Drosophila. Representative SEM images of thorax from PINK1WT, (upper panels) and PINK1KO (lower panels) flies combined with dATP7 deficiency (dATG7KO) or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1 (Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D (Mhc>hDrp1S616D) are shown (a). Quantitation of crushed thorax in flies with indicated genotypes is presented (b). > 100 flies for each experiment are analyzed. Scale bar=200µm. One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( c , d ) Drp1 rescues PINK1KO induced mitochondrial defects in dATG7KO Drosophila . Representative of indirect flight muscle from PINK1WT ( upper panels ) PINK1KO ( lower panels ) flies combined with dATG7KO or their controls ( dATG7WT ) , followed by expressing either mhc - gal4 ( Mhc / + ) alone , or Mhc - gal4 driven expression of human Drp1 ( Mhc > hDrp1WT ) , mhc - gal4 driven expression of human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven expression of human Drp1S616D ( Mhc > hDrp1S616D ) are shown ( c ) . Scale bar = 1 μm . Normal abnormal mitochondrial number per mm2 from each experimental group were quantified . > 3 flies / group 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Bars represent mean ± of three independent experiments ( d ) .

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 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 B-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 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

(c, d) Drp1 rescues PINK1KO induced mitochondrial defects in dATG7KO Drosophila. Representative TEM images of indirect flight muscle from PINK1WT (upper panels) and PINK1KO (lower panels) flies combined with dATG7KO or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1(Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A(Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D(Mhc>hDrp1S616D) are shown (c). Scale bar=1 μm. Normal and abnormal mitochondrial number per mm2 from each experimental group were and quantified. >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (d).


( e ) . Drp1 rescues PINK1KO induced ATP reduction in Drosophila . ATP contents of thorax muscle tissues from indicated genotypes were measured normalized against One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O B-MEASURED_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

(e). Drp1 rescues PINK1KO induced ATP reduction in Drosophila. ATP contents of thorax muscle tissues from the indicated genotypes were measured and normalized against the protein levels One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( g , h ) . Reduction of Drp1S616 phosphorylation in dermal fibroblasts derived from PD with PINK1 mutations . Lysates of derived from three unrelated normal control individuals ( Control , C ) four familial PD with PINK1 mutations ( PINK1mutant ) were immunoblotted with antibodies recognizing either phosphor - Drp1S616 ( pS616 ) or Drp1 . β - actin was detected as a loading control ( g ) . A quantitative of phospho - Drp1S616 ( pS616 / Drp1 ) is shown ( h ) . Student ' s . * * p < 0 . 01 , ns : no significance . Data was presented as mean ± of three independent experiments .

O O O O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_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

(g, h). Reduction of Drp1S616 phosphorylation in dermal fibroblasts derived from PD patients with PINK1 mutations. Lysates of cells derived from three unrelated normal control individuals (Control, C) and four familial PD patients with PINK1 mutations (PINK1mutant) were immunoblotted with antibodies recognizing either phosphor-Drp1S616 (pS616) or Drp1 protein. β-actin was detected as a loading control (g). A quantitative analysis of phospho-Drp1S616 (pS616/Drp1) is shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD . Lysates of derived from five unrelated normal control individuals ( Control , C ) seven unrelated sporadic PD ( Sporadic PD , SP ) were immunoblotted with antibodies recognizing either phospho - Drp1S616 ( pS616 ) or Drp1 . β - actin was detected as a loading control ( i ) .

B-MEASURED_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-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients. Lysates of cells derived from five unrelated normal control individuals (Control, C) and seven unrelated sporadic PD patients (Sporadic PD, SP) were immunoblotted with antibodies recognizing either phospho-Drp1S616 (pS616) or Drp1 protein. β-actin was detected as a loading control (i).


Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD . A quantitative of phospho - Drp1S616 ( pS616 / Drp1 ) in sporadic cases is shown ( j ) . Student ' s . * p < 0 . 05 . Data was presented as mean ± of three independent experiments .

B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_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

Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients. A quantitative analysis of phospho-Drp1S616 (pS616/Drp1) in sporadic cases is shown (j). Student's test. *p<0.05. Data was presented as mean ± SEM of three independent experiments.


B . Volcano plot showing magnitude of differential gene up - regulation , as measured by respective log 2 - fold change in expression level , at day ( positive ) or night ( negative ) versus statistical significance , as quantified by a negative binomial exact ( red dots , dashed p < 0 . 05 adjusted for multiple testing using Benjamin - Hochberg ) . Known circadian - regulated genes are labelled .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Volcano plot showing the magnitude of differential gene up-regulation, as measured by the respective log 2-fold change in expression level, at day (positive) or night (negative) versus statistical significance, as quantified by a negative binomial exact test (red dots, dashed line p<0.05 adjusted for multiple testing using Benjamin-Hochberg). Known circadian-regulated genes are labelled.


B . Violin plots depicting distribution of quantified average expression of Prok2 , ProkR2 , Vip , Vipr2 , Avp , Avpr1a , Grp Grpr in each sub - cluster in CT7 . 5 ( left ) CT15 . 5 ( right ) datasets .

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

B. Violin plots depicting the distribution of the quantified average cellular expression of Prok2, ProkR2, Vip, Vipr2, Avp, Avpr1a, Grp and Grpr in each sub-cluster in CT7.5 (left) and CT15.5 (right) datasets.


C . period of Cry1 - luc bioluminescence rhythms in SCN slices transfected with either pProk2 . Cre . T2A . mCherry or pProkR2 . Cre . T2A . Venus does not differ between treatments ( p = 0 . 71 , n = 10 , 9 SCN slices per group , respectively ) . D . Similarly , amplitude of Cry1 - luc bioluminescence rhythms from Prok2 + SCN ProkR2 + SCN , does not differ between treatments ( p = 0 . 67 ) .

O O O O B-MEASURED_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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

C. The period of Cry1-luc bioluminescence rhythms in SCN slices transfected with either pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus does not differ between treatments (p=0.71, n= 10, 9 SCN slices per group, respectively). D. Similarly, the amplitude of Cry1-luc bioluminescence rhythms from Prok2+ SCN cells and ProkR2+ SCN cells, does not differ between treatments (p=0.67).


C . period of Cry1 - luc bioluminescence rhythms in SCN slices transfected with either pProk2 . Cre . T2A . mCherry or pProkR2 . Cre . T2A . Venus does not differ between treatments ( p = 0 . 71 , n = 10 , 9 SCN slices per group , respectively ) .

O O O O B-MEASURED_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

C. The period of Cry1-luc bioluminescence rhythms in SCN slices transfected with either pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus does not differ between treatments (p=0.71, n= 10, 9 SCN slices per group, respectively).


H . Left : confocal of representative SCN slices dual - transfected with pProk2 . Cre . T2A . mCherry Syn . GCaMP6f ( top ) , or pProkR2 . Cre . T2A . Venus Syn . jRCaMP1a ( below ) ( scale bar = 500µm ) . Right , above : representative detrended plots of signal from GCaMP6f ( green ) traces pCry1 - DIO - Luc , emitted from Prok2 + SCN ( red ) . Right , below : representative detrended plots of jRCaMP1a ( purple ) pCry1 - DIO - Luc emitted from ProkR2 + ( green ) . Orientation bars depict horizontal axis of optic chiasm vertical axis of third ventricle for each SCN .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

H. Left: confocal images of representative SCN slices dual-transfected with pProk2.Cre.T2A.mCherry and Syn.GCaMP6f (top), or pProkR2.Cre.T2A.Venus and Syn.jRCaMP1a (below) (scale bar=500µm). Right, above: representative detrended plots of signal from GCaMP6f (green) traces and pCry1-DIO-Luc, emitted from Prok2+ SCN cells (red). Right, below: representative detrended plots of jRCaMP1a (purple) and pCry1-DIO-Luc emitted from ProkR2+ cells (light green). Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN.


B . Comparison of Per2 : : Luciferase rhythm amplitude of cycle preceding first second cycles succeeding vehicle ( applied CT23 - 20 grey n = 12 SCN slices , CT - 20 - 23 grey n = 6 SCN slices ) or Prok2RA ( applied CT23 - 20 purple n = 13 SCN slices , CT20 - 23 pink n = 11 SCN slices ) treatment ( interaction , p < 0 . 0001 ) . A significant decline in amplitude occurred when Prok2RA signalling was blocked between CT20 - 23 ( * * * * p < 0 . 0001 ) .

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

B. Comparison of the Per2::Luciferase rhythm amplitude of the cycle preceding and the first and second cycles succeeding vehicle (applied CT23-20 dark grey n=12 SCN slices, CT-20-23 light grey n=6 SCN slices) or Prok2RA (applied CT23-20 purple n=13 SCN slices, CT20-23 pink n=11 SCN slices) treatment (interaction, p<0.0001). A significant decline in amplitude occurred when Prok2RA signalling was blocked between CT20-23 (****p<0.0001).


H . Period lengthening occurred at same rate when Prok2 . Cre or ProkR2 . Cre was first AAV to be applied ( p = 0 . 1 ; Prok2 . Cre - baseline vs . 1 AAV , * p = 0 . 01 ; baseline vs . both AAVs , * p = 0 . 04 ; ProkR2 . Cre - baseline vs . 1 AAV , * p = 0 . 03 ; baseline vs . both AAVs , * * p = 0 . 01 .

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 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 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

H. Period lengthening occurred at the same rate when Prok2.Cre or ProkR2.Cre was the first AAV to be applied (p=0.1; Prok2.Cre - baseline vs. 1 AAV, *p=0.01; baseline vs. both AAVs, *p=0.04; ProkR2.Cre - baseline vs. 1 AAV, *p=0.03; baseline vs. both AAVs, **p=0.01.


A . Representative Per2 : : Luciferase traces of Cry1 , 2 - / - SCN slices at baseline ( black , arrhythmic ) , after Cry1 complementation in Prok2 + ( red ) further complementation in ProkR2 + ( yellow , rhythmic ) ( cohort n = 8 ) . B . as in A but order of complementation reversed ( green = Cry1 complementation in ProkR2 + ) ( cohort n = 8 ) .

O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O B-MEASURED_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 B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O

A. Representative Per2::Luciferase traces of Cry1,2-/- SCN slices at baseline (black, arrhythmic), after Cry1 complementation in Prok2+ cells (red) and further complementation in ProkR2+ cells (yellow, rhythmic) (cohort n=8). B. as in A but order of complementation reversed (green =Cry1 complementation in ProkR2+ cells) (cohort n=8).


A . Representative Per2 : : Luciferase traces of Cry1 , 2 - / - SCN slices at baseline ( black , arrhythmic ) , after Cry1 complementation in Prok2 + ( red ) further complementation in ProkR2 + ( yellow , rhythmic ) ( cohort n = 8 ) .

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

A. Representative Per2::Luciferase traces of Cry1,2-/- SCN slices at baseline (black, arrhythmic), after Cry1 complementation in Prok2+ cells (red) and further complementation in ProkR2+ cells (yellow, rhythmic) (cohort n=8).


( A , Neutrophils isolated from tamoxifen - treated Nlrp3A350V / wt CreT + Nlrp3A350V / wt CreT - mice were incubated with LPS ( 3 hr ) before culture media were analysed for secreted IL - 1β ( A ) Cytokine values represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA P - values were corrected for multiple comparisons ( Bonferroni ) . * * , p ≤ 0 . 01 .

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

(A, Neutrophils isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice were incubated with LPS (3 hr) before culture media were analysed for secreted IL-1β (A) Cytokine values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA and P-values were corrected for multiple comparisons (Bonferroni). **, p ≤ 0.01.


B ) . Neutrophils isolated from tamoxifen - treated Nlrp3A350V / wt CreT + Nlrp3A350V / wt CreT - mice were incubated with LPS lysates culture media were immunoblotted for β - actin , caspase - 1 IL - 1β ( B ) .

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

B). Neutrophils isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice were incubated with LPS and cell lysates and culture media were immunoblotted for β-actin, caspase-1 and IL-1β (B).


( C ) Representative pictures of Nlrp3A350V / wt MRP8 - CreTg Nlrp3A350V / wt MRP8 - Cre - mice on days 4 8 after birth . Left side in each picture : Nlrp3A350V / wt MRP8 - Cre - mice ; right side in each picture : Nlrp3A350V / wt MRP8 - CreTg . Abscesses ( day 4 ) scaling erythema ( day 8 ) are observed in Nlrp3A350V / wt MRP8 - CreTg mice .

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

(C) Representative pictures of Nlrp3A350V/wt MRP8-CreTg and Nlrp3A350V/wt MRP8-Cre- mice on days 4 and 8 after birth. Left side in each picture: Nlrp3A350V/wt MRP8-Cre- mice; right side in each picture: Nlrp3A350V/wt MRP8-CreTg. Abscesses (day 4) and scaling erythema (day 8) are observed in Nlrp3A350V/wt MRP8-CreTg mice.


( D ) Survival curves of Nlrp3A350V / wt MRP8 - CreTg ( n = 22 ) littermate Nlrp3A350V / wt MRP8 - Cre - ( n = 21 ) mice . Statistical significance was analysed by log - rank Mantel - Cox . * * * * , p ≤ 0 . 0001 .

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

(D) Survival curves of Nlrp3A350V/wt MRP8-CreTg (n=22) and littermate Nlrp3A350V/wt MRP8-Cre- (n=21) mice. Statistical significance was analysed by the log-rank Mantel-Cox test. ****, p ≤0.0001.


( E ) Representative H & E - stained sections of spleen ( top , scale bar : 100 µM ) liver ( bottom , scale bar : 200 µM ) of Nlrp3A350V / wt MRP8 - CreTg littermate Nlrp3A350V / wt MRP8 - Cre - mice . Spleens of Nlrp3A350V / wt MRP8 - Cre - control mice ( top left ) display characteristic pulp ( WP ) regions ( example shown surrounded by dashed ) composed of lymphoid follicles periarteriolar lymphoid sheaths ( PALS ) , which at this magnification are recognized by - purple , large of lymphocytes surrounding central artery ( CA ) their distinctive separation from red - red pulp ( RP ) . WP was largely absent in spleens of Nlrp3A350V / wt MRP8 - CreTg ( top middle ) . Megakaryocytes ( MK ) are indicated for easier recognition of areas of extramedullary hematopoiesis ( EMH ) . Most of spleen parenchyma of Nlrp3A350V / wt MRP8 - CreTg was replaced by EMH of mostly myeloid lineage , which can be recognized by medium - sized with more cytoplasm , giving a lighter appearance ( inset ) . In both overview picture ( top middle picture ) inset ( top right ) of Nlrp3A350V / wt MRP8 - CreTg spleen , a small amount of EMH of erythroid lineage is present that can be recognized by small of darker - stained , smaller ( asterix , * ) . Liver sections of both Nlrp3A350V / wtMRP8 - Cre - control mice diseased Nlrp3A350V / wt MRP8 - CreTg pups displayed comparable EMH of erythroid lineage ( bottom left picture , indicated with yellow arrows ) . Liver sections of Nlrp3A350V / wt MRP8 - CreTg pups also showed a modest increase in EMH of myeloid lineage ( bottom middle picture inset ( scale bar : 100 µM ) in bottom right , indicated with yellow arrowheads ) , as well as hepatocyte enlargement . ( F - I ) H & E - stained histological sections of spleen ( F , G ) or liver ( H , I ) of Nlrp3A350V / wt MRP8 - Cre - ( Cre - , n = 8 ) Nlrp3A350V / wt MRP8 - CreTg ( CreTg , n = 6 ) mice were assigned a score of 0 - 5 for lymphoid depletion ( F ) or EMH ( G , H ) or hepatocyte enlargement ( I ) by a board - certified pathologist . Values represent mean ± of n = 6 - 8 biological repeats . Statistical significance was analysed by Mann - Whitney U - . * * * , p ≤ 0 . 001 .

O O O O O O O O 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 B-CONTROLLED_VAR 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 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 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 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 B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O 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) Representative H&E-stained sections of spleen (top, scale bar: 100 µM) and liver (bottom, scale bar: 200 µM) of Nlrp3A350V/wt MRP8-CreTg and littermate Nlrp3A350V/wt MRP8-Cre- mice. Spleens of Nlrp3A350V/wt MRP8-Cre- control mice (top left) display characteristic white pulp (WP) regions (example shown surrounded by dashed line) composed of lymphoid follicles and periarteriolar lymphoid sheaths (PALS), which at this magnification are recognized by dark-purple staining, large aggregates of lymphocytes surrounding the central artery (CA) and their distinctive separation from the red-staining red pulp (RP). WP was largely absent in spleens of Nlrp3A350V/wt MRP8-CreTg (top middle). Megakaryocytes (MK) are indicated for easier recognition of areas of extramedullary hematopoiesis (EMH). Most of the spleen parenchyma of Nlrp3A350V/wt MRP8-CreTg animals was replaced by EMH of mostly myeloid lineage, which can be recognized by the medium-sized cells with more cytoplasm, giving a lighter appearance (inset). In both the overview picture (top middle picture) and inset (top right) of Nlrp3A350V/wt MRP8-CreTg spleen, a small amount of EMH of the erythroid lineage is present that can be recognized by small aggregates of darker-stained, smaller cells (asterix,*). Liver sections of both Nlrp3A350V/wtMRP8-Cre- control mice and diseased Nlrp3A350V/wt MRP8-CreTg pups displayed comparable EMH of the erythroid lineage (bottom left picture, indicated with yellow arrows). Liver sections of Nlrp3A350V/wt MRP8-CreTg pups also showed a modest increase in EMH of the myeloid lineage (bottom middle picture and inset (scale bar: 100 µM) in bottom right, indicated with yellow arrowheads), as well as hepatocyte enlargement. (F-I) H&E-stained histological sections of spleen (F, G) or liver (H, I) of Nlrp3A350V/wt MRP8-Cre- (Cre-, n=8) and Nlrp3A350V/wt MRP8-CreTg (CreTg, n=6) mice were assigned a score of 0-5 for lymphoid depletion (F) or EMH (G, H) or hepatocyte enlargement (I) by a board-certified pathologist. Values represent mean ± SEM of n=6-8 biological repeats. Statistical significance was analysed by the Mann-Whitney U-test. ***, p ≤0.001.


( J - L ) Cytokine secretion ( Luminex ) of serum samples obtained at day 7 from Nlrp3A350V / wt MRP8 - Cre - ( Cre - , n = 8 ) Nlrp3A350V / wt MRP8 - CreTg ( CreTg , n = 6 ) pups . Values represent mean ± of n = 6 - 8 biological repeats . Statistical of serum cytokine was performed by Welch ' s t - . * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

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 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

(J-L) Cytokine secretion analysis (Luminex) of serum samples obtained at day 7 from Nlrp3A350V/wt MRP8-Cre- (Cre-, n=8) and Nlrp3A350V/wt MRP8-CreTg (CreTg, n=6) pups. Values represent mean ± SEM of n=6-8 biological repeats. Statistical analysis of serum cytokine levels was performed by the Welch's t-test. *, p ≤0.05; **, p ≤0.01.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with LeTx ( 3 hr ) ( A , B ) IL - 1β secretion were determined in culture media ( A , combined lysates culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) ( B , Cytokine values represent mean ± of n = 3 biological repeats immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with LeTx (3 hr) (A, B) IL-1β secretion levels were determined in culture media (A, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) (B, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) ( C , D ) IL - 1β secretion were determined in culture media C , combined lysates culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) D , Cytokine values represent mean ± of n = 3 biological repeats immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) (C, D) IL-1β secretion levels were determined in culture media C, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) D, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or infected with F . tularensis ( 18 hr ) ( E , F ) IL - 1β secretion were determined in culture media E , combined lysates culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) F , Cytokine values represent mean ± of n = 3 biological repeats immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or infected with F. tularensis (18 hr) (E, F) IL-1β secretion levels were determined in culture media E, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) F, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with Flatox ( 3 hr ) ( G , H ) . IL - 1β secretion were determined in culture media G ) ; combined lysates culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) H ) . Cytokine values represent mean ± of n = 3 biological repeats immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with Flatox (3 hr) (G, H). IL-1β secretion levels were determined in culture media G); and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) H). Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with LeTx ( A ) , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . data are representative of n = 3 biological repeats .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with LeTx (A), PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( B ) PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . data are representative of n = 3 biological repeats .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (B) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with ATP ( C ) PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . data are representative of n = 3 biological repeats .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with ATP (C) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( D ) . PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . data are representative of n = 3 biological repeats .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (D). PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


( E ) LPS - primed B6 BMNs were left untreated ( LPS ) or subsequently stimulated with FlaTox ( LPS + FlaTox ) for 3 hr . Data are representative of n = 3 biological repeats . Culture media ( Sup ) whole lysates ( Lys ) were immunoblotted for release of GAPDH into culture media . Data are representative of n = 3 biological repeats .

O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(E) LPS-primed B6 BMNs were left untreated (LPS) or subsequently stimulated with FlaTox (LPS+FlaTox) for 3 hr. Data are representative of n=3 biological repeats. Culture media (Sup) and whole cell lysates (Lys) were immunoblotted for release of cytosolic GAPDH into culture media. Data are representative of n=3 biological repeats.


( F ) B6 BMNs were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . Culture media ( Sup ) whole lysates ( Lys ) were immunoblotted for release of GAPDH into culture media . Data are representative of n = 3 biological repeats .

O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O

(F) B6 BMNs were left untreated (Mock) or infected with S. Typhimurium at MOI 25. Culture media (Sup) and whole cell lysates (Lys) were immunoblotted for release of cytosolic GAPDH into culture media. Data are representative of n=3 biological repeats.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) ( A Whole lysates ( Lys ) culture media ( Sup ) were immunoblotted separately for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) GSDMD ( p30 ) . * with Western blots indicates non - specific cross - reactivity of antibody . data are representative of n = 3 biological repeats .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) (A Whole cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( 3 hr ) ( B , Whole lysates ( Lys ) culture media ( Sup ) were immunoblotted separately for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) GSDMD ( p30 ) . * with Western blots indicates non - specific cross - reactivity of antibody . data are representative of n = 3 biological repeats .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (3 hr) (B, Whole cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with nigericin ( 100 mins ) ( C , Whole lysates ( Lys ) culture media ( Sup ) were immunoblotted separately for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) GSDMD ( p30 ) . * with Western blots indicates non - specific cross - reactivity of antibody . data are representative of n = 3 biological repeats .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with nigericin (100 mins) (C, Whole cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) D , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Values represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) D, PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( 3 hr ) E , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Values represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (3 hr) E, PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with nigericin ( 100 mins ) F , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Values represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with nigericin (100 mins) F, PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) G ) , IL - 1β secretion were determined by Luminex . Values represent mean ± of n = 3 ( G , biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) G), IL-1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=3 (G, biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( 3 hr ) , H ) IL - 1β secretion were determined by Luminex . Values represent mean ± of n = 4 ( H ) biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (3 hr) , H) IL-1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=4 (H) biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with nigericin ( 100 mins ) I ) . IL - 1β secretion were determined by Luminex . Values represent mean ± of n = 3 I ) biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with nigericin (100 mins) I). IL-1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=3 I) biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) , stimulated with PMA ( 3 hr ) Whole lysates ( Lys ) culture media ( Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) GSDMD ( p30 ) ( A ) * with Western blots indicates non - specific bands .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30) (A) * with Western blots indicates non-specific bands.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) , stimulated with PMA ( 3 hr ) ( B , PMP was assessed by measurement of SYTOX Green incorporation over time . Values represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * * , p ≤ 0 . 001 ; ns = non - significant . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) (B, PMP was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) , stimulated with PMA ( 3 hr ) Whole lysates ( Lys ) culture media ( Sup ) were immunoblotted for extracellular release of β - actin

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDH


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) , stimulated with nigericin ( 100 mins ) Whole lysates ( Lys ) culture media ( Sup ) were immunoblotted for extracellular release of β - actin

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with nigericin (100 mins) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDH


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) , stimulated with nigericin ( 100 mins ) G ) PMP was assessed by measurement of SYTOX Green incorporation over time . Values represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * * , p ≤ 0 . 001 ; ns = non - significant . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with nigericin (100 mins) G) PMP was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) , stimulated with TcDA ( 3 hr ) ( H ) . Whole lysates ( Lys ) culture media ( Sup ) were immunoblotted for extracellular release of β - actin GAPDH H ) .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with TcDA (3 hr) (H). Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDH H).


Neutrophils of indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( A ) Representative confocal taken at different time points were stained with DAPI ( blue nuclei ) SYTOX Green ( blue ) . Scale bar : 20 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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (A) Representative confocal micrographs taken at different time points were stained with DAPI (dark blue nuclei) and SYTOX Green (light blue). Scale bar: 20 μM.


( B ) Representative confocal of untreated ( Mock ) , S . Typhimurium - infected or PMA - stimulated wildtype BMNs stained for neutrophil elastase ( Red ) SYTOX Green ( Green ) ( Scale bar : 10 μM ) .

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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O

(B) Representative confocal micrographs of untreated (Mock), S. Typhimurium-infected or PMA-stimulated wildtype BMNs stained for neutrophil elastase (Red) and SYTOX Green (Green) (Scale bar: 10 μM).


Neutrophils of indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( C , lysates ( Lys ) culture media ( Sup ) were immunoblotted for indicated at 1h 3 hr post - infection . Data information : S . Typh . : S . Typhimurium .

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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (C, Cell lysates (Lys) and culture media (Sup) were immunoblotted for the indicated proteins at 1h and 3 hr post-infection. Data information: S. Typh.: S. Typhimurium.


Neutrophils of indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . D ) lysates ( Lys ) culture media ( Sup ) were immunoblotted for indicated at 1h 3 hr post - infection . Data information : S . Typh . : S . Typhimurium .

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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. D) Cell lysates (Lys) and culture media (Sup) were immunoblotted for the indicated proteins at 1h and 3 hr post-infection. Data information: S. Typh.: S. Typhimurium.


Neutrophils of indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( E ) was measured by LDH 3 h post - infection . Data information : In E , values represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (E) Cell death was measured by LDH assay 3 h post-infection. Data information: In E, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


Neutrophils of indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( F ) IL - 1β secretion were determined by Luminex 1 hr post - infection . Data information : In F , values represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (F) IL-1β secretion levels were determined by Luminex assay 1 hr post-infection. Data information: In F, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


Neutrophils of indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( G ) Representative confocal of neutrophils stained with DAPI ( blue nuclei ) SYTOX Green ( blue ) taken at different time points post - infection ( scale bar : 20 μM ) . ( H ) PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : In H , values represent mean ± of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (G) Representative confocal micrographs of neutrophils stained with DAPI (dark blue nuclei) and SYTOX Green (light blue) taken at different time points post-infection (scale bar: 20 μM). (H) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: In H, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


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

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

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

O O O O O 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 B-CONTROLLED_VAR 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) 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 of choroidal explants cultured for 5 days in presence or absence of COCO . Scale bar , 500 μm . ( F ) of sprouting surface of shown in ( C ; n = 6 ) .

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

(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 ) of wound closure .

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

(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 ) of EdU incorporation .

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


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

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

(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 of signal ) . Scale bar , 100 μm . ( C ) of vascular number of branchpoints ( p - values are versus PBS treatment ) . Vessel : * * 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 ) .

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 O O O O O 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 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

(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 ) of % of IsoB4 + vessels to Coll IV + vessels ; ( n = 9 ) .

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

(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 ) of of IsoB4 vascular covered by NG2 ( % 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 ) of apoptotic in control COCO - injected mice ( n = 3 ) .

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

(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 of signal ) . Scale bar , 100 μm . ( C ) of vascular number of branchpoints ( p - values are versus PBS treatment ) . Vessel : * * 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 B-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 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 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

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

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_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

(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 of signal ) . Scale bar , 100 μm . ( C ) of vascular number of branchpoints ; ( n = 4 ) .

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

(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 ) of vascular number of branchpoints ; ( n = 4 ) .

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

(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 ) of neovascular tuft in P17 OIR . * * P = 0 . 0021 ( PBS vs COCO ) ; * P = 0 . 0122 ( PBS vs Flt1Fc ) ; ( n = 6 ) . ( D ) of central avascular 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 prior to - CNV injected with PBS , COCO or Flt1Fc stained with IB4 ( green ) phalloidin ( red ) . Scale bar , 100 μm . ( G ) of CNV surface . * P = 0 . 0210 ( PBS vs COCO ) ; * P = 0 . 0113 ( PBS vs Flt1Fc ) ; ( n = 12 - 14 burns from 5 / group ) .

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 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

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

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(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 of western blot experiments ; ( n = 3 ) .

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

(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 revealing a unique band at ~ 36 kDa ( arrow ) . Recombinant human COCO was used as positive control .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

(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 incubated with an anti - mouse COCO antibody revealing a unique band at ~ 36 - 38 kDa ( arrow ) . Western blots are representative of 3 independent experiments .

O O O O O O O O O O O O O O O O O B-MEASURED_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

(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 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 secondary antibody .

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(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 ) hematoxylin of P5 mouse retinas .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

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


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

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


( F ) Immunofluoresence 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 generated by DESeq2 of top - altered genes of HUVECs treated with COCO compared to PBS - treated control ( 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 of mitochondrial respiration ( OCR ) glycolysis ( ECAR ) in HUVECs stimulated with COCO for 1 , 2 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 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

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

O O O O B-MEASURED_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

(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 ) .

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

(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 in NADH for 6 or 24 hours with COCO . * * P = 0 . 0028 ( 6hours ) ; * P = 0 . 0207 ( 24hours ) ; ( n = 3 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_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 O O O O O O O O O

(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 ) .

O O O O B-MEASURED_VAR 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

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 ROS using DCFDA in HUVECs treated with COCO / 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 B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR 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 in HUVECs treated with COCO . * P = 0 . 0372 ( - vs 24hours ) ; * P = 0 . 0137 ( - vs 3 - NP ) ; ( n = 3 ) .

O O O O O O B-MEASURED_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 O O O O O O O O O O O O O O

(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 of choroidal explants cultured for 4 days in presence or absence of COCO / or N - acetylcystein . Scale bar , 500 μm . ( I ) of sprouting surface of 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

(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).


A . A549 were transfected with 2 μg of poly ( I : C ) for 6 hrs then infected with ZIKV ( MOI = 3 ) for 18 hrs ( Pre - treatment ) or infected with ZIKV first for 6 hrs then transfected with poly ( I : C ) for 12 hrs ( Post - treatment ) . of ZIKVgenomic were determined by qRT - PCR . Values are expressed as mean ± standard error . * * * P < 0 . 001 ( Student ' s t - ) , N =

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 O 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 O O O O O O O O O O O O O O O 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. A549 cells were transfected with 2 μg of poly(I:C) for 6 hrs and then infected with ZIKV (MOI=3) for 18 hrs (Pre-treatment) or infected with ZIKV first for 6 hrs and then transfected with poly(I:C) for 12 hrs (Post-treatment). Levels of ZIKVgenomic RNA were determined by qRT-PCR. Values are expressed as mean ± standard error. ***P<0.001 (Student's t-test), N=3


B . A549 infected with ZIKV ( MOI = 3 ) for 6 hrs were transfected with 2 μg poly ( I : C ) for 12 hrs after which of ifn -

O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR

B. A549 cells infected with ZIKV (MOI=3) for 6 hrs were transfected with 2 μg poly(I:C) for 12 hrs after which levels of ifn-


C . A549 were infected with ZIKV ( MOI = 5 ) for 16 hrs then transfected with an IFIT1 promoter - driven firefly luciferase plasmid ( pGL3B / 561 ) a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 1 μg of poly ( I : C ) . Eight hours later lysates were harvested subjected to luciferase . Values are expressed as mean of two independent experiments , N =

O O O O O O O O O O O O O O O O O O O B-MEASURED_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

C. A549 cells were infected with ZIKV (MOI=5) for 16 hrs and then transfected with an IFIT1 promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N=2


D . HEK293T were transfected with plasmids encoding each of 10 ZIKV . Sixteen hours later they were transfected with an IFIT1 promoter - driven firefly luciferase plasmid ( pGL3B / 561 ) a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 1 μg of poly ( I : C ) . Eight hours later lysates were harvested subjected to luciferase . C = capsid ; E = envelope . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) , N =

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_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 O O O O O O O O O O O O O O

D. HEK293T cells were transfected with plasmids encoding each of the 10 ZIKV proteins. Sixteen hours later they were transfected with an IFIT1 promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test), N=4


E . A549 were infected with ZIKV ( MOI = 5 ) for 16 hrs then transfected with indicated promoter - driven firefly luciferase plasmids a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 1 μg of poly ( I : C ) . Eight hours later lysates were harvested subjected to luciferase . Values are expressed as mean of two independent experiments , N =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A549 cells were infected with ZIKV (MOI=5) for 16 hrs and then transfected with the indicated promoter-driven firefly luciferase plasmids and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N=2