Cancer sphere showing reduced sphere - forming capacity of Panc1 hPaca1 in miR - 203 overexpression . Colonies with a diameter greater than 75 μM for Panc1 greater 30 μM for hPaca1 were counted as spheres . n = 3 , mean ± , Mann - Whitney U - .

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Cancer stem cell sphere assay showing reduced sphere-forming capacity of Panc1 and hPaca1 in miR-203 overexpression cells. Colonies with a diameter greater than 75 μM for Panc1 and greater 30 μM for hPaca1 cells were counted as spheres. n = 3, mean ± SEM, Mann-Whitney U-test.


B , C Histone marks were analyzed using ChIP coupled to qRT - PCR for Panc1 control versus shZEB , MDA - MB - 231 control versus shZEB ( B ) , BxPC3 control versus gemcitabine resistant ( gr ) ( C ) . In MDA - MB - 231 Panc1 , active histone marks H3K4me3 , H3ac , H4ac , H3K9ac were enriched . Vice versa , in drug - resistant clones of BxPC3 , active marks were reduced in CpG islands . repressive histone mark H3K27me3 was not detectable in miR - 200 loci , but in loci of miR - 203 E - cadherin in Panc1 MDA - MB - 231 . methylation status was determined by bisulfite sequencing . Depletion of ZEB1 in MDA - MB - 231 resulted in almost complete demethylation , whereas selection of drug - resistant , ZEB1 - expressing clones in BxPC3 induced complete methylation . n = 2 ( Panc1 ) or 3 ( MDA - MB - 231 BxPC3 ) , mean ± ; unpaired Student ' s t - .

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B, C Histone marks were analyzed using ChIP coupled to qRT-PCR for Panc1 control versus shZEB, MDA-MB-231 control versus shZEB (B), and BxPC3 control versus gemcitabine resistant (gr) (C). In MDA-MB-231 and Panc1, the active histone marks H3K4me3, H3ac, H4ac, and H3K9ac were enriched. Vice versa, in the drug-resistant clones of BxPC3, the active marks were reduced in the CpG islands. The repressive histone mark H3K27me3 was not detectable in the miR-200 loci, but in the loci of miR-203 and E-cadherin in Panc1 and MDA-MB-231. DNA methylation status was determined by bisulfite sequencing. Depletion of ZEB1 in MDA-MB-231 resulted in almost complete demethylation, whereas the selection of drug-resistant, ZEB1-expressing clones in BxPC3 induced complete methylation. n = 2 (Panc1) or 3 (MDA-MB-231 and BxPC3), mean ± SEM; unpaired Student's t-test.


Heat map showing relative expression after drug treatment for 48 h in Panc1 . Values measured by qRT - PCR were depicted with software GENE - E . Only mocetinostat upregulated miRNAs downregulated ZEB1 .

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Heat map showing the relative expression levels after drug treatment for 48 h in Panc1. Values measured by qRT-PCR were depicted with the software GENE-E. Only mocetinostat upregulated the miRNAs and downregulated ZEB1.


Relative expression of indicated genes in Panc1 measured by qRT - PCR after treatment with different HDAC inhibitors . Note downregulation of ZEB1 upregulation of miR - 203 , miR - 200 , E - cadherin by mocetinostat . n = 3 , mean ± ; unpaired Student ' s t - . For significance , see Supplementary Table S1 .

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Relative expression of indicated genes in Panc1 measured by qRT-PCR after treatment with different HDAC inhibitors. Note the downregulation of ZEB1 and upregulation of miR-203, miR-200, and E-cadherin by mocetinostat. n = 3, mean ± SEM; unpaired Student's t-test. For significance, see Supplementary Table S1.


Immunoblot immunofluorescence showing that mocetinostat treatment ( 1 μM , 48 h ) reduced ZEB1 expression induced E - cadherin in Panc1 . Expression of histone deacetylases was not altered by mocetinostat , but histone acetylation was induced . Scale bar 10 μm .

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Immunoblot and immunofluorescence showing that mocetinostat treatment (1 μM, 48 h) reduced ZEB1 expression and induced E-cadherin in Panc1. Expression of histone deacetylases was not altered by mocetinostat, but histone acetylation was induced. Scale bar 10 μm.


immunoprecipitation validated mocetinostat - induced ( 1 μM , 48 h ) enrichment of active histone marks H3ac , H4ac , H3K9ac , H3K4me3 at ZEB1 target gene loci in Panc1 . n = 3 , mean ± ; unpaired Student ' s t - .

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Chromatin immunoprecipitation analysis validated mocetinostat-induced (1 μM, 48 h) enrichment of the active histone marks H3ac, H4ac, H3K9ac, and H3K4me3 at ZEB1 target gene loci in Panc1. n = 3, mean ± SEM; unpaired Student's t-test.


Mocetinostat treatment reduced expression of anti - apoptotic miR - 203 target survivin sphere - forming capacity in Panc1 when pre - treated with mocetinostat for 48 h . n = 3 , mean ± ; Mann - Whitney U - .

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Mocetinostat treatment reduced expression of the anti-apoptotic miR-203 target survivin and sphere-forming capacity in Panc1 when pre-treated with mocetinostat for 48 h. n = 3, mean ± SEM; Mann-Whitney U-test.


Immunoblot immunofluorescence for caspase - 3 showing strong increase in apoptosis in gemcitabine - resistant Panc1 after combined treatment with mocetinostat ( 1 μM ) gemcitabine ( 50 nM ) . Scale bar 20 μm .

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Immunoblot and immunofluorescence for cleaved caspase-3 showing strong increase in apoptosis in gemcitabine-resistant Panc1 after combined treatment with mocetinostat (1 μM) and gemcitabine (50 nM). Scale bar 20 μm.


MTT for Panc1 treated with indicated concentrations of mocetinostat ( left ) or SAHA ( middle ) gemcitabine ( 72 h ) . Combined treatment of mocetinostat gemcitabine significantly reduced viability . In contrast , a combination with SAHA had no effect . For calculation of CI synergy between drugs , see Table2 . Comparison of mocetinostat SAHA alone ( right ) . n = 3 , mean ± , Dunnett ' s multiple comparisons ( P - values in graphs are * * * P < 0 . 001 * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

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MTT assay for Panc1 treated with the indicated concentrations of mocetinostat (left) or SAHA (middle) and gemcitabine (72 h). Combined treatment of mocetinostat and gemcitabine significantly reduced cell viability. In contrast, a combination with SAHA had no effect. For calculation of the CI and synergy between the drugs, see Table2. Comparison of mocetinostat and SAHA alone (right). n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are ***P < 0.001 and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


Effects of mocetinostat on caspase expression susceptibility to gemcitabine in - derived pancreatic cancer . Note that hPaca1 behaves similar to Panc1 , but mocetinostat had no significant effect in hPaca2 . Scale bar 20 μm . n = 3 , mean ± , Dunnett ' s multiple comparisons ( P - values in graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

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Effects of mocetinostat on cleaved caspase expression and susceptibility to gemcitabine in the patient-derived pancreatic cancer cells. Note that hPaca1 behaves similar to Panc1, but mocetinostat had no significant effect in hPaca2. Scale bar 20 μm. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


MTT assays comparing effects of docetaxel mocetinostat in prostate cancer DU - 145 docetaxel - resistant subclone DU - 145 DR ( left ) . Mocetinostat treatment of DU - 145 DR downregulates ZEB1 , upregulates E - cadherin , miR - 200 , miR - 203 expression . For relative miRNA expression , expression in original DU - 145 were set to 1 ( middle panels , immunoblot panel derives from same experiment shown in Fig1A ) . Mocetinostat sensitizes DU - 145 DR to docetaxel ( right ) . For calculation of CI synergy between drugs , see Table2 . n = 3 , mean ± , Dunnett ' s multiple comparisons ( P - values in graphs are * P = 0 . 01 - 0 . 05 ; for exact P - values , see Supplementary Table S4 ) .

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MTT assays comparing the effects of docetaxel and mocetinostat in the prostate cancer cell line DU-145 and the docetaxel-resistant subclone DU-145 DR (left). Mocetinostat treatment of DU-145 DR downregulates ZEB1, upregulates E-cadherin, miR-200, and miR-203 expression. For relative miRNA expression, the expression levels in original DU-145 were set to 1 (middle panels, the immunoblot panel derives from the same experiment shown in Fig1A). Mocetinostat sensitizes DU-145 DR to docetaxel (right). For calculation of the CI and synergy between the drugs, see Table2. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05; for exact P-values, see Supplementary Table S4).


Relative volume ( RTV ) of Panc1 - derived tumors in NMRI nu / nu mice . Eleven days after implantation , mice were randomized according to volume . Treatment with mocetinostat ( 60 or 90 mg / kg / day ) gemcitabine ( 25 mg / kg / day ) was implemented ( day 0 ) as depicted in scheme . Shown are group medians of RTVs over time ( left ) RTVs on day 32 ( right ) . n = 5 for each treatment group ; nonparametric Mann - Whitney U - .

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Relative tumor volume (RTV) of Panc1-derived tumors in NMRI nu/nu mice. Eleven days after implantation, mice were randomized according to tumor volume. Treatment with mocetinostat (60 or 90 mg/kg/day) and gemcitabine (25 mg/kg/day) was implemented (day 0) as depicted in the scheme. Shown are the group medians of the RTVs over time (left) and the individual RTVs on day 32 (right). n = 5 for each treatment group; nonparametric Mann-Whitney U-test.


Representative immunohistochemical stainings of serial sections showing reduced ZEB1 increased E - cadherin in tissues of mice treated with mocetinostat . Scale bar 40 μm , inserts for higher magnifications 10 μm . Squares indicate magnified regions .

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Representative immunohistochemical stainings of serial sections showing reduced ZEB1 and increased E-cadherin in tumor tissues of mice treated with mocetinostat. Scale bar 40 μm, inserts for higher magnifications 10 μm. Squares indicate magnified regions.


Representative pictures of in situ hybridization for miR - 203 control probe showing gain of miR - 203 associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat - treated xenograft tumors . Scale bar 40 μm , inserts for higher magnifications 5 μm . Squares indicate magnified regions .

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Representative pictures of in situ hybridization for miR-203 and control probe showing gain of miR-203 and associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat-treated xenograft tumors. Scale bar 40 μm, inserts for higher magnifications 5 μm. Squares indicate magnified regions.


Schematic outline results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice . Panc1 were pre - treated with mocetinostat ( 1 μM ) / or gemcitabine ( 50 nM ) for 48 h , followed by a 7 - day recovery period before being injected subcutaneously ( left ) . Equal numbers of viable were injected in 75 μl volume . At day 9 after injection , growth was detectable in groups ( lower right ) . To better visualize compare growth , volume at day 9 was set to 1 increasing slope of volume to day 37 is depicted ( upper right ) . absolute volumes on day 37 ( lower left ) group medians of absolute volumes over time ( lower right ) are shown . For pre - treated with combination of mocetinostat gemcitabine , growth was arrested . n = 4 for each treatment group ; nonparametric Mann - Whitney U - .

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Schematic outline and results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice. Panc1 cells were pre-treated with mocetinostat (1 μM) and/or gemcitabine (50 nM) for 48 h, followed by a 7-day recovery period before being injected subcutaneously (left). Equal numbers of viable cells were injected in 75 μl volume. At day 9 after injection, tumor growth was detectable in all groups (lower right). To better visualize and compare tumor growth, the tumor volume at day 9 was set to 1 and the increasing slope of the tumor volume to day 37 is depicted (upper right). The individual absolute tumor volumes on day 37 (lower left) and the group medians of the absolute tumor volumes over time (lower right) are shown. For cells pre-treated with the combination of mocetinostat and gemcitabine, tumor growth was arrested. n = 4 for each treatment group; nonparametric Mann-Whitney U-test.


A , B Relative expression of miR - 203 miR - 200c in pancreatic adenocarcinomas . ( A ) In normal versus tissue of same case ( n = 6 cases ) , ( B ) in non - recurrence ( n = 10 cases ) versus recurrence group ( n = 11 cases ) . mean value of lower group in each figure was set to 1 . Nonparametric Mann - Whitney U - .

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A, B Relative expression levels of miR-203 and miR-200c in pancreatic adenocarcinomas. (A) In normal versus tumor tissue of the same case (n = 6 cases), (B) in the non-recurrence (n = 10 cases) versus the recurrence group (n = 11 cases). The mean value of the lower group in each figure was set to 1. Nonparametric Mann-Whitney U-test.


B Na + K + contents Na + / K + in roots shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d .

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B The Na+ and K+ contents and Na+/K+ ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d.


C Linear regression of root Na + / K + under salt stress ( 150 mM NaCl for 1 d ) fruit in 195 accessions from three groups . R2 , coefficient of determination . n is accession number of three groups .

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C Linear regression of root Na+/K+ ratio under salt stress (150 mM NaCl for 1 d) and fruit weight in 195 accessions from three groups. R2, coefficient of determination. n is the accession number of the three groups.


D Na + / K + ratios in roots of TS - 21 , TS - 422 , TS - 577 , TS - 670 after treatment with 150 mM NaCl for indicated time ( n = 3 biological repeats ) .

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D The Na+/K+ ratios in roots of TS-21, TS-422, TS-577, and TS-670 after treatment with 150 mM NaCl for the indicated time (n = 3 biological repeats).


E , F Salt - resistance of two PIM accessions ( TS - 21 TS - 422 ) two BIG ( TS - 577 TS - 670 ) . 20 - day - old seedlings grown in soil were treated with 200 mM NaCl for 3 ( E ) . survival rates were obtained from at least 12 in three repeated experiments ( F ) .

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E, F Salt-resistance assay of two PIM accessions (TS-21 and TS-422) and two BIG (TS-577 and TS-670). 20-day-old seedlings grown in soil were treated with 200 mM NaCl for 3 weeks (E). The survival rates were obtained from at least 12 plants in three repeated experiments (F).


A Manhattan plot displaying GWAS results of Na + / K + in root . red - dashed indicates Bonferroni - adjusted significance threshold ( P = 1 . 0 × 10 - 7 ) . Red arrow indicates significant SNP signal of Na + / K + associated with SlHAK20 .

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A Manhattan plot displaying the GWAS results of Na+/K+ ratio in root. The red-dashed line indicates the Bonferroni-adjusted significance threshold (P = 1.0 × 10-7). Red arrow indicates the significant SNP signal of Na+/K+ ratio associated with SlHAK20.


B nucleotide diversity ratios between PIM CER , between CER BIG on chromosome 4 . black - dashed horizontal lines indicate top 10 % threshold for entire chromosome 4 ( 1 . 82 πPIM / πCER for domestication 4 . 27 πCER / πBIG for improvement ) . red arrows indicate position of SlHAK20 in sweeps . π , nucleotide diversity .

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B The nucleotide diversity ratios between PIM and CER, and between CER and BIG on chromosome 4. The black-dashed horizontal lines indicate top 10% threshold for entire chromosome 4 (1.82 πPIM/πCER for domestication and 4.27 πCER/πBIG for improvement). The red arrows indicate the position of SlHAK20 in the sweeps. π, nucleotide diversity.


C SlHAK20 - association mapping pairwise LD . Dots represent SNPs . Indel48 is highlighted in red . indel - 2381 , indel - 1818 in promoter region five nonsynonymous variants are marked in blue . These eight variants are related to pairwise LD diagram with dashed lines .

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C SlHAK20-based association mapping and pairwise LD analysis. Dots represent SNPs. Indel48 is highlighted in red. The indel-2381, indel-1818 in the promoter region and five nonsynonymous variants are marked in blue. These eight variants are related to the pairwise LD diagram with dashed lines.


D Haplotypes of SlHAK20 among tomato natural variations . distribution of Na + / K + in each haplotype group is exhibited by a box plot . n indicates number of accessions belonging to each haplotype . In box plots , middle indicates median , box indicates range of percentiles of total data using Turkey method , whiskers indicate interquartile range outer dots are outliers . Significant difference was determined by Student ' s t - .

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D Haplotypes of SlHAK20 among tomato natural variations. The distribution of Na+/K+ ratio in each haplotype group is exhibited by a box plot. The n indicates the number of accessions belonging to each haplotype. In the box plots, the middle line indicates the median, the box indicates the range of the percentiles of the total data using Turkey method, the whiskers indicate the interquartile range and the outer dots are outliers. Significant difference was determined by Student's t-test.


E Allele distribution of SlHAK20 locus at position in PIM , CER , BIG groups . n indicates accession number .

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E Allele distribution of the SlHAK20 locus at position in PIM, CER, and BIG groups. The n indicates the accession number.


expression pattern of SlHAK20 in tomato . GUS activity was examined in seedling ( left ) root ( right ) of 21 - days - old SlHAK20TS - 21pro : GUS transgenic tomato ( B ) . Bars , 2 cm .

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The expression pattern of SlHAK20 in tomato. The GUS activity was examined in seedling (left) and root (right) of 21-days-old SlHAK20TS-21pro:GUS transgenic tomato (B). Bars, 2 cm.


expression pattern of SlHAK20 in tomato . Cross - sections of hypocotyl ( C ) of SlHAK20TS - 21pro : GUS transgenic . scale bar is 400 μm in ( C )

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The expression pattern of SlHAK20 in tomato. Cross-sections of hypocotyl (C) of SlHAK20TS-21pro:GUS transgenic plants. The scale bar is 400 μm in (C)


expression pattern of SlHAK20 in tomato . Cross - sections of root he scale bar is 200 μm in ( D ) .

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The expression pattern of SlHAK20 in tomato. Cross-sections of root he scale bar is 200 μm in (D).


E Subcellular localization of SlHAK20 - YFP in tomato . SlHAK20Hap1 - YFP ( left ) SlHAK20Hap2 - YFP ( right ) were localized in plasma . YFP , yellow florescence . FM4 - 64 , a lipophilic styryl compound as a red marker of plasma . Bar , 20 μm .

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E Subcellular localization of SlHAK20-YFP in tomato. The SlHAK20Hap1-YFP (left) and SlHAK20Hap2-YFP (right) were localized in the plasma membrane. YFP, yellow florescence protein. FM4-64, a lipophilic styryl compound as a red fluorescent marker of plasma membrane. Bar, 20 μm.


F SlHAK20 complements K + uptake - defective yeast mutant assayed using a dose - response method . K + uptake - defective yeast mutant R5421 transformed with SlHAK20Hap1 , SlHAK20Hap2 , vector ( p416 - GPD ) were cultured in AP medium containing indicated concentrations of K + for 24 h . data points represent three replicates .

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F SlHAK20 complements the K+ uptake-defective yeast mutant assayed using a dose-response method. The K+ uptake-defective yeast mutant R5421 transformed with SlHAK20Hap1, SlHAK20Hap2, and vector (p416-GPD) were cultured in AP medium containing indicated concentrations of K+ for 24 h. The data points represent three replicates.


G Na + uptake of yeast expressing SlHAK20 . Shown are Na + concentrations in medium over time of culture of ANT3 yeast transformed with SlHAK20Hap1 , SlHAK20Hap2 , or empty vector . initial NaCl in medium was 60 μM . data are shown as means ± SD ( n = 4 ) .

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G Na+ uptake analysis of yeast cells expressing SlHAK20. Shown are the Na+ concentrations in the medium over the time of culture of the ANT3 yeast cells transformed with SlHAK20Hap1, SlHAK20Hap2, or empty vector. The initial NaCl concentration in the medium was 60 μM. The data are shown as the means ± SD (n = 4).


H Na + uptake kinetic of SlHAK20 in yeast . curves show - dependent Na + inﬂux in yeast expressing SlHAK20Hap1 or SlHAK20Hap2 in presence of 0 , 5 , 10 , 20 , 30 , 40 , 50 , 60 , 100 , 150 or 200 μM NaCl in medium . data are expressed as mean ± SD ( n = 3 ) .

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H Na+ uptake kinetic analysis of SlHAK20 in yeast cells. The curves show concentration- dependent Na+ inﬂux in yeast expressing SlHAK20Hap1 or SlHAK20Hap2 in the presence of 0, 5, 10, 20, 30, 40, 50, 60, 100, 150 or 200 μM NaCl in the medium. The data are expressed as mean ± SD (n = 3).


Na + / K + contents in roots of TS - 21 wild type slhak20 mutants . Data are shown as means ± SD ( n = 4 ) .

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Na+/K+ ratio contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).


Na + contents in roots of TS - 21 wild type slhak20 mutants . Data are shown as means ± SD ( n = 4 ) .

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Na+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).


K + contents in roots of TS - 21 wild type slhak20 mutants . Data are shown as means ± SD ( n = 4 ) .

K+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).


Na + contents in xylem sap of TS - 21 slhak20 mutants after 50 mM NaCl treatment for indicated days . Data represent means ± SD ( n = 4 ) .

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Na+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4).


K + contents in xylem sap of TS - 21 slhak20 mutants after 50 mM NaCl treatment for indicated days . Data represent means ± SD ( n = 4 ) .

K+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4).


G Shoot height of TS - 21 , slhak20 - 1 , slhak20 - 2 . height of 24 - day - old TS - 21 , slhak20 - 1 , slhak20 - 2 were measured after growing under normal conditions for 14 days , followed by salt treatment for 14 days , recovery for 10 days . Values are means ± SD ( n = 10 of each genotype ) .

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G Shoot height of TS-21, slhak20-1, and slhak20-2. Plant height of 24-day-old TS-21, slhak20-1, and slhak20-2 were measured after growing under normal conditions for 14 days, followed by salt treatment for 14 days, and recovery for 10 days. Values are means ± SD (n = 10 plants of each genotype).


H Salt resistance of TS - 21 wild type slhak20 mutants . TS - 21 , slhak20 - 1 , slhak20 - 2 grown under normal growth conditions for 24 days , followed by a treatment with 200 mM NaCl for 14 days , then recovered for 10 days . Control grown under normal growth conditions for 38 days . Scale bars , 5 cm .

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H Salt resistance of TS-21 wild type and slhak20 mutants. TS-21, slhak20-1, and slhak20-2 grown under normal growth conditions for 24 days, followed by a treatment with 200 mM NaCl for 14 days, and then recovered for 10 days. Control plants grown under normal growth conditions for 38 days. Scale bars, 5 cm.


Salt - tolerance of TS - 670 slhak20 mutant . TS - 670 , slhak20 - 3 , slhak20 - 4 grown under normal growth conditions for 23 days , then treated with 150 mM NaCl for 5 days . Control grown under normal growth conditions for 28 days .

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Salt-tolerance assay of TS-670 and slhak20 mutant plants. TS-670, slhak20-3, and slhak20-4 grown under normal growth conditions for 23 days, and then treated with 150 mM NaCl for 5 days. Control plants grown under normal growth conditions for 28 days.


height fresh of TS - 670 , slhak20 - 3 , slhak20 - 4 under normal salt stress conditions . Shoot height f 23 - day - old TS - 670 , slhak20 - 3 , slhak20 - 4 were quantified after growing under normal conditions for 5 days , or salt treatment with 150 mM NaCl for 5 days . Values are means ± SD ( n = 10 of each genotype ) .

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Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. Shoot height f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype).


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Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress


height fresh of TS - 670 , slhak20 - 3 , slhak20 - 4 under normal salt stress conditions . resh f 23 - day - old TS - 670 , slhak20 - 3 , slhak20 - 4 were quantified after growing under normal conditions for 5 days , or salt treatment with 150 mM NaCl for 5 days . Values are means ± SD ( n = 10 of each genotype ) .

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Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. resh weight f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype).


Phenotype of NIP oshak4 / 17 double mutant under normal salt stress conditions . 20 - day - old seedlings of NIP oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . pictures show representative ere analyzed among these genotypes . ontrol were grown under normal growth conditions for 28 days

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Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative plants ere analyzed among these genotypes. ontrol plants were grown under normal growth conditions for 28 days


Phenotype of NIP oshak4 / 17 double mutant under normal salt stress conditions . 20 - day - old seedlings of NIP oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . pictures show representative ere analyzed among these genotypes . ontrol were grown under normal growth conditions for 28

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Phenotype of NIP oshak4 / 17 double mutant under normal salt stress conditions . 20 - day - old seedlings of NIP oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . pictures show representative fresh of shoots were analyzed among these genotypes . Control were grown under normal growth conditions for 28 days fresh weights of shoots were measured . Values are means ± SD ( n = 12 of each genotype for fresh

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Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative fresh weight of shoots were analyzed among these genotypes. Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured. Values are means ± SD (n = 12 plants of each genotype for fresh weight


Phenotype of NIP oshak4 / 17 double mutant under normal salt stress conditions . 20 - day - old seedlings of NIP oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . pictures show representative survival rate were analyzed among these genotypes .

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Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative survival rate were analyzed among these genotypes.


A transcript of SlHAK20 in TS - 670 three independent SlHAK20Hap1 - YFP transgenic lines grown under normal growth conditions .

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A The transcript levels of SlHAK20 in TS-670 and three independent SlHAK20Hap1-YFP transgenic lines grown under normal growth conditions.


B of SlHAK20Hap1 - YFP in TS - 670 transgenic . ACTIN was used as loading control .

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B The protein levels of SlHAK20Hap1-YFP in TS-670 and transgenic plants. ACTIN was used as loading control.


of transgenic lines in soil under salt stress . survival rates were obtained from at least 8 in four repeated

Phenotypes of transgenic lines in soil under salt stress. The survival rates were obtained from at least 8 plants in four repeated experiments


38 - day - old grown in soil as control . 20 - day - old TS - 670 Hap1OE - 1 to - 3 were treated with 175 mM NaCl for 3 recovered for 2

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38-day-old plants grown in soil as control. 20-day-old TS-670 and Hap1OE-1 to -3 were treated with 175 mM NaCl for 3 weeks and recovered for 2 weeks


Na + in shoots roots of TS - 670 three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 Hap1OE - 3 grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 7 days .

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The Na+ in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.


K + in shoots roots of TS - 670 three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 Hap1OE - 3 grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 7 days .

The K+ in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.


Na + / K + in shoots roots of TS - 670 three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 Hap1OE - 3 grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 7 days .

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The Na+/K+ ratio in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.


( C ) Immunohistochemistry for C . burnetii in lung on d7 after i . t . infection . Representative quantitation of lung tissue positive for C . burnetii ( n = 12 - 13 mice , pooled from three experiments , each normalized to mean of WT or Acod1 + / - infected mice ) . Each dot represents one mouse .

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(C) Immunohistochemistry for C. burnetii in the lung on d7 after i.t. infection. Representative images and quantitation of lung tissue positive for C. burnetii (n=12-13 mice, pooled from three experiments, each normalized to the mean of WT or Acod1+/- infected mice). Each dot represents one mouse.


( E ) Immunohistochemistry for C . burnetii on day 7 after intraperitoneal infection . Each dot represents one mouse .

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(E) Immunohistochemistry for C. burnetii on day 7 after intraperitoneal infection. Each dot represents one mouse.


representative of liver sections of immunohistochemistry for C . burnetii in liver sections ( G ) . n = 6 - 11 mice ( for NMII infections ) , pooled from two experiments . Each dot represents one mouse . ( Mann - Whitney comparing treatment control group of NMII infected Acod1 - / - mice in specified organs , *

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representative images of liver sections and quantification of immunohistochemistry for C. burnetii in liver sections (G). n=6-11 mice (for NMII infections), pooled from two experiments. Each dot represents one mouse. (Mann-Whitney test comparing treatment and control group of NMII infected Acod1-/- mice in specified organs, *


( B ) BAX / BAK - deficient HeLa reconstituted with BAX where indicated HA - Parkin were treated with antimycin A oligomycin ( AO ) for 2 h then 1 µM of each of BH3 mimetics ABT - 737 S63485 for 6 h . was assessed at indicated times by LDH . Data is mean + / - SD of four independent experiments .

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(B) BAX/BAK-deficient HeLa cells reconstituted with BAX and where indicated HA-Parkin were treated with antimycin A and oligomycin (AO) for 2 h and then 1 µM of each of the BH3 mimetics ABT-737 and S63485 for 6 h. Cell death was assessed at the indicated times by LDH assay. Data is mean +/- SD of four independent experiments.


( C ) BAX / BAK - deficient HeLa ectopically expressing BAX Parkin were treated with antimycin A oligomycin ( AO , 3 h ) , MG132 ( 20 µM , 3 . 5 h ) BH3 mimetics ( 1 µM of each of ABT - 737 S63485 , 1 h ) . Cytosol fractions were immunoblotted as indicated . Graph shows densitometric of non - ubiquitinated ( top , relative to untreated control ) or cytochrome c release ( bottom ; cytosol / cytosol + , relative to untreated controls ) from three independent experiments . Error bars represent mean + / - SD .

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(C) BAX/BAK-deficient HeLa cells ectopically expressing BAX and Parkin were treated with antimycin A and oligomycin (AO, 3 h), MG132 (20 µM, 3.5 h) and BH3 mimetics (1 µM of each of ABT-737 and S63485, 1 h). Cytosol and membrane fractions were immunoblotted as indicated. Graph shows densitometric analysis of non-ubiquitinated membrane proteins (top, relative to untreated control) or cytochrome c release (bottom; cytosol/cytosol+membrane, relative to untreated controls) from three independent experiments. Error bars represent mean +/- SD.


( D ) BAX / BAK - deficient HeLa ectopically expressing BAX were treated

(D) BAX/BAK-deficient HeLa cells ectopically expressing BAX were treated and analysed


( E ) UBA pull - down of ubiquitinated following antimycin A oligomycin ( AO ) treatment of BAX / BAK - deficient HeLa ectopically expressing HA - Parkin BAX for 3 h . Representative immunoblot from two independent experiments .

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(E) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment of BAX/BAK-deficient HeLa cells ectopically expressing HA-Parkin and BAX for 3 h. Representative immunoblot from two independent experiments.


( A ) Subcellular fractionation of cytosol fractions of HeLa expressing HA - Parkin following 2 h of antimycin A oligomycin ( AO ) treatment .

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(A) Subcellular fractionation of cytosol and membrane fractions of HeLa cells expressing HA-Parkin following 2 h of antimycin A and oligomycin (AO) treatment.


( B ) Immunoblotting of whole lysates immunoblotted following treatment with antimycin A oligomycin ( AO ) for 3 h with MG132 ( 20 µM ) . Graph shows densitometric of non - ubiquitinated relative to untreated control from three independent experiments . Error bars represent mean + / - SD .

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(B) Immunoblotting of whole cell lysates immunoblotted following treatment with antimycin A and oligomycin (AO) for 3 h with MG132 (20 µM). Graph shows densitometric analysis of non-ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean +/- SD.


( C ) UBA pull - down of ubiquitinated following antimycin A oligomycin ( AO ) treatment for 3 h treatment with non - specific DUB USP2 for 30 min at 37 ° C , representative immunoblot from two independent experiments .

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(C) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment for 3 h and treatment with the non-specific DUB USP2 for 30 min at 37°C, representative immunoblot from two independent experiments.


( E ) Whole lysates of HA - Parkin BAK variant - expressing HeLa following 3 h antimycin A oligomycin ( AO ) treatment with 30 min pre - treatment with MG132 ( 20 µM ) , representative immunoblots from three independent experiments .

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(E) Whole cell lysates of HA-Parkin and BAK variant-expressing HeLa cells following 3 h antimycin A and oligomycin (AO) treatment with 30 min pre-treatment with MG132 (20 µM), representative immunoblots from three independent experiments.


( A ) HeLaParkin were treated with CCCP ( 10 μM ) , antimycin A oligomycin ( AO ) for 2h or GTPP ( 10 μM ) for 6h . Immunoblots are representative of three independent experiments . Graph shows densitometric of non - ubiquitinated relative to untreated control from three independent experiments . Error bars represent mean + / -

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(A) HeLaParkin cells were treated with CCCP (10 μM), antimycin A and oligomycin (AO) for 2h or GTPP (10 μM) for 6h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non-ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean +/-


( B ) SH - SY5Y neuroblastoma expressing Cas9 together with a sgRNA targeting PRKN ( sgPRKN ) or a non - targeting control ( sgCon ) were assessed for Parkin expression by immunoblotting of fractions .

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(B) SH-SY5Y neuroblastoma cells expressing Cas9 together with a sgRNA targeting PRKN (sgPRKN) or a non-targeting control (sgCon) were assessed for Parkin expression by immunoblotting of cytosolic fractions.


SH - SY5Y generated in ( B ) were treated with antimycin A oligomycin ( AO ) for 3 h prior to lysis immunoblotting . Mono di - ubiquitinated BAK in Parkin - expressing following AO are indicated ( asterisk ) . Data is representative of three independent experiments .

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SH-SY5Y cells generated in (B) were treated with antimycin A and oligomycin (AO) for 3 h prior to cell lysis and immunoblotting. Mono and di-ubiquitinated BAK in Parkin-expressing cells following AO are indicated (asterisk). Data is representative of three independent experiments.


( D ) Domain architecture of human Parkin with positions of selected PD - associated mutations . Ubl , ubiquitin - like ; IBR , in - between - RING HeLa expressing wild - type Parkin or indicated PD - associated Parkin mutants were treated with antimycin A oligomycin ( AO ) for 2h . Immunoblots are representative of three independent experiments . Graph shows densitometric of non - ubiquitinated in AO - treated samples relative to untreated control from three independent experiments . Error bars represent mean + / - SD . Parkin constructs were N - terminally FLAG - tagged except R104W .

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(D) Domain architecture of human Parkin with positions of selected PD-associated mutations. Ubl, ubiquitin-like; IBR, in-between-RING HeLa cells expressing wild-type Parkin or the indicated PD-associated Parkin mutants were treated with antimycin A and oligomycin (AO) for 2h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non-ubiquitinated proteins in AO-treated samples relative to untreated control from three independent experiments. Error bars represent mean +/- SD. All Parkin constructs were N-terminally FLAG-tagged except R104W.


( A ) BN - PAGE SDS - PAGE of HeLa + HA - Parkin treated with 1 µM of each of ABT - 737 S63485 for 3 h ( BH3 ) , antimycin A oligomycin ( AO ) for 2 h or a combination of two . Experiment was performed in presence of 10 µM QVD . oph immunoblots are representative of three independent experiments .

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(A) BN-PAGE and SDS-PAGE of HeLa+HA-Parkin cells treated with 1 µM of each of ABT-737 and S63485 for 3 h (BH3), antimycin A and oligomycin (AO) for 2 h or a combination of the two. Experiment was performed in the presence of 10 µM QVD.oph and immunoblots are representative of three independent experiments.


( B ) UBA enrichment of ubiquitinated from HeLa + HA - Parkin , BAX - / - BAK - / - ( DKO ) HeLa or wild - type ( WT ) HeLa expressing HA - Parkin in response to 1 µM of each of ABT - 737 S63485 for 3 h ( BH3 ) in presence of QVD . oph ( 10 μM ) . Representative of three independent experiments .

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(B) UBA enrichment of ubiquitinated proteins from HeLa+HA-Parkin cells, BAX-/- BAK-/- (DKO) HeLa cells or wild-type (WT) HeLa cells expressing HA-Parkin in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of QVD.oph (10 μM). Representative of three independent experiments.


( C ) HeLa expressing HA - Parkin Cas9 were transduced with sgRNA targeting PINK1 or a non - targeting sgRNA control , treated with AO ( 2 h ) to stabilise PINK1 expression immunoblotted for PINK1 .

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(C) HeLa cells expressing HA-Parkin and Cas9 were transduced with sgRNA targeting PINK1 or a non-targeting sgRNA control, treated with AO (2 h) to stabilise PINK1 expression and immunoblotted for PINK1.


( D ) Parkin activity induced by apoptotic mitochondrial damage is PINK1 - dependent . UBA enrichment of ubiquitinated from HeLa generated in response to 1 µM of each of ABT - 737 S63485 for 3 h ( BH3 ) in presence of Q - VD . oph ( 10 μM ) . * , ubiquitinated . Representative of two independent experiments .

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(D) Parkin activity induced by apoptotic mitochondrial damage is PINK1-dependent. UBA enrichment of ubiquitinated proteins from HeLa cells generated in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of Q-VD.oph (10 μM). *, ubiquitinated protein. Representative of two independent experiments.


( B ) Non - reducing SDS - PAGE immunoblot of recombinant BAK BAK - Ub following treatment with 25 mM

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(B) Non-reducing SDS-PAGE and immunoblot of recombinant BAK and BAK-Ub following treatment with 25 mM TCEP


( C ) Timecourse monitoring increase on liposomes following addition of 4 µM BID - BH3 peptide to 50 nM BAK or BAK - Ub in presence or absence of 25 mM TCEP . Data is mean + / - SD of three independent experiments .

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(C) Timecourse monitoring fluorescence increase on liposomes following addition of 4 µM BID-BH3 peptide to 50 nM BAK or BAK-Ub in the presence or absence of 25 mM TCEP. Data is mean +/- SD of three independent experiments.


( D ) BN - PAGE of BAK BAK - Ub on liposomes following incubation with 4 µM BID - BH3 peptide in presence or absence of 25 mM TCEP , immunoblotted for BAK . Data representative of three independent experiments . Graphs shows densitometric of BAK tetramer ( 4x ) from three or two independent experiments . Error bars represent mean + / - SD for n = 3 or mean range for n = 2 .

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(D) BN-PAGE of BAK and BAK-Ub on liposomes following incubation with 4 µM BID-BH3 peptide in the presence or absence of 25 mM TCEP, immunoblotted for BAK. Data representative of three independent experiments. Graphs shows densitometric analysis of the BAK tetramer (4x) from three or two independent experiments. Error bars represent mean +/- SD for n=3 or mean and range for n=2.


( A ) Immunoblot of whole lysates of MEFs stably expressing ΔCys variants of BAK .

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(A) Immunoblot of whole cell lysates of MEFs stably expressing ΔCys variants of BAK.


( B ) Conjugation of ubiquitin to BAK K113C impairs BAK apoptotic function . Mitochondrially - enriched membranes from MEFs expressing BAK or BAK K113C were cross - linked ( CuPhe ) with UbG76C treated with cBID for 30 min at 30 ° C . Supernatant ( S ) pellet ( P ) fractions were then immunoblotted for BAK under non - reducing conditions , or cytochrome c or ubiquitin under reducing conditions . Representative of three independent experiments . Note that incorporation of recombinant UbG76C on higher molecular is likely due to incomplete reduction on reducing SDS - PAGE .

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(B) Conjugation of ubiquitin to BAK K113C impairs BAK apoptotic function. Mitochondrially-enriched membranes from MEFs expressing BAK or BAK K113C were cross-linked (CuPhe) with UbG76C and treated with cBID for 30 min at 30°C. Supernatant (S) and pellet (P) fractions were then immunoblotted for BAK under non-reducing conditions, or cytochrome c or ubiquitin under reducing conditions. Representative of three independent experiments. Note that the incorporation of recombinant UbG76C on higher molecular weight proteins is likely due to incomplete reduction on reducing SDS-PAGE.


( C ) Parkin - mediated ubiquitination limits BAK activation . HeLa + HA - Parkin were treated with antimycin A oligomycin ( AO ) for 2 h prior to induction of apoptosis with 1 µM of each of BH3 mimetics ABT - 737 S63485 ( BH3 mim ) for 1 h . Intracellular flow cytometry was performed using conformation - specific anti - BAK antibody G317 - 2 . Representative histograms are shown fold increase in mean ( MFI ) is plotted from three independent experiments with error bars showing SD . Data are normalised to untreated control .

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(C) Parkin-mediated ubiquitination limits BAK activation. HeLa+HA-Parkin cells were treated with antimycin A and oligomycin (AO) for 2 h prior to induction of apoptosis with 1 µM of each of the BH3 mimetics ABT-737 and S63485 (BH3 mim) for 1 h. Intracellular flow cytometry was performed using the conformation-specific anti-BAK antibody G317-2. Representative histograms are shown and the fold increase in mean fluorescence intensity (MFI) is plotted from three independent experiments with error bars showing SD. Data are normalised to untreated control.


( D ) Parkin - mediated ubiquitination limits BAK apoptotic activity . BAK - / - BAX - / - HeLa expressing Parkin BAK were treated with AO ( 2 h ) prior to isolation of mitochondria treatment with recombinant cBID of cytochrome c release . Immunoblots are representative of three independent experiments . Graph shows densitometric of three independent experiments or cytochrome c in supernatant fraction . Error bars represent mean + / - SD .

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(D) Parkin-mediated ubiquitination limits BAK apoptotic activity. BAK-/-BAX-/- HeLa cells expressing Parkin and BAK were treated with AO (2 h) prior to isolation of mitochondria and treatment with recombinant cBID and analysis of cytochrome c release. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of three independent experiments or cytochrome c in the supernatant fraction. Error bars represent mean +/- SD.


( E ) Parkin activity limits cytochrome c release . HeLa or HeLa expressing Parkin were treated analysed Immunoblots are representative of three independent experiments . Ubiquitinated BAK indicated ( arrow ) . Graph shows densitometric of three independent experiments showing cytochrome c release ( supernatant / supernatant + ) relative to 100 nM cBID as 100 % release . Error bars represent mean + / - SD .

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(E) Parkin activity limits cytochrome c release. HeLa cells or HeLa cells expressing Parkin were treated and analysed Immunoblots are representative of three independent experiments. Ubiquitinated BAK indicated (arrow). Graph shows densitometric analysis of three independent experiments showing cytochrome c release (supernatant/supernatant+membrane) relative to 100 nM cBID as 100% release. Error bars represent mean +/- SD.


B - C . Graphs showing relative ( B ; n = 15 WT , 12 ECko ; curve comparison ) survival ( C ; n = 15 WT , 11 ECko ; Log - Rank ) of mice upon tamoxifen treatment . Data information : Data shown as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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B-C. Graphs showing the relative weight (B; n= 15 WT, 12 ECko; curve comparison) and the survival (C; n= 15 WT, 11 ECko; Log-Rank test) of mice upon tamoxifen treatment. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


D . Representative of intestine of Casp8WT Casp8ECko mice , with mild severe hemorrhages at a late disease stage .

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D. Representative images of the intestine of Casp8WT and Casp8ECko mice, with mild and severe hemorrhages at a late disease stage.


E - F . Representative of H & E ( E ) of intestinal pathology ( F ) of small intestine at an early disease stage in Casp8WT Casp8ECko mice ( enteropathy score ; n = 6 WT , 6 ECko ; two unpaired Student ' s t - ) . Scale bars : 100µm ; insets 50µm . Data information : Data shown as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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E-F. Representative images of H&E staining (E) and quantification of intestinal pathology (F) of the small intestine at an early disease stage in Casp8WT and Casp8ECko mice (enteropathy score; n= 6 WT, 6 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm; insets 50µm. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


G - H . of intestinal pathology ( enteropathy score , G ; n = 8 WT , 7 ECko ; two unpaired Student ' s t - ) hemorrhages into mucosa ( H ; n = 4 WT , 5 ECko ; two unpaired Student ' s t - ) at a late disease stage in Casp8WT Casp8ECko mice . Data information : Data shown as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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G-H. Quantification of intestinal pathology (enteropathy score, G; n= 8 WT, 7 ECko; two tailed unpaired Student's t-test) and hemorrhages into the mucosa (H; n= 4 WT, 5 ECko; two tailed unpaired Student's t-test) at a late disease stage in Casp8WT and Casp8ECko mice. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


I . Overview of H & E stained swiss roll preparations of Casp8WT Casp8ECko mice . Scale bars : 2mm .

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I. Overview of H&E stained swiss roll preparations of Casp8WT and Casp8ECko mice. Scale bars: 2mm.


J . Representative of H & E of small intestine of Casp8WT ( j ' ) Casp8ECko mice with mild ( j ' ' ) more severely ( j ' ' ' ) affected areas at a late disease stage ( arrows point to hemorrhages , asterisk points to focal erosion of epithelium ) . Scale bars : 100µm .

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J. Representative images of H&E staining of the small intestine of Casp8WT (j') and Casp8ECko mice with mild (j'') and more severely (j''') affected areas at a late disease stage (white arrows point to hemorrhages, asterisk points to focal erosion of the epithelium). Scale bars: 100µm.


A - B . QPCR of genes associated to immune response ( A ) , vessel permeability EC activation ( B ) in small intestine lysates at a late disease timepoint in Casp8WT Casp8ECko mice ( n = 4 WT , 3 - 4 ECko , biological replicates , multiple t - with Holm - Sidak method ) . Data information : Data shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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A-B. QPCR analysis of genes associated to immune and bacterial response (A), vessel permeability and EC activation (B) in small intestine lysates at a late disease timepoint in Casp8WT and Casp8ECko mice (n= 4 WT, 3-4 ECko, biological replicates, multiple t-test with Holm-Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.


Graphs revealing that significantly upregulated cytokines in proteome profiler in Casp8ECko mice are associated to complement cascade ( C ) ( n = 4 WT , 4 ECko , multiple t - with Holm - Sidak method ) . Data information : Data shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the complement cascade (C) (n= 4 WT, 4 ECko, multiple t-test with Holm- Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.


Graphs revealing that significantly upregulated cytokines in proteome profiler in Casp8ECko mice are associated to mucosal healing ( D ) ( n = 4 WT , 4 ECko , multiple t - with Holm - Sidak method ) . Data information : Data shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the mucosal healing (D) (n= 4 WT, 4 ECko, multiple t-test with Holm- Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.


Representative ( E ; between dotted lines shows crypts ) of Ki67 in small intestine at an early disease stage in Casp8WT Casp8ECko mice ( n = 5 WT , 6 ECko Scale bars : 100µm .

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Representative images (E; area between dotted lines shows crypts) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice (n= 5 WT, 6 ECko Scale bars: 100µm.


( F ) of Ki67 in small intestine at an early disease stage in Casp8WT Casp8ECko mice ( n = 5 WT , 6 ECko two unpaired Student ' s t - ) . Data information : Data shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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quantification (F) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice (n= 5 WT, 6 ECko two tailed unpaired Student's t-test). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.


G . QPCR of proliferation markers Ki67 c - myc in ileal lysates from late disease stage Casp8WT Casp8ECko mice ( n = 4 WT , 3 - 4ECko , two - way ANOVA with Sidak method ) . Data information : Data shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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G. QPCR analysis of the proliferation markers Ki67 and c-myc in ileal lysates from late disease stage Casp8WT and Casp8ECko mice (n= 4 WT, 3-4ECko, two-way ANOVA with Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.


H . Representative of E - Cadherin , showing intact epithelial barrier integrity in Casp8ECko mice in healthy areas ( h ' ) , disrupted barrier integrity in intestinal lesions ( h ' ' ) at late disease stages compared to Casp8WT mice ( n = 4 WT , 4 ECko ) . Scale bars : 100µm .

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H. Representative images of E-Cadherin staining, showing intact epithelial barrier integrity in Casp8ECko mice in healthy areas (h'), and disrupted barrier integrity in intestinal lesions (h'') at late disease stages compared to Casp8WT mice (n= 4 WT, 4 ECko). Scale bars: 100µm.


I . Representative pictures of CD45 NIMPR14 at late disease stages ( n = 5 WT , 5 ECko ) , showing accumulation of CD45 + NIMPR14 + ( black arrowheads ) in Casp8ECko intestine at sites of impaired epithelial barrier integrity tissue necrosis ( red asterisks ) . Scale bars : 100µm .

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I. Representative pictures of CD45 and NIMPR14 staining at late disease stages (n= 5 WT, 5 ECko), showing accumulation of CD45+ and NIMPR14+ cells (black arrowheads) in Casp8ECko intestine at sites of impaired epithelial barrier integrity and tissue necrosis (red asterisks). Scale bars: 100µm.


J . Representative of CD4 , F4 / 80 MPO stainings in small intestines at a late disease stage showing accumulation of these at crypt base in Casp8ECko mice ( arrowheads ) ( n = 4 WT , 4 ECko ) . Scale bars : 50µm .

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J. Representative images of CD4, F4/80 and MPO stainings in small intestines at a late disease stage showing accumulation of these cells at the crypt base in Casp8ECko mice (arrowheads) (n= 4 WT, 4 ECko). Scale bars: 50µm.


A . Representative pictures showing Peyer ´ s Patches in Casp8WT mice , different severities of hemorrhage formation in Casp8ECko mice at an early disease stage . Scale bars : 1mm .

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A. Representative pictures showing Peyer´s Patches in Casp8WT mice, and different severities of hemorrhage formation in Casp8ECko mice at an early disease stage. Scale bars: 1mm.


of lymphatic vessel density ( B ) in Peyer ' s Patches in Casp8WT Casp8ECko mice at an early disease stage ( n = 9 WT , 7 - 10 ECko Peyer ' s Patches are pooled from 4 - 6 mice per genotype ; one - way ANOVA with tukey ' s multiple comparison ) . Data information : Data shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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Quantification of lymphatic vessel density (B) in Peyer's Patches in Casp8WT and Casp8ECko mice at an early disease stage (n = 9 WT, 7-10 ECko Peyer's Patches are pooled from 4-6 mice per genotype; one-way ANOVA with tukey's multiple comparison). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


representative pictures of CD31 ( pan EC ) Lyve1 ( lymphatic ) ( C ) in Peyer ' s Patches in Casp8WT Casp8ECko mice at an early disease stage ( n = 9 WT , 7 - 10 ECko Peyer ' s Patches are pooled from 4 - 6 mice per genotype Scale bars : 200µm .

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representative pictures of CD31 (pan EC) and Lyve1 (lymphatic) staining (C) in Peyer's Patches in Casp8WT and Casp8ECko mice at an early disease stage (n = 9 WT, 7-10 ECko Peyer's Patches are pooled from 4-6 mice per genotype Scale bars: 200µm.


D - F . Representative ( D ) quantifications ( E , F ) of stainings for CD31 , Lyve1 DAPI ( nuclei ) in small intestinal wholemounts at an early disease stage ( blue arrowheads point to defective lacteals , arrowheads indicate edema ) in Casp8ECko mice ( E ; n = 7 WT , 6 ECko . F ; n = 7 WT , 10 ECko ; two unpaired Student ' s t - ) . Scale bars : 100µm . Data information : Data shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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D-F. Representative images (D) and quantifications (E, F) of stainings for CD31, Lyve1 and DAPI (nuclei) in small intestinal wholemounts at an early disease stage (blue arrowheads point to defective lacteals, white arrowheads indicate edema) in Casp8ECko mice (E; n= 7 WT, 6 ECko. F; n= 7 WT, 10 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm. Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


G . Representative of small intestine sections of mice injected with a 70kDa fluorescently labeled dextran stained for CD31 at an early disease stage . arrowheads point to extravasated dextran in Casp8ECko mice ( n = 5 WT , 5 ECko ) . Scale bars : 50µm . H . of extravasated dextran in small intestine brain lysates in Casp8WT Casp8ECko mice at an early disease stage ( n = 4 WT , 4 - 5 ECko ; two - way ANOVA with tukey ' s multiple comparison ) . RFU : relative units . Data information : Data shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of small intestine sections of mice injected with a 70kDa fluorescently labeled dextran and stained for CD31 at an early disease stage. White arrowheads point to extravasated dextran in Casp8ECko mice (n= 5 WT, 5 ECko). Scale bars: 50µm. H. Quantification of extravasated dextran in small intestine and brain lysates in Casp8WT and Casp8ECko mice at an early disease stage (n= 4 WT, 4-5 ECko; two-way ANOVA with tukey's multiple comparison). RFU: relative fluorescent units. Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


I . Representative of CD31 VE - Cadherin in intestinal wholemounts at an early disease stage ( n = 5 WT , 5 ECko ) in Casp8WT Casp8ECko mice ( red arrowheads point to VE - Cadherin ' empty ' vessel patches ) . Scale bars : 25µm .

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I. Representative images of CD31 and VE-Cadherin staining in intestinal wholemounts at an early disease stage (n= 5 WT, 5 ECko) in Casp8WT and Casp8ECko mice (red arrowheads point to VE-Cadherin 'empty' vessel patches). Scale bars: 25µm.


A - B . Representative ( A ) ( B ) of CD31 , TUNEL DAPI in small intestine sections of Casp8WT Casp8ECko mice at an early disease stage ( red dotted lines : epithelium ; dotted lines : endothelium ) in ( n = 5 WT , 5 ECko , multiple t - with Holm - Sidak method ) . Scale bars : 100µm . Data information : data is shown as mean ± . ns : not significant .

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A-B. Representative images (A) and quantification (B) of CD31, TUNEL and DAPI staining in small intestine sections of Casp8WT and Casp8ECko mice at an early disease stage (red dotted lines: epithelium; white dotted lines: endothelium) in (n= 5 WT, 5 ECko, multiple t-tests with Holm-Sidak method). Scale bars: 100µm. Data information: All data is shown as mean ± SEM. ns: not significant.


D - E . Graphs showing survival relative of Casp8WT / MLKLko Casp8ECko / MLKLko mice upon tamoxifen treatment . ( D ; n = 5 - 8 WT / MLKLko , 5 - 8 ECko / MLKLko ; Log - Rank , E ; n = 7 WT , 8 ECko ; two unpaired Student ' s t - ) .

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D-E. Graphs showing survival and relative weight of Casp8WT/MLKLko and Casp8ECko/MLKLko mice upon tamoxifen treatment. (D; n= 5-8 WT/ MLKLko, 5-8 ECko/MLKLko; Log-Rank test, E; n= 7 WT, 8 ECko; two tailed unpaired Student's t-test).


F . Representative pictures of intestines of Casp8WT / MLKLko Casp8ECko / MLKLko mice at 30 days after tamoxifen treatment ( n = 8 WT , 8 ECko ) .

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F. Representative pictures of intestines of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 8 WT, 8 ECko).


G . Representative of stainings for CD31 , Lyve1 DAPI in small intestinal wholemounts from Casp8WT / MLKLko Casp8ECko / MLKLko mice at 30 days after tamoxifen treatment . Scale bars : 100µm .

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G. Representative images of stainings for CD31, Lyve1 and DAPI in small intestinal wholemounts from Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment. Scale bars: 100µm.


H - K . of vessel ( H , I ) lacteal ( J , K ) at two different timepoints after tamoxifen treatment in Casp8WT / MLKLko Casp8ECko / MLKLko mice ( n = 4 - 6 WT / MLKLko , 5 ECko / MLKLko ; two unpaired Student ' s t - ) . Data information : data is shown as mean ± . ns : not significant .

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H-K. Quantification of vessel area (H, I) and lacteal length (J, K) at two different timepoints after tamoxifen treatment in Casp8WT/MLKLko and Casp8ECko/MLKLko mice (n= 4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.


L . Graph showing of extravasated 70kDa Dextran from small intestine tissue of Casp8WT / MLKLko Casp8ECko / MLKLko mice at 30 days after tamoxifen treatment ( n = 7 WT / MLKLko , 6 ECko / MLKLko ; two unpaired Student ' s t - ) . Data information : data is shown as mean ± . ns : not significant .

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L. Graph showing quantification of extravasated 70kDa Dextran from small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 7 WT/MLKLko, 6 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.


M - N . Representative pictures ( M ) ( N ) of lymphatic vessel density in Peyer ' s Patches of Casp8WT / MLKLko Casp8ECko / MLKLko mice at 140 days after tamoxifen treatment ( n = 8 WT , 7 ECko Peyer ' s Patches are pooled from 4 mice per genotype ; two - unpaired students t - ) . Scale bars : 200µm . Data information : data is shown as mean ± . ns : not significant .

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M-N. Representative pictures (M) and quantification (N) of lymphatic vessel density in Peyer's Patches of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 140 days after tamoxifen treatment (n = 8 WT, 7 ECko Peyer's Patches are pooled from 4 mice per genotype; two-tailed unpaired students t-test). Scale bars: 200µm. Data information: All data is shown as mean ± SEM. ns: not significant.


O . Representative pictures of H & E stainings of small intestine tissue of Casp8WT / MLKLko Casp8ECko / MLKLko mice ( n = 4 - 6 WT , 4 - 5 ECko ) . Scale bars : 100µm . P - Q . of intestinal pathology in Casp8WT / MLKLko Casp8ECko / MLKLko mice at two different timepoints after tamoxifen treatment ( enteropathy score ; n = 4 - 6 WT / MLKLko , 5 ECko / MLKLko ; two unpaired Student ' s t - ) . Data information : data is shown as mean ± . ns : not significant .

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O. Representative pictures of H&E stainings of small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice (n= 4-6 WT, 4-5 ECko). Scale bars: 100µm. P-Q. Quantification of intestinal pathology in Casp8WT/MLKLko and Casp8ECko/MLKLko mice at two different timepoints after tamoxifen treatment (enteropathy score; n=4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.


A . Representative pictures of TUNEL together with CD31 Lyve - 1 in Peyer ' s Patches of Casp8WT Casp8ECko mice . TUNEL + LECs , but not BECs could already be seen in non - hemorrhagic Peyer ' s Patches ( a ' ) , further accumulated in hemorrhagic Peyer ' s Patches ( a ' ' ) . dotted areas outline location of lymphatic plexus . arrowheads point to TUNEL + LECs ( note that necroptotic LECs only weakly express Lyve - 1 ) . Scale bars : 50µm , insets : 10µm .

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A. Representative pictures of TUNEL staining together with CD31 and Lyve-1 in Peyer's Patches of Casp8WT and Casp8ECko mice. TUNEL+ LECs, but not BECs could already be seen in non-hemorrhagic Peyer's Patches (a'), and further accumulated in hemorrhagic Peyer's Patches (a''). White dotted areas outline location of the lymphatic plexus. White arrowheads point to TUNEL+ LECs (note that necroptotic LECs only weakly express Lyve-1). Scale bars: 50µm, insets: 10µm.


C . Survival ( n = 5 WT , 6 BECko ; Log - Rank ) of Casp8WT Casp8ECko mice upon tamoxifen treatment .

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C. Survival (n= 5 WT, 6 BECko; Log-Rank test) of Casp8WT and Casp8ECko mice upon tamoxifen treatment.


D . Representative pictures of intestines of Casp8WT Casp8BECko mice .

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D. Representative pictures of intestines of Casp8WT and Casp8BECko mice.


Representative pictures of H & E of small intestine of Casp8WT Casp8BECko mice ( E ) , enteropathy score ( F ; n = 5 WT , 6 ECko , two unpaired Student ' s t - ) Scale bars : 100µm , insets : 50µm . Data information : Data shown as mean ± . ns : not significant .

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Representative pictures of H&E staining of small intestine of Casp8WT and Casp8BECko mice (E), enteropathy score (F; n= 5 WT, 6 ECko, two tailed unpaired Student's t-test) Scale bars: 100µm, insets: 50µm. Data information: Data shown as mean ± SEM. ns: not significant.


mucosal hemorrhages ( G ; n = 5 WT , 6 ECko , two unpaired Student ' s t - ) . Data information : Data shown as mean ± . ns : not significant .

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mucosal hemorrhages (G; n= 5 WT, 6 ECko, two tailed unpaired Student's t-test). Data information: Data shown as mean ± SEM. ns: not significant.


Representative of stainings for CD31 , Lyve1 DAPI in small intestinal wholemounts ( H ) of vessel density ( I ) in Casp8BECko compared to Casp8WT mice ( I ; n = 5 WT , 6 BECko ; two unpaired Student ' s t - , Scale bars : 100µm . Data information : Data shown as mean ± . ns : not significant .

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Representative images of stainings for CD31, Lyve1 and DAPI in small intestinal wholemounts (H) and quantification of vessel density (I) in Casp8BECko compared to Casp8WT mice (I; n= 5 WT, 6 BECko; two tailed unpaired Student's t-test, Scale bars: 100µm. Data information: Data shown as mean ± SEM. ns: not significant.


of lacteal ( J ) in Casp8BECko compared to Casp8WT mice ; n = 5 WT , 5 BECko ; two unpaired Student ' s t - with Welch ' s correction ) . Data information : Data shown as mean ± . ns : not significant .

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quantification of lacteal length (J) in Casp8BECko compared to Casp8WT mice ; n= 5 WT, 5 BECko; two tailed unpaired Student's t-test with Welch's correction). Data information: Data shown as mean ± SEM. ns: not significant.


K - L . Representative pictures ( K ) ( L ) of lymphatic vessel density Peyer ' s Patches in Casp8WT Casp8BECko mice ( n = 10 WT , 11 BECko Peyer ' s Patches from 5 - 6 mice per genotype ; two unpaired Student ' s t - ) . Scale bars : 200µm . Data information : Data shown as mean ± . ns : not significant .

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K-L. Representative pictures (K) and quantification (L) of lymphatic vessel density Peyer's Patches in Casp8WT and Casp8BECko mice (n = 10 WT, 11 BECko Peyer's Patches from 5-6 mice per genotype; two tailed unpaired Student's t-test). Scale bars: 200µm. Data information: Data shown as mean ± SEM. ns: not significant.


B . Survival graph after intravenous TNF - α injection of Casp8WT vs . Casp8ECko mice within first 24h ( n = 11 WT , 11 ECko ; Log - Rank ) .

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B. Survival graph after intravenous TNF-α injection of Casp8WT vs. Casp8ECko mice within the first 24h (n=11 WT, 11 ECko; Log-Rank test).


C . Representative of small intestines 24h after TNF - α injection in Casp8WT Casp8ECko mice ( blue arrowheads point to Peyer ' s Patches ) .

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C. Representative images of the small intestines 24h after TNF-α injection in Casp8WT and Casp8ECko mice (blue arrowheads point to Peyer's Patches).


D - E . Representative pictures of Peyer ' s Patches ( D ) of lymphatic vessel density ( E ) in Casp8WT , Casp8ECko Casp8ECko / MLKLko mice injected with TNF - α ( n = 7 WT , 5 ECko , 15 ECko / MLKLko . Peyer ' s Patches are pooled from 3 - 5 mice per genotype ) . Scale bars : 200µm . Data information : data is shown as mean ± . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; ns : not significant .

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D-E. Representative pictures of Peyer's Patches (D) and quantification of lymphatic vessel density (E) in Casp8WT, Casp8ECko and Casp8ECko/MLKLko mice injected with TNF-α (n=7 WT, 5 ECko, 15 ECko/MLKLko. Peyer's Patches are pooled from 3-5 mice per genotype). Scale bars: 200µm. Data information: All data is shown as mean ± SEM. * P<0.05, *** P <0.001, **** P <0.0001; ns: not significant.


F . Representative pictures of TUNEL together with CD31 Lyve - 1 in Peyer ' s Patches of Casp8WT , Casp8ECko Casp8ECko / MLKLko mice injected with TNF - α . TUNEL + LECs , but not BECs could already be seen in Casp8ECko , but neither in Casp8WT nor in Casp8ECko / MLKLko mice . dotted areas outline location of lymphatic plexus . Yellow arrowheads point to TUNEL + ECs ) . Scale bars : 50µm .

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F. Representative pictures of TUNEL staining together with CD31 and Lyve-1 in Peyer's Patches of Casp8WT, Casp8ECko and Casp8ECko/MLKLko mice injected with TNF-α. TUNEL+ LECs, but not BECs could already be seen in Casp8ECko, but neither in Casp8WT nor in Casp8ECko/MLKLko mice. White dotted areas outline location of the lymphatic plexus. Yellow arrowheads point to TUNEL+ECs). Scale bars: 50µm.


H . Survival graph of Casp8WT Casp8ECko mice after Enbrel ® treatment ( n = 7 WT , 5 ECko + IgG , 8 ECko + Enbrel ; Log - Rank ) .

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H. Survival graph of Casp8WT and Casp8ECko mice after Enbrel® treatment (n=7 WT, 5 ECko + IgG, 8 ECko + Enbrel; Log-Rank test).


Representative of small intestines ( I ) of Casp8ECko mice after Enbrel ® treatment Scale bars : 100µm .

Representative images of small intestines (I) of Casp8ECko mice after Enbrel® treatment Scale bars: 100µm.


Representative of H & E ( J ) with histopathological scoring ( K ) of Casp8WT Casp8ECko mice after Enbrel ® treatment ( n = 6 WT , 4 ECko + IgG ; 8 ECko + Enbrel ( from which 3 mice were diseased , 5 mice healthy ) ; one - way ANOVA with tukey ' s multiple comparison ) . Scale bars : 100µm . Data information : data is shown as mean ± . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; ns : not significant .

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Representative images of H&E staining (J) with histopathological scoring (K) of Casp8WT and Casp8ECko mice after Enbrel® treatment (n=6 WT, 4 ECko + IgG; 8 ECko + Enbrel (from which 3 mice were diseased, 5 mice healthy); one-way ANOVA with tukey's multiple comparison). Scale bars: 100µm. Data information: All data is shown as mean ± SEM. * P<0.05, *** P <0.001, **** P <0.0001; ns: not significant.


of most prominent phyla ( B ; n = 4 Control , 6 - 8 ; multiple t - with Holm - Sidak method ) by qPCR , In B , cycle number of 16S rDNA expression compared to total number of cycles is shown . Data information : Data shown as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; ns : not significant . Scale bars : 100μm .

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analysis of the most prominent bacterial phyla (B; n= 4 Control, 6-8 Antibiotics; multiple t-tests with Holm-Sidak method) by qPCR analysis, In B, the cycle number of 16S rDNA expression compared to the total number of cycles is shown. Data information: Data shown as mean ± SEM. ** P <0.01, *** P <0.001, **** P <0.0001; ns: not significant. Scale bars: 100μm.


Graphs showing relative ( C ; n = 11 WT , 7 ECko , 10 ECko + one - way ANOVA with tukey ' s multiple comparison ) upon tamoxifen treatment in presence of an antibiotic cocktail .

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Graphs showing the relative weight (C; n= 11 WT, 7 ECko, 10 ECko+ antibiotics one-way ANOVA with tukey's multiple comparison) upon tamoxifen treatment in the presence of an antibiotic cocktail.


Graphs showing survival of mice ( D ; n = 6 WT , 7 ECko , 7 ECko + ; Log - Rank ) upon tamoxifen treatment in presence of an antibiotic cocktail .

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Graphs showing the survival of mice (D; n= 6 WT, 7 ECko, 7 ECko+ antibiotics; Log-Rank test) upon tamoxifen treatment in the presence of an antibiotic cocktail.


E . Representative of gut of Casp8WT Casp8ECko mice with antibiotic treatment 30 days after tamoxifen .

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E. Representative images of the gut of Casp8WT and Casp8ECko mice with antibiotic treatment 30 days after tamoxifen.


F - H . Representative of H & E ( F ) , of intestinal pathology ( G ) mucosal hemorrhages ( H ) in Casp8WT Casp8ECko mice treated with compared to untreated Casp8ECko mice ( G ; n = 6 WT , 4 ECko + , 5 ECko ; one - way ANOVA with tukey ' s multiple comparison , H ; n = 6 WT , 4 ECko + , 4 ECko ; one - way ANOVA with tukey ' s multiple comparison ) . Data information : Data shown as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; ns : not significant . Scale bars : 100μm .

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F-H. Representative images of H&E staining (F), quantification of intestinal pathology (G) and mucosal hemorrhages (H) in Casp8WT and Casp8ECko mice treated with antibiotics compared to untreated Casp8ECko mice (G; n= 6 WT, 4 ECko+ antibiotics, 5 ECko; one-way ANOVA with tukey's multiple comparison, H; n= 6 WT, 4 ECko+ antibiotics, 4 ECko; one-way ANOVA with tukey's multiple comparison). Data information: Data shown as mean ± SEM. ** P <0.01, *** P <0.001, **** P <0.0001; ns: not significant. Scale bars: 100μm.


Polygon distribution of biological tissues compared with Voronoi diagrams . A ) Segmentation of a Drosophila prepupa wing imaginal disc epithelium ( dWP ) .

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Polygon distribution of biological tissues compared with Voronoi diagrams. A) Segmentation of a Drosophila prepupa wing imaginal disc epithelium (dWP).


B ) Adult Biceps ( BCA ) biopsy stained with anti - collagen VI ( green ) anti - Myosin Heavy Chain ( red ) antibodies . C ) Segmentation of image showed in ( B ) .

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B) Adult Biceps (BCA) biopsy stained with anti-collagen VI (green) and anti-Myosin Heavy Chain (red) antibodies. C) Segmentation of the image showed in (B).


D ) Segmented image from a chicken neural tube epithelium ( cNT ) .

D) Segmented image from a chicken embryo neural tube epithelium (cNT).


E ) Polygon distribution of different biological tissues analysed . Two types of distribution can be distinguished : Poisson Voronoi polygon distribution for Diagram 1 ( D1 ) cNT , conserved polygon distribution for of image categories .

E) Polygon distribution of the different biological tissues analysed. Two types of distribution can be distinguished: The Poisson Voronoi polygon distribution for Diagram 1 (D1) and cNT, and the conserved polygon distribution for the rest of image categories.


CVT path matches polygon distribution of natural tissues . A ) Polygon distribution Voronoi diagrams resulting from iterative application of Lloyd ´ s algorithm .

The CVT path matches the polygon distribution of natural tissues. A) Polygon distribution Voronoi diagrams resulting from the iterative application of Lloyd´s algorithm.


B - E ) Density plot showing frequency of a value for four , five , seven eight sided respectively , depending of of hexagons ( P6 ) in CVT diagrams . darker region reflects higher probability of a determine value . Coloured circles represent average values of each polygon class for different sets of analysed in this study . dWL , Drosophila larva wing disc , green . dMWP , Drosophila mutant wing disc , violet . EYE , Drosophila eye disc , orange . BCA , biceps control adult , green . BNA , biceps neurogenic atrophy , blue .

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B-E) Density plot showing the frequency of a value for four, five, seven and eight sided cells respectively, depending of the percentage of hexagons (P6) in the CVT diagrams. The darker region reflects the higher probability of a determine value. Coloured circles represent the average values of each polygon class for the different sets of images analysed in this study. dWL, Drosophila larva wing disc, light green. dMWP, Drosophila mutant wing disc, violet. EYE, Drosophila eye disc, orange. BCA, biceps control adult, dark green. BNA, biceps neurogenic atrophy, dark blue.


Relationship between polygon distribution . A ) distribution of several Voronoi diagrams along CVT path . areas became progressively more homogeneous after each Lloyd

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Relationship between area and polygon distribution. A) Area distribution of several Voronoi diagrams along the CVT path. Cell areas became progressively more homogeneous after each Lloyd iteration


B ) Comparison of distribution for proliferating epithelia cNT ( blue ) , dWL ( green ) dWP ( red ) . At right of image Biceps ( green ) Diagram 5 ( grey ) .

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B) Comparison of area distribution for proliferating epithelia cNT (blue), dWL (green) and dWP (red). At the right of the image Biceps (green) and Diagram 5 (grey) images.


A ) Segmentation of a prepupal eye imaginal disc from Drosophila ( anterior to left ) . analysed developing photoreceptors are labelled in green . adjacent to photoreceptors are labelled in blue ( these are " cone " in most posterior clusters ) .

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A) Segmentation of a prepupal eye imaginal disc from Drosophila (anterior to the left). The analysed developing photoreceptors are labelled in green. The cells adjacent to the photoreceptors are labelled in blue (these are "cone cells" in the most posterior clusters).


B ) Segmentation of a prepupa wing imaginal disc epithelium where myosin II have been

B) Segmentation of a prepupa wing imaginal disc epithelium where myosin II levels have been reduced


. C ) Adult Biceps biopsy from a with neurogenic atrophy pathology . Fibres outlined with collagen VI antibody ( green ) . D ) Segmentation of image showed in ( C ) highlighting two atrophic elongated fibres that present seven neighbours .

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. C) Adult Biceps biopsy from a patient with neurogenic atrophy pathology. Fibres outlined with collagen VI antibody (green). D) Segmentation of the image showed in (C) highlighting two atrophic elongated fibres that present seven neighbours.


Western blot for indicated below blot , comparing input MCF - 7 , IP with IgG or indicated antibody . arrows indicate expected of of interest .

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Western blot for the protein indicated below the blot, comparing input MCF-7 cells, and IP with IgG or the indicated antibody. The arrows indicate the expected size of the protein of interest.


RIP - qPCR for indicated ( AsGFP or NORAD ) in transfected with AcGFP bearing NucLibB in 3 ' UTR , with RIP performed with indicated antibody determined by qRT - PCR . N = 5 . Error bars - s . e . m .

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RIP-qPCR for the indicated RNA (AsGFP or NORAD) in cells transfected with the AcGFP bearing NucLibB in the 3' UTR, with RIP performed with the indicated antibody and RNA levels determined by qRT-PCR. N=5. Error bars - s.e.m.


Nuc / Cyto ratios for NucLibB tiles , bound ( + , log2 ( IP / Input ) ≥ 0 . 5 ) or not bound ( - , log2 ( IP / Input ) < 0 . 5 ) by indicated factors . Boxplots are as in Figure 1C . P values computed using two - sized Wilcoxon rank - sum .

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Nuc/Cyto ratios for NucLibB tiles, bound (+, log2(IP/Input)≥0.5) or not bound (-, log2(IP/Input)<0.5) by the indicated factors. Boxplots are as in Figure 1C. P values computed using two-sized Wilcoxon rank-sum test.