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 ) .

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

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 .

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

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 .

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

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 ) .

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

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).


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

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 ) .

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

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

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

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

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


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

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

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 .

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

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 .

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

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 .

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

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

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

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 .

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

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 .

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

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 .

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

(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 .

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

(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 , *

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

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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 + / -

O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O 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) 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 .

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

(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 .

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

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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

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

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 .

O O O 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

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 .

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

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 .

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

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 .

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

J. 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 .

O O O O O O O O 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

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 .

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

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 .

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

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 .

O O O O B-MEASURED_VAR 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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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 O O O O O O O O

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 .

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

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 .

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

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 .

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

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 .

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

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 - ) .

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

D-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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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

M-N. 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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

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 ) .

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

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 .

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

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 .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

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 ) .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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

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

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 ) .

O O O O O O O O O O O O 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) 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 ) .

O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

. 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 .

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

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 .

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

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 .

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

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

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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 were acquired longitudinally at one , two three months post injection .

(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 mice treated with DFO for five months stained by TUNEL to detect . Hoechst was used for . Untreated mice of similar were used as healthy controls . Flat mount were captured by 10 × confocal microscopy stitched together .

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

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

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

(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 ) microscopic examination of iRPE 24 48 hours post treatment . Scale bar : 50 µm .

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

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


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

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

(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 ) among iRPE population was examined by TUNEL 24 hours 48 hours post treatment . Untreated iRPE were included as control . were counterstained with Hoechst .

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

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

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

(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 . statistics are analyzed by one - way ANOVA with Tukey . results are presented as mean ± S . E . M . , n = 3 iRPE lines for each group . * p < 0 . 05 .

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

(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 ) transcripts of ferroptosis - associated markers were determined by qPCR using iRPE as template . extract from untreated iRPE was included as control . level of each transcript was normalized to ACTB . statistics are analyzed by paired student ' s t - for each target gene . 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 .

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

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

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

(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 iRPE with DFO treatment was determined by seahorse extracellular flux . 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 .

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

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

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

(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 mice treated with DFO for up to 10 months in conjunction with supplementation of AKG for seven months .

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

(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 DFO - treated ( 100 mM ) iRPE with AKG supplementation ( 10 mM ) for 24 hours was determined by seahorse extracellular flux . 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 .

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

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

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

(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 for multiple comparisons was performed to determine statistical significance Each symbol represents value obtained for from an independent donor in an independent experiment . ( a ) n = 4 ; Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) when values were compared with

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

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 for multiple comparisons was performed to determine statistical significance Each symbol represents value obtained for from an independent donor in an independent experiment . ( b ) n = 5 Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) when values were compared with

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

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 for multiple comparisons was performed to determine statistical significance Each symbol represents value obtained for from an independent donor in an independent experiment . ( c ) n = 3 Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) when values were compared with

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

(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 addition of RBC , iRBC or LPS . hemozoin crystals can be observed within macrophages . Bar is 30 µm .

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

(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 - was performed to determine statistical significance . Each symbol represents value obtained for from an independent donor in an independent experiment . ( e ) n = 3 . Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) when values were compared RBCL ( e ) .

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

(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 indicated stimuli for 24 h . RBCL , iRBCL , XO were added to macrophages for 24 h . Each symbol represents value obtained for 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 for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) .

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

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 indicated stimuli for 24 h . , XO , Hypoxanthine ( Hyx ) were added to macrophages for 24 h . Each symbol represents value obtained for 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 for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) .

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

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 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 value obtained for 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 for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) .

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

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).