A . The graph shows the number of differentially expressed ( DE ) genes 24h following SNA or DCA compared to their respective controls .

O 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. The graph shows the number of differentially expressed (DE) genes 24h following SNA or DCA compared to their respective controls.


B . RNAseq read counts show differential occupancy of H3K9ac on specific gene regions , including an increased occupancy close to the transcriptional start site ( TSS ) after SNA only , when compared to corresponding controls .

O O O O O O O O O O O O O O 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. RNAseq read counts show differential occupancy of H3K9ac on specific gene regions, including an increased occupancy close to the transcriptional start site (TSS) after SNA only, when compared to corresponding controls.


C . Bar graph shows the number of genes that are upregulated or downregulated following SNA or DCA ( RNAseq ) with an increased or decreased H3K9ac occupancy ( ChIPseq ) , respectively . D . Odds ratio analysis of the enrichment / depletion of genes with differential H3K9ac occupancy across each upregulated and downregulated gene cluster in ( C ) ; the numbers in red represent the p value given by Fisher ' s exact test ( ns = not significant ) .

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

C. Bar graph shows the number of genes that are upregulated or downregulated following SNA or DCA (RNAseq) with an increased or decreased H3K9ac occupancy (ChIPseq), respectively. D. Odds ratio analysis of the enrichment/depletion of genes with differential H3K9ac occupancy across each upregulated and downregulated gene cluster in (C); the numbers in red represent the p value given by Fisher's exact test (ns= not significant).


A , B . Co - immunofluorescence of anti - neuronal and anti - regeneration associated proteins in DRG following SCI and treatment with 966 or vehicle . Shown is increased protein expression for regeneration - associated targets in NF200 positive DRG neurons ( arrows ) after 966 treatment ( 5 weeks following SCI ) . Scale bar , 50 μm . ( B ) Data is expressed as fold change of fluorescence intensity in NF200 + cells , 966 versus vehicle ± s . e . m . N = 4 - 7 biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 indicate significant difference versus vehicle ( Student ' s t - test ) .

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 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 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

A, B. Co-immunofluorescence of anti-neuronal and anti-regeneration associated proteins in DRG following SCI and treatment with 966 or vehicle. Shown is increased protein expression for regeneration-associated targets in NF200 positive DRG neurons (arrows) after 966 treatment (5 weeks following SCI). Scale bar, 50 μm. (B) Data is expressed as fold change of fluorescence intensity in NF200+ cells, 966 versus vehicle ± s.e.m. N=4-7 biological replicates * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference versus vehicle (Student's t-test).


A - B . AAV - HDAC3 mutant ( Y298H ; dominant negative deacetylase inactive ) promoted DRG regenerative growth after spinal cord injury vs AAV - V5 control ( injected 5 weeks prior to SCI in the sciatic nerve bilaterally ) as shown by dextran - red traced axons across and beyond the lesion site ( GFAP , green ) ( dotted lines denote the rostral and caudal margins of the scar around the lesion ) . Scale bar , ( A - B ) 200 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. AAV-HDAC3 mutant (Y298H; dominant negative deacetylase inactive) promoted DRG regenerative growth after spinal cord injury vs AAV-V5 control (injected 5 weeks prior to SCI in the sciatic nerve bilaterally) as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, (A-B) 200 μm.


C - F . Immunofluorescence for H3K9ac . AAV - HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons , but not in surrounding nuclei of satellite cells . Scale bar , ( C - F ) 50 μm .

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

C-F. Immunofluorescence for H3K9ac. AAV-HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons, but not in surrounding nuclei of satellite cells. Scale bar, (C-F) 50 μm.


G - J . New synaptic formations ( VGlut1 + ) from regrowing axons are shown beyond the lesion site after injection of AAV - HDAC3mut . ( G ) Scale bar 200 μm , ( H - J ) High magnification insets depict co - localization of Dextran and vGlut1 staining . Scale bar 50 μm .

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

G-J. New synaptic formations (VGlut1+) from regrowing axons are shown beyond the lesion site after injection of AAV-HDAC3mut. (G) Scale bar 200 μm, (H-J) High magnification insets depict co-localization of Dextran and vGlut1 staining. Scale bar 50 μm.


K - L . Quantification of dextran positive axons shows that AAV - HDAC3mut promotes DRG regenerative growth across and beyond the spinal lesion site . Data is expressed as percentage of dextran + axons at each distance vs dextran + axons at - 700μm from the lesion margin ( K ) or distance from the caudal margin of the lesion to the most rostral dextran + axon tip for each animal ( L ) . N = 10 animals per condition , ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 indicate a significant difference ( ANOVA followed by Bonferroni test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of dextran positive axons shows that AAV-HDAC3mut promotes DRG regenerative growth across and beyond the spinal lesion site. Data is expressed as percentage of dextran+ axons at each distance vs dextran+ axons at -700μm from the lesion margin (K) or distance from the caudal margin of the lesion to the most rostral dextran+ axon tip for each animal (L). N= 10 animals per condition, ± s.e.m. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 indicate a significant difference (ANOVA followed by Bonferroni test).


A - B . Intrathecally administered RGFP966 ( 966 ) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran - red traced axons across and beyond the lesion site ( GFAP , green ) ( dotted lines denote the rostral and caudal margins of the scar around the lesion ) . Scale bar , 200 μm .

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

A-B. Intrathecally administered RGFP966 (966) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, 200 μm.


C - D . Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site . Data is expressed as ratio of dextran + axons vs dextran + axons at - 700 μm from the lesion site ± s . e . m ( C ) or distance from the caudal margin of the lesion to the last dextran + axon tip ( D ) . N = 6 - 10 animals per condition . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 ) indicate a significant difference ( ANOVA followed by Bonferroni test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site. Data is expressed as ratio of dextran+ axons vs dextran+ axons at -700 μm from the lesion site ± s.e.m (C) or distance from the caudal margin of the lesion to the last dextran+ axon tip (D). N= 6-10 animals per condition. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).


E . Immunofluorescence for H3K9ac and Neurofilament 200 ( NF200 ) shows upregulated H3K9ac in vivo in DRG neurons after 966 . Scale bar , 50 µm .

O O O O O O O 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

E. Immunofluorescence for H3K9ac and Neurofilament 200 (NF200) shows upregulated H3K9ac in vivo in DRG neurons after 966. Scale bar, 50 µm.


F - G . Data is expressed as fold change of fluorescence intensity mean levels ( F ) or % of H3K9ac + cells ( G ) ( H3K9ac + threshold was set at the mean intensity level of vehicle treated animals , DAPI counter staining was used to label nuclei from NF200 + or NF200 - cells ) vs veh ± s . e . m . N = 11 - 12 biological replicates . ( * * * p < 0 . 005 ) indicate a significant difference vs respective Veh ( ANOVA followed by Bonferroni test ) .

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

F-G. Data is expressed as fold change of fluorescence intensity mean levels (F) or % of H3K9ac+ cells (G) (H3K9ac+ threshold was set at the mean intensity level of vehicle treated animals, DAPI counter staining was used to label nuclei from NF200+ or NF200- cells) vs veh± s.e.m. N= 11-12 biological replicates. (***p<0.005) indicate a significant difference vs respective Veh (ANOVA followed by Bonferroni test).


( D ) HeLa cells were transfected with 10 nM control ( n = 84 ) or Nup50 siRNA ( n = 171 ) , 20 nM control ( n = 101 ) or Nup50 siRNA ( n = 157 ) and 40 nM control ( n = 86 ) or Nup50 siRNA ( n = 204 ) . 72 h post transfection , cells were fixed with 4 % PFA and stained with antibodies against Nup50 and mAB414 , chromatin was labeled with DAPI . Scale bars : 50 µm . Quantitation of the mAB414 rim intensity at three different Nup50 RNAi and control oligo concentrations . The means are indicated as diamonds , error bars show the standard deviations . P - values have been calculated from a student t - test comparing the mean between the experimental conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with 10 nM control (n=84) or Nup50 siRNA (n=171), 20 nM control (n=101) or Nup50 siRNA (n=157) and 40 nM control (n=86) or Nup50 siRNA (n=204). 72 h post transfection, cells were fixed with 4% PFA and stained with antibodies against Nup50 and mAB414, chromatin was labeled with DAPI. Scale bars: 50 µm. Quantitation of the mAB414 rim intensity at three different Nup50 RNAi and control oligo concentrations. The means are indicated as diamonds, error bars show the standard deviations. P-values have been calculated from a student t-test comparing the mean between the experimental conditions.


( A ) Mouse 3T3 - NIH cells were transfected with 20 nM control , Nup50A , Nup50B or a combination of Nup50A and Nup50B siRNA . After 72h cells were fixed and stained with mAB414 ( green ) and antibodies against mouse Nup50 ( red ) . Chromatin is stained with DAPI ( blue ) . The merge of the three channels is shown on the left column . Scale bar : 50 µm . An insert shows a zoom on a representative nucleus for each picture . Scale bar : 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mouse 3T3-NIH cells were transfected with 20 nM control, Nup50A, Nup50B or a combination of Nup50A and Nup50B siRNA. After 72h cells were fixed and stained with mAB414 (green) and antibodies against mouse Nup50 (red). Chromatin is stained with DAPI (blue). The merge of the three channels is shown on the left column. Scale bar: 50 µm. An insert shows a zoom on a representative nucleus for each picture. Scale bar: 10 µm.


( B ) Quantitation of the mAB414 rim intensity from n = 4 independent experiments done as in ( A ) , each represented by a different color . The mean of each individual experiment and condition is represented by a diamond , the mean of all four experiments by a horizontal black line . P - values have been calculated from a paired ratio t - test comparing the mean between the experimental conditions . Error bars show the standard deviations of the means .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitation of the mAB414 rim intensity from n=4 independent experiments done as in (A), each represented by a different color. The mean of each individual experiment and condition is represented by a diamond, the mean of all four experiments by a horizontal black line. P-values have been calculated from a paired ratio t-test comparing the mean between the experimental conditions. Error bars show the standard deviations of the means.


( A , B ) Demembranated sperm chromatin was preincubated in Xenopus egg extract , depleted for MEL28 / ELYS or Nup50 ( B ) or control treated ( Mock , A ) . After 10 min , membranes were added to the reaction . Reactions were stopped at the indicated time points by fixation and analyzed by confocal microscopy after immunostaining with α - MEL28 / ELYS ( A ) , α - Nup50 ( B ) , and mAb414 ( A , B ) . Chromatin was stained with DAPI ( blue in the overlay ) . Scale bar : 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Demembranated sperm chromatin was preincubated in Xenopus egg extract, depleted for MEL28/ELYS or Nup50 (B) or control treated (Mock, A). After 10 min, membranes were added to the reaction. Reactions were stopped at the indicated time points by fixation and analyzed by confocal microscopy after immunostaining with α-MEL28/ELYS (A), α-Nup50 (B), and mAb414 (A, B). Chromatin was stained with DAPI (blue in the overlay). Scale bar: 10 μm.


3 µM recombinant EGFP , EGFP - tagged MEL28 / ELYS ( aa 2290 - 2408 ) or EGFP - tagged Nup50 were incubated with empty or DNA - coated magnetic beads , which were chromatinized with Xenopus egg extracts ( right panel ) or unchromatinized DNA - beads ( left panel ) . After 3h the beads were re - isolated , washed , co - stained with DAPI and analyzed by confocal microscopy . Scale bar : 10 µm .

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

3 µM recombinant EGFP, EGFP-tagged MEL28/ELYS (aa 2290-2408) or EGFP-tagged Nup50 were incubated with empty or DNA-coated magnetic beads, which were chromatinized with Xenopus egg extracts (right panel) or unchromatinized DNA-beads (left panel). After 3h the beads were re-isolated, washed, co-stained with DAPI and analyzed by confocal microscopy. Scale bar: 10 µm.


( C ) HeLa cells were transfected with EGFP - Nup50 comprising different point mutations in the minimal NPC binding region and analyzed as in ( A ) . Scale Bar : 10 µm .

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

(C) HeLa cells were transfected with EGFP-Nup50 comprising different point mutations in the minimal NPC binding region and analyzed as in (A). Scale Bar: 10 µm.


( D ) GST - fusion constructs of the Xenopus Nup50 minimal NPC binding fragment ( aa 144 - 189 ) comprising no or single point mutations were incubated with Xenopus egg extracts . Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies . The quantitation shows the average MEL28 / ELYS and Nup153 bead bound signal from three independent experiments , normalized to the signal of their respective wild - type GST fusion Data points from individual experiments are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GST-fusion constructs of the Xenopus Nup50 minimal NPC binding fragment (aa 144-189) comprising no or single point mutations were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies. The quantitation shows the average MEL28/ELYS and Nup153 bead bound signal from three independent experiments, normalized to the signal of their respective wild-type GST fusion Data points from individual experiments are indicated.


( A ) Confocal microscopy images of nuclei assembled for 120 min in mock depleted , Nup50 depleted ( ∆ Nup50 ) , and Nup50 depleted Xenopus egg extracts supplemented with recombinant wild - type Nup50 or different mutants . Nuclei were fixed in 4 % PFA and 0 . 5 % glutaraldehyde , stained for NPCs ( mAB414 ) and the chromatin ( DAPI ) . Scale bar : 10 µm . ( B ) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in ( A ) . Data points from the individual experiments are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant wild-type Nup50 or different mutants. Nuclei were fixed in 4% PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. (B) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (A). Data points from the individual experiments are indicated.


( C ) Confocal microscopy images of nuclei assembled with N - terminal Nup50 truncations and an minimal N - terminal fragment . Samples were analyzed as in ( A ) . ( D ) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in ( C ) . Data points from the individual experiments are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of nuclei assembled with N-terminal Nup50 truncations and an minimal N-terminal fragment. Samples were analyzed as in (A). (D) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (C). Data points from the individual experiments are indicated.


( B ) Confocal microscopy images of fixed nuclei assembled for 120 min in mock depleted ( mock ) and Nup50 depleted ( ∆ Nup50 ) Xenopus egg extracts supplemented with recombinant mouse Nup50 orthologues . Nuclei were stained for Nup50 ( Nup50 antibody for Xenopus protein , His6 for mouse protein ) and NPCs ( mAB414 , red ) on the chromatin ( DAPI ) . Scale bar : 10 µm . ( C ) The average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments ( performed as in B ) is shown . Data points from individual experiments are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of fixed nuclei assembled for 120 min in mock depleted (mock) and Nup50 depleted (∆Nup50) Xenopus egg extracts supplemented with recombinant mouse Nup50 orthologues. Nuclei were stained for Nup50 (Nup50 antibody for Xenopus protein, His6 for mouse protein) and NPCs (mAB414, red) on the chromatin (DAPI). Scale bar: 10 µm. (C) The average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments (performed as in B) is shown. Data points from individual experiments are indicated.


( E ) GST - fusion constructs of the Xenopus Nup53 RRM domain ( aa 162 - 267 , control ) full - length Xenopus Nup50 and the two mouse Nup50 orthologues were incubated with Xenopus egg extracts . Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies The right panel shows the average MEL28 / ELYS , Nup153 , importin β and ran bead bound signal from two independent experiments normalized to their respective Xenopus signal . Data points from individual experiments are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GST-fusion constructs of the Xenopus Nup53 RRM domain (aa 162-267, control) full-length Xenopus Nup50 and the two mouse Nup50 orthologues were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies The right panel shows the average MEL28/ELYS, Nup153, importin β and ran bead bound signal from two independent experiments normalized to their respective Xenopus signal. Data points from individual experiments are indicated.


HEK293T cells were transfected with empty FLAG - tag vector as a control or FLAG - Nup50 N - terminal fragment ( aa 1 - 120 of the human sequence ) . 24 h post transfection cells were lysed , FLAG - tagged proteins immuno - isolated and analyzed by mass spectrometry with the Volcano blot showing the identified interactors of Nup50 ( yellow , data from three independent experiments ) Data points from individual experiments are indicated . KPNA2 is the gene name of importin α .

HEK293T cells were transfected with empty FLAG-tag vector as a control or FLAG-Nup50 N-terminal fragment (aa 1-120 of the human sequence). 24 h post transfection cells were lysed, FLAG-tagged proteins immuno-isolated and analyzed by mass spectrometry with the Volcano blot showing the identified interactors of Nup50 (yellow, data from three independent experiments) Data points from individual experiments are indicated. KPNA2 is the gene name of importin α.


HEK293T cells were transfected with empty FLAG - tag vector as a control or FLAG - Nup50 N - terminal fragment ( aa 1 - 120 of the human sequence ) . 24 h post transfection cells were lysed , FLAG - tagged proteins immuno - isolated and analyzed by western blotting ( B ) , with 10 % of the inputs loaded . The quantification of the western blot ( B , lower panel ) shows the mean signal intensity normalized on the input of at least two independent experiments . Data points from individual experiments are indicated .

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

HEK293T cells were transfected with empty FLAG-tag vector as a control or FLAG-Nup50 N-terminal fragment (aa 1-120 of the human sequence). 24 h post transfection cells were lysed, FLAG-tagged proteins immuno-isolated and analyzed by western blotting (B), with 10% of the inputs loaded. The quantification of the western blot (B, lower panel) shows the mean signal intensity normalized on the input of at least two independent experiments. Data points from individual experiments are indicated.


( E ) 2 µM recombinant Ran , loaded with MANT - GDP was incubated with 2 mM GppNHp in buffer control , supplemented with 2 nM recombinant RCC1 , 20 nM recombinant Xenopus Nup50 proteins , or RCC1 and Nup50 together . GDP to GppNHp exchanged was monitored by the decrease in MANT fluorescence of the liberated GDP - MANT . The lower panel shows the rate constant of each experimental condition with n = 4 independent experiment per condition , bars represent the mean , individual data points are indicated .

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 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) 2 µM recombinant Ran, loaded with MANT-GDP was incubated with 2 mM GppNHp in buffer control, supplemented with 2 nM recombinant RCC1, 20 nM recombinant Xenopus Nup50 proteins, or RCC1 and Nup50 together. GDP to GppNHp exchanged was monitored by the decrease in MANT fluorescence of the liberated GDP-MANT. The lower panel shows the rate constant of each experimental condition with n=4 independent experiment per condition, bars represent the mean, individual data points are indicated.


( G ) Confocal microscopy images of nuclei assembled for 120 min in mock depleted , Nup50 depleted ( ∆ Nup50 ) , and Nup50 depleted Xenopus egg extracts supplemented with recombinant Xenopus wild - type Nup50 or RCC1 binding mutants . Nuclei were fixed in 4 % PFA and 0 . 5 % glutaraldehyde , stained for NPCs ( mAB414 ) and the chromatin ( DAPI ) . Scale bar : 10 µm . Quantitation shows the average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments . Individual data points are indicated ( H ) Confocal microscopy images of nuclei assembled for 120 min in mock depleted , Nup50 depleted ( ∆ Nup50 ) , and Nup50 depleted Xenopus egg extracts supplemented with RCC1 excess as indicated . Nuclei were fixed in 4 % PFA and 0 . 5 % glutaraldehyde , stained for NPCs ( mAB414 ) and the chromatin ( DAPI ) . Scale bar : 10 µm . Quantitation shows the average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments . Individual data points are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant Xenopus wild-type Nup50 or RCC1 binding mutants. Nuclei were fixed in 4 % PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated (H) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with RCC1 excess as indicated. Nuclei were fixed in 4 % PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated.


Effects of basic and bulky hydrophobic amino acids on LT - induced pyroptosis and caspase - 1 activation in RAWRA cells . Cells were pre - treated with 10 mM indicated amino acids prior to LT stimulation . Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates ( A ) . Mock , DMEM medium .

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

Effects of basic and bulky hydrophobic amino acids on LT-induced pyroptosis and caspase-1 activation in RAWRA cells. Cells were pre-treated with 10 mM indicated amino acids prior to LT stimulation. Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates (A). Mock, DMEM medium.


Cell supernatants were subjected to anti - caspase - 1 immunoblotting and the lysates were blotted with the anti - tubulin antibody ( B ) . p10 , mature caspase - 1 ; p45 , caspase - 1 precursor .

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

Cell supernatants were subjected to anti-caspase-1 immunoblotting and the lysates were blotted with the anti-tubulin antibody (B). p10, mature caspase-1; p45, caspase-1 precursor.


C , D Effects of basic and bulky hydrophobic amino acids on LT - induced RFP - ASC specks formation in RAWRA cells . Fluorescence images were taken on a confocal microscopy ( C ) . The percentages of cells showing RFP - ASC specks ( mean values ± SD from three replicates ) are in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Effects of basic and bulky hydrophobic amino acids on LT-induced RFP-ASC specks formation in RAWRA cells. Fluorescence images were taken on a confocal microscopy (C). The percentages of cells showing RFP-ASC specks (mean values ± SD from three replicates) are in (D).


E Effects of basic and bulky hydrophobic amino acids on LT - induced caspase - 1 activation in Tlr4 - / - iBMDMs . Cells were treated as that in ( A ) . Cell supernatants were subjected to anti - caspase - 1 immunoblotting and the lysates were blotted with the anti - tubulin or anti - MEK3 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Effects of basic and bulky hydrophobic amino acids on LT-induced caspase-1 activation in Tlr4-/- iBMDMs. Cells were treated as that in (A). Cell supernatants were subjected to anti-caspase-1 immunoblotting and the lysates were blotted with the anti-tubulin or anti-MEK3 antibody.


A RFP - ASC specks formation assay of the effect of Ubr2 knockdown on LT - induced inflammasome activation . RAWRA cells were transfected with a control siRNA or Ubr2 - targeting siRNA mixtures for 60 h followed by LT treatment . The numbers in the merged panel are the percentages of the cells showing RFP - ASC specks .

A RFP-ASC specks formation assay of the effect of Ubr2 knockdown on LT-induced inflammasome activation. RAWRA cells were transfected with a control siRNA or Ubr2-targeting siRNA mixtures for 60 h followed by LT treatment. The numbers in the merged panel are the percentages of the cells showing RFP-ASC specks.


B Effect of Ubr2 stable knockdown on caspase - 1 activation in RAWRA cells . Cells stably expressing a control or Ubr2 - targeting shRNA were incubated with WT LT ( + ) or its E687C mutant ( - ) for 3 h .

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

B Effect of Ubr2 stable knockdown on caspase-1 activation in RAWRA cells. Cells stably expressing a control or Ubr2-targeting shRNA were incubated with WT LT (+) or its E687C mutant (-) for 3 h.


C Effects of Ubr2 knockdown ( siRNA # 05 ) on LT - induced NLRP1B inflammasome activation reconstituted in 293T cells . Cells were treated with WT LT ( + ) or its E687C mutant ( - ) .

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

C Effects of Ubr2 knockdown (siRNA #05) on LT-induced NLRP1B inflammasome activation reconstituted in 293T cells. Cells were treated with WT LT (+) or its E687C mutant (-).


D , E Effect of Ubr2 knockout on NLRP1B or NAIP2 - NLRC4 inflammasome activation . WT and Ubr2 - / - iBMDMs were treated with LT or BsaK for 3 h . Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E Effect of Ubr2 knockout on NLRP1B or NAIP2-NLRC4 inflammasome activation. WT and Ubr2-/- iBMDMs were treated with LT or BsaK for 3 h. Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates (D).


A Immunoblotting of caspase - 1 activation in Ubrs - knockdown BMDMs derived from the 129 / Sv mice . Cells were transfected with the indicated siRNA for 60 h , and then treated with WT LT ( + ) or its E687C mutant ( - ) for 3 h .

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

A Immunoblotting of caspase-1 activation in Ubrs-knockdown BMDMs derived from the 129/Sv mice. Cells were transfected with the indicated siRNA for 60 h, and then treated with WT LT (+) or its E687C mutant (-) for 3 h.


Immunoblotting of caspase - 1 activation in E2s - knockdown BMDMs derived from the 129 / Sv mice .

Immunoblotting of caspase-1 activation in E2s-knockdown BMDMs derived from the 129/Sv mice.


Additional Ube2o siRNAs were used to confirm the inhibitory effect of Ube2o knockdown on LT - induced caspase - 1 activation ( C ) . Ube2o knockdown efficiency was measured by qPCR and are shown as mean values ± SD from three replicates .

Additional Ube2o siRNAs were used to confirm the inhibitory effect of Ube2o knockdown on LT-induced caspase-1 activation (C). Ube2o knockdown efficiency was measured by qPCR and are shown as mean values ± SD from three replicates.


RFP - ASC specks formation assay of the effect of Ube2o knockdown ( siRNA Ube2o - 01 ) on LT - induced inflammasome activation in RAWRA cells . The percentages of cells showing RFP - ASC specks ( mean values ± SD from three replicates ) are in ( D ) .

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

RFP-ASC specks formation assay of the effect of Ube2o knockdown (siRNA Ube2o-01) on LT-induced inflammasome activation in RAWRA cells. The percentages of cells showing RFP-ASC specks (mean values ± SD from three replicates) are in (D).


F Co - immunoprecipitation interaction of UBE2O and UBR2 . Myc - UBE2O and Flag - UBR2 were co - expressed in 293T cells and cell lysates were subjected to anti - Flag immunoprecipitation followed by immunoblotting analyses as shown .

O O O O O O O O O O O O O O O O O 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 Co-immunoprecipitation interaction of UBE2O and UBR2. Myc-UBE2O and Flag-UBR2 were co-expressed in 293T cells and cell lysates were subjected to anti-Flag immunoprecipitation followed by immunoblotting analyses as shown.


B Effect of LT treatment on NLRP1B protein level . 293T cells were transfected with Flag - NLRP1B expression plasmid for 24 h , and cells were then treated with WT LT ( + ) or its E687C mutant ( - ) for 4 h . Shown is the anti - Flag immunoblot of the total cell lysates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Effect of LT treatment on NLRP1B protein level. 293T cells were transfected with Flag-NLRP1B expression plasmid for 24 h, and cells were then treated with WT LT (+) or its E687C mutant (-) for 4 h. Shown is the anti-Flag immunoblot of the total cell lysates.


C RFP - ASC specks formation in 293T cells expressing an indicated NLRP1B variant . Cells were treated with LT for 3 h . Scale bar , 20 μm . 1 - 983 and 984 - C ( residues 984 to the C terminus ) are the FIIND - mediated autocleaved fragments of NLRP1B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RFP-ASC specks formation in 293T cells expressing an indicated NLRP1B variant. Cells were treated with LT for 3 h. Scale bar, 20 μm. 1-983 and 984-C (residues 984 to the C terminus) are the FIIND-mediated autocleaved fragments of NLRP1B.


Effect of MG132 on LT - induced NLRP1B degradation . 293T cells expressing NLRP1B ( 1 - 983 ) - HA were treated with LT and MG132 for 8 h . Shown are the anti - HA and anti - tubulin immunoblots of the total cell lysates .

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

Effect of MG132 on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates.


E Effect of MG132 and Leu on LT - induced NLRP1B degradation . 293T cells expressing NLRP1B ( 1 - 983 ) - HA were treated with LT and MG132 for 8 h . 10 mM Leu was added to cells 30 min prior to LT treatment . Shown are the anti - HA and anti - tubulin immunoblots of the total cell lysates .

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

E Effect of MG132 and Leu on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. 10 mM Leu was added to cells 30 min prior to LT treatment. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates.


F LT - induced ubiquitination of NLRP1B . 293T cells expressing NLRP1B ( 1 - 983 ) - HA were treated with LT and / or MG132 for 6 h . Cells were then harvested for anti - HA immunoprecipitation and subjected to immunoblotting analyses using indicated antibodies .

O O 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

F LT-induced ubiquitination of NLRP1B. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and/or MG132 for 6 h. Cells were then harvested for anti-HA immunoprecipitation and subjected to immunoblotting analyses using indicated antibodies.


A Pup survival analysis between P0 - P10 , by sex . Each dot represents the absolute percent of remaining animals per group ( Jag1CTRL n = 211 , male Jag1Ndr / Ndr n = 26 , female Jag1Ndr / Ndr n = 20 , log - rank test , * * * * P < 0 . 0001 ) .

A Pup survival analysis between P0-P10, by sex. Each dot represents the absolute percent of remaining animals per group (Jag1CTRL n=211, male Jag1Ndr/Ndr n=26, female Jag1Ndr/Ndr n= 20, log-rank test, ****P<0.0001).


B P10 plasma total bilirubin levels ( n = 6 per group , unpaired t test , * * P = 0 . 0091 ) . C P10 plasma thrombin antithrombin levels ( n = 6 per group , unpaired t test , ns , P = 0 . 2788 ) .

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

B P10 plasma total bilirubin levels (n=6 per group, unpaired t test, **P=0.0091). C P10 plasma thrombin antithrombin levels (n=6 per group, unpaired t test, ns, P=0.2788).


D P10 serum fibrinogen levels ( n = 6 per group , unpaired t test , ns , P = 0 . 7585 ) .

O 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 P10 serum fibrinogen levels (n=6 per group, unpaired t test, ns, P=0.7585).


E P10 whole blood prothrombin time ( INR ) ( n = 6 per group , unpaired t test , ns , P > 0 . 9999 ) .

O O O O O 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 P10 whole blood prothrombin time (INR) (n=6 per group, unpaired t test, ns, P>0.9999).


F P10 tail bleeding time ( n = 6 per group , unpaired t test , ns , P = 0 . 8916 ) .

F P10 tail bleeding time (n=6 per group, unpaired t test, ns, P=0.8916).


G - I µCT of P30 skulls . ( G ) Color map depicting wall thickness . Scale bar 3 . 5 mm . ( H ) Skull full thickness volume ( n = 5 - 6 per group , two - way ANOVA . Interaction P = 0 . 7595 ; Sex P = 0 . 1164 , Genotype * * P = 0 . 0012 . Šídák ' s multiple comparisons test : Male Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0227 ; : Female Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 034 ) , ( I ) Compact bone total volume ( n = 5 - 6 per group , two - way ANOVA . Interaction P = 0 . 8631 ; Sex P = 0 . 0901 , Genotype * * P = 0 . 0014 . Šídák ' s multiple comparisons test : Male Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0305 ; Female Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0307 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I µCT of P30 skulls. (G) Color map depicting wall thickness. Scale bar 3.5 mm. (H) Skull full thickness volume (n=5-6 per group, two-way ANOVA. Interaction P= 0.7595; Sex P= 0.1164, Genotype **P= 0.0012. Šídák's multiple comparisons test: Male Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0227; : Female Jag1+/+ vs Jag1Ndr/Ndr *P= 0.034), (I) Compact bone total volume (n=5-6 per group, two-way ANOVA. Interaction P= 0.8631; Sex P= 0.0901, Genotype **P= 0.0014. Šídák's multiple comparisons test: Male Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0305; Female Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0307).


J - K ( J ) Provoked vascular accidents induced by portal vein resin injection , with ( K ) resin leakage quantification outside of blood vessels ( red ) in adult mice ( n = 6 per group , unpaired t test , ns , P = 0 . 1088 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K (J) Provoked vascular accidents induced by portal vein resin injection, with (K) resin leakage quantification outside of blood vessels (red) in adult mice (n=6 per group, unpaired t test, ns, P=0.1088).


L Adult brain Evans blue assay ( Jag1CTRL n = 7 , Jag1Ndr / Ndr n = 8 , unpaired t test , ns , P = 0 . 3807 ) .

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

L Adult brain Evans blue assay (Jag1CTRL n=7, Jag1Ndr/Ndr n=8, unpaired t test, ns, P=0.3807).


M Hemorrhagic P3 Jag1Ndr / Ndr brain ( n = 2 / 83 Jag1Ndr / Ndr mice , sex n . d . ) .

M Hemorrhagic P3 Jag1Ndr/Ndr brain (n= 2/83 Jag1Ndr/Ndr mice, sex n.d.).


N Hemorrhagic P15 Jag1Ndr / Ndr retina . Scale bar 20 µm . Sex n . d .

N Hemorrhagic P15 Jag1Ndr/Ndr retina. Scale bar 20 µm. Sex n.d.


O Red blood cells outside P10 Jag1Ndr / Ndr retinal arteriole . Scale bar 10 µm .

O Red blood cells outside P10 Jag1Ndr/Ndr retinal arteriole. Scale bar 10 µm.


( A ) Radial arrangement of arterioles ( magenta ) and venules ( green ) from the optic nerve . Blue arrows label arteriovenous crossings . Magnification of crossing shows compression / narrowing of the underlying vein . Scale bar left and middle panels 50 µm , and right panel 20 µm .

(A) Radial arrangement of arterioles (magenta) and venules (green) from the optic nerve. Blue arrows label arteriovenous crossings. Magnification of crossing shows compression/narrowing of the underlying vein. Scale bar left and middle panels 50 µm, and right panel 20 µm.


( B ) Arteriovenous crossings number per retina ( n = 6 per group , two - way ANOVA with Tukey ' s multiple comparisons test . Interaction P = 0 . 7074 , Sex P = > 0 . 9999 , Genotype * * * * P = < 0 . 0001 , Tukey ' s multiple comparisons test : Male : Jag1 + / + vs . Male : Jag1Ndr / Ndr * P = 0 . 0201 ; Female : Jag1 + / + vs . Female : Jag1Ndr / Ndr * * P = 0 . 0061 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Arteriovenous crossings number per retina (n=6 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.7074, Sex P =>0.9999, Genotype ****P= <0.0001, Tukey's multiple comparisons test: Male:Jag1+/+ vs. Male:Jag1Ndr/Ndr *P= 0.0201; Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr **P= 0.0061).


C - D ( C ) Number of major arterioles ( n = 6 per group , two - way ANOVA with Tukey ' s multiple comparisons test . Interaction P = 0 . 4353 , Sex P = 0 . 1995 , Genotype * * * * P = < 0 . 0001 , Tukey ' s multiple comparisons test : Male : Jag1 + / + vs . Male : Jag1Ndr / Ndr * P = 0 . 0146 ; Female : Jag1 + / + vs . Female : Jag1Ndr / Ndr * * P = 0 . 0011 ) and ( D ) venules ( n = 6 per group , two - way ANOVA with Tukey ' s multiple comparisons test . Interaction P = 0 . 044 , Sex P = 0 . 044 , Genotype * * * * P = < 0 . 0001 , Tukey ' s multiple comparisons test : Male : Jag1Ndr / Ndr vs . Female : Jag1Ndr / Ndr * P = 0 . 0302 ; Female : Jag1 + / + vs . Female : Jag1Ndr / Ndr * * * P = 0 . 0002 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (C) Number of major arterioles (n=6 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.4353, Sex P =0.1995, Genotype ****P= <0.0001, Tukey's multiple comparisons test: Male:Jag1+/+ vs. Male:Jag1Ndr/Ndr *P= 0.0146; Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr **P= 0.0011) and (D) venules (n=6 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.044, Sex P =0.044, Genotype ****P= <0.0001, Tukey's multiple comparisons test: Male:Jag1Ndr/Ndr vs. Female:Jag1Ndr/Ndr *P=0.0302; Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr ***P= 0.0002).


E - F ( E ) Arterial tortuosity at P30 and 1 year , irrespective of sex ( n = 6 per group , two - way ANOVA , not significant ) . ( F ) Arterial tortuosity in male and female mice , irrespective of age ( n = 5 - 7 , two - way ANOVA , not significant ) . G - H ( G ) Venous tortuosity at P30 and 1 year , irrespective of sex ( n = 6 - 8 per group , two - way ANOVA with Tukey ' s multiple comparisons test . Interaction P = 0 . 1166 , Age P = 0 . 0787 , Genotype * * P = 0 . 0013 . Tukey ' s multiple comparisons test : P30 : Jag1 + / + vs . P30 : Jag1Ndr / Ndr * * P = 0 . 004 ; P30 : Jag1Ndr / Ndr vs . 1 year : Jag1Ndr / Ndr P = 0 . 0802 ; 1 year : Jag1 + / + vs . 1 year : Jag1Ndr / Ndr P = 0 . 5066 ) . ( H ) Venous tortuosity in male and female mice , irrespective of age ( n = 5 - 7 , two - way ANOVA with Tukey ' s multiple comparisons test . Interaction * P = 0 . 0223 , Sex * * P = 0 . 007 , Genotype * * * P = < 0 . 0001 . Tukey ' s multiple comparisons test : Male : Jag1 + / + vs . Male : Jag1Ndr / Ndr P = 0 . 3247 , Male : Jag1Ndr / Ndr vs . Female : Jag1Ndr / Ndr * * P = 0 . 0031 , Female : Jag1 + / + vs . Female : Jag1Ndr / Ndr * * * * P = 0 . 0001 ) .

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

E-F (E) Arterial tortuosity at P30 and 1 year, irrespective of sex (n=6 per group, two-way ANOVA, not significant). (F) Arterial tortuosity in male and female mice, irrespective of age (n=5-7, two-way ANOVA, not significant). G-H (G) Venous tortuosity at P30 and 1 year, irrespective of sex (n=6-8 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.1166, Age P = 0.0787, Genotype **P=0.0013. Tukey's multiple comparisons test: P30:Jag1+/+ vs. P30:Jag1Ndr/Ndr **P=0.004; P30:Jag1Ndr/Ndr vs. 1 year:Jag1Ndr/Ndr P=0.0802; 1 year:Jag1+/+ vs. 1 year:Jag1Ndr/Ndr P= 0.5066). (H) Venous tortuosity in male and female mice, irrespective of age (n=5-7, two-way ANOVA with Tukey's multiple comparisons test. Interaction *P = 0.0223, Sex **P = 0.007, Genotype ***P= <0.0001. Tukey's multiple comparisons test: Male:Jag1+/+ vs. Male:Jag1Ndr/Ndr P= 0.3247, Male:Jag1Ndr/Ndr vs. Female:Jag1Ndr/Ndr **P= 0.0031, Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr ****P= 0.0001).


( J ) Tortuosity of MCA , as assessed by number of diversions per length ( n = 6 , t - test ns ) .

O O 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) Tortuosity of MCA, as assessed by number of diversions per length (n=6, t-test ns).


( K ) Visualization of MCA with alpha smooth muscle cell actin ( αSMA ) showed stereotype vasculature in 6 of 6 Jag1 + / + animals , but 2 of 6 Jag1Ndr / Ndr animals showed highly divergent MCA architecture ( blue arrows ) .

O O O O O O O O O O O O O O O O O O O O O O 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) Visualization of MCA with alpha smooth muscle cell actin (αSMA) showed stereotype vasculature in 6 of 6 Jag1+/+ animals, but 2 of 6 Jag1Ndr/Ndr animals showed highly divergent MCA architecture (blue arrows).


( L ) Distances between arterial branches in MCA at P10 ( unpaired t - test , not significant ) .

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

(L) Distances between arterial branches in MCA at P10 (unpaired t-test, not significant).


A - E αSMA coverage of ( A ) P10 , ( B ) P15 , ( C ) P30 , ( D ) 3 - 6 month old and € 1 - year - old retinal arterioles . Brackets in A , B denote perpendicular VSMCs around arteriole . Yellow arrows in A , B , C . indicate VSMCs with an orientation parallel to the blood vessel . Green arrowheads in D denote stenosis . White arrowheads in E label αSMA - negative gaps and white arrows an aneurysm . Cropped images were placed on black background for esthetic purposes .

A-E αSMA coverage of (A) P10, (B) P15, (C) P30, (D) 3-6 month old and € 1-year-old retinal arterioles. Brackets in A, B denote perpendicular VSMCs around arteriole. Yellow arrows in A, B, C. indicate VSMCs with an orientation parallel to the blood vessel. Green arrowheads in D denote stenosis. White arrowheads in E label αSMA-negative gaps and white arrows an aneurysm. Cropped images were placed on black background for esthetic purposes.


( F ) Incidence of α SMA negative gaps on arteries by age ( Two - way ANOVA with Sidak ' s multiple comparisons test ) .

O O O 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) Incidence of α SMA negative gaps on arteries by age (Two-way ANOVA with Sidak's multiple comparisons test).


( G ) Number of α SMA negative gaps per retina and mouse by age ( Two - way ANOVA with Sidak ' s multiple comparisons test . Dots represent biological replicates / individual retinas / mice . Two - way ANOVA not significant . Šídák ' s multiple comparisons test : 1 year Jag1 + / + vs 1 year Jag1Ndr / Ndr * P = 0 . 0189 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Number of α SMA negative gaps per retina and mouse by age (Two-way ANOVA with Sidak's multiple comparisons test. Dots represent biological replicates/individual retinas/mice. Two-way ANOVA not significant. Šídák's multiple comparisons test: 1 year Jag1+/+ vs 1 year Jag1Ndr/Ndr *P= 0.0189).


H Transmission electron microscopy of retinal arterioles . VSMCs color - coded in magenta , EC in green . Scale bar 5 µm , boxed region 1 µm . Blue arrowheads denote the edges of VSMCs .

O O O O O O O O O O O O O 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 Transmission electron microscopy of retinal arterioles. VSMCs color-coded in magenta, EC in green. Scale bar 5 µm, boxed region 1 µm. Blue arrowheads denote the edges of VSMCs.


I - J ( I ) One - year - old retinal arteriolar cCasp3 + apoptotic cells . Scale bar 10 µm . ( J ) cCasp3 + cells per field ( n = 6 per group , unpaired t test , * P = 0 . 0109 ) .

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

I-J (I) One-year-old retinal arteriolar cCasp3+ apoptotic cells. Scale bar 10 µm. (J) cCasp3+ cells per field (n=6 per group, unpaired t test, *P=0.0109).


( K ) Left panel aSMA coverage of MCA of P10 mice . Scale bar 20 µm . Right panel magnification of area where number of nuclei was counted . Nuclei were color - coded blue .

(K) Left panel aSMA coverage of MCA of P10 mice. Scale bar 20 µm. Right panel magnification of area where number of nuclei was counted. Nuclei were color-coded blue.


( L ) MCA vascular smooth muscle cell nuclei quantification ( n = 6 per group , unpaired t test , * P = 0 . 0421 ) .

(L) MCA vascular smooth muscle cell nuclei quantification (n=6 per group, unpaired t test, *P=0.0421).


( N ) Blood pressure ( BP ) before treatment in male and female Jag1CTRL and Jag1Ndr / Ndr mice . Each dot represents one animal / one biological replicate . ( Two - way ANOVA Interaction P = 0 . 9407 , Sex P = 0 . 4957 , Genotype * P = 0 . 0207 . Šídák ' s multiple comparisons test not significant ) .

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

(N) Blood pressure (BP) before treatment in male and female Jag1CTRL and Jag1Ndr/Ndr mice. Each dot represents one animal/one biological replicate. (Two-way ANOVA Interaction P=0.9407, Sex P=0.4957, Genotype *P= 0.0207. Šídák's multiple comparisons test not significant).


( O ) Blood pressure increase in Jag1CTRL and Jag1Ndr / Ndr mice treated with vehicle or AngII for two weeks ( Jag1 + / + ctrl n = 4 , Jag1 + / + AngII n = 4 , Jag1Ndr / Ndr ctrl n = 5 , Jag1Ndr / Ndr AngII n = 5 , two - way ANOVA , with Sidak ' s multiple comparisons ) .

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 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) Blood pressure increase in Jag1CTRL and Jag1Ndr/Ndr mice treated with vehicle or AngII for two weeks (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparisons).


( P ) Detection of Evans blue leakage in brain in Jag1CTRL and Jag1Ndr / Ndr mice with or without AngII treatment . ( Jag1 + / + ctrl n = 4 , Jag1 + / + AngII n = 4 , Jag1Ndr / Ndr ctrl n = 5 , Jag1Ndr / Ndr AngII n = 5 , two - way ANOVA , with Sidak ' s multiple comparison . Interaction P = 0 . 9687 , Treatment * * P = 0 . 007 , Genotype P = 0 . 5058 ) .

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 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

(P) Detection of Evans blue leakage in brain in Jag1CTRL and Jag1Ndr/Ndr mice with or without AngII treatment. (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P=0.9687, Treatment **P=0.007, Genotype P=0.5058).


( Q ) One Jag1Ndr / Ndr mouse ( male ) displayed a macroscopic EB leakage , upon AngII treatment .

(Q) One Jag1Ndr/Ndr mouse (male) displayed a macroscopic EB leakage, upon AngII treatment.


® Retinal arteriolar αSMA coverage in vehicle or AngII - treated mice . Green arrowheads denote stenosis , blue arrowheads label αSMA - negative gap . ( S ) αSMA - negative gaps in arterioles per retina ( Jag1 + / + ctrl n = 4 , Jag1 + / + AngII n = 4 , Jag1Ndr / Ndr ctrl n = 3 - 5 , Jag1Ndr / Ndr AngII n = 5 , two - way ANOVA , with Sidak ' s multiple comparison . Interaction P = 0 . 1054 , treatment P = 0 . 0815 ; Genotype P = 0 . 0747 . Ší ' ák ' s multiple comparisons test : AngII treated Jag1 + / + vs AngII treated Jag1Ndr / Ndr * P = 0 . 0315 ) . ) .

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

® Retinal arteriolar αSMA coverage in vehicle or AngII-treated mice. Green arrowheads denote stenosis, blue arrowheads label αSMA-negative gap. (S) αSMA-negative gaps in arterioles per retina (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=3-5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P = 0.1054, treatment P=0.0815; Genotype P=0.0747. Ší'ák's multiple comparisons test: AngII treated Jag1+/+ vs AngII treated Jag1Ndr/Ndr *P= 0.0315).).


A - D ( A ) P30 retina , three CD31 + capillary layers pseudo - colored for superficial , intermediate and deep layers . Scale bar 50 µm . ( B ) P30 SCP branching point number ( n = 6 - 8 per group , unpaired t test , ns , P = 0 . 5346 ) . ( C ) P30 ICP vascular length ( n = 6 per group , unpaired t test , * P = 0 . 0124 ) . ( D ) P30 ICP branching point number ( n = 6 per group , unpaired t test , * * P = 0 . 0057 ) . E - H ( E ) 3 - 6 months old retina , three pseudo - colored CD31 + capillary layers . Scale bar 50 µm . ( F ) Adult SCP branching point number ( n = 6 per group , unpaired t test , ns , P = 0 . 5365 ) . ( G ) Adult ICP vascular length ( n = 6 - 7 per group , unpaired t test , * * P = 0 . 0078 ) . ( H ) Adult ICP branching point number ( n = 6 - 7 per group , unpaired t test , * * P = 0 .

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

A-D (A) P30 retina, three CD31+ capillary layers pseudo-colored for superficial, intermediate and deep layers. Scale bar 50 µm. (B) P30 SCP branching point number (n=6-8 per group, unpaired t test, ns, P=0.5346). (C) P30 ICP vascular length (n=6 per group, unpaired t test, *P=0.0124). (D) P30 ICP branching point number (n=6 per group, unpaired t test, **P=0.0057). E-H (E) 3-6 months old retina, three pseudo-colored CD31+ capillary layers. Scale bar 50 µm. (F) Adult SCP branching point number (n=6 per group, unpaired t test, ns, P=0.5365). (G) Adult ICP vascular length (n=6-7 per group, unpaired t test, **P=0.0078). (H) Adult ICP branching point number (n=6-7 per group, unpaired t test, **P=0.001


I - J ( I ) ICP COLIV and CD31 ( arrowhead labels COLIV + CD31 - capillary ) . Scale bar 50 µm . ( J ) Empty COLIV sleeve length in ICP per field ( n = 6 per group , unpaired t test , ns , P = 0 . 1583 ) .

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

I-J (I) ICP COLIV and CD31 (arrowhead labels COLIV+ CD31- capillary). Scale bar 50 µm. (J) Empty COLIV sleeve length in ICP per field (n=6 per group, unpaired t test, ns, P=0.1583).


( K ) Retinal capillary plexuses in 3 - 6 months old ( top panels ) and one - year - old mice ( bottom panels ) . White arrowheads label discontinuous ICP . Blue arrowhead labels SCP , green ICP and magenta DCP .

O O O O O O O O O O O O O O O O O O O O O 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) Retinal capillary plexuses in 3-6 months old (top panels) and one-year-old mice (bottom panels). White arrowheads label discontinuous ICP. Blue arrowhead labels SCP, green ICP and magenta DCP.


( L - M ) Vertical sprout number between SCP ( S ) and ICP ( I ) and ICP ( I ) and DCP ( D ) in ( L ) adult ( n = 6 per group , two - way ANOVA , genotype * * * P = 0 . 0004 , Sidak ' s multiple comparisons test S - I ns , P = 0 . 0592 , I - D * * * * P < 0 . 0001 ) and ( M ) one - year - old mice ( n = 8 per group , two - way ANOVA , genotype ns , P = 0 . 4507 , Sidak ' s multiple comparisons test S - I ns , P = 0 . 9483 , I - D ns , P = 0 . 6046 ) .

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

(L-M) Vertical sprout number between SCP (S) and ICP (I) and ICP (I) and DCP (D) in (L) adult (n=6 per group, two-way ANOVA, genotype ***P=0.0004, Sidak's multiple comparisons test S-I ns, P=0.0592, I-D ****P<0.0001) and (M) one-year-old mice (n=8 per group, two-way ANOVA, genotype ns, P=0.4507, Sidak's multiple comparisons test S-I ns, P=0.9483, I-D ns, P=0.6046).


N - P ( N ) P10 and ( O ) P40 neurofilament ( NF ) and CD31 staining . Scale bar 50 µm . ( P ) % area positive for NF in P10 and P40 mice ( n = 6 per group , two - way ANOVA : Interaction * * * P = 0 . 0001 ; Age * P = 0 . 0428 ; Genotype * * * P = 0 . 0004 . Sidak ' s multiple comparisons test : P10 Jag1 + / + vs P10 Jag1Ndr / Ndr P = 0 . 9246 ; P40 Jag1 + / + vs P40 Jag1Ndr / Ndr * * * * P = < 0 . 0001 ) .

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

N-P (N) P10 and (O) P40 neurofilament (NF) and CD31 staining. Scale bar 50 µm. (P) % area positive for NF in P10 and P40 mice (n=6 per group, two-way ANOVA: Interaction ***P= 0.0001; Age *P=0.0428; Genotype ***P=0.0004. Sidak's multiple comparisons test: P10 Jag1+/+ vs P10 Jag1Ndr/Ndr P= 0.9246; P40 Jag1+/+ vs P40 Jag1Ndr/Ndr ****P= <0.0001).


A Representative retinographs from a healthy adult , patient with CADASIL , healthy pediatric individual , patient with ALGS and patient with BA . Boxed regions and black arrowheads magnify arteriovenous crossing .

O O O O O O O O O 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 Representative retinographs from a healthy adult, patient with CADASIL, healthy pediatric individual, patient with ALGS and patient with BA. Boxed regions and black arrowheads magnify arteriovenous crossing.


B - F ( B ) Arteriovenous crossings per retina ( n = 14 per group , unpaired t test , ns , P = 0 . 3854 . ( C ) Major arterioles ( n = 14 per group , unpaired t test , ns , P = 0 . 2902 ) and ( D ) venules ( n = 14 per group , unpaired t test , ns , P = 0 . 1536 ) . ( E ) Arterial ( n = 14 per group , unpaired t test , ns , P = 0 . 0759 ) and ( F ) venous tortuosity ( n = 14 per group , unpaired t test , ns , P = 0 . 1050 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F (B) Arteriovenous crossings per retina (n=14 per group, unpaired t test, ns, P=0.3854. (C) Major arterioles (n=14 per group, unpaired t test, ns, P=0.2902) and (D) venules (n=14 per group, unpaired t test, ns, P=0.1536). (E) Arterial (n=14 per group, unpaired t test, ns, P=0.0759) and (F) venous tortuosity (n=14 per group, unpaired t test, ns, P=0.1050).


G - K ( G ) Arteriovenous crossings per retina ( CTRL n = 5 , ALGS n = 3 , BA n = 4 , one - way ANOVA , ns , P = 0 . 0608 , Sidak ' s multiple comparisons ALGS vs . CTRL * P = 0 . 0492 , ALGS vs . BA ns , P = 0 . 0999 ) . ( H ) Major arterioles ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * * P = 0 . 0026 , Sidak ' s multiple comparisons ALGS vs . CTRL * * P = 0 . 0039 , ALGS vs . BA ns , P = 0 . 9799 ) and ( I ) venules ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * * * P = 0 . 0008 , Sidak ' s multiple comparisons ALGS vs . CTRL * * P = 0 . 0011 , ALGS vs . BA ns , P > 0 . 9999 ) . ( J ) Arterial ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * P = 0 . 0486 , Sidak ' s multiple comparisons ALGS vs . CTRL * P = 0 . 0392 , ALGS vs . BA ns , P = 0 . 1204 ) and ( K ) venous tortuosity ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * * P = 0 . 009 , Sidak ' s multiple comparisons ALGS vs . CTRL * * P = 0 . 0076 , ALGS vs . BA * P = 0 . 0327 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K (G) Arteriovenous crossings per retina (CTRL n=5, ALGS n=3, BA n=4, one-way ANOVA, ns, P=0.0608, Sidak's multiple comparisons ALGS vs. CTRL *P=0.0492, ALGS vs. BA ns, P=0.0999). (H) Major arterioles (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, **P=0.0026, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0039, ALGS vs. BA ns, P=0.9799) and (I) venules (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, ***P=0.0008, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0011, ALGS vs. BA ns, P>0.9999). (J) Arterial (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, *P=0.0486, Sidak's multiple comparisons ALGS vs. CTRL *P=0.0392, ALGS vs. BA ns, P=0.1204) and (K) venous tortuosity (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, **P=0.009, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0076, ALGS vs. BA *P=0.0327).


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were treated with various doses of ATP . CBA analysis of IL - 1β ( A ) in the culture supernatants .

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

LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. CBA analysis of IL-1β (A) in the culture supernatants.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were treated with various doses of ATP . ELISA of IL - 18 ( B ) in the culture supernatants .

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

LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. ELISA of IL-18 (B) in the culture supernatants.


CBA analysis of IL - 1β secretion from Abro1 + / + and Abro1 − / − BMDMs transduced with GFP - or ABRO1 - GFP - expressing lentiviruses prior to stimulation with LPS and ATP . Cell lysates were immunoblotted with anti - ABRO1 antibody .

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

CBA analysis of IL-1β secretion from Abro1+/+ and Abro1−/− BMDMs transduced with GFP- or ABRO1-GFP-expressing lentiviruses prior to stimulation with LPS and ATP. Cell lysates were immunoblotted with anti-ABRO1 antibody.


ELISA analysis of IL - 1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium ( Con ) , LPS or LPS plus ATP . Cell lysates were immunoblotted with anti - ABRO1 and anti - GAPDH antibodies .

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

ELISA analysis of IL-1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium (Con), LPS or LPS plus ATP. Cell lysates were immunoblotted with anti-ABRO1 and anti-GAPDH antibodies.


Abro1 + / + and Abro1 − / − BMDMs were left untreated ( Con ) , treated with LPS alone ( LPS ) or pretreated with LPS and then stimulated with nigericin , silica , MSU , MDP , Alum , poly ( dA : dT ) , flagellin or LTx ( Anthrax Lethal Factor ) . CBA analysis of IL - 1β secretion in the culture supernatants .

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

Abro1+/+ and Abro1−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with nigericin, silica, MSU, MDP, Alum, poly (dA: dT), flagellin or LTx (Anthrax Lethal Factor). CBA analysis of IL-1β secretion in the culture supernatants.


Representative immunoblot analysis of cleaved caspase - 1 and IL - 1β in culture supernatants ( SN ) of LPS - primed Abro1 + / + and Abro1 − / − BMDMs treated with ATP , nigericin , MSU , flagellin or poly ( dA : dT ) .

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

Representative immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Abro1+/+ and Abro1−/− BMDMs treated with ATP, nigericin, MSU, flagellin or poly (dA: dT).


Flow cytometric analysis of caspase - 1 activity in LPS - primed Abro1 + / + and Abro1 − / − BMDMs stimulated with nigericin for indicated times .

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

Flow cytometric analysis of caspase-1 activity in LPS-primed Abro1+/+ and Abro1−/− BMDMs stimulated with nigericin for indicated times.


Immunostaining of endogenous ASC specks in LPS - primed Abro1 + / + and Abro1 − / − BMDMs left untreated or treated with nigericin or poly ( dA : dT ) . Scale bars , 10 μm . Quantification of ASC specks from ( H ) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei . At least 100 cells from each treatment condition were quantified . UD , undetectable .

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

Immunostaining of endogenous ASC specks in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with nigericin or poly (dA:dT). Scale bars, 10 μm. Quantification of ASC specks from (H) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei. At least 100 cells from each treatment condition were quantified. UD, undetectable.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were left untreated or treated with ATP . Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti - ASC antibody .

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

LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti-ASC antibody.


WT , Abro1 − / − and Nlrp3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β and TNF - α in the peritoneal lavage fluid ( A ) . n = 6 for PBS groups and n = 12 for MSU groups .

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 B-CONTROLLED_VAR O O

WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (A). n = 6 for PBS groups and n = 12 for MSU groups.


WT , Abro1 − / − and Nlrp3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . Flow cytometric analysis of peritoneal cell exudates ( B ) . n = 6 for PBS groups and n = 12 for MSU groups .

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 B-CONTROLLED_VAR O O

WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. Flow cytometric analysis of peritoneal cell exudates (B). n = 6 for PBS groups and n = 12 for MSU groups.


Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) . Flow cytometric analysis of caspase - 1 activity in peritoneal exudate monocytes and neutrophils . n = 12 per group .

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

Abro1+/+ and Abro1−/− mice were intraperitoneally injected with MSU (1 mg per mouse). Flow cytometric analysis of caspase-1 activity in peritoneal exudate monocytes and neutrophils. n=12 per group.


Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with Alum ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β in the peritoneal lavage fluid ( D ) n = 6 for PBS groups and n = 12 for Alum groups .

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

Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β in the peritoneal lavage fluid (D) n = 6 for PBS groups and n = 12 for Alum groups.


Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with Alum ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . flow cytometric analysis of peritoneal cell exudates ( E ) . n = 6 for PBS groups and n = 12 for Alum groups .

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 B-CONTROLLED_VAR O O

Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (E). n = 6 for PBS groups and n = 12 for Alum groups.


Survival of Abro1 + / + and Abro1 − / − mice subjected to LPS ( 15 mg / kg ) . n = 20 per group .

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

Survival of Abro1+/+ and Abro1−/− mice subjected to LPS (15 mg/kg). n=20 per group.


ELISA of serum IL - 1β from Abro1 + / + and Abro1 − / − mice 3 h after intraperitoneal injection of LPS ( 15mg / kg ) or vehicle ( PBS ) . n = 6 for PBS groups and n = 12 for LPS groups .

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 B-CONTROLLED_VAR O O

ELISA of serum IL-1β from Abro1+/+ and Abro1−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 and Myc - tagged NLRP3 , ASC or pro - casapase - 1 ( Pro - Casp - 1 ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3, ASC or pro-casapase-1 (Pro-Casp-1). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.


BMDMs were treated with indicated stimuli . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 and Myc - tagged NLRP3 or NLRP3 deletion mutants as indicated in ( F ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3 or NLRP3 deletion mutants as indicated in (F). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 and Flag - tagged ABRO1 or ABRO1 deletion mutants as indicated in ( H ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3 and Flag-tagged ABRO1 or ABRO1 deletion mutants as indicated in (H). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - tagged NLRP3 ( WT ) , NLRP3 ( S194D ) , or NLRP3 ( S194A ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-tagged NLRP3 (WT), NLRP3 (S194D), or NLRP3 (S194A). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


WT BMDMs were treated with or without 5 μM Anisomycin for 2 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT BMDMs were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194A ) were treated with or without 5 μM Anisomycin for 2 h . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194A) were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194D ) were treated with or without 100 nM SP600125 for 2 h . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194D) were treated with or without 100 nM SP600125 for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


WT and Nlrp3S194A / S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT and Nlrp3S194A/S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 , Flag - ABRO1 and HA - ubiquitin ( HA - Ub ) , HA - Ub K48 only or K63 only . Before collection , cells were treated with MG132 ( 10 μM ) for 6 h . Immunoblot analysis of NLRP3 ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3, Flag-ABRO1 and HA-ubiquitin (HA-Ub), HA-Ub K48 only or K63 only. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding HA - Ub , Flag - ABRO1 , and NLRP3 truncations as indicated . Before collection , cells were treated with MG132 ( 10 μM ) for 6 h . Immunoblot analysis of ubiquitination of NLRP3 truncations ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding HA-Ub, Flag-ABRO1, and NLRP3 truncations as indicated. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of ubiquitination of NLRP3 truncations (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were left untreated or treated with ATP ( left ) or nigericin ( right ) . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O 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 Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP (left) or nigericin (right). Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


Nlrp3 - / - BMDMs transduced with lentiviruses expressing Flag - NLRP3 ( WT ) or Flag - NLRP3 ( A350V ) were left unstimulated or stimulated with LPS or LPS plus ATP . Immunoblot analysis of NLRP3 and NLRP3 ( A350V ) ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

O O O O 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 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

Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were left unstimulated or stimulated with LPS or LPS plus ATP. Immunoblot analysis of NLRP3 and NLRP3 (A350V) ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 ( A350V ) and HA - ubiquitin ( HA - Ub ) . Immunoblot analysis of NLRP3 ( A350V ) ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3 (A350V) and HA-ubiquitin (HA-Ub). Immunoblot analysis of NLRP3 (A350V) ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


Nlrp3 - / - BMDMs transduced with lentiviruses expressing Flag - NLRP3 ( WT ) or Flag - NLRP3 ( A350V ) were stimulated with or without 100ng / ml LPS for 1 h . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti - ABRO1 antibody .

O O O O O O 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

Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were stimulated with or without 100ng/ml LPS for 1 h. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 , and HA - ubiquitin ( HA - Ub ) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h . Immunoblot analysis of NLRP3 ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3, and HA-ubiquitin (HA-Ub) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


LPS - primed WT and Nlrp3S194A / S194A BMDMs were left untreated or treated with ATP . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed WT and Nlrp3S194A/S194A BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


WT BMDMs were stimulated with LPS for 1 h . Immunoblot analysis of NLRP3 , ABRO1 , and BRCC3 from the cell lysates immunoprecipitated with control IgG , anti - ABRO1 , anti - BRCC3 or anti - NLRP3 antibody .

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

WT BMDMs were stimulated with LPS for 1 h. Immunoblot analysis of NLRP3, ABRO1, and BRCC3 from the cell lysates immunoprecipitated with control IgG, anti-ABRO1, anti-BRCC3 or anti-NLRP3 antibody.


Brcc3 + / + and Brcc3 − / − BMDMs were treated with or without LPS for 1 h . Before LPS treatment , Brcc3 − / − BMDMs were pretreated with MG132 ( 10 μM ) for 6 h to rescue the expression of ABRO1 . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti - ABRO1 antibody .

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

Brcc3+/+ and Brcc3−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Brcc3−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of ABRO1. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.


Abro1 + / + and Abro1 − / − BMDMs were treated with or without LPS for 1 h . Before LPS treatment , Abro1 − / − BMDMs were pretreated with MG132 ( 10 μM ) for 6 h to rescue the expression of BRCC3 . Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti - BRCC3 antibody .

Abro1+/+ and Abro1−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Abro1−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of BRCC3. Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti-BRCC3 antibody.


A constant amount of Myc - NLRP3 plasmid together with an increasing amount of Myc - ABRO1 plasmid were co - transfected in HEK - 293T cells . Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti - BRCC3 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O 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 constant amount of Myc-NLRP3 plasmid together with an increasing amount of Myc-ABRO1 plasmid were co-transfected in HEK-293T cells. Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti-BRCC3 antibody.


Brcc3 + / + and Brcc3 − / − BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP were treated with LPS and nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - NLRP3 antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3

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

Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody


Abro1 + / + , Abro1 − / − , BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP were treated with LPS and nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - NLRP3 antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3

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

Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody


Brcc3 + / + and Brcc3 − / − BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP CBA analysis of IL - 1β in culture

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

Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants


Abro1 + / + , Abro1 − / − , BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP CBA analysis of IL - 1β in culture

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

Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants


Brcc3 + / + and Brcc3 − / − BMDMs were left untreated ( Con ) , treated with LPS alone ( LPS ) or pretreated with LPS and then stimulated with ATP , nigericin , MSU , Alum , poly ( dA : dT ) or flagellin . CBA analysis of IL - 1β secretion in the culture supernatants .

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

Brcc3+/+ and Brcc3−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with ATP, nigericin, MSU, Alum, poly (dA:dT) or flagellin. CBA analysis of IL-1β secretion in the culture supernatants.


Immunoblot analysis of cleaved caspase - 1 and IL - 1β in culture supernatants ( SN ) of LPS - primed Brcc3 + / + and Brcc3 − / − BMDMs treated with ATP , nigericin , MSU , flagellin , and poly ( dA : dT ) .

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

Immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Brcc3+/+ and Brcc3−/− BMDMs treated with ATP, nigericin, MSU, flagellin, and poly (dA: dT).


Immunostaining of endogenous ASC specks in LPS - primed Brcc3 + / + and Brcc3 - / - BMDMs left untreated or treated with nigericin or poly ( dA : dT ) . ASC speck positive cells were counted and analyzed as described in Fig . 1I . UD , undetectable .

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 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

Immunostaining of endogenous ASC specks in LPS-primed Brcc3+/+ and Brcc3-/- BMDMs left untreated or treated with nigericin or poly (dA:dT). ASC speck positive cells were counted and analyzed as described in Fig. 1I. UD, undetectable.


LPS - primed Brcc3 + / + and Brcc3 − / − BMDMs were left unstimulated or stimulated with nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed Brcc3+/+ and Brcc3−/− BMDMs were left unstimulated or stimulated with nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


Survival of Brcc3 + / + and Brcc3 − / − mice subjected to LPS ( 15 mg / kg ) . n = 20 per group .

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

Survival of Brcc3+/+ and Brcc3−/− mice subjected to LPS (15 mg/kg). n=20 per group.


ELISA of serum IL - 1β from Brcc3 + / + and Brcc3 − / − mice 3 h after intraperitoneal injection of LPS ( 15mg / kg ) or vehicle ( PBS ) . n = 6 for PBS groups and n = 12 for LPS groups .

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 B-CONTROLLED_VAR O O

ELISA of serum IL-1β from Brcc3+/+ and Brcc3−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.


Brcc3 + / + and Brcc3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β and TNF - α in the peritoneal lavage fluid ( G )

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

Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (G)


Brcc3 + / + and Brcc3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . flow cytometric analysis of peritoneal cell exudates ( H ) . n = 6 for PBS groups and n = 12 for MSU groups .

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 B-CONTROLLED_VAR O O

Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (H). n = 6 for PBS groups and n = 12 for MSU groups.


A , B , Immunostaining for CD31 + blood vessels in control ( ctrl , A ) and Gpr124KO ( B ) forebrains . Note the normal vasculature in the cortical hem ( asterisk ) of Gpr124KO mice .

O O O O O O O O O O O O O O O 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, Immunostaining for CD31+ blood vessels in control (ctrl, A) and Gpr124KO (B) forebrains. Note the normal vasculature in the cortical hem (asterisk) of Gpr124KO mice.


C , D , Immunostaining for Pax6 in control ( C ) and Gpr124KO ( D ) forebrains to reveal the lateral extension of the neocortex ( dashed line ) between the cortical hem ( arrowhead ) and the lateral ganglionic eminence ( arrow ) . E , Quantification of the lateral extension of the cortex in control and Gpr124KO brains ( mean ± SEM ; N = 10 ; * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunostaining for Pax6 in control (C) and Gpr124KO (D) forebrains to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). E, Quantification of the lateral extension of the cortex in control and Gpr124KO brains (mean±SEM; N=10; *** p<0.001).