( 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 .

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(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 ) .

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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, 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 .

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(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 .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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

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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 .

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

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 .

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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 .

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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 .

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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 .

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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 .

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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.


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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 .

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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 .

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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.


Immunoprecipitation and biotinylation of SUMM2NB - FLAG ( A ) , RPP4NB - FLAG ( B ) , or CHS1NB - FLAG ( C ) by SNIPER1 H129Y - HATurboID in N . benthamiana . Two biological repeats were carried out with similar results .

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Immunoprecipitation and biotinylation of SUMM2NB-FLAG (A), RPP4NB-FLAG (B), or CHS1NB-FLAG (C) by SNIPER1 H129Y-HATurboID in N. benthamiana. Two biological repeats were carried out with similar results.


Interaction of SNIPER1 H129Y - CLuc and SUMM2NB - NLuc ( D ) , RPP4NB - NLuc ( E ) or CHS1NB - NLuc ( F ) as tested by split - luciferase complementary assay in N . benthamiana . Three biological repeats with four technical repeats each were carried out with similar results .

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Interaction of SNIPER1 H129Y-CLuc and SUMM2NB-NLuc (D), RPP4NB-NLuc (E) or CHS1NB-NLuc (F) as tested by split-luciferase complementary assay in N. benthamiana. Three biological repeats with four technical repeats each were carried out with similar results.


Ubiquitination of the NB domains from SUMM2 ( A ) , RPP4 ( B ) and CHS1 ( C ) . Immunoblot of bacterial lysates form E . coil strains expressing AtUBA1 - S , MBP - NB - HA , AtUBC8 - S , HIS - FLAG - UBQ10 and SNIPER1 - myc , or strains without one of these components . Two biological repeats were carried out with similar results .

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Ubiquitination of the NB domains from SUMM2 (A), RPP4 (B) and CHS1 (C). Immunoblot of bacterial lysates form E. coil strains expressing AtUBA1-S, MBP-NB-HA, AtUBC8-S, HIS-FLAG-UBQ10 and SNIPER1-myc, or strains without one of these components. Two biological repeats were carried out with similar results.


( a , b ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP ( a ) . The high and low 20 % were sorted ( a ) ,

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

(a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a),


( a , b ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP ( a ) . The high and low 20 % were sorted ( a ) , re - plated and treated with lysosomal protease inhibitors pepstatin and E - 64d for 1 h ; lysates were then immunoblotted for the indicated proteins ( b ) . ( c ) Densitometry of LC3 - II and p62 western blots ( normalized to actin and hour 0 , mean ± s . e . m . , n = 3 blots from 2 independent experiments , * P = 0 . 051 , * * P = 0 . 0091 ) .

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

(a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a), re-plated and treated with lysosomal protease inhibitors pepstatin and E-64d for 1 h; lysates were then immunoblotted for the indicated proteins (b). (c) Densitometry of LC3-II and p62 western blots (normalized to actin and hour 0, mean ± s.e.m., n = 3 blots from 2 independent experiments, *P = 0.051, **P = 0.0091).


( d ) ; autophagic LC3 puncta were assessed by quantitative

(d); autophagic LC3 puncta were assessed by quantitative microscopy


( d ) ; autophagic LC3 puncta were assessed by quantitative microscopy ( e ; punctate area per cell , mean ± s . e . m . , n = 50 fields , * P = 0 . 010 , * * P = 0 . 053 ) .

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

(d); autophagic LC3 puncta were assessed by quantitative microscopy (e; punctate area per cell, mean ± s.e.m., n = 50 fields, *P = 0.010, **P = 0.053).


f ) Electron micrographs of HeLa mCherry - GFP - LC3 cells sorted for autophagic flux as in a . Yellow arrows denote autophagosomes ; red arrows indicate autolysosomes . ( g ) Quantification of autophagosomes and autolysomes from electron micrographs ( mean ± s . e . m . , n = 50 fields ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

f) Electron micrographs of HeLa mCherry-GFP-LC3 cells sorted for autophagic flux as in a. Yellow arrows denote autophagosomes; red arrows indicate autolysosomes. (g) Quantification of autophagosomes and autolysomes from electron micrographs (mean ± s.e.m., n = 50 fields). Uncropped images of blots are shown in Supplementary Fig. 6.


a - c ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP . Cells were then re - plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points ( median ratio of mCherry / GFP fluorescence ± s . e . m . , n = 3 wells ) . Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies ( b ) . Representative flow cytometry histograms for cells at 0 h and 24 h after sorting ( c ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-c) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP. Cells were then re-plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points (median ratio of mCherry/GFP fluorescence ± s.e.m., n = 3 wells). Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies (b). Representative flow cytometry histograms for cells at 0 h and 24 h after sorting (c).


( d ) BJAB mCherry - GFP - LC3 cells were sorted for autophagic flux as in a ( top and bottom 20 % ) . Following treatment with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) , cell viability was determined by MTS assay ( a tetrazolium reduction - based metabolic measurement ) at 24 h ( percentage of untreated control , mean ± s . e . m . , n = 3 wells , * P = 2 . 3 × 10 − 4 , * * P = 0 . 0036 ) .

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

(d) BJAB mCherry-GFP-LC3 cells were sorted for autophagic flux as in a (top and bottom 20%). Following treatment with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1), cell viability was determined by MTS assay (a tetrazolium reduction-based metabolic measurement) at 24 h (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 2.3×10−4, **P = 0.0036).


( e ) Long - term growth of BJAB mCherry - GFP - LC3 cells sorted for autophagic flux followed by treatment with Fas ligand ( 4 ng ml − 1 ) or TRAIL ( 15 ng ml − 1 ) for 24 h . Cells were then re - plated and allowed to recover for 5 ( Fas ligand ) or 6 ( TRAIL ) days then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 012 ) .

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

(e) Long-term growth of BJAB mCherry-GFP-LC3 cells sorted for autophagic flux followed by treatment with Fas ligand (4 ng ml−1) or TRAIL (15 ng ml−1) for 24 h. Cells were then re-plated and allowed to recover for 5 (Fas ligand) or 6 (TRAIL) days then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.012).


( f ) BJAB mCherry - GFP - LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI ( mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) .

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

(f) BJAB mCherry-GFP-LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI (mean ± s.e.m., n = 3 wells, *P = 0.0046).


( g ) Cells sorted as in a , were re - plated and , starting at the indicated times following sorting , treated with Fas ligand ( 4 ng ml − 1 ) for 24 h ; cell viability was then determined by MTS ( percentage of untreated control , mean ± s . e . m . , n = 3 wells ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

O 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

(g) Cells sorted as in a, were re-plated and, starting at the indicated times following sorting, treated with Fas ligand (4 ng ml−1) for 24 h; cell viability was then determined by MTS (percentage of untreated control, mean ± s.e.m., n = 3 wells). Uncropped images of blots are shown in Supplementary Fig. 6.


( a ) BJAB and Jurkat cells were treated with vehicle or chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by Fas ligand ( 1 . 5 ng ml − 1 ) . Cell viability was determined by MTS 24 h later ( percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 0 . 0058 ) .

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

(a) BJAB and Jurkat cells were treated with vehicle or chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (1.5 ng ml−1). Cell viability was determined by MTS 24 h later (percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 0.0058).


( b ) Immunoblots of cells in a , c probed for the indicated antibodies . CQ ,

(b) Immunoblots of cells in a,c probed for the indicated antibodies. CQ, chloroquine


( c ) BJAB and Jurkat cells were treated with chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by treatment with Fas ligand ( 4 ng ml − 1 ) for 24 h ( same technical replicate as a ) . Cells were then re - plated at low density in growth media and allowed to recover for 5 days , and then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0024 ) .

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

(c) BJAB and Jurkat cells were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by treatment with Fas ligand (4 ng ml−1) for 24 h (same technical replicate as a). Cells were then re-plated at low density in growth media and allowed to recover for 5 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0024).


( d ) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs , followed by 3 days of puromycin selection . Selected cells were plated and treated with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) for 24 h and viability was assessed by MTS ( percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 , * P = 1 . 7 × 10 − 4 , * * P = 0 . 024 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs, followed by 3 days of puromycin selection. Selected cells were plated and treated with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1) for 24 h and viability was assessed by MTS (percentage of control (no ligand), mean ± s.e.m., n = 3, *P = 1.7×10−4, **P = 0.024).


( e ) Immunoblots for Atg5 , Atg7 and p62 confirm protein depletion and autophagy inhibition in d , f .

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

(e) Immunoblots for Atg5, Atg7 and p62 confirm protein depletion and autophagy inhibition in d,f.


( f ) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand ( 15 ng ml − 1 ) or TRAIL ( 12 . 5 ng ml − 1 ) for 24 h ( same technical replicate as d ) . Cells were then re - plated at low density and allowed to recover for 5 - 6 days , and then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0089 ) . Atg7 knockdown data were not included for long - term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(f) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand (15 ng ml−1) or TRAIL (12.5 ng ml−1) for 24 h (same technical replicate as d). Cells were then re-plated at low density and allowed to recover for 5-6 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0089). Atg7 knockdown data were not included for long-term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment. Uncropped images of blots are shown in Supplementary Fig. 6.


( a , b ) BJAB and Jurkat cells stably expressing mCherry - GFP - LC3 were flow sorted for high and low autophagic flux , treated with Fas ligand ( 1 . 5 ng ml − 1 ) for 24 h and viability was assessed by MTS ( a ; percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) . ( b ) Immunoblots following sorting of the samples in a .

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

(a,b) BJAB and Jurkat cells stably expressing mCherry-GFP-LC3 were flow sorted for high and low autophagic flux, treated with Fas ligand (1.5 ng ml−1) for 24 h and viability was assessed by MTS (a; percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0046). (b) Immunoblots following sorting of the samples in a.


( c , d ) The indicated cell lines were treated with chloroquine ( BJAB , CEM 20 μM ; SKW6 . 4 , 25 μM ; Jurkat , 10 μM ) for 16 h , followed by Fas ligand ( BJAB , 12 . 5 ng ml − 1 ; SKW6 . 4 , 50 ng ml − 1 ; Jurkat , 0 . 4 ng ml − 1 ; CEM , 40 ng ml − 1 ) . Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies ( c ) .

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

(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c).


( c , d ) The indicated cell lines were treated with chloroquine ( BJAB , CEM 20 μM ; SKW6 . 4 , 25 μM ; Jurkat , 10 μM ) for 16 h , followed by Fas ligand ( BJAB , 12 . 5 ng ml − 1 ; SKW6 . 4 , 50 ng ml − 1 ; Jurkat , 0 . 4 ng ml − 1 ; CEM , 40 ng ml − 1 ) . Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies ( c ) . Cell viability was assessed by MTS 24 h following Fas ligand treatment ( d ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 1 . 6 × 10 − 4 ) .

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 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

(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c). Cell viability was assessed by MTS 24 h following Fas ligand treatment (d; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 1.6×10−4).


( e , f ) BJAB and Jurkat cells were transduced with control , Atg5 , Atg7 or Vps34 shRNA lentiviruses , followed by three days of puromycin selection . Cells were then treated with Fas ligand ( 1 . 25 ng ml − 1 ) or TRAIL ( 1 . 25 ng ml − 1 ) for 24 h and viability was assessed by MTS ( e ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 1 . 4 × 10 − 6 , * * P = 2 . 3 × 10 − 5 , * * * P = 3 . 9 × 10 − 5 , § P = 9 . 2 × 10 − 6 , § § P = 3 . 4 × 10 − 6 , § § § P = 1 . 7 × 10 − 5 ) . Immunoblots demonstrate Atg5 , Atg7 and Vps34 knockdown , autophagy inhibition and altered Fap - 1 levels . The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other ; the Fap - 1 blot is not separated to demonstrate the lack of Fap - 1 protein in Type II Jurkat cells ( f ) . Fap - 1 blots ( b , c , f ) were run on separate gels owing to the quantity of protein required for detection ( see Methods ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(e,f) BJAB and Jurkat cells were transduced with control, Atg5, Atg7 or Vps34 shRNA lentiviruses, followed by three days of puromycin selection. Cells were then treated with Fas ligand (1.25 ng ml−1) or TRAIL (1.25 ng ml−1) for 24 h and viability was assessed by MTS (e; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 1.4×10−6, **P = 2.3×10−5, ***P = 3.9×10−5, §P = 9.2×10−6, §§P = 3.4×10−6, §§§P = 1.7×10−5). Immunoblots demonstrate Atg5, Atg7 and Vps34 knockdown, autophagy inhibition and altered Fap-1 levels. The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other; the Fap-1 blot is not separated to demonstrate the lack of Fap-1 protein in Type II Jurkat cells (f). Fap-1 blots (b,c,f) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.


( a , b ) BJAB and Jurkat cells expressing the indicated Fap - 1 wild - type ( WT ) and catalytically inactive ( ΔCD ) constructs were treated with chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by Fas ligand ( BJAB , 1 . 5 ng ml − 1 ; Jurkat 15 ng ml − 1 ) for 24 h . Cell viability was determined by MTS ( a ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 2 . 4 × 10 − 5 , * * * P = 0 . 0024 ) . Cell lysates were blotted and probed with the indicated antibodies ( b ) .

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

(a,b) BJAB and Jurkat cells expressing the indicated Fap-1 wild-type (WT) and catalytically inactive (ΔCD) constructs were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (BJAB, 1.5 ng ml−1; Jurkat 15 ng ml−1) for 24 h. Cell viability was determined by MTS (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 2.4×10−5, ***P = 0.0024). Cell lysates were blotted and probed with the indicated antibodies (b).


c - e ) BJAB cells expressing control or Fap - 1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand ( 12 . 5 ng ml − 1 ) or TRAIL ( 25 ng ml − 1 ) for 24 h ; cell viability was then determined by MTS ( c ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 4 . 8 × 10 − 6 , * * P = 8 . 1 × 10 − 5 , * * * P = 8 . 0 × 10 − 4 , § P = 0 . 013 , NS P > 0 . 05 ) .

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

c-e) BJAB cells expressing control or Fap-1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand (12.5 ng ml−1) or TRAIL (25 ng ml−1) for 24 h; cell viability was then determined by MTS (c; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 4.8×10−6, **P = 8.1×10−5, ***P = 8.0×10−4, §P = 0.013, NS P>0.05).


( d ) Immunoblots of lysates collected following chloroquine treatment . Long - term growth was assessed in cells from c by re - plating them at low density and allowing them to recover for 5 days , followed by viability assay ( percentage of untreated control , mean ± s . e . m . , n = 3 wells , * P = 0 . 043 ; e ) . Fap - 1 blots ( b , d ) were run on separate gels owing to the quantity of protein required for detection ( see Methods ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(d) Immunoblots of lysates collected following chloroquine treatment. Long-term growth was assessed in cells from c by re-plating them at low density and allowing them to recover for 5 days, followed by viability assay (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 0.043; e). Fap-1 blots (b,d) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.


a , b ) Cell viability was determined by MTS assay following Fas ligand ( 4 ng ml − 1 ) treatment in BJAB cells expressing control or p62 lentiviral shRNAs ( a ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 3 . 2 × 10 − 5 , * * P = 1 . 8 × 10 − 6 ) . Immunoblots confirm p62 depletion and Fap - 1 levels ( b ) .

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a,b) Cell viability was determined by MTS assay following Fas ligand (4 ng ml−1) treatment in BJAB cells expressing control or p62 lentiviral shRNAs (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 3.2×10−5, **P = 1.8×10−6). Immunoblots confirm p62 depletion and Fap-1 levels (b).


( c ) Co - immunoprecipitation of endogenous p62 and Fap - 1 in BJAB cells treated with 20 μM chloroquine for 16 h , followed by treatment with Fas ligand ( 50 ng ml − 1 ) for 2 h at 4 ° C .

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(c) Co-immunoprecipitation of endogenous p62 and Fap-1 in BJAB cells treated with 20 μM chloroquine for 16 h, followed by treatment with Fas ligand (50 ng ml−1) for 2 h at 4°C.


( A ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) the TAK1 inhibitor NG25 ( 2 μM ) , the IKKβ inhibitor BI605906 ( 5 μM ) or the IKKβ inhibitor PS1145 ( 10 μM ) . The cells were then co - stimulated for 30 min with 100 ng / ml LPS and 4 mM ATP . Cell lysates ( 10 μg protein ) were denatured in SDS , subjected to SDS - PAGE and immunoblotted with the antibodies indicated . ( B ) As in A , except that the cells were co - stimulated for 1 h with 100 ng / ml LPS and 5 μM nigericin . Similar results were obtained in three independent experiments in A and B .

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(A) WT BMDM were incubated for 1 h without (-) or with (+) the TAK1 inhibitor NG25 (2 μM), the IKKβ inhibitor BI605906 (5 μM) or the IKKβ inhibitor PS1145 (10 μM). The cells were then co-stimulated for 30 min with 100 ng/ml LPS and 4 mM ATP. Cell lysates (10 μg protein) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. (B) As in A, except that the cells were co-stimulated for 1 h with 100 ng/ml LPS and 5 μM nigericin. Similar results were obtained in three independent experiments in A and B.


( C , D ) As in A , B , except that primary human monocyte - derived macrophages were used instead of mouse BMDM . Similar results were obtained in two independent experiments .

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(C, D) As in A, B, except that primary human monocyte-derived macrophages were used instead of mouse BMDM. Similar results were obtained in two independent experiments.


( A , B ) The Mouse macrophage J774A . 1 cell line was transfected with control siRNA or siRNA against IKKβ ( A ) or IKKα ( B ) . 72 h post - transfection , the cells were co - stimulated for 60 min without ( - ) or with ( + ) 100 ng / ml LPS and / or 5 μM nigericin . Cell lysates ( 10 μg ) were denatured in SDS , subjected to SDS - PAGE and immunoblotted with the antibodies indicated . Similar results were obtained in three ( A ) or two ( B ) independent experiments .

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(A, B) The Mouse macrophage J774A.1 cell line was transfected with control siRNA or siRNA against IKKβ (A) or IKKα (B). 72 h post-transfection, the cells were co-stimulated for 60 min without (-) or with (+) 100 ng/ml LPS and/or 5 μM nigericin. Cell lysates (10 μg) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. Similar results were obtained in three (A) or two (B) independent experiments.


( C , D ) As in ( A , B ) except that BMDM from IKKβ - LysM - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT control mice were co - stimulated without ( - ) or with ( + ) 100 ng / ml LPS and / or 4 mM ATP ( C ) or with 100 ng / ml LPS and / or 5 μM nigericin ( D ) . Similar results were obtained in two independent experiments .

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(C, D) As in (A, B) except that BMDM from IKKβ-LysM-Cre (flox/flox) (IKKβ (fl/fl)) or WT control mice were co-stimulated without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (C) or with 100 ng/ml LPS and/or 5 μM nigericin (D). Similar results were obtained in two independent experiments.


( A , B ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) , the IKKβ inhibitors BI605906 ( 5 μM ) , TPCA - 1 ( 5 μM ) or PS1145 ( 10 μM ) , the TAK1 inhibitor NG25 ( 2 μM ) or the NLRP3 inhibitor MCC950 . The cells were then co - stimulated for 30 min without ( - ) or with ( + ) 100 ng / ml LPS and / or 4 mM ATP ( A ) and / or 5 μM nigericin ( B ) . The cell culture medium was removed and the cells lysed . Protein in the culture medium was precipitated ( see Methods ) , dissolved in SDS . and subjected to SDS - PAGE , along with cell lysates . After transfer to PVDF membranes , immunoblotting was performed with antibodies recognizing both full length ( FL ) and cleaved ( CL ) gasdermin D ( GSDMD ) , the p20 and p10 fragments of caspase - 1 and IL - 18 . Similar results were obtained in two independent experiments . ( C ) As in A , except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) and control WT cells were used . Similar results were obtained in three independent experiments .

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(A, B) WT BMDM were incubated for 1 h without (-) or with (+), the IKKβ inhibitors BI605906 (5 μM), TPCA-1 (5 μM) or PS1145 (10 μM), the TAK1 inhibitor NG25 (2 μM) or the NLRP3 inhibitor MCC950. The cells were then co-stimulated for 30 min without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (A) and/or 5 μM nigericin (B). The cell culture medium was removed and the cells lysed. Protein in the culture medium was precipitated (see Methods), dissolved in SDS. and subjected to SDS-PAGE, along with cell lysates. After transfer to PVDF membranes, immunoblotting was performed with antibodies recognizing both full length (FL) and cleaved (CL) gasdermin D (GSDMD), the p20 and p10 fragments of caspase-1 and IL-18. Similar results were obtained in two independent experiments. (C) As in A, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) and control WT cells were used. Similar results were obtained in three independent experiments.


( A ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) 5 μM BI605906 , then stimulated for 1 h with ( + ) 100 ng / ml LPS and / or 5 μM nigericin or left unstimulated . The cells were lysed in buffer containing 1 % ( v / v ) Triton - X - 100 , and the Triton - X - 100 - soluble and Triton - X - 100 - insoluble fractions were prepared as in Methods . The Triton - X - 100 - insoluble fraction was dissolved SDS subjected to SDS - PAGE and immunoblotted with the antibodies indicated . The nuclear protein Histone H3B ( H3B ) was used as a loading control in the Tritin X - 100 insoluble fraction . Similar results were obtained in three independent experiments . ( B ) As in A , except that the Triton - X - 100 - insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2 . 0 mM DSS .

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

(A) WT BMDM were incubated for 1 h without (-) or with (+) 5 μM BI605906, then stimulated for 1 h with (+) 100 ng/ml LPS and/or 5 μM nigericin or left unstimulated. The cells were lysed in buffer containing 1% (v/v) Triton-X-100, and the Triton-X-100-soluble and Triton-X-100-insoluble fractions were prepared as in Methods. The Triton-X-100-insoluble fraction was dissolved SDS subjected to SDS-PAGE and immunoblotted with the antibodies indicated. The nuclear protein Histone H3B (H3B) was used as a loading control in the Tritin X-100 insoluble fraction. Similar results were obtained in three independent experiments. (B) As in A, except that the Triton-X-100-insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2.0 mM DSS.


( C , As in A , B except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) mice ( IKKβ ( fl / fl ) ) were used . The U2 Small Nuclear RNA Auxiliary Factor 1 ( U2AF1 ) was used as a loading control . Similar results were obtained in two independent experiments .

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(C, As in A, B except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) mice (IKKβ (fl/fl)) were used. The U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) was used as a loading control. Similar results were obtained in two independent experiments.


( E , F ) As in A , B except that BMDM from caspase - 1 KO mice were used . Similar results were obtained in two independent experiments .

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(E, F) As in A, B except that BMDM from caspase-1 KO mice were used. Similar results were obtained in two independent experiments.


( A - H ) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA - 1 ( 5 μM ) ( C , G ) or BI605906 ( 5 μM ) ( D , H ) . The cells were then stimulated for 1 h with 100 ng / ml LPS ( E ) or 5 μM nigericin ( F , G , H ) , or co - stimulated with 100 ng / ml LPS and 5 μM nigericin ( B , C , D ) or left unstimulated ( control ) ( A ) . The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3 . A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity . ( A - H ) the panel images show the PLA signal ( red ) and DAPI staining for nuclei ( blue ) . Images were acquired by sequential laser scanning on a confocal microscope . Similar results were obtained in three independent experiments and representative images are shown . Scale bar = 50 μm .

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(A-H) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA-1 (5 μM) (C, G) or BI605906 (5 μM) (D, H). The cells were then stimulated for 1 h with 100 ng/ml LPS (E) or 5 μM nigericin (F, G, H), or co-stimulated with 100 ng/ml LPS and 5 μM nigericin (B, C, D) or left unstimulated (control) (A). The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3. A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity. (A-H) the panel images show the PLA signal (red) and DAPI staining for nuclei (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were obtained in three independent experiments and representative images are shown. Scale bar = 50 μm.


( I - L ) As in A - H , except that IKKβ inhibitors were omitted and iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS and 10 μM nigericin or left unstimulated ( control ) . Scale bar = 50 μm .

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(I-L) As in A-H, except that IKKβ inhibitors were omitted and iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl)) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated (control). Scale bar = 50 μm.


( A - H ) The experiments were performed as in Figs 5A - 5H , except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody , which was visualized using a secondary antibody ( red ) . Nuclei were counterstained with DAPI ( blue ) . Images were acquired by sequential laser scanning on a confocal microscope . Similar results were observed in many fields and obtained in three independent experiments . Representative images are shown Data information : In all panels , scale bar = 50 μm .

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

(A-H) The experiments were performed as in Figs 5A-5H, except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody, which was visualized using a secondary antibody (red). Nuclei were counterstained with DAPI (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were observed in many fields and obtained in three independent experiments. Representative images are shown Data information: In all panels, scale bar = 50 μm.


( I - L ) As in A - H , except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS and 10 μM nigericin or left unstimulated . Similar results were obtained in two independent experiments . Data information : In all panels , scale bar = 50 μm .

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

(I-L) As in A-H, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated. Similar results were obtained in two independent experiments. Data information: In all panels, scale bar = 50 μm.


( A ) Western blotting of Mcrs1 at the GV ( 0 h ) , GVBD ( 4 h ) , MI ( 8 h ) , and MII ( 12 h ) stages of mouse oocyte meiosis . The blots were probed with anti - Mcrs1 and anti - actin antibodies .

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(A) Western blotting of Mcrs1 at the GV (0 h), GVBD (4 h), MI (8 h), and MII (12 h) stages of mouse oocyte meiosis. The blots were probed with anti-Mcrs1 and anti-actin antibodies.


( G ) Percentages of control ( n = 210 ) , Mcrs1 - KD ( n = 182 ) , and Mcrs1 - rescue ( n = 201 ) oocytes that underwent GVBD and PBE . The bars are representing the mean ± SEM . The P - values were calculated using Student ' s t - test . GVBD % : * * * P = 0 . 0003 ; * P = 0 . 01 , PB1 % : * P = 0 . 03 ; * P = 0 . 02 .

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(G) Percentages of control (n = 210), Mcrs1-KD (n = 182), and Mcrs1-rescue (n = 201) oocytes that underwent GVBD and PBE. The bars are representing the mean ± SEM. The P-values were calculated using Student's t-test. GVBD%: ***P = 0.0003; *P = 0.01, PB1%: *P = 0.03; *P = 0.02.


( A ) Representative images of the occurrence of GVBD in control , Mcrs1 - KD , and Mcrs1 - rescue ( Mcrs1 siRNA + Mcrs1 mRNA ) oocytes at 3 h following release from IBMX . Scale bar , 100 μm . ( B ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control ( n = 155 ) , Mcrs1 - KD ( n = 157 ) , and Mcrs1 - rescue ( n = 150 ) oocytes .

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(A) Representative images of the occurrence of GVBD in control, Mcrs1-KD, and Mcrs1-rescue (Mcrs1 siRNA + Mcrs1 mRNA) oocytes at 3 h following release from IBMX. Scale bar, 100 μm. (B) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 155), Mcrs1-KD (n = 157), and Mcrs1-rescue (n = 150) oocytes.


( D ) Expression levels of CDK1 in Mcrs1 - KD and control oocytes . The bars represent the mean ( ± SEM ) of 5 biological replicates . The P - values were calculated using Student ' s t - test . * P = 0 . 01 . ( E ) Expression levels of p - CDK1 ( Y15 ) in Mcrs1 - KD and control oocytes at 1 h following release from IBMX ( 200 oocytes per sample ) . * * P = 0 . 006 . ( F ) Expression levels of cyclin B1 in control and Mcrs1 - KD oocytes at 1 h following release from IBMX ( 200 oocytes per sample ) . The bars represent the mean ( ± SEM ) of 3 biological replicates . The P - values were calculated using Student ' s t - test . * * P = 0 . 0042 .

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(D) Expression levels of CDK1 in Mcrs1-KD and control oocytes. The bars represent the mean (± SEM) of 5 biological replicates. The P-values were calculated using Student's t-test. *P = 0.01. (E) Expression levels of p-CDK1(Y15) in Mcrs1-KD and control oocytes at 1 h following release from IBMX (200 oocytes per sample). **P = 0.006. (F) Expression levels of cyclin B1 in control and Mcrs1-KD oocytes at 1 h following release from IBMX (200 oocytes per sample). The bars represent the mean (± SEM) of 3 biological replicates. The P-values were calculated using Student's t-test. **P = 0.0042.


( I ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control oocytes ( n = 112 ) , Mcrs1 - KD oocytes ( n = 98 ) , and Mcrs1 - KD oocytes treated with 5 mM WEE1 - 1N - 4 ( n = 102 ) . ( J ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control ( n = 123 ) , Mcrs1 - KD ( n = 134 ) , and Mcrs1 siRNA + cyclin B1 - GFP ( n = 95 ) oocytes . ( K ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control ( n = 92 ) , Mcrs1 - KD ( n = 97 ) , and Mcrs1 siRNA + cyclin B1 - GFP oocytes treated with 5 mM WEE1 - 1N - 4 ( n = 106 ) .

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(I) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control oocytes (n = 112), Mcrs1-KD oocytes (n = 98), and Mcrs1-KD oocytes treated with 5 mM WEE1-1N-4 (n = 102). (J) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 123), Mcrs1-KD (n = 134), and Mcrs1 siRNA + cyclin B1-GFP (n = 95) oocytes. (K) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 92), Mcrs1-KD (n = 97), and Mcrs1 siRNA + cyclin B1-GFP oocytes treated with 5 mM WEE1-1N-4 (n = 106).


( B ) Detection of newly synthesized RNAs by EU incorporation in control , Mcrs1 - KD , and Mcrs1 - rescue GV oocytes . The scattergram shows the relative fluorescence intensity of EU in control ( n = 43 ) , Mcrs1 - KD ( n = 52 ) , and Mcrs1 - rescue ( n = 30 ) GV oocytes . Scale bar , 10 μm . * * * P = 0 . 0001 .

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(B) Detection of newly synthesized RNAs by EU incorporation in control, Mcrs1-KD, and Mcrs1-rescue GV oocytes. The scattergram shows the relative fluorescence intensity of EU in control (n = 43), Mcrs1-KD (n = 52), and Mcrs1-rescue (n = 30) GV oocytes. Scale bar, 10 μm. ***P = 0.0001.


( C ) Immunofluorescence analysis of H3K4me2 in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of H3K4me2 signals in control ( n = 26 ) and Mcrs1 - KD ( n = 30 ) GV oocytes . * * * P = 0 . 0001 .

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

(C) Immunofluorescence analysis of H3K4me2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H3K4me2 signals in control (n = 26) and Mcrs1-KD (n = 30) GV oocytes. ***P = 0.0001.


( E ) Immunofluorescence analysis of H3K9me2 in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of H3K9me2 signals in control ( n = 29 ) and Mcrs1 - KD ( n = 22 ) GV oocytes . * * P = 0 . 0035 . ( F ) Immunofluorescence analysis of H4K16ac in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of H4K16ac signals in control ( n = 30 ) and Mcrs1 - KD ( n = 28 ) GV oocytes . * P = 0 . 022 .

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

(E) Immunofluorescence analysis of H3K9me2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H3K9me2 signals in control (n = 29) and Mcrs1-KD (n = 22) GV oocytes. **P = 0.0035. (F) Immunofluorescence analysis of H4K16ac in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H4K16ac signals in control (n = 30) and Mcrs1-KD (n = 28) GV oocytes. *P = 0.022.


( G ) Immunofluorescence analysis of HDAC2 in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of HDAC2 signals in control ( n = 27 ) and Mcrs1 - KD ( n = 24 ) GV oocytes . * * * P = 0 . 0005 .

(G) Immunofluorescence analysis of HDAC2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of HDAC2 signals in control (n = 27) and Mcrs1-KD (n = 24) GV oocytes. ***P = 0.0005.


( A ) Representative images of spindle assembly at 3 h post - release from IBMX in control and Mcrs1 - KD oocytes . Scale bar , 20 μm .

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

(A) Representative images of spindle assembly at 3 h post-release from IBMX in control and Mcrs1-KD oocytes. Scale bar, 20 μm.


( G ) Immunofluorescence analysis of H3S10ph and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The scattergram shows the relative fluorescence intensity of H3S10ph signals in control ( n = 21 ) and Mcrs1 - KD ( n = 23 ) oocytes . * P = 0 . 017 .

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

(G) Immunofluorescence analysis of H3S10ph and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of H3S10ph signals in control (n = 21) and Mcrs1-KD (n = 23) oocytes. *P = 0.017.