( F ) Kaplan - Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib ( black ) and vehicle ( grey ) . Statistical analysis of the survival curves ( F ) was performed with the log - rank ( Mantel - Cox ) test ; * * P < 0 . 01 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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) Kaplan-Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib (black) and vehicle (grey). Statistical analysis of the survival curves (F) was performed with the log-rank (Mantel-Cox) test; **P < 0.01.


( B ) Genome - wide binding of Pax5 - Jak2 in in vitro cultured pro - B cells from Pax5Jak2 / + Rosa26BirA / + mice at the age of 3 weeks ( expressing Pax5 ) and ex vivo Pax5Jak2 / + Rosa26BirA / + B - ALL tumors ( lacking Pax5 ) , as determined by Bio - ChIP - seq analysis The DNA - binding pattern of full - length Pax5 was determined by Bio - ChIP - seq analysis of ex vivo sorted Pax5Bio / Bio pro - B cells , which carried a C ‐ terminal biotin acceptor sequence together with an IRES ‐ BirA gene insertion in the 3 ' untranslated region of Pax5 Two independent Bio - ChIP - seq experiments were performed for each cell type . Representative binding patterns of Pax5 and Pax5 - Jak2 in the three B cell types are shown for a selected genomic region , with horizontal bars indicating Pax5 or Pax5 - Jak2 peaks that were identified by MACS peak calling ( left ) . The number of Pax5 ( white ) and Pax5 - Jak2 ( grey or black ) peaks , which were defined by stringent MACS peak calling with a P value of < 10 - 10 in the three B cell types , are shown to the right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Genome-wide binding of Pax5-Jak2 in in vitro cultured pro-B cells from Pax5Jak2/+ Rosa26BirA/+ mice at the age of 3 weeks (expressing Pax5) and ex vivo Pax5Jak2/+ Rosa26BirA/+ B-ALL tumors (lacking Pax5), as determined by Bio-ChIP-seq analysis The DNA-binding pattern of full-length Pax5 was determined by Bio-ChIP-seq analysis of ex vivo sorted Pax5Bio/Bio pro-B cells, which carried a C‐terminal biotin acceptor sequence together with an IRES‐BirA gene insertion in the 3' untranslated region of Pax5 Two independent Bio-ChIP-seq experiments were performed for each cell type. Representative binding patterns of Pax5 and Pax5-Jak2 in the three B cell types are shown for a selected genomic region, with horizontal bars indicating Pax5 or Pax5-Jak2 peaks that were identified by MACS peak calling (left). The number of Pax5 (white) and Pax5-Jak2 (grey or black) peaks, which were defined by stringent MACS peak calling with a P value of < 10-10 in the three B cell types, are shown to the right.


( F , G ) Flow - cytometric analysis of the bone marrow and spleen from Pax5Prd * - Jak2 / + ( black ; n = 6 ) and control Pax5 + / + ( grey ; n = 5 ) mice at the age of 6 - 7 weeks . The frequency of the indicated B cell types in each organ is indicated in ( F ) , and the flow - cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in ( G ) . Statistical data are shown as mean percentages with SEM and were analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Šídák ' s correction ) : ns > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 . Each dot corresponds to one mouse .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow-cytometric analysis of the bone marrow and spleen from Pax5Prd*-Jak2 /+ (black; n = 6) and control Pax5+/+ (grey; n = 5) mice at the age of 6-7 weeks. The frequency of the indicated B cell types in each organ is indicated in (F), and the flow-cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in (G). Statistical data are shown as mean percentages with SEM and were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction): ns > 0.05; *P < 0.05; **P < 0.01. Each dot corresponds to one mouse.


( H ) Kaplan - Meier survival analysis of Pax5Prd * - Jak2 / + ( black ) and Pax5 + / + ( grey ) mice . A P value of < 0 . 0001 was determined for the survival curves by statistical analysis with the log - rank ( Mantel - Cox ) test . n , number of mice analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier survival analysis of Pax5Prd*-Jak2 /+ (black) and Pax5+/+ (grey) mice. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test. n, number of mice analyzed.


( C ) Specificity of the anti - H3Y41ph antibody , as shown by immunoblot analysis . One phosphorylated ( pY41 ) or non - phosphorylated peptide was coupled to ubiquitin ( Ubi , 8 . 5 kDa ) , followed by separation of the protein conjugates on SDS - PAGE and immunoblot detection with the anti - H3Y41ph antibody present in the 8B2 - C1 cell supernatant . Following treatment with calf intestinal alkaline phosphatase ( CIP ) , the dephosphorylated Ubi - pY41 peptide conjugate could no longer be detected with the anti - H3Y41ph antibody .

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

(C) Specificity of the anti-H3Y41ph antibody, as shown by immunoblot analysis. One phosphorylated (pY41) or non-phosphorylated peptide was coupled to ubiquitin (Ubi, 8.5 kDa), followed by separation of the protein conjugates on SDS-PAGE and immunoblot detection with the anti-H3Y41ph antibody present in the 8B2-C1 cell supernatant. Following treatment with calf intestinal alkaline phosphatase (CIP), the dephosphorylated Ubi-pY41 peptide conjugate could no longer be detected with the anti-H3Y41ph antibody.


( D ) Immunoblot analysis of whole - cell extracts prepared from Pax5Jak2 / + and control Pax5Etv6 / + Cdkn2ab + / - B - ALL cells as well as from the human HEL , TMD8 and K1106 cell lines . Phosphorylated ( p ) STAT5 was detected with an anti - STAT5 ( pY694 ) antibody and the pY41 - peptide with the purified anti H3Y41ph antibody One to five pY41 - peptides were coupled to ubiquitin , which was added in the range of 50 ng per well . The Gapdh and histone H3 proteins were analyzed as loading control . An unspecific protein is denoted by an asterisk . One representative of 5 immunoblot experiments is shown .

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

(D) Immunoblot analysis of whole-cell extracts prepared from Pax5Jak2/+ and control Pax5Etv6/+ Cdkn2ab+/- B-ALL cells as well as from the human HEL, TMD8 and K1106 cell lines. Phosphorylated (p) STAT5 was detected with an anti-STAT5 (pY694) antibody and the pY41-peptide with the purified anti H3Y41ph antibody One to five pY41-peptides were coupled to ubiquitin, which was added in the range of 50 ng per well. The Gapdh and histone H3 proteins were analyzed as loading control. An unspecific protein is denoted by an asterisk. One representative of 5 immunoblot experiments is shown.


( A , Tumor progression in Il7 + / + ( grey ) , Il7 + / - ( dashed ) and Il7 - / - ( black ) mice transplanted with Pax5Jak2 - Luc / + tumor cells , as determined by bioluminescence measurements at the indicated days after cell transfer . Images of three transplanted mice for each Il7 genotype are shown at the indicated days after cell

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Tumor progression in Il7+/+ (grey), Il7+/- (dashed) and Il7-/- (black) mice transplanted with Pax5Jak2-Luc/+ tumor cells, as determined by bioluminescence measurements at the indicated days after cell transfer. Images of three transplanted mice for each Il7 genotype are shown at the indicated days after cell transfer


( C ) Kaplan - Meier survival analysis of Il7 + / + , Il7 + / - and Il7 - / - mice transplanted with Pax5Jak2 - Luc / + tumors cells . Statistical analysis of the survival curves was performed with the log - rank ( Mantel - Cox ) test ; ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier survival analysis of Il7+/+, Il7+/- and Il7-/- mice transplanted with Pax5Jak2-Luc/+ tumors cells. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ns P > 0.05, *P < 0.05, **P < 0.01.


( E ) Quantification of the median fluorescence intensity ( MFI ) values of the p - STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5 + / + ( grey ) , Pax5Jak2 / + ( black ) and Pax5Jak2 - KD / + ( white ) mice at the age of 3 - 10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two - tailed unpaired Student ' s t - test : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot corresponds to one mouse .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the median fluorescence intensity (MFI) values of the p-STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5+/+ (grey), Pax5Jak2/+ (black) and Pax5Jak2-KD/+ (white) mice at the age of 3-10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.


( F ) Flow cytometric analysis of p - STAT5 levels in pro - B cells ( B220 + CD19 + Kit + IgM - ) and leukemic B220low B cells of Pax5Jak2 / + mice ( black ) as well as in control pro - B cells of Pax5 + / + ( WT ) mice ( grey ) at the age of 4 - 5 weeks . A representative flow cytometric analysis of the bone marrow of a Pax5Jak2 / + mouse ( left ) and representative intracellular p - STAT5 stainings ( middle ) are shown . The p - STAT5 levels in pro - B and leukemic B220low B cells ( right ) are quantified as MFI values relative to that of Pax5 + / + pro - B cells . Statistical data are presented as mean values with SEM and were analyzed by the two - tailed unpaired Student ' s t - test : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot corresponds to one mouse .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometric analysis of p-STAT5 levels in pro-B cells (B220+CD19+Kit+IgM-) and leukemic B220low B cells of Pax5Jak2/+ mice (black) as well as in control pro-B cells of Pax5+/+ (WT) mice (grey) at the age of 4-5 weeks. A representative flow cytometric analysis of the bone marrow of a Pax5Jak2/+ mouse (left) and representative intracellular p-STAT5 stainings (middle) are shown. The p-STAT5 levels in pro-B and leukemic B220low B cells (right) are quantified as MFI values relative to that of Pax5+/+ pro-B cells. Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.


( I ) Upregulation of the genes Cxcr5 , Syndig1l and Sema4a in Pax5Jak2 / + ( PJ ) B - ALL cells . Mean TPM values with SEM are shown for the following RNA - seq experiments : 2 ( Ctrl B - ALL ) , 4 ( PJ B - ALL )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Upregulation of the genes Cxcr5, Syndig1l and Sema4a in Pax5Jak2/+ (PJ) B-ALL cells. Mean TPM values with SEM are shown for the following RNA-seq experiments: 2 (Ctrl B-ALL), 4 (PJ B-ALL)


K ) Identification of Cxcr5 , Syndig1l and Sema4a as activated STAT5 target genes . ChIP - seq analysis identified STAT5 - binding regions at all three loci ( K ) . Horizontal bars below the ChIP - seq track indicate STAT5 - binding regions identified by MACS peak calling . RPM , reads per million .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Identification of Cxcr5, Syndig1l and Sema4a as activated STAT5 target genes. ChIP-seq analysis identified STAT5-binding regions at all three loci (K). Horizontal bars below the ChIP-seq track indicate STAT5-binding regions identified by MACS peak calling. RPM, reads per million.


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


( A ) Number of cell corpses at the L1 stage of development per 100 animals carrying the neurotoxic mec ‐ 4 ( d ) or deg ‐ 3 ( d ) alleles in SNT ‐ 1 ‐ deficient mutants . For mec ‐ 4 ( d ) mutant animals , bars denote touch receptor neuron corpses . For deg ‐ 3 ( d ) mutants , bars denote inner labial neuron 1 ( L1 ) sensory neuron and PVC interneuron corpses .

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(A) Number of cell corpses at the L1 stage of development per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles in SNT‐1‐deficient mutants. For mec‐4(d) mutant animals, bars denote touch receptor neuron corpses. For deg‐3(d)mutants, bars denote inner labial neuron 1 (L1) sensory neuron and PVC interneuron corpses.


( B ) Expression of a full ‐ length MEC ‐ 4 : : GFP reporter fusion under the control of the mec ‐ 4 promoter in touch receptor neurons of wild ‐ type , snt ‐ 1 and unc ‐ 57 mutant animals . Representative photos of touch receptor neuron cell bodies are shown . Bar denotes 6 μm . The quantification ( % ) of GFP signal intensity from the animals examined is graphed below ( n = 100 neurons per assay ; P0 . 05 compared with wild type ; t ‐ test ) .

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(B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, snt‐1 and unc‐57 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test).


( C ) Percentage of SNT ‐ 1 ‐ deficient animals that survive near ‐ lethal treatment with sodium azide . This chemical inhibits the activity of the respiratory electron transport complex IV ( cytochrome C oxidase ) and simulates hypoxia .

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(C) Percentage of SNT‐1‐deficient animals that survive near‐lethal treatment with sodium azide. This chemical inhibits the activity of the respiratory electron transport complex IV (cytochrome C oxidase) and simulates hypoxia.


( D ) Number of touch receptor neuron corpses , at the L1 stage of development , per 100 animals carrying the neurotoxic mec - 4 ( d ) or deg ‐ 3 ( d ) alleles in UNC ‐ 57 ‐ deficient animals .

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(D) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec-4(d) or deg‐3(d) alleles in UNC‐57‐deficient animals.


( E ) Percentage of UNC ‐ 57 ‐ deficient animals that survive after the hypoxia ‐ inducing treatment with sodium azide .

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(E) Percentage of UNC‐57‐deficient animals that survive after the hypoxia‐inducing treatment with sodium azide.


( F ) Number of touch receptor neuron corpses , at the L1 stage of development , per 100 animals carrying the neurotoxic mec ‐ 4 ( d ) or deg ‐ 3 ( d ) alleles together with lesions in genes encoding key proteins that participate in all four steps of clathrin ‐ mediated endocytosis . UNC ‐ 11 and DPY ‐ 23 are adaptor proteins required for the formation of clathrin ‐ coated pits , DYN ‐ 1 is a GTPase necessary for fission of clathrin ‐ coated vesicles and UNC ‐ 26 participates in the uncoating of vesicles . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 001 , compared with wild ‐ type animals , unpaired t ‐ test ) .

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(F) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles together with lesions in genes encoding key proteins that participate in all four steps of clathrin‐mediated endocytosis. UNC‐11 and DPY‐23 are adaptor proteins required for the formation of clathrin‐coated pits, DYN‐1 is a GTPase necessary for fission of clathrin‐coated vesicles and UNC‐26 participates in the uncoating of vesicles. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with wild‐type animals, unpaired t‐test).


( A ) Confocal images of touch receptor neurons expressing a pmec ‐ 17APS ‐ 2GFP reporter transgene . During early necrosis , the number of APS ‐ 2 : : GFP puncta , corresponding to clathrin ‐ coated pits and vesicles , in touch receptor neurons ofmec ‐ 4 ( d ) animals does not change significantly , whereas at later stages it decreases . A similar decrease is also observed in unc ‐ 57 ( e1190 ) mutants , defective for clathrin ‐ mediated endocytosis .

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(A) Confocal images of touch receptor neurons expressing a pmec‐17APS‐2GFP reporter transgene. During early necrosis, the number of APS‐2::GFP puncta, corresponding to clathrin‐coated pits and vesicles, in touch receptor neurons ofmec‐4(d) animals does not change significantly, whereas at later stages it decreases. A similar decrease is also observed in unc‐57(e1190) mutants, defective for clathrin‐mediated endocytosis.


( B ) Confocal images of touch receptor neurons expressing a pmec ‐ 7GFP : : RAB ‐ 5 reporter transgene . The number of fluorescent puncta that correspond to early endosomes in touch receptor neurons of mec ‐ 4 ( d ) animals significantly increases during early necrosis , whereas it declines later .

(B) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐5 reporter transgene. The number of fluorescent puncta that correspond to early endosomes in touch receptor neurons of mec‐4(d) animals significantly increases during early necrosis, whereas it declines later.


( C ) Confocal images of touch receptor neurons expressing a pmec ‐ 7GFP : : RAB ‐ 11 reporter transgene . The number of GFP : : RAB ‐ 11 puncta that correspond to recycling endosomes in touch receptor neurons of mec ‐ 4 ( d ) animals increases early during cell death , while it declines as degeneration proceeds . Both early and recycling endosomes tend to accumulate around a swollen structure that is probably the nucleus . Bars denote 4 μm . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 001 , compared with control animals , unpaired t ‐ test ) .

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(C) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐11 reporter transgene. The number of GFP::RAB‐11 puncta that correspond to recycling endosomes in touch receptor neurons of mec‐4(d) animals increases early during cell death, while it declines as degeneration proceeds. Both early and recycling endosomes tend to accumulate around a swollen structure that is probably the nucleus. Bars denote 4 μm. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).


Endocytosis functions together with lysosomal proteolysis to facilitate necrotic cell death . ( A ) Depletion of synaptotagmin ( SNT ‐ 1 ) or endophilin ( UNC ‐ 57 ) does not further suppress MEC ‐ 4 ( d ) ‐ induced necrosis in cad ‐ 1 mutants with reduced cathepsin activity or with V ‐ ATPase dysfunction ( B , C ) .

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Endocytosis functions together with lysosomal proteolysis to facilitate necrotic cell death. (A) Depletion of synaptotagmin (SNT‐1) or endophilin (UNC‐57) does not further suppress MEC‐4(d)‐induced necrosis in cad‐1 mutants with reduced cathepsin activity or with V‐ATPase dysfunction (B, C).


Similarly , no synthetic reduction of necrotic cell death is observed in calpain ‐ deficient animals that also carry lesions in the synaptotagmin ( D ) or endophilin ( E ) genes . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P > 0 . 5 , compared with single mutant control animals , unpaired t ‐ test ) .

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Similarly, no synthetic reduction of necrotic cell death is observed in calpain‐deficient animals that also carry lesions in the synaptotagmin (D) or endophilin (E) genes. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).


( A ) Synaptotagmin ( SNT ‐ 1 ) deficiency further suppresses MEC ‐ 4 ( d ) ‐ induced necrosis in animals with impaired autophagy .

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(A) Synaptotagmin (SNT‐1) deficiency further suppresses MEC‐4(d)‐induced necrosis in animals with impaired autophagy.


( B ) Endophilin dysfunction enhances suppression of necrosis in mec ‐ 4 ( d ) ; lgg ‐ 1 ( RNAi ) animals , where autophagy is impaired . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 01 , compared with single mutant control animals , unpaired t ‐ test ) .

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(B) Endophilin dysfunction enhances suppression of necrosis in mec‐4(d);lgg‐1(RNAi) animals, where autophagy is impaired. Error bars denote s.e.m. values (n>250 for all populations examined; P0.01, compared with single mutant control animals, unpaired t‐test).


( A ) Depletion of the kinesin 1 heavy chain UNC ‐ 116 suppresses necrosis induced by both mec ‐ 4 ( d ) and deg ‐ 3 ( d ) toxic alleles .

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(A) Depletion of the kinesin 1 heavy chain UNC‐116 suppresses necrosis induced by both mec‐4(d) and deg‐3(d) toxic alleles.


( B ) Expression of a full ‐ length MEC ‐ 4 : : GFP reporter fusion under the control of the mec ‐ 4 promoter in touch receptor neurons of wild ‐ type , unc ‐ 116 and unc ‐ 104 mutant animals . Representative photos of touch receptor neuron cell bodies are shown . Bar denotes 6 μm . The quantification ( % ) of GFP signal intensity from the animals examined is graphed below ( n = 100 neurons per assay ; P0 . 05 compared with wild type ; t ‐ test ) .

(B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, unc‐116 and unc‐104 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test).


( C ) Percentage of unc ‐ 116 animals that survive after treatment with sodium azide .

(C) Percentage of unc‐116 animals that survive after treatment with sodium azide.


( D ) Depletion of the monomeric kinesin UNC ‐ 104 protects neurons against MEC ‐ 4 ( d ) ‐ induced degeneration .

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(D) Depletion of the monomeric kinesin UNC‐104 protects neurons against MEC‐4(d)‐induced degeneration.


( E ) unc ‐ 104 mutant animals are more resistant to hypoxic death induced by sodium azide .

(E) unc‐104 mutant animals are more resistant to hypoxic death induced by sodium azide.


( F ) Combined inactivation of endocytosis and kinesin ‐ mediated intracellular trafficking does not enhance protection of touch receptor neurons against MEC ‐ 4 ( d ) ‐ induced degeneration . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 001 , compared with control animals , unpaired t ‐ test ) .

(F) Combined inactivation of endocytosis and kinesin‐mediated intracellular trafficking does not enhance protection of touch receptor neurons against MEC‐4(d)‐induced degeneration. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).


Genetic interaction between kinesin ‐ mediated intracellular trafficking and cellular proteolytic pathways mediating necrotic cell death . Dysfunction of either kinesin 1 heavy chain ( UNC ‐ 116 ) or the monomeric kinesin UNC ‐ 104 does not significantly enhance suppression of neurodegeneration in aspartyl protease ‐ deficient mutant animals ( A , B ) , in animals with compromised lysosomal acidification ( C , D ) , or in calpain protease ‐ deficient mutants animals ( E , F ) . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P > 0 . 5 , compared with single mutant control animals , unpaired t ‐ test ) .

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Genetic interaction between kinesin‐mediated intracellular trafficking and cellular proteolytic pathways mediating necrotic cell death. Dysfunction of either kinesin 1 heavy chain (UNC‐116) or the monomeric kinesin UNC‐104 does not significantly enhance suppression of neurodegeneration in aspartyl protease‐deficient mutant animals (A, B), in animals with compromised lysosomal acidification (C, D), or in calpain protease‐deficient mutants animals (E, F). Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).


( A ) Kinesin 1 heavy chain ( UNC ‐ 116 ) deficiency .

(A) Kinesin 1 heavy chain (UNC‐116) deficiency.


( B ) Monomeric kinesin UNC ‐ 104 deficiency . In both cases , no significant synthetic protection of neurons is observed in LGG ‐ 1 / LC3 ‐ depleted animals with impaired autophagy . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P > 0 . 5 , compared with single mutant control animals , unpaired t ‐ test ) .

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(B) Monomeric kinesin UNC‐104 deficiency. In both cases, no significant synthetic protection of neurons is observed in LGG‐1/LC3‐depleted animals with impaired autophagy. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).


( B ) Enrichment profiles of TEAD4 and AP - 1 consensus motifs in 600 bp regions centred on the ZEB1 peak summits . The two grey dashed lines highlight the 200 bp regions around the ZEB1 peak summits which were used for HOMER known motif

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(B) Enrichment profiles of TEAD4 and AP-1 consensus motifs in 600 bp regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the 200 bp regions around the ZEB1 peak summits which were used for HOMER known motif analysis


( D ) Enrichment profile of JUN peak summit positions in 1 kb regions centred on the ZEB1 peak summits . The two grey dashed lines highlight the region of + / - 200 bp around the ZEB1 peak summit positions where JUN peaks are counted as overlapping with ZEB1 peaks .

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(D) Enrichment profile of JUN peak summit positions in 1 kb regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the region of +/- 200 bp around the ZEB1 peak summit positions where JUN peaks are counted as overlapping with ZEB1 peaks.


( E ) Heatmap representing the signal enrichment of JUN ChIP - seq reads in 2 kb regions centred on the ZEB1 peak summits .

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(E) Heatmap representing the signal enrichment of JUN ChIP-seq reads in 2 kb regions centred on the ZEB1 peak summits.


( A ) In situ proximity ligation assay ( PLA ) of ZEB1 with JUN , FOSL1 and TAZ shows close proximity of ZEB1 with JUN and FOSL1 but not TAZ in the nucleus of MDA - MB - 231 cells indicated by red fluorescent dots . As negative control ZEB1 was transiently knocked down by siRNA . In the left panel representative microscopic images are shown . Scale bar = 20 µm . Quantification of the PLA is shown on the right .

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(A) In situ proximity ligation assay (PLA) of ZEB1 with JUN, FOSL1 and TAZ shows close proximity of ZEB1 with JUN and FOSL1 but not TAZ in the nucleus of MDA-MB-231 cells indicated by red fluorescent dots. As negative control ZEB1 was transiently knocked down by siRNA. In the left panel representative microscopic images are shown. Scale bar = 20 µm. Quantification of the PLA is shown on the right.


( B ) Co - immunoprecipitation ( Co - IP ) of endogenous ZEB1 and FOSL1 or JUN in MDA - MB - 231 cells shows co - precipitation of ZEB1 with FOSL1 and JUN .

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(B) Co-immunoprecipitation (Co-IP) of endogenous ZEB1 and FOSL1 or JUN in MDA-MB-231 cells shows co-precipitation of ZEB1 with FOSL1 and JUN.


( A ) Definition of two different ZEB1 peak subsets based on the presence or absence of overlapping YAP and JUN peaks . The two illustrative genome browser images show one example of a ZEB1 - only peak and one example of a ZEB1 / YAP / JUN peak ( at the EFEMP1 gene and within the SHROOM3 gene , respectively ) . Control ( ctrl ) is the input for ZEB1 and IgG for the YAP and JUN ChIP - seqs . ChIP - seqs and respective controls were scaled accordingly .

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(A) Definition of two different ZEB1 peak subsets based on the presence or absence of overlapping YAP and JUN peaks. The two illustrative genome browser images show one example of a ZEB1-only peak and one example of a ZEB1/YAP/JUN peak (at the EFEMP1 gene and within the SHROOM3 gene, respectively). Control (ctrl) is the input for ZEB1 and IgG for the YAP and JUN ChIP-seqs. ChIP-seqs and respective controls were scaled accordingly.


( C ) Localisation of ZEB1 peak summits relative to the closest TSS .

(C) Localisation of ZEB1 peak summits relative to the closest TSS.


( A ) qRT - PCR in TGFß treated MCF10A cells after siRNA mediated knockdown of ZEB1 , FOSL1 or YAP compared to control knockdown cells . Knockdown efficiency of all three factors is shown on the left . The right panel shows their effects on 11 ZEB1 / YAP / AP - 1 activated target genes .

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(A) qRT-PCR in TGFß treated MCF10A cells after siRNA mediated knockdown of ZEB1, FOSL1 or YAP compared to control knockdown cells. Knockdown efficiency of all three factors is shown on the left. The right panel shows their effects on 11 ZEB1/YAP/AP-1 activated target genes.


( B ) Overlap of ZEB1 , YAP and JUN peaks at the promoter of CYR61 , CTGF and ANKRD1 and in a known enhancer region of the DOCK9 gene .

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(B) Overlap of ZEB1, YAP and JUN peaks at the promoter of CYR61, CTGF and ANKRD1 and in a known enhancer region of the DOCK9 gene.


( C ) ZEB1 - only peak at the promoter of LLGL2 , a well - known ZEB1 repressed target .

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(C) ZEB1-only peak at the promoter of LLGL2, a well-known ZEB1 repressed target.


( D ) Luciferase reporter assay in MDA - MB - 231 cells comparing control and transient siRNA - mediated knockdown of ZEB1 . Reporter constructs of regulatory regions of genes For reporter assays , firefly luciferase activity was normalized to co - transfected renilla luciferase .

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(D) Luciferase reporter assay in MDA-MB-231 cells comparing control and transient siRNA-mediated knockdown of ZEB1. Reporter constructs of regulatory regions of genes For reporter assays, firefly luciferase activity was normalized to co-transfected renilla luciferase.


( E ) Luciferase reporter assays with the ANKRD1 promoter and DOCK9 enhancer constructs in MCF7 cells upon overexpression of ZEB1 , YAP and JUN / FOSL1 ( AP - 1 ) or different combinations of the factors . n = 5 ( ANKRD1 ) and n = 3 ( DOCK9 ) ; shown is mean ± s . e . m . ; * P ≤ 0 . 05 ; ratio paired t - test . For reporter assays , firefly luciferase activity was normalized to co - transfected renilla luciferase .

(E) Luciferase reporter assays with the ANKRD1 promoter and DOCK9 enhancer constructs in MCF7 cells upon overexpression of ZEB1, YAP and JUN/FOSL1 (AP-1) or different combinations of the factors. n = 5 (ANKRD1) and n = 3 (DOCK9); shown is mean ± s.e.m.; *P ≤ 0.05; ratio paired t-test. For reporter assays, firefly luciferase activity was normalized to co-transfected renilla luciferase.


( C ) The upper panel shows a schematic representation of the ANKRD1 luciferase reporter constructs . The lower panel shows luciferase reporter assays with these different ANKRD1 promoter constructs in MCF7 cells upon overexpression of ZEB1 , YAP and JUN / FOSL1 or different combinations of the factors . n = 4 - 5 ; shown is mean ± s . e . m . ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; ratio paired t - test . Firefly luciferase activity was normalized to co - transfected renilla luciferase .

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(C) The upper panel shows a schematic representation of the ANKRD1 luciferase reporter constructs. The lower panel shows luciferase reporter assays with these different ANKRD1 promoter constructs in MCF7 cells upon overexpression of ZEB1, YAP and JUN/FOSL1 or different combinations of the factors. n = 4-5; shown is mean ± s.e.m.; *P ≤ 0.05; **P ≤ 0.01; ratio paired t-test. Firefly luciferase activity was normalized to co-transfected renilla luciferase.


( D ) Left panel : EMSA using nuclear extracts from MDA - MB - 231 and labelled TEAD binding side as probe ( black arrow ) . Competition of specific binding complex ( read arrow ) with cold probe ( c ) , anti - JUN , - panTEAD , and - ZEB1 antisera . Right panel : recombinant GST - ZEB1 , but not GST - Ctrl , only binds to Z - box .

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(D) Left panel: EMSA using nuclear extracts from MDA-MB-231 and labelled TEAD binding side as probe (black arrow). Competition of specific binding complex (read arrow) with cold probe (c), anti-JUN, -panTEAD, and -ZEB1 antisera. Right panel: recombinant GST-ZEB1, but not GST-Ctrl, only binds to Z-box.


( B ) Comparison of ZEB1 ChIP - seq peaks with ZEB1 - dependent changes in chromatin activation status identified by ATAC - seq on control and ZEB1 knock - down MDA - MB - 231 cells . Percentage of ZEB1 - only ( n = 5 , 963 ) and ZEB1 / YAP / JUN peaks ( n = 1 , 993 ) falling into ZEB1 repressed or activated genomic regions are shown .

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(B) Comparison of ZEB1 ChIP-seq peaks with ZEB1-dependent changes in chromatin activation status identified by ATAC-seq on control and ZEB1 knock-down MDA-MB-231 cells. Percentage of ZEB1-only (n=5,963) and ZEB1/YAP/JUN peaks (n=1,993) falling into ZEB1 repressed or activated genomic regions are shown.


( C ) Genome browser tracks of ZEB1 , YAP and JUN ChIP - seq signal intensity in wild type cells overlaid to ATAC - seq signal intensity in wild type , control ( shCtrl ) and ZEB1 ( shZEB1 ) knock - down MDA - MB - 231 cells . One known ZEB1 repressed target ( CDH1 ) and one activated target ( ANKRD1 ) are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Genome browser tracks of ZEB1, YAP and JUN ChIP-seq signal intensity in wild type cells overlaid to ATAC-seq signal intensity in wild type, control (shCtrl) and ZEB1 (shZEB1) knock-down MDA-MB-231 cells. One known ZEB1 repressed target (CDH1) and one activated target (ANKRD1) are shown.


( A ) Correlation of ZEB1 mRNA expression with the expression of YAP , FOSL1 and JUN across all breast cancer cell lines included in the cancer cell line encyclopedia ( CCLE ) . rs : Spearman ' s rank correlation coefficient .

O O O O O O O O O O O O O O O O O O O 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) Correlation of ZEB1 mRNA expression with the expression of YAP, FOSL1 and JUN across all breast cancer cell lines included in the cancer cell line encyclopedia (CCLE). rs: Spearman's rank correlation coefficient.


( B ) Heatmap of RNA - seq expression data of all CCLE breast cancer cell lines .

(B) Heatmap of RNA-seq expression data of all CCLE breast cancer cell lines.


( A ) Kaplan Meier plots from survival analyses of human ER - / PR - breast cancers showing distant metastasis free survival ( DMFS ) based on the expression of the indicated genes or gene combinations . HR : hazard ratio . p - value from log - rank test .

(A) Kaplan Meier plots from survival analyses of human ER-/PR- breast cancers showing distant metastasis free survival (DMFS) based on the expression of the indicated genes or gene combinations. HR: hazard ratio. p-value from log-rank test.


( B ) Hypergeometric testing showed a significant association of nuclear ZEB1 and FOSL1 expression on the protein level as determined by immunohistochemistry on 81 human breast cancer samples . The upper panel shows staining of a serial section of a negative and a positive case . Note that stromal cells ( asterisks ) are negative for FOSL1 , and partially positive for ZEB1 and YAP , and that expression of ZEB1 in tumour cells ( arrows ) of positive cases is weaker than in stromal cells . Odds ratio ( OR ) : measure of association between categorical variables with OR > 1 indicating a positive association .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hypergeometric testing showed a significant association of nuclear ZEB1 and FOSL1 expression on the protein level as determined by immunohistochemistry on 81 human breast cancer samples. The upper panel shows staining of a serial section of a negative and a positive case. Note that stromal cells (asterisks) are negative for FOSL1, and partially positive for ZEB1 and YAP, and that expression of ZEB1 in tumour cells (arrows) of positive cases is weaker than in stromal cells. Odds ratio (OR): measure of association between categorical variables with OR > 1 indicating a positive association.


( C ) Heatmap of mRNA expression data of ZEB1 , YAP , JUN and 9 selected activated ZEB1 / YAP / AP - 1 target genes in breast cancer patient samples ( GSE18229 ) . Genes were selected for known roles in tumour promoting properties like cell migration . Tumours in which ≥ 6 out of 9 genes of the selected ZEB1 / YAP / AP - 1 target gene set were expressed higher than the 60th percentile of expression were defined as " tumour promoting gene set high " ; when expression of ≥ 6 out of the 9 genes was below the 40th percentile , tumours were defined as " tumour promoting gene set low " . Tumours were annotated according to their molecular subtype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap of mRNA expression data of ZEB1, YAP, JUN and 9 selected activated ZEB1/YAP/AP-1 target genes in breast cancer patient samples (GSE18229). Genes were selected for known roles in tumour promoting properties like cell migration. Tumours in which ≥ 6 out of 9 genes of the selected ZEB1/YAP/AP-1 target gene set were expressed higher than the 60th percentile of expression were defined as "tumour promoting gene set high"; when expression of ≥ 6 out of the 9 genes was below the 40th percentile, tumours were defined as "tumour promoting gene set low". Tumours were annotated according to their molecular subtype.


( E ) Kaplan Meier plots from survival analyses of human breast cancers patient samples ( GSE18229 ) showing relapse free survival ( RFS ) based on the combined expression of the 9 selected ZEB1 / YAP / AP - 1 activated targets HR : hazard ratio . p - value from log - rank test . ( F ) Kaplan Meier plots from survival analyses of human ER - / PR - breast cancers showing distant metastasis free survival ( DMFS ) based on the combined expression of the 9 selected ZEB1 / YAP / AP - 1 activated targets HR : hazard ratio . p - value from log - rank test .

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

(E) Kaplan Meier plots from survival analyses of human breast cancers patient samples (GSE18229) showing relapse free survival (RFS) based on the combined expression of the 9 selected ZEB1/YAP/AP-1 activated targets HR: hazard ratio. p-value from log-rank test. (F) Kaplan Meier plots from survival analyses of human ER-/PR- breast cancers showing distant metastasis free survival (DMFS) based on the combined expression of the 9 selected ZEB1/YAP/AP-1 activated targets HR: hazard ratio. p-value from log-rank test.


Morphology of four - week - old soil - grown plants of Col - 0 , snc1 , and two independent transgenic lines of SNIPER1 OE into snc1 background . OE stands for overexpression of SNIPER1 . Scale bar = 1 cm . SNIPER1 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . Quantification of H . a . Noco2 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . m . ES4326 on four - week - old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0 . 001 . Error bars represent mean ± SD ( n = 4 for day 0 ; n = 5 for day 3 ) . Three independent experiments were carried out with similar results .

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

Morphology of four-week-old soil-grown plants of Col-0, snc1, and two independent transgenic lines of SNIPER1 OE into snc1 background. OE stands for overexpression of SNIPER1. Scale bar = 1 cm. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.


Morphology of four - week - old soil - grown plants of two independent transgenic lines of SNIPER1 OE in Col - 0 background . Scale bar = 1 cm . No difference in growth and development was observed . SNIPER1 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . Quantification of H . a . Noco2 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . t . DC3000 on four - week - old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0 . 001 . Error bars represent mean ± SD ( n = 4 for day 0 ; n = 5 for day 3 ) . Three independent experiments were carried out with similar results .

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

Morphology of four-week-old soil-grown plants of two independent transgenic lines of SNIPER1 OE in Col-0 background. Scale bar = 1 cm. No difference in growth and development was observed. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.t. DC3000 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.


Morphology of four - week - old soil - grown plants of Col - 0 , chs1 - 2 ( A ) , chs2 - 1 ( B ) and two independent transgenic lines of SNIPER1 OE into chs1 - 2 ( A ) and chs2 - 1 ( B ) backgrounds . Plants were grown at 16 ° C under long day conditions . Scale bar = 1 cm .

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

Morphology of four-week-old soil-grown plants of Col-0, chs1-2 (A), chs2-1 (B) and two independent transgenic lines of SNIPER1 OE into chs1-2 (A) and chs2-1 (B) backgrounds. Plants were grown at 16 °C under long day conditions. Scale bar = 1 cm.


Morphology of four - week - old soil - grown plants of Col - 0 , chs3 - 2D ( C ) , snc2 - 1D eds5 npr1 ( D ) and two independent transgenic lines of SNIPER1 OE into chs3 - 2D ( C ) and snc2 - 1D eds5 npr1 ( D ) backgrounds . Scale bar = 1 cm .

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

Morphology of four-week-old soil-grown plants of Col-0, chs3-2D (C), snc2-1D eds5 npr1 (D) and two independent transgenic lines of SNIPER1 OE into chs3-2D (C) and snc2-1D eds5 npr1 (D) backgrounds. Scale bar = 1 cm.


Morphology of four - week - old soil - grown plants of Col - 0 , mekk1 - 5 ndr1 , and two independent transgenic lines of SNIPER1 OE into mekk1 - 5 ndr1 background . Scale bar = 1 cm . The mekk1 ndr1 double mutant was used instead of the mekk1 - 5 single mutant is to overcome the sterility . SNIPER1 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . Quantification of H . a . Noco2 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . m . ES4326 on four - week - old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0 . 001 . mn stands for mekk1 - 5 ndr1 double mutant . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results .

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

Morphology of four-week-old soil-grown plants of Col-0, mekk1-5 ndr1, and two independent transgenic lines of SNIPER1 OE into mekk1-5 ndr1 background. Scale bar = 1 cm. The mekk1 ndr1 double mutant was used instead of the mekk1-5 single mutant is to overcome the sterility. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. mn stands for mekk1-5 ndr1 double mutant. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.


In vitro ubiquitination assay using E . coli - expressed SNIPER1 - myc ( E3 ) , AtUBC8 ( E2 ) , AtUBA1 ( E1 ) and / or HIS - FLAG - Ubiquitin . The molecular mass markers are indicated on the left ( kDa ) . Asterisk indicates SNIPER1 - myc . Arrow points to His - FLAG - Ub and straight lines on the right indicate self - ubiquitinated SNIPER1 .

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

In vitro ubiquitination assay using E. coli-expressed SNIPER1-myc (E3), AtUBC8 (E2), AtUBA1 (E1) and/or HIS-FLAG-Ubiquitin. The molecular mass markers are indicated on the left (kDa). Asterisk indicates SNIPER1-myc. Arrow points to His-FLAG-Ub and straight lines on the right indicate self-ubiquitinated SNIPER1.


Immunoblot analysis of SNC1 ( A - B ) , SUMM2 - 3HA ( C ) , RPS4HA ( D ) , RPP4myc ( E ) , RPS2HA ( F ) and RPM1myc ( G ) protein levels in the indicated genotypes . For all panels , equal loading is shown by Ponceau S staining of a non - specific band . The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD ( n = 3 ) . Molecular mass marker in kilo Daltons is indicated on the left . Three independent experiments were carried out with similar results .

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

Immunoblot analysis of SNC1 (A-B), SUMM2-3HA (C), RPS4HA (D), RPP4myc (E), RPS2HA (F) and RPM1myc (G) protein levels in the indicated genotypes. For all panels, equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.


Growth of P . s . t . avrRps4 ( A ) , avrRpt2 ( B ) and avrRpm1 ( C ) on four - week - old leaves of the indicated genotypes at 3 dpi with bacterial inoculum of OD600 = 0 . 0001 . Error bars represent mean ± SD ( n = 5 ) . Three independent experiments were carried out with similar results . Appendix Figure S4 shows the growth of P . s . t . avrRps4 , avrRpt2 and avrRpm1 at 0 dpi . Quantification of H . a . Emwa1 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results .

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

Growth of P.s.t. avrRps4 (A), avrRpt2 (B) and avrRpm1 (C) on four-week-old leaves of the indicated genotypes at 3 dpi with bacterial inoculum of OD600 = 0.0001. Error bars represent mean ± SD (n=5). Three independent experiments were carried out with similar results. Appendix Figure S4 shows the growth of P.s.t. avrRps4, avrRpt2 and avrRpm1 at 0 dpi. Quantification of H.a. Emwa1 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.


Morphology of four - week - old soil - grown plants of Col - 0 and two independent transgenic lines of 35S - SNIPER1H129Y . Scale bar = 1 cm .

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

Morphology of four-week-old soil-grown plants of Col-0 and two independent transgenic lines of 35S-SNIPER1H129Y. Scale bar = 1 cm.


PR1 and PR2 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results .

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

PR1 and PR2 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results.


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

Morphology of four-week-old soil-grown plants of Col-0, sniper1-1, sniper2-1, eds1-2, SNIPER1H129Y, SNIPER1H129Y eds1-2, sniper1-1 sniper2-1 eds1-2 and sniper1-c1 sniper2-1 eds1-2. sniper1-1 is an exonic T-DNA insertional mutant. sniper1-c1 is a SNIPER1 knockout mutant containing 807bp deletion that was generated by CRISPR-Cas9 system. Scale bar = 1 cm.


Quantification of H . a . Noco2 sporulation in the indicated plants 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Two independent experiments were carried out with similar results .

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

Quantification of H.a. Noco2 sporulation in the indicated plants 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Two independent experiments were carried out with similar results.


Immunoblot of RPS4HA ( E ) , SNC1 ( F ) , RPS2HA ( G ) , and RPM1myc ( H ) in SNIPER1H129Y background . Equal loading is shown by Ponceau S staining of a non - specific band . The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD ( n = 3 ) . Molecular mass marker in kilo Daltons is indicated on the left . Three independent experiments were carried out with similar results .

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

Immunoblot of RPS4HA (E), SNC1 (F), RPS2HA (G), and RPM1myc (H) in SNIPER1H129Y background. Equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.