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 .

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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 .

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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 .

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(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 .

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(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 .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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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 ( - ) .

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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 ) .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 . ) .

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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 .

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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 .

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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 ) .

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

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

(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 ) .

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(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 .

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

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 ) .

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

(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 .

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

(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 ) .

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(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 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

(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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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 .

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

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

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

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 .

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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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 .

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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 .

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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 .

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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 .

B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 ) .

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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 .

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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 B-MEASURED_VAR 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 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

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

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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 .

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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 .

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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 .

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

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 .

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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 .

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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 .

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

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

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 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

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

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

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

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 .

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

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 .

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

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 .

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

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

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

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

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

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

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

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

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

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 .

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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 .

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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 )

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

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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 .

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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 ) .

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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).


F , G Staining for EdU ( red ) and Ki67 ( green ) in control ( F ) and Gpr124KO ( G ) cortices at E13 . 5 , 24 hours after EdU injection . H , Quantification of Ki67 + NPCs in the VZ ( blue ) , SVZ / CP ( grey ) and of Ki67 - neurons ( cyan ) generated from EdU - labeled NPCs in control and Gpr124KO embryos ( mean ± SEM ; N = 4 ( control ) and N = 3 ( Gpr124KO ) ; * p < 0 . 05 , * * p < 0 . 01 ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

F,G Staining for EdU (red) and Ki67 (green) in control (F) and Gpr124KO (G) cortices at E13.5, 24 hours after EdU injection. H, Quantification of Ki67+ NPCs in the VZ (blue), SVZ/CP (grey) and of Ki67- neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124KO embryos (mean±SEM; N=4 (control) and N=3 (Gpr124KO); * p<0.05, ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


I , J , Immunostaining for Tbr2 ( red ) and Pax6 ( green ) in control ( I ) and Gpr124KO ( J ) cortices . K , Quantification of newborn BPs in the VZ of control and Gpr124KO cortices ( mean ± SEM ; N = 4 ; * p < 0 . 05 ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

I,J, Immunostaining for Tbr2 (red) and Pax6 (green ) in control (I) and Gpr124KO (J) cortices. K, Quantification of newborn BPs in the VZ of control and Gpr124KO cortices (mean±SEM; N=4; * p<0.05). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


L , M , Immunostaining for Tbr2 ( red ) and Ngn1 ( green ) in control ( L ) and Gpr124KO ( M ) cortices . N , O Quantification of neurogenic ( Ngn1 + ) RGs and BPs ( N ) or expanding ( Tbr2 - Ngn1 - ) RGs ( O ) in control and Gpr124KO cortices ( mean ± SEM ; N = 4 ; * p < 0 . 05 , * * * p < 0 . 001 ) . Note that the fraction of total BPs is significantly reduced ( Fig 1K ) while the fraction of Ngn1 + BPs is insignificantly reduced ( Fig 1N ) , because the proportion of Ngn1 + Tbr2 + cells within all BPs is slightly , though statisically insignificantly higher in the Gpr124KObrains ( see Fig S2M ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

L,M, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (L) and Gpr124KO (M) cortices. N,O Quantification of neurogenic (Ngn1+) RGs and BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124KO cortices (mean±SEM; N=4; * p<0.05, *** p<0.001). Note that the fraction of total BPs is significantly reduced (Fig 1K) while the fraction of Ngn1+BPs is insignificantly reduced (Fig 1N), because the proportion of Ngn1+Tbr2+ cells within all BPs is slightly, though statisically insignificantly higher in the Gpr124KObrains (see Fig S2M). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


P , Q Stainings for Tbr2 ( red ) and for Tis21 - GFP expression ( green ) in control ( P ) and Gpr124KO ( Q ) cortices . R , S Quantification of neurogenic ( Tis21 - GFP + ) RGs and BPs ( R ) or expanding ( Tbr2 - Tis21 - GFP - ) RGs ( S ) in control and Gpr124KO cortices ( mean ± SEM ; N = 4 ; * * * p < 0 . 001 ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

P,Q Stainings for Tbr2 (red) and for Tis21-GFP expression (green) in control (P) and Gpr124KO (Q) cortices. R,S Quantification of neurogenic (Tis21-GFP+) RGs and BPs (R) or expanding (Tbr2- Tis21-GFP-) RGs (S) in control and Gpr124KO cortices (mean±SEM; N=4; *** p<0.001). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


A , B , Staining for Isolectin B4 + blood vessels in control ( A ) and Gpr124ECcKO ( B ) forebrains . Asterisks mark the lateral ventricles .

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A,B, Staining for Isolectin B4+ blood vessels in control (A) and Gpr124ECcKO (B) forebrains. Asterisks mark the lateral ventricles.


C , D , Staining for pimonidazole ( PIMO , green ) and Isolectin B4 + blood vessels ( red ) in ctrl ( C ) and Gpr124ECcKO ( D ) cortices .

O O O 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

C,D, Staining for pimonidazole (PIMO, green) and Isolectin B4+ blood vessels (red) in ctrl (C) and Gpr124ECcKO (D) cortices.


E , F , Immunostaining for Pax6 in control ( E ) and Gpr124ECcKO ( F ) forebrain sections to reveal the lateral extension of the neocortex ( dashed line ) between the cortical hem ( arrowhead ) and the lateral ganglionic eminence ( arrow ) . G , Quantification the lateral extension of the cortex in control and Gpr124ECcKObrains ( mean ± SEM ; N = 6 for ctrl and N = 8 for Gpr124ECcKO ; * p < 0 . 05 ) .

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

E,F, Immunostaining for Pax6 in control (E) and Gpr124ECcKO (F) forebrain sections to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). G, Quantification the lateral extension of the cortex in control and Gpr124ECcKObrains (mean±SEM; N=6 for ctrl and N=8 for Gpr124ECcKO; * p<0.05).


H , I , Staining for EdU ( red ) and Ki67 ( green ) in control ( H ) and Gpr124ECcKO ( I ) cortices at E13 . 5 , 24 hours after EdU injection . J , Quantification of Ki67 + NPCs in the in VZ ( blue ) , SVZ / CP ( grey ) and of Ki67 - neurons ( cyan ) generated from EdU - labeled NPCs in control and Gpr124ECcKO embryos ( mean ± SEM ; N = 4 ; * p < 0 . 05 , * * P < 0 . 01 ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

H,I, Staining for EdU (red) and Ki67 (green) in control (H) and Gpr124ECcKO (I) cortices at E13.5, 24 hours after EdU injection. J, Quantification of Ki67+ NPCs in the in VZ (blue), SVZ/CP (grey) and of Ki67- neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124ECcKO embryos (mean±SEM; N=4; * p<0.05, ** P<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


K , L , Immunostaining for Tbr2 ( red ) and Ngn1 ( green ) in control ( K ) and Gpr124KO ( L ) cortices . M - O , Quantification of neurogenic ( Ngn1 + ) RGs and BPs ( M ) , newborn BPs ( N ) or expanding ( Tbr2 - Ngn1 - ) RGs ( O ) in control and Gpr124ECcKO cortices ( mean ± SEM ; N = 4 ; * p < 0 . 05 , * * p < 0 . 01 ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

K,L, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (K) and Gpr124KO (L) cortices. M-O, Quantification of neurogenic (Ngn1+) RGs and BPs (M), newborn BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124ECcKO cortices (mean±SEM; N=4; * p<0.05, ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


A , qRT - PCR analysis of NSC - specific markers ( Fabp7 , Hes5 ) and neuronal markers ( Dcx , Tubb3 ) in the Prom1 + fraction , presented as fold change compared to the Prom1 - fraction ) from WT cortices at E14 . 5 ( mean ± SEM ; N = 3 for Dcx ; N = 6 for all other genes ) .

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A, qRT-PCR analysis of NSC-specific markers (Fabp7, Hes5) and neuronal markers (Dcx, Tubb3) in the Prom1+ fraction, presented as fold change compared to the Prom1- fraction) from WT cortices at E14.5 (mean±SEM; N=3 for Dcx; N=6 for all other genes).


F , Western blot for HIF - 1 ( top ) , HIF - 2 ( middle ) and a - tubulin as loading control ( bottom ) using forebrain lysates from E13 . 5 control ( left ) or Gpr124KOembryos ( middle ) . HIF - 1 / 2 - transfected HEK293 cells served as positive control ( right ) . G , Quantification of HIF - 1 band intensities shown in panel F ( mean ± SEM ; N = 3 ; * * p < 0 . 01 ) .

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F, Western blot for HIF-1 (top), HIF-2 (middle) and a-tubulin as loading control (bottom) using forebrain lysates from E13.5 control (left) or Gpr124KOembryos (middle). HIF-1/2-transfected HEK293 cells served as positive control (right). G, Quantification of HIF-1 band intensities shown in panel F (mean±SEM; N=3; ** p<0.01).


H , I , Immunostaining for HIF - 1α ( red ) and DAPI ( blue ) in control ( H , H ' , H ' ' ) and Gpr124KO ( I , I ' , I ' ' ) cortices . Panels H ' , H ' ' , I ' and I ' ' are magnifications of the boxed areas in panels I and J , showing HIF - 1α signal alone ( H ' , I ' ) or together with DAPI ( H ' ' , I ' ' ) . Asterisks indicate autofluorescent blood cells . The dashed line indicates the basal boundary of the VZ . Scale bar = 100 µm . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

H,I, Immunostaining for HIF-1α (red) and DAPI (blue) in control (H, H', H'') and Gpr124KO (I,I',I'') cortices. Panels H',H'',I' and I'' are magnifications of the boxed areas in panels I and J, showing HIF-1α signal alone (H',I') or together with DAPI (H'',I''). Asterisks indicate autofluorescent blood cells. The dashed line indicates the basal boundary of the VZ. Scale bar = 100 µm. Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


J , qRT - PCR analysis of HIF - 1 target gene expression in VZ cells from the cortex of control and Gpr124KO E13 . 5 embryos ( mean ± SEM ; N = 4 ; * * * p < 0 . 001 ) . SVZ , subventricular zone ; VZ , ventricular zone .

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J, qRT-PCR analysis of HIF-1 target gene expression in VZ cells from the cortex of control and Gpr124KO E13.5 embryos (mean±SEM; N=4; *** p<0.001). SVZ, subventricular zone; VZ, ventricular zone.


A - F , Staining for Isolectin B4 ( A - C ) and oxidized thiols ( D - F ) in WT cortices at E10 . 5 ( A , D ) , E11 . 5 ( B , E ) and E12 . 5 ( C , F ) . Panels D - F are pseudocolor conversions of staining intensity ( from low [ purple ] to high [ red ] ) . Arrowheads denote the position of the vascular front .

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A-F, Staining for Isolectin B4 (A-C) and oxidized thiols (D-F) in WT cortices at E10.5 (A,D), E11.5 (B,E) and E12.5 (C,F). Panels D-F are pseudocolor conversions of staining intensity (from low [purple] to high [red]). Arrowheads denote the position of the vascular front.


G - I , Immunostaining for HIF - 1α ( red ) and DAPI ( blue ) in WT cortices at E10 . 5 ( G - G ' ' ) , E11 . 5 ( H - H ' ' ) and E12 . 5 ( I - I ' ' ) . Panels G ' , G ' ' , H ' , H ' ' , I ' and I ' ' are magnifications of the boxed areas in their respective original panels . Arrows point to the border of lateral and dorsal cortex .

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G-I, Immunostaining for HIF-1α (red) and DAPI (blue) in WT cortices at E10.5 (G-G''), E11.5 (H-H'') and E12.5 (I-I''). Panels G',G'',H',H'',I' and I'' are magnifications of the boxed areas in their respective original panels. Arrows point to the border of lateral and dorsal cortex.


J - L , Immunostaining for Glut1 of WT cortices at E10 . 0 ( J ) , E11 . 5 ( K ) and E12 . 5 ( L ) . Dashed lines indicate the apical border of the cortex . Scale bar : 100 µm ( D , F , G , H , I , J - L ) or 50 µm ( I ' ' ) , respectively .

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J-L, Immunostaining for Glut1 of WT cortices at E10.0 (J), E11.5 (K) and E12.5 (L). Dashed lines indicate the apical border of the cortex. Scale bar: 100 µm (D,F,G,H,I, J-L) or 50 µm (I''), respectively.