D . Confocal fluorescence images of Proteostat signal ( 1 : 2000 , red spots ) and DAPI staining ( blue ) , scale bar is 10µm . E . Quantitation of protein aggregates in IGRs by immunofluorescence analysis using Fiji software . ( T - test analysis , * * = p - value < 0 . 01 , N = 3 biological replicates , data are mean ± SD ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Confocal fluorescence images of Proteostat signal (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm. E. Quantitation of protein aggregates in IGRs by immunofluorescence analysis using Fiji software. (T-test analysis, ** = p-value<0.01, N=3 biological replicates, data are mean ±SD).


F . Quantitation , by immunofluorescence analysis , of YAP in different cellular compartments ( T - test analysis , * = p - value < 0 . 05 , N = 3 biological replicates , data are mean ± SD ) . Images were quantified by subdividing cells into mostly cytosolic YAP ( Cytosol ) , mostly nuclear YAP ( Nuclear ) , or equal distribution ( Total ) from three biological replicates .

O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O B-SUBCELLULAR O O O B-SUBCELLULAR B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O

F. Quantitation, by immunofluorescence analysis, of YAP in different cellular compartments (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD). Images were quantified by subdividing cells into mostly cytosolic YAP (Cytosol), mostly nuclear YAP (Nuclear), or equal distribution (Total) from three biological replicates.


G . mRNA levels of CTGF measured by real - time PCR in IGR37 treated with DMSO or NB - 360 ( T - test analysis , * = p - value < 0 . 05 , N = 3 biological replicates , data are mean ± SD ) .

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

G. mRNA levels of CTGF measured by real-time PCR in IGR37 treated with DMSO or NB-360 (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD).


H . CTGF mRNA level measured by real - time PCR in IGR39 treated with IGR39 conditioned medium ( CTRL ) or with IGR37 conditioned medium ( CM ) , N = 4 biological replicates . T - test analysis , * * * p - value < 0 . 001 , data are mean ± SD .

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

H. CTGF mRNA level measured by real-time PCR in IGR39 treated with IGR39 conditioned medium (CTRL) or with IGR37 conditioned medium (CM), N=4 biological replicates. T-test analysis, *** p-value <0.001, data are mean ±SD.


I . CTGF mRNA level measured by real - time PCR in IGR39 supplemented with recombinant PMEL amyloid fibrils ( 0 . 5µM ) , N = 3 biological replicates . T - test analysis : * * * p - value < 0 . 001 , data are mean ± SD .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O 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. CTGF mRNA level measured by real-time PCR in IGR39 supplemented with recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis: *** p-value <0.001, data are mean ±SD.


J . CTGF mRNA level measured by real - time PCR in IGR37 treated with DMSO , NB - 360 or NB - 360 plus recombinant PMEL amyloid fibrils ( 0 . 5µM ) , N = 3 biological replicates . T - test analysis * = p - value < 0 . 05 , * * = p - value < 0 . 01 . Data are mean ± SD .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O 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. CTGF mRNA level measured by real-time PCR in IGR37 treated with DMSO, NB-360 or NB-360 plus recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis* = p-value<0.05, ** = p-value<0.01. Data are mean ±SD.


A . MTT assay for IGRs treated with DMSO or NB - 360 ( 25µM ) . Biological replicates N = 4 biological replicates . T - test analysis * * = p - value < 0 . 01 , * * * p - value < 0 . 001 . Data are mean ± SD . B . MTT assay for IGRs cells treated with DMSO or inhibitor 3I ( 3µM ) . Biological replicates N = 3 biological replicates . T - test analysis * * = p - value < 0 . 01 . Data are mean ± SD .

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

A. MTT assay for IGRs treated with DMSO or NB-360 (25µM). Biological replicates N=4 biological replicates. T-test analysis ** = p-value<0.01, *** p-value <0.001. Data are mean ±SD. B. MTT assay for IGRs cells treated with DMSO or inhibitor 3I (3µM). Biological replicates N=3 biological replicates. T-test analysis ** = p-value<0.01. Data are mean ±SD.


C - D . MTT assay of IGRs treated with NB - 360 ( 25µM ) and different concentration of doxorubicin as indicated . Biological replicates N = 4 biological replicates . T - test analysis : * = 0 . 01 < p - value < 0 . 05 , * * = p - value < 0 . 01 . Data are mean ± SD .

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

C-D. MTT assay of IGRs treated with NB-360 (25µM) and different concentration of doxorubicin as indicated. Biological replicates N=4 biological replicates. T-test analysis: * = 0.01<p-value<0.05, ** = p-value<0.01. Data are mean ±SD.


E . T - test analysis of IC50 values for doxorubicin used alone or in combination with NB - 360 ( combo ) . Data are mean ± SD . T - test analysis : * = 0 . 01 < p - value < 0 . 05 ; * * = 0 . 001 < p - value < 0 . 01 .

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

E. T-test analysis of IC50 values for doxorubicin used alone or in combination with NB-360 (combo). Data are mean ±SD. T-test analysis: * = 0.01<p-value<0.05; ** = 0.001<p-value<0.01.


A . RGFP966 ( 966 ) - dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth ( 24h in culture ) compared to HDAC class II , HDAC1 / 2 and HDAC1 or 3 inhibitions via 69A , 963 and 233 respectively . Data information : Data expressed as mean fold change N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni test ) .

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

A. RGFP966 (966)-dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth (24h in culture) compared to HDAC class II, HDAC1/2 and HDAC1 or 3 inhibitions via 69A, 963 and 233 respectively. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni test).


B , C . Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth ( average neurite outgrowth after 24h in culture , Tuj1 positive neurons ) on PDL - laminin or Myelin substrates . Scale bars , 100 μm . Data information : Data expressed as average neurite length ± s . e . m . N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle Student ' s t - test )

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

B, C. Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth (average neurite outgrowth after 24h in culture, Tuj1 positive neurons) on PDL-laminin or Myelin substrates. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)


D , E . Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle . Data information : Data expressed as mean fold change N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle Student ' s t - test )

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

D, E. Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)


F , G . AAV - mediated mutant HDAC3 ( Y298H , V5 ) infection of DRG neurons induces neurite outgrowth ( Tuj - 1 / V5 double positive neurons ) after 36h in culture , which is repressed by wildtype HDAC3 overexpression . Scale bars , 100 μm . Data information : Data expressed as average neurite length ± s . e . m . N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus V5 vs AAV - GFP ANOVA followed by Bonferroni test ) .

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

F, G. AAV-mediated mutant HDAC3 (Y298H, V5) infection of DRG neurons induces neurite outgrowth (Tuj-1/V5 double positive neurons) after 36h in culture, which is repressed by wildtype HDAC3 overexpression. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus V5 vs AAV-GFP ANOVA followed by Bonferroni test).


A . IHC in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased , primarily in the nucleus 24h after sciatic nerve axotomy ( SNA ) but not spinal dorsal column axotomy ( DCA ) . Scale bars , 100 μm .

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

A. IHC in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased, primarily in the nucleus 24h after sciatic nerve axotomy (SNA) but not spinal dorsal column axotomy (DCA). Scale bars, 100 μm.


B - C . Bar graphs show mean fold change levels ( B ) or % of pHDAC3 + cells ( C ) ( pHDAC3 + threshold was set at the minimum intensity level of sham cells ) ± s . e . m . DAPI counter staining was used to label nuclei N = 9 - 10 biological replicates . ( * * * p < 0 . 005 ) indicate significant difference versus sham or laminectomy ( lam ) ( ANOVA followed by Bonferroni test ) .

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

B-C. Bar graphs show mean fold change levels (B) or % of pHDAC3+ cells (C) (pHDAC3+ threshold was set at the minimum intensity level of sham cells) ± s.e.m. DAPI counter staining was used to label nuclei N= 9-10 biological replicates. (***p<0.005) indicate significant difference versus sham or laminectomy (lam) (ANOVA followed by Bonferroni test).


D . Western blot revealed that HDAC3 phosphorylation is decreased in DRG nuclear extracts but not in the cytoplasm 24h after SNA . E . Bar graphs show intensity measurement of nuclear vs cytoplasm immunoblot . Data is expressed as mean ± s . e . m . N = 3 biological replicates . ( * * * p < 0 . 005 ) indicate significant difference versus sham ( Student ´ s t - test ) .

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

D. Western blot revealed that HDAC3 phosphorylation is decreased in DRG nuclear extracts but not in the cytoplasm 24h after SNA. E. Bar graphs show intensity measurement of nuclear vs cytoplasm immunoblot. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (***p<0.005) indicate significant difference versus sham (Student´s t-test).


F . HDAC3 activity ( HDAC3 IP and activity assay ) is strongly reduced after SNA but not DCA . Data is expressed as mean ± s . e . m . N = 3 biological replicates . ( * p < 0 . 05 ) indicate significant difference versus sham ( ANOVA followed by Bonferroni test ) .

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

F. HDAC3 activity (HDAC3 IP and activity assay) is strongly reduced after SNA but not DCA. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (*p<0.05) indicate significant difference versus sham (ANOVA followed by Bonferroni test).


PP4 activities are induced after SNA but not DCA , and are blocked by the calcium chelator EGTA . Data is expressed as mean ± s . e . m . N = 3 - 4 biological replicates . ( * * * p < 0 . 001 ) indicate significant difference versus sham or lam ( Two - way ANOVA followed by Bonferroni test ) .

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

PP4 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).


PP2 activities are induced after SNA but not DCA , and are blocked by the calcium chelator EGTA . Data is expressed as mean ± s . e . m . N = 3 - 4 biological replicates . ( * * * p < 0 . 001 ) indicate significant difference versus sham or lam ( Two - way ANOVA followed by Bonferroni test ) .

PP2 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).


C . SNA but not DCA significantly enhances calcium levels in DRG . Data is expressed as mean ± s . e . m . N = 4 biological replicates . ( * p < 0 . 05 ) indicate significant difference versus sham or lam ( ANOVA followed by Bonferroni test ) .

O O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O

C. SNA but not DCA significantly enhances calcium levels in DRG. Data is expressed as mean ± s.e.m. N= 4 biological replicates. (*p<0.05) indicate significant difference versus sham or lam (ANOVA followed by Bonferroni test).


D - F . Immunoblotting of DRG homogenates shows that nerve injury - dependent nuclear HDAC3 dephosphorylation is reduced by the calcium scavenger EGTA upon in vivo delivery on the injured sciatic nerve ( Axo = Axotomy ) . Cytoplasmic pERK levels are used as control of axotomy - dependent signalling . ( E - F ) . Data is expressed as mean fold change of band intensity levels ± s . e . m . N = 3 biological replicates . ( * * p < 0 . 01 , * * * p < 0 . 001 ) indicate a significant difference ( ANOVA followed by Bonferroni test ) .

O O O O B-EXP_ASSAY O B-TISSUE O O O B-DISEASE I-DISEASE O O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-DISEASE B-TISSUE I-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-F. Immunoblotting of DRG homogenates shows that nerve injury-dependent nuclear HDAC3 dephosphorylation is reduced by the calcium scavenger EGTA upon in vivo delivery on the injured sciatic nerve (Axo=Axotomy). Cytoplasmic pERK levels are used as control of axotomy-dependent signalling. (E-F). Data is expressed as mean fold change of band intensity levels ± s.e.m. N= 3 biological replicates. (**p<0.01, ***p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).


Significant Pearson correlations between calcium levels ( Fluo4AM ) and H3K9ac , r2 : 0 . 7802 ( G ) in cultured DRG neurons after KCl . The Pearson correlations were disrupted by administration of Fostriecin ( calcium and H3K9ac , r2 : 0 . 0007 Data is expressed as single cell fluorescence levels . N = 50 cells per condition from 4 biological replicates .

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

Significant Pearson correlations between calcium levels (Fluo4AM) and H3K9ac, r2: 0.7802 (G) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin (calcium and H3K9ac, r2: 0.0007 Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.


Significant Pearson correlations between calcium levels ( Fluo4AM ) ; or pHDAC3 , r2 : 0 . 6366 ( H ) in cultured DRG neurons after KCl . The Pearson correlations were disrupted by administration of Fostriecin calcium and pHDAC3 , r2 : 0 . 3025 ) . Data is expressed as single cell fluorescence levels . N = 50 cells per condition from 4 biological replicates .

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

Significant Pearson correlations between calcium levels (Fluo4AM) ; or pHDAC3, r2: 0.6366 (H) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin calcium and pHDAC3, r2: 0.3025). Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.


J - L . Immunofluorescence ( J ) in DRG tissue sections 24h after injury in vivo shows that the PP2a / PP4 inhibitor Fostriecin ( F ) inhibits the injury - induced decrease in pHDAC3 ( K ) and the increase in H3K9ac ( L ) . Scale bar , 10 μm . ( K - L ) . Data is expressed as mean fluorescence intensity in arbitrary units ± s . e . m . N = 3 ( L ) or 4 ( K ) biological replicates ( * p < 0 . 05 ; * * p < 0 . 01 ) indicate significant difference versus sham ( ANOVA followed by Bonferroni test ) .

O O O O B-EXP_ASSAY O O O O B-TISSUE O O O O B-DISEASE O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-DISEASE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J-L. Immunofluorescence (J) in DRG tissue sections 24h after injury in vivo shows that the PP2a/PP4 inhibitor Fostriecin (F) inhibits the injury-induced decrease in pHDAC3 (K) and the increase in H3K9ac (L). Scale bar, 10 μm. (K-L). Data is expressed as mean fluorescence intensity in arbitrary units ± s.e.m. N= 3 (L) or 4 (K) biological replicates (*p<0.05; **p<0.01) indicate significant difference versus sham (ANOVA followed by Bonferroni test).


M - N . Pharmacological inhibition of PP2a / PP4 impairs conditioning - induced DRG neurite outgrowth ex vivo ( 12h in culture ) . M . DRG neurite outgrowth ( TUJ1 positive cells ) 24h after sham or sciatic nerve axotomy ( SNA ) prior to i . t . administration of vehicle ( V ) or Fostriecin ( F ) ( 240µM ) . Scale bar , 100µm . ( N ) Data is expressed as average neurite length per neuron ± s . e . m . N = 3 biological replicates per condition , technical triplicate . * * * p < 0 . 005 indicate significant difference versus sham / vehicle ( One way ANOVA followed by Bonferroni ) .

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

M-N. Pharmacological inhibition of PP2a/PP4 impairs conditioning-induced DRG neurite outgrowth ex vivo (12h in culture). M. DRG neurite outgrowth (TUJ1 positive cells) 24h after sham or sciatic nerve axotomy (SNA) prior to i.t. administration of vehicle (V) or Fostriecin (F) (240µM). Scale bar, 100µm. (N) Data is expressed as average neurite length per neuron ± s.e.m. N= 3 biological replicates per condition, technical triplicate. *** p<0.005 indicate significant difference versus sham/vehicle (One way ANOVA followed by Bonferroni).


A - E . PP4c gene silencing promotes HDAC3 phosphorylation in the presence of KCl ( A - C , arrows ) , both in the nucleus and the cytoplasm ( D ) . Scale bar , 20 μm . ( C , D ) Data is expressed as mean fold change vs scrambled ( scr ) siRNA of fluorescent intensity of pHDAC3 or PP4c in GFP positive cells ± s . e . m . N = 3 biological replicates . ( * * p < 0 . 01 ; * * * p < 0 . 005 ) indicate significant difference versus scrambled siRNA ( Student ' s t - test ) . ( E ) PCR against PP4c demonstrates silencing of PP4c mRNA . F - G . PP2a gene silencing inhibits the KCl induced dephosphorylation of HDAC3 as shown by immunoblotting of pHDAC3 ( F ) . ( G ) Data is expressed as mean fold change of immunoblot band intensity ± s . e . m . N = 3 biological replicates ( * p < 0 . 05 ; * * * * p < 0 . 001 ) indicate significant difference versus control siRNA ( Student ' s t - test ) .

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

A-E. PP4c gene silencing promotes HDAC3 phosphorylation in the presence of KCl (A-C, arrows), both in the nucleus and the cytoplasm (D). Scale bar, 20 μm. (C,D) Data is expressed as mean fold change vs scrambled (scr) siRNA of fluorescent intensity of pHDAC3 or PP4c in GFP positive cells ± s.e.m. N=3 biological replicates. (**p<0.01; ***p<0.005) indicate significant difference versus scrambled siRNA (Student's t-test). (E) PCR against PP4c demonstrates silencing of PP4c mRNA. F-G. PP2a gene silencing inhibits the KCl induced dephosphorylation of HDAC3 as shown by immunoblotting of pHDAC3 (F). (G) Data is expressed as mean fold change of immunoblot band intensity ± s.e.m. N= 3 biological replicates (*p<0.05; ****p<0.001) indicate significant difference versus control siRNA (Student's t-test).


H - I . Genetic modification of activity - dependent phosphorylation of serine 424 of HDAC3 affects DRG neurite outgrowth . ( H ) DRG neurite outgrowth after transfection of control ( V5 control ) , WT HDAC3 - V5 , phospho - dead HDAC3 ( S424A ) - V5 or phospho - mimetic HDAC3 ( S424D ) - V5 and after 24h in culture . Scale bar , 50µm . ( I ) Data is expressed as mean ± s . e . m . N = 8 - 9 biological replicates , approximately 35 V5 positive cells each . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 indicate significant difference ( One way ANOVA followed by Bonferroni ) .

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

H-I. Genetic modification of activity-dependent phosphorylation of serine 424 of HDAC3 affects DRG neurite outgrowth. (H) DRG neurite outgrowth after transfection of control (V5 control), WT HDAC3-V5, phospho-dead HDAC3 (S424A)-V5 or phospho-mimetic HDAC3 (S424D)-V5 and after 24h in culture. Scale bar, 50µm. (I) Data is expressed as mean ± s.e.m. N= 8-9 biological replicates, approximately 35 V5 positive cells each. * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference (One way ANOVA followed by Bonferroni).


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

O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

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


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

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

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


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

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

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


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

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

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


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

O O O O B-ORGANISM O B-GENEPROD O O O O O O B-SUBCELLULAR O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O B-DISEASE I-DISEASE I-DISEASE O B-ORGANISM O B-GENEPROD O O O O O O O B-DISEASE O O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

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

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


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

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

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


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

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O B-SUBCELLULAR B-EXP_ASSAY O B-DISEASE I-DISEASE I-DISEASE O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE B-SUBCELLULAR O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O 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-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

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

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


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

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

HEK293T cells were transfected with empty FLAG-tag vector as a control or FLAG-Nup50 N-terminal fragment (aa 1-120 of the human sequence). 24 h post transfection cells were lysed, FLAG-tagged proteins immuno-isolated and analyzed by 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 .

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

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


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

O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O B-ORGANISM B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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 .

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

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 .

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

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 ) .

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

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


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

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

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


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

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

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


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

O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O

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


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

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

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


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

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

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


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

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

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


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

O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-TISSUE B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O B-TISSUE B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

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 . ) .

O B-DISEASE O B-GENEPROD O O B-TISSUE O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O

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 .

O B-DISEASE O B-GENEPROD O O B-TISSUE O O O O O O O O O O O

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


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

O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O

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 .

O O O O O O B-TISSUE O O O O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O

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


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

O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O

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


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

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

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


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

O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O

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


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

O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O B-CELL_TYPE O O O O O O B-TISSUE I-TISSUE O O O O O O B-DISEASE O O O O O O B-GENEPROD O O O O O O O B-DISEASE 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 ) .

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

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


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

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

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-GENEPROD O O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR 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 ) .

O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

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

(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-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 B-TISSUE I-TISSUE B-GENEPROD O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O B-DISEASE O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-TISSUE O B-TISSUE O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD 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 B-TISSUE O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE 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 B-TISSUE B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O

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

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


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

O O B-EXP_ASSAY O O O O O O O B-DISEASE O O O O O O O B-DISEASE O O O B-DISEASE O O O O O O O O O O

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