( 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 .

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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 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 .

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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 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 .

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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 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 + / -

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 and immunoblot of recombinant BAK and 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 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 .

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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 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 .

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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 cell 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 images of the intestine of Casp8WT and Casp8ECko mice , with mild and 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 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 .

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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 . 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 .

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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 and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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 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 .

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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.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 , and 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.


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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 and 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 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 .

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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 . 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 test ) of Casp8WT and 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 and Casp8BECko mice .

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


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 .

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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 tailed unpaired Student ' s t - test ) . Data information : Data shown as mean ± SEM . 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 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 .

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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.


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 .

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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 ) 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 .

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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 the first 24h ( n = 11 WT , 11 ECko ; Log - Rank test ) .

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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 images of the small intestines 24h after TNF - α injection in Casp8WT and 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 ) 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 .

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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 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 .

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

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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 images 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 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 .

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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.


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 .

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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 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 .

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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 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 .

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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 images of the gut of Casp8WT and 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 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 .

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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 ) and anti - Myosin Heavy Chain ( red ) antibodies . C ) Segmentation of the 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 embryo neural tube epithelium ( cNT ) .

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


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 .

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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.


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

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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 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 .

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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 area and polygon distribution . A ) Area distribution of several Voronoi diagrams along the CVT path . Cell 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 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 .

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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 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 ) .

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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 levels have been

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


. 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 .

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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 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 .

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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 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 .

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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 the indicated factors . Boxplots are as in Figure 1C . P values computed using two - sized Wilcoxon rank - sum test .

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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.


mRNA ( top ) and protein ( bottom ) levels of the indicated genes in cells transfected with the indicated siRNAs . n = 8 . Error bars are s . e . m . P - values computed two - sided t - test . * * P ≤ 0 . 01 * * * * P ≤ 0 . 0001 .

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mRNA (top) and protein (bottom) levels of the indicated genes in cells transfected with the indicated siRNAs. n=8. Error bars are s.e.m. P-values computed two-sided t-test. ** P≤0.01 **** P≤0.0001.


Differences in the Nuc / Cyto ( log2 ) ratios for tiles containing the SIRLOIN element ( up to 6 mismatches ) and other NucLibB tiles ( only WT sequence tiles were considered ) . Boxplots are as in Figure 1C . P - values computed using two - sided Wilcoxon rank - sum test .

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Differences in the Nuc/Cyto (log2) ratios for tiles containing the SIRLOIN element (up to 6 mismatches) and other NucLibB tiles (only WT sequence tiles were considered). Boxplots are as in Figure 1C. P-values computed using two-sided Wilcoxon rank-sum test.


IP / Input ( top ) and changes in Nuc / Cyto ( log2 ) ratios upon siRNA targeting of the indicated factor ( top ) for WT NucLibB tiles with the indicated number of the indicated motifs . Number of tiles in each group is indicated in parentheses . * - P < 0 . 05 * * P < 0 . 005 * * * P < 0 . 0005 for comparing the group with the indicated number of matches of the motif with all tiles containing a lower motif count . Boxplots are as in Figure 1C . P - values computed using two - sided Wilcoxon rank - sum test . * - P < 0 . 05 , * * - P < 0 . 005 , * * * - P < 0 . 0005 .

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IP/Input (top) and changes in Nuc/Cyto (log2) ratios upon siRNA targeting of the indicated factor (top) for WT NucLibB tiles with the indicated number of the indicated motifs. Number of tiles in each group is indicated in parentheses. *- P<0.05 ** P<0.005 *** P<0.0005 for comparing the group with the indicated number of matches of the motif with all tiles containing a lower motif count. Boxplots are as in Figure 1C. P-values computed using two-sided Wilcoxon rank-sum test. * - P<0.05, ** - P<0.005, *** - P<0.0005.


( A - D ) Color fundus photographs and SW - AF examinations on four patients of β - thalassemia subject to chelation therapy by DFO for at least 16 years . Fundus phenotypes , including RPE mottling and depigmentation in the macula ( Case Ⅰ ) ( Gelman et al , 2014 ) , choroidal sclerosis in the perimacular areas ( Case Ⅱ ) , peripapillary ( Case Ⅲ and Ⅳ ) , and subretinal pigmentation ( Case Ⅳ ) were evaluated . RPE lesions including concentric distribution of stippled hyper - autofluorescence at the macula ( Case Ⅰ ) , large areas of hypo - autofluorescent regions at parapapillary or perimacular area ( Case Ⅱ and III ) , and extensive RPE loss in both eyes ( Case Ⅳ ) were observed by SW - AF .

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(A - D) Color fundus photographs and SW-AF examinations on four patients of β-thalassemia subject to chelation therapy by DFO for at least 16 years. Fundus phenotypes, including RPE mottling and depigmentation in the macula (Case Ⅰ) (Gelman et al, 2014), choroidal sclerosis in the perimacular areas (Case Ⅱ), peripapillary (Case Ⅲ and Ⅳ), and subretinal pigmentation (Case Ⅳ) were evaluated. RPE lesions including concentric distribution of stippled hyper-autofluorescence at the macula (Case Ⅰ), large areas of hypo-autofluorescent regions at parapapillary or perimacular area (Case Ⅱ and III), and extensive RPE loss in both eyes (Case Ⅳ) were observed by SW-AF.


( A - C ) Adult C57BL / 6J mice ( four months old ) were intraperitoneally injected with DFO at 100 mg / kg three times a week for three months in a row . SW - AF images were acquired longitudinally at one , two and three months post injection .

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(A - C) Adult C57BL/6J mice (four months old) were intraperitoneally injected with DFO at 100 mg/kg three times a week for three months in a row. SW-AF images were acquired longitudinally at one, two and three months post injection.


( I , J ) Flat mount RPE was harvested from the mice treated with DFO for five months and stained by TUNEL to detect cell death . Hoechst was used for nuclear staining . Untreated mice of similar age were used as the healthy controls . Flat mount images were captured by 10 × confocal microscopy and stitched together .

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(I, J) Flat mount RPE was harvested from the mice treated with DFO for five months and stained by TUNEL to detect cell death. Hoechst was used for nuclear staining. Untreated mice of similar age were used as the healthy controls. Flat mount images were captured by 10× confocal microscopy and stitched together.


( K - N ) The RPE was dissected from the mice injected with DFO for three and nine months respectively and stained with phalloidin . Age - matched untreated mice were included as the controls . The images were taken at 40 × magnification .

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(K - N) The RPE was dissected from the mice injected with DFO for three and nine months respectively and stained with phalloidin. Age-matched untreated mice were included as the controls. The images were taken at 40× magnification.


( A - D ) Light microscopic examination of the iRPE cells 24 and 48 hours post treatment . Scale bar : 50 µm .

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(A - D) Light microscopic examination of the iRPE cells 24 and 48 hours post treatment. Scale bar: 50 µm.


( E - H ) iRPE cells were stained with ZO - 1 24 and 48 hours post treatment . Untreated iRPE cells were used as the control .

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(E - H) iRPE cells were stained with ZO-1 24 and 48 hours post treatment. Untreated iRPE cells were used as the control.


( I - L ) Cell death among the iRPE population was examined by TUNEL assay 24 hours and 48 hours post treatment . Untreated iRPE cells were included as the control . The cells were counterstained with Hoechst .

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(I - L) Cell death among the iRPE population was examined by TUNEL assay 24 hours and 48 hours post treatment. Untreated iRPE cells were included as the control. The cells were counterstained with Hoechst.


( A ) iRPE cells treated with DFO were collected and lysed to measure the expression of HIF1α and HIF2α 24 and 48 hours after the treatment . The iRPE cells supplemented with 0 . 1 % DMSO in the culture media were used as the control . Actin was used as the loading control . HIF1α ( post - translationally modified ) was predicted to be ~ 100 kDa ; HIF2α ( post translationally modified ) was predicted to be ~ 120 kDa . ( B ) iRPE cells treated with DFO were lysed and fractionated by centrifugation to measure the expression of HIF1α and HIF2α in the nuclei 24 and 48 hours after the treatment . Untreated iRPE cells were used as the normal control . Histone H3 was used as the loading control . (

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(A) iRPE cells treated with DFO were collected and lysed to measure the expression of HIF1α and HIF2α 24 and 48 hours after the treatment. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the control. Actin was used as the loading control. HIF1α (post-translationally modified) was predicted to be ~100 kDa; HIF2α (post translationally modified) was predicted to be ~ 120 kDa. (B) iRPE cells treated with DFO were lysed and fractionated by centrifugation to measure the expression of HIF1α and HIF2α in the nuclei 24 and 48 hours after the treatment. Untreated iRPE cells were used as the normal control. Histone H3 was used as the loading control. (


( A ) Ferrous iron was measured . The statistics are analyzed by one - way ANOVA with the Tukey test . The results are presented as mean ± S . E . M . , n = 3 iRPE lines for each group . * p < 0 . 05 .

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(A) Ferrous iron was measured. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05.


( B ) The transcripts of ferroptosis - associated markers were determined by qPCR using iRPE cells as the template . The RNA extract from untreated iRPE was included as the control . The level of each transcript was normalized to ACTB . The statistics are analyzed by ratio paired student ' s t - test for each target gene . The results are presented as mean ± S . E . M . , n = 3 iRPE lines for each group . * p < 0 . 05 ; * * p < 0 . 01 . Round dots : untreated iRPE ; square dots : DFO - treated iRPE .

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(B) The transcripts of ferroptosis-associated markers were determined by qPCR using iRPE cells as the template. The RNA extract from untreated iRPE was included as the control. The level of each transcript was normalized to ACTB. The statistics are analyzed by ratio paired student's t-test for each target gene. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05; ** p < 0.01. Round dots: untreated iRPE; square dots: DFO-treated iRPE.


( C ) The level of ROS was measured . The statistics are analyzed by one - way ANOVA with the Tukey test . The results are presented as mean ± S . E . M . , n = 6 iRPE lines for each group . * p < 0 . 05 , * * p < 0 . 01 . DHE : Dihydroethidium .

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(C) The level of ROS was measured. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 6 iRPE lines for each group. * p < 0.05, ** p < 0.01. DHE: Dihydroethidium.


( D ) Mitochondrial respiration of the iRPE cells with DFO treatment was determined by seahorse extracellular flux assay . OCR was determined over a course of time . Each data point is shown as mean ± S . E . M . , n = 4 iRPE lines for each group . OCR : oxygen consumption rate ; FCCP : carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ; Rot : rotenone ; AA : antimycin A .

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(D) Mitochondrial respiration of the iRPE cells with DFO treatment was determined by seahorse extracellular flux assay. OCR was determined over a course of time. Each data point is shown as mean ± S.E.M., n = 4 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.


( A - C ) Light microscopy of morphological protection of AKG against DFO toxicity on iRPE . The cells were treated with DFO at 100 mM for 48 hours ( B ) . AKG was supplemented at 10 mM simultaneously ( C ) . The control group was supplemented with 0 . 1 % DMSO ( A ) . Scale bar : 100 µm . ( D - F ) The DFO - treated iRPE cells with or without AKG supplementation were subject to phalloidin staining ( E and F ) . The iRPE cells treated with 0 . 1 % DMSO - containing media were included as the controls ( D ) . The images were captured at 40 × magnification . Scale bar : 30 µm . ( G - I ) Detection of cell death in the DFO - treated iRPE cells with or without AKG supplementation by TUNEL staining . Hoechst was used for nuclear staining . The images were captured at 40 × magnification . Scale bar : 30 µm . (

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(A - C) Light microscopy of morphological protection of AKG against DFO toxicity on iRPE. The cells were treated with DFO at 100 mM for 48 hours (B). AKG was supplemented at 10 mM simultaneously (C). The control group was supplemented with 0.1% DMSO (A). Scale bar: 100 µm. (D - F) The DFO-treated iRPE cells with or without AKG supplementation were subject to phalloidin staining (E and F). The iRPE cells treated with 0.1% DMSO-containing media were included as the controls (D). The images were captured at 40× magnification. Scale bar: 30 µm. (G - I) Detection of cell death in the DFO-treated iRPE cells with or without AKG supplementation by TUNEL staining. Hoechst was used for nuclear staining. The images were captured at 40× magnification. Scale bar: 30 µm. (J


( J - L ) SW - AF was performed longitudinally on the mice treated with DFO for up to 10 months in conjunction with supplementation of AKG for seven months .

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(J - L) SW-AF was performed longitudinally on the mice treated with DFO for up to 10 months in conjunction with supplementation of AKG for seven months.


( A ) Mitochondrial respiration of the DFO - treated ( 100 mM ) iRPE cells with AKG supplementation ( 10 mM ) for 24 hours was determined by seahorse extracellular flux assay . OCR was measured over a course of time . Each dot is shown as mean ± S . E . M . , n = 5 iRPE lines for each group . OCR : oxygen consumption rate ; FCCP : carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ; Rot : rotenone ; AA : antimycin A .

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(A) Mitochondrial respiration of the DFO-treated (100 mM) iRPE cells with AKG supplementation (10 mM) for 24 hours was determined by seahorse extracellular flux assay. OCR was measured over a course of time. Each dot is shown as mean ± S.E.M., n = 5 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.


( G ) The DFO - treated ( 100 mM ) iRPE cells supplemented with AKG ( 10 mM ) for 48 hours were lysed for immunoblotting against HIF2α . The iRPE cells supplemented with 0 . 1 % DMSO in the culture media were used as the controls . HIF2α ( post translationally modified ) was predicted to be ~ 120 kDa . Actin was included as the loading control . ( H ) RPE lysate from the one - year - old mice subject to 10 - month DFO treatment with or without concomitant supplementation of AKG was used for immunoblotting against HIF2α . The RPE of the age - matched untreated mice was included as the control . HIF2α ( degraded ) was predicted to be 40 - 80 kDa . Actin was used as the loading control .

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(G) The DFO-treated (100 mM) iRPE cells supplemented with AKG (10 mM) for 48 hours were lysed for immunoblotting against HIF2α. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the controls. HIF2α (post translationally modified) was predicted to be ~ 120 kDa. Actin was included as the loading control. (H) RPE lysate from the one-year-old mice subject to 10-month DFO treatment with or without concomitant supplementation of AKG was used for immunoblotting against HIF2α. The RPE of the age-matched untreated mice was included as the control. HIF2α (degraded) was predicted to be 40 - 80 kDa. Actin was used as the loading control.


Macrophages produce little to no inflammatory cytokines when incubated with either ( a ) whole iRBC for 24 h . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( a ) n = 4 ; Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared with

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Macrophages produce little to no inflammatory cytokines when incubated with either (a) whole iRBC for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC


Macrophages produce little to no inflammatory cytokines when incubated with either ( b ) fresh iRBCL for 24 h . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( b ) n = 5 Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared with

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Macrophages produce little to no inflammatory cytokines when incubated with either (b) fresh iRBCL for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (b) n=5 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC


( c ) Macrophages were pre - incubated with IFNγ ( 1000U / ml ) for 18 h before iRBCL stimulation . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( c ) n = 3 Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared with

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(c) Macrophages were pre-incubated with IFNγ (1000U/ml) for 18 h before iRBCL stimulation. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=3 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC


( d ) Phase - contrast microscopy of macrophages 24 h after the addition of RBC , iRBC or LPS . Dark hemozoin crystals can be observed within macrophages . Bar is 30 µm .

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(d) Phase-contrast microscopy of macrophages 24 h after the addition of RBC, iRBC or LPS. Dark hemozoin crystals can be observed within macrophages. Bar is 30 µm.


( e ) Macrophages were incubated with iRBCL 24 h before addition of LPS . Paired t - test was performed to determine statistical significance . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( e ) n = 3 . Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared RBCL ( e ) .

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(e) Macrophages were incubated with iRBCL 24 h before addition of LPS. Paired t-test was performed to determine statistical significance. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (e) n=3. Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared RBCL (e).


Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . RBCL , iRBCL , XO were added to macrophages for 24 h . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with RBCL ( a ) n = 7 One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. RBCL, iRBCL, XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL (a) n=7 One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . , XO , Hypoxanthine ( Hyx ) were added to macrophages for 24 h . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO, Hypoxanthine (Hyx) were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . XO added to macrophages for 24 h . Febuxostat ( Feb ) was pre - incubated for 30 minutes with XO before addition to macrophages . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. XO added to macrophages for 24 h. Febuxostat (Feb) was pre-incubated for 30 minutes with XO before addition to macrophages. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . , XO the anti - oxidants N - acetyl - L - cysteine ( NAC ) and 1 - Thioglycerol ( 1 - TG ) , and heat inactivated ( HI ) XO were added to macrophages for 24 h . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO the anti-oxidants N-acetyl-L-cysteine (NAC) and 1-Thioglycerol (1-TG), and heat inactivated (HI) XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of PBMC ( e ) incubated with the indicated stimuli for 24 h . RBC , iRBC , RBCL , iRBCL , XO Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with RBCL n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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Cytokine secretion of PBMC (e) incubated with the indicated stimuli for 24 h. RBC, iRBC, RBCL, iRBCL, XO Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


( a ) XO was incubated with macrophages for the indicated times , when wells were washed twice and media was replaced . Supernatants were collected for cytokine determinations 24 h later ( n = 3 ) . Kruskal - Wallis statistical method was used for statistical analysis . Blue asterisks indicate significance when values were compared with time 0 ( * p < 0 . 05 ) .

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

(a) XO was incubated with macrophages for the indicated times, when wells were washed twice and media was replaced. Supernatants were collected for cytokine determinations 24 h later (n=3). Kruskal-Wallis statistical method was used for statistical analysis. Blue asterisks indicate significance when values were compared with time 0 (*p<0.05).


( b ) Diagram shows experimental set up with macrophages in the bottom of the well and XO added either in the dialysis cassette ( DC , blue ) or the well ( black ) . XO activity panels show that the enzyme did not leak through the DC membrane . Secretion of IL6 and TNF was measured 24 h after addition of XO in each compartment . A representative result of two independent experiments is shown .

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(b) Diagram shows experimental set up with macrophages in the bottom of the well and XO added either in the dialysis cassette (DC, blue) or the well (black). XO activity panels show that the enzyme did not leak through the DC membrane. Secretion of IL6 and TNF was measured 24 h after addition of XO in each compartment. A representative result of two independent experiments is shown.


( a ) Plasma levels of TNF and IL8 from patients with uncomplicated ( black circles , n = 14 ) or cerebral ( red circles , n = 9 ) malaria correlate with levels of XO - produced ROS detected in each sample . ROS is expressed as fold change over plasma from healthy controls . Uncomplicated malaria , gray circles , cerebral malaria , red circles . Linear regression ( a ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(a) Plasma levels of TNF and IL8 from patients with uncomplicated (black circles, n=14) or cerebral (red circles, n=9) malaria correlate with levels of XO-produced ROS detected in each sample. ROS is expressed as fold change over plasma from healthy controls. Uncomplicated malaria, gray circles, cerebral malaria, red circles. Linear regression (a) were performed to determine statistical significance (*p<0.05, **p<0.01).


( b ) Levels of XO activity in malaria patients correlate with disease severity UM ( uncomplicated malaria ) , CM ( cerebral malaria ) . Mann - Whitney test ( b ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(b) Levels of XO activity in malaria patients correlate with disease severity UM (uncomplicated malaria), CM (cerebral malaria). Mann-Whitney test (b) were performed to determine statistical significance (*p<0.05, **p<0.01).


( c ) Plasma from malaria patients induce macrophages to secrete cytokines , which are inhibited by a XO specific inhibitor . Plasma from a healthy control ( HP ) and from three patients : P1 ( with cerebral malaria ) , P2 and P3 ( both with uncomplicated malaria ) was pre - incubated for 30 min with febuxostat ( Feb ) or alone at 37ºC before addition to macrophages at 1 : 2 dilution in media for 30 min . Cells were washed and cytokine secretion measured in triplicates after 24 h of incubation . unpaired t - tests ( c ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(c) Plasma from malaria patients induce macrophages to secrete cytokines, which are inhibited by a XO specific inhibitor. Plasma from a healthy control (HP) and from three patients: P1 (with cerebral malaria), P2 and P3 (both with uncomplicated malaria) was pre-incubated for 30 min with febuxostat (Feb) or alone at 37ºC before addition to macrophages at 1:2 dilution in media for 30 min. Cells were washed and cytokine secretion measured in triplicates after 24 h of incubation. unpaired t-tests (c) were performed to determine statistical significance (*p<0.05, **p<0.01).


Cytokine secretion by macrophages after 24 h incubation with the indicated stimuli . ( b ) IL1β levels after addition of increasing concentration of RBCL or iRBCL ( 1 : 2 , 1 : 4 and 1 : 8 macrophage : iRBCL ) . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance . Asterisks indicate significance when values are compared with XO + RBCL ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( a ) n = 4 ; ( b ) n = 2 Insets show paired samples by donor in two different conditions .

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Cytokine secretion by macrophages after 24 h incubation with the indicated stimuli. (b) IL1β levels after addition of increasing concentration of RBCL or iRBCL (1:2, 1:4 and 1:8 macrophage:iRBCL). One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; (b) n=2 Insets show paired samples by donor in two different conditions.


chemokine secretion by macrophages after 24 h incubation with the indicated stimuli . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance . Asterisks indicate significance when values are compared with XO + RBCL ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( c ) n = 4 . Insets show paired samples by donor in two different conditions .

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chemokine secretion by macrophages after 24 h incubation with the indicated stimuli. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=4. Insets show paired samples by donor in two different conditions.