figf4 ( b ) RPE cells with stable expression of cherry - LC3 were transiently transfected with the GFP - myosin VI cargo - binding tail domain containing various mutations in the protein - interaction ( ΔWWY and ΔRRL ) and ubiquitin - binding motifs ( A1013G ) , followed by treatment with 250 nM Torin1 for 3 h to induce autophagy . Immunofluorescence microscopy was performed either in the absence or presence of saponin extraction . The arrowheads indicate areas of co - localization . Scale bar , 20 μm . ( c ) A Pearson ' s coefficient was calculated on the basis of the degree of co - localization between the different GFP - myosin VI mutant tails and cherry - LC3 from confocal immunofluorescence micrographs . The graph represents data from more than 20 transfected cells from n = 2 independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(b) RPE cells with stable expression of cherry-LC3 were transiently transfected with the GFP-myosin VI cargo-binding tail domain containing various mutations in the protein-interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 h to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin extraction. The arrowheads indicate areas of co-localization. Scale bar, 20 μm. (c) A Pearson's coefficient was calculated on the basis of the degree of co-localization between the different GFP-myosin VI mutant tails and cherry-LC3 from confocal immunofluorescence micrographs. The graph represents data from more than 20 transfected cells from n = 2 independent experiments.


figf5 ( a ) Left , immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h . Immunolabelling was performed against the indicated endogenous proteins optineurin , T6BP and NDP52 ( red ) , and LC3 ( green ) . The insets show higher magnifications of the areas outlined in the main images . Right , from the confocal images , a Pearson ' s coefficient was calculated to estimate the degree of co - localization of the different autophagy adaptors with LC3 . As a negative control , a Pearson ' s coefficient was calculated after rotation of one colour image by 90 ° . The results represent > 20 cells from n = 2 independent experiments . ( b ) Left , stable cherry - LC3 - expressing RPE cells were transiently transfected with GFP - myosin VI , treated with Torin1 for 3 h to induce autophagy , and subsequently processed for confocal immunofluorescence microscopy . Areas of co - localization are white in the merged three - colour images and the insets show higher magnifications of the areas outlined in the main images . Right , a Pearson ' s coefficient was calculated for the degree of co - localization between the three colours . As a negative control , a Pearson ' s coefficient was calculated after rotating one colour image by 90 ° . Results represent > 10 cells from n = 2 independent experiments . ( c ) HeLa cells were either mock transfected or co - transfected with siRNA targeted against TNO . Cells were either left untreated or treated with100 nM bafilomycin A1 ( BfnA1 ) for 4 h . Western blot analysis was performed against the indicated proteins . ( d ) Quantification of LC3 - II intensity ( ± s . d . ) was performed from western blot data of TNO - siRNA treated HeLa cells . The results represent n = 3independent experiments . * * P0 . 01 , * * * P0 . 001 ( e ) Immunofluorescence microscopy of RFP - GFP - LC3 - expressing HeLa cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin . The results are calculated from > 100 cells from n = 2 independent experiments represented as the Pearson ' s coefficient of GFP / RFP signal overlap . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) Left, immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h. Immunolabelling was performed against the indicated endogenous proteins optineurin, T6BP and NDP52 (red), and LC3 (green). The insets show higher magnifications of the areas outlined in the main images. Right, from the confocal images, a Pearson's coefficient was calculated to estimate the degree of co-localization of the different autophagy adaptors with LC3. As a negative control, a Pearson's coefficient was calculated after rotation of one colour image by 90°. The results represent >20 cells from n = 2 independent experiments.(b) Left, stable cherry-LC3-expressing RPE cells were transiently transfected with GFP-myosin VI, treated with Torin1 for 3 h to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of co-localization are white in the merged three-colour images and the insets show higher magnifications of the areas outlined in the main images. Right, a Pearson's coefficient was calculated for the degree of co-localization between the three colours. As a negative control, a Pearson's coefficient was calculated after rotating one colour image by 90°. Results represent >10 cells from n = 2 independent experiments.(c) HeLa cells were either mock transfected or co-transfected with siRNA targeted against TNO. Cells were either left untreated or treated with100 nM bafilomycin A1 (BfnA1) for 4 h. Western blot analysis was performed against the indicated proteins. (d) Quantification of LC3-II intensity (±s.d.) was performed from western blot data of TNO-siRNA treated HeLa cells. The results represent n = 3independent experiments. **P0.01, ***P0.001(e) Immunofluorescence microscopy of RFP-GFP-LC3-expressing HeLa cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin. The results are calculated from >100 cells from n = 2 independent experiments represented as the Pearson's coefficient of GFP/RFP signal overlap. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


figf6 ( a ) Myosin VI and Tom1 / Tom1L2 co - immunoprecipitate from LNCAP cell lysates . Immunoprecipitation of myosin VI with three separate affinity - purified rabbit polyclonal antibodies ( B4 , B7 , 2401 ) was performed alongside an IgG control immunoprecipitation . The immunoprecipitates were analysed by western blotting with antibodies against Tom1 / Tom1L2 . ( b ) Binding of myosin VI to Tom1 requires the WWY motif . Bottom , the mammalian two - hybrid assay was used to test binding of full - length Tom1 and Tom1L2 or various truncated versions of Tom1 , annotated by amino acid numbers , against wild - type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail . The graph shows the mean values from n = 2 independent experiments . Top , Tom1 domain structure and truncation mutants . VHS , Vps - 27 , Hrs and STAM ; GAT , GGA and Tom1 . ( c ) RPE cells stably expressing GFP - myosin VI were processed for immunofluorescence microscopy to evaluate GFP - myosin VI and endogenous Tom1 / Tom1L2 or Rab5 co - localization . The insets show higher magnifications of the areas outlined in the main panels . The arrows indicate areas of co - localization . ( d ) RPE cells with stable expression of GFP - myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot analysis ( left ) and immunofluorescence microscopy ( middle ) to evaluate GFP - myosin VI tail localization . Cells were either fixed directly or saponin - extracted before fixation . Quantification ( right ) was performed on RPE cells stably expressing GFP - myosin VI tail and transfected with mock , Tom1 or TNO siRNA . Cells were processed for immunocytochemistry , immunolabelled for GFP and nuclei labelled with Hoechst followed by quantification of GFP - myosin VI tail punctae per cell using an automated Cellomics VTi microscope . More than 600 cells per group from n = 3 independent experiments were analysed and are represented as the mean number of GFP - myosin VI tail punctae per cell ( ± s . d . ) . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(a) Myosin VI and Tom1/Tom1L2 co-immunoprecipitate from LNCAP cell lysates. Immunoprecipitation of myosin VI with three separate affinity-purified rabbit polyclonal antibodies (B4, B7, 2401) was performed alongside an IgG control immunoprecipitation. The immunoprecipitates were analysed by western blotting with antibodies against Tom1/Tom1L2.(b) Binding of myosin VI to Tom1 requires the WWY motif. Bottom, the mammalian two-hybrid assay was used to test binding of full-length Tom1 and Tom1L2 or various truncated versions of Tom1, annotated by amino acid numbers, against wild-type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail. The graph shows the mean values from n = 2 independent experiments. Top, Tom1 domain structure and truncation mutants. VHS, Vps-27, Hrs and STAM; GAT, GGA and Tom1.(c) RPE cells stably expressing GFP-myosin VI were processed for immunofluorescence microscopy to evaluate GFP-myosin VI and endogenous Tom1/Tom1L2 or Rab5 co-localization. The insets show higher magnifications of the areas outlined in the main panels. The arrows indicate areas of co-localization.(d) RPE cells with stable expression of GFP-myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot analysis (left) and immunofluorescence microscopy (middle) to evaluate GFP-myosin VI tail localization. Cells were either fixed directly or saponin-extracted before fixation. Quantification (right) was performed on RPE cells stably expressing GFP-myosin VI tail and transfected with mock, Tom1 or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labelled with Hoechst followed by quantification of GFP-myosin VI tail punctae per cell using an automated Cellomics VTi microscope. More than 600 cells per group from n = 3 independent experiments were analysed and are represented as the mean number of GFP-myosin VI tail punctae per cell (±s.d.). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


figf7 ( a ) HeLa cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM bafilomycin A1 for 4 h . Western blot analysis was performed on whole - cell lysates against the indicated proteins . ( b ) Quantification of LC3 - II intensity of western blots was performed by an infrared imaging system . ( ± s . d . ; n = 3 ) . * P0 . 05 , * * P0 . 01 . ( c , left ) Confocal immunofluorescence microscopy was performed on HeLa cells transfected with Tom1 siRNA to evaluate LC3 punctae formation ( green ) . The insets show higher magnifications of the areas outlined in the main images . Nuclei are labelled with Hoechst ( blue ) . ( c , right , d ) Quantification of LC3 - positive punctae ( c , right ) and p62 - positive punctae ( d ) was performed in a 96 - well format on an Arrayscan VTi HCS microscope . Cells were identified by Hoechst staining of nuclei ( blue ) . The results are represented as the average punctae fluorescence intensity per cell ( ± s . d . ) from n = 3 independent experiments each performed in triplicate wells , each with > 500 cells . ( c , left ) Confocal immunofluorescence microscopy was performed on HeLa cells transfected with Tom1 siRNA to evaluate LC3 punctae formation ( green ) . The insets show higher magnifications of the areas outlined in the main images . Nuclei are labelled with Hoechst ( blue ) . ( c , right , d ) Quantification of LC3 - positive punctae ( c , right ) and p62 - positive punctae ( d ) was performed in a 96 - well format on an Arrayscan VTi HCS microscope . Cells were identified by Hoechst staining of nuclei ( blue ) . The results are represented as the average punctae fluorescence intensity per cell ( ± s . d . ) from n = 3 independent experiments each performed in triplicate wells , each with > 500 cells . ( e ) HeLa cells with stable expression of the RFP - GFP - LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown ( left ) . Confocal immunofluorescence microscopy ( middle ) was performed and images were subsequently quantified for the correlation between the GFP and RFP signals using the ImageJ JACoP plugin ( right ) . The results are for at least 100 individual cells from n = 3 independent experiments and are represented as the Pearson ' s coefficient of GFP / RFP overlap ( ± s . d . ) . A higher Pearson ' s coefficient of GFP / RFP signal overlap represents a greater number of autophagosomes when compared with autolysosomes . ( f ) Confocal immunofluorescence microscopy of Tom1 - depleted RPE cells stably expressing GFP - LC3 immunostained against GFP ( green ) and cathepsin D ( red ) . Hoechst labels the nuclei ( blue ) . The insets show higher magnifications of the areas outlined in the main images . ( g ) HeLa cells with stable expression of HttQ72 - GFP were transiently transfected with Tom1 siRNA ( top right ) , saponin - extracted , and processed for immunofluorescence microscopy ( left ) to quantify HttQ72 - GFP punctae ( bottom right ) . Immunolabelling against GFP ( green ) and p62 ( red ) was performed . Nuclei ( blue ) were labelled with Hoechst . The results are represented as the number of GFP - expressing cells with greater than 15 GFP - positive spots per cell ( ± s . d . ; n = 3 ) . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) HeLa cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM bafilomycin A1 for 4 h. Western blot analysis was performed on whole-cell lysates against the indicated proteins. (b) Quantification of LC3-II intensity of western blots was performed by an infrared imaging system. (±s.d.; n = 3). *P0.05, **P0.01.(c, left) Confocal immunofluorescence microscopy was performed on HeLa cells transfected with Tom1 siRNA to evaluate LC3 punctae formation (green). The insets show higher magnifications of the areas outlined in the main images. Nuclei are labelled with Hoechst (blue). (c,right,d) Quantification of LC3-positive punctae (c, right) and p62-positive punctae (d) was performed in a 96-well format on an Arrayscan VTi HCS microscope. Cells were identified by Hoechst staining of nuclei (blue). The results are represented as the average punctae fluorescence intensity per cell (±s.d.) from n = 3 independent experiments each performed in triplicate wells, each with >500 cells.(c, left) Confocal immunofluorescence microscopy was performed on HeLa cells transfected with Tom1 siRNA to evaluate LC3 punctae formation (green). The insets show higher magnifications of the areas outlined in the main images. Nuclei are labelled with Hoechst (blue). (c,right,d) Quantification of LC3-positive punctae (c, right) and p62-positive punctae (d) was performed in a 96-well format on an Arrayscan VTi HCS microscope. Cells were identified by Hoechst staining of nuclei (blue). The results are represented as the average punctae fluorescence intensity per cell (±s.d.) from n = 3 independent experiments each performed in triplicate wells, each with >500 cells.(e) HeLa cells with stable expression of the RFP-GFP-LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown (left). Confocal immunofluorescence microscopy (middle) was performed and images were subsequently quantified for the correlation between the GFP and RFP signals using the ImageJ JACoP plugin (right). The results are for at least 100 individual cells from n = 3 independent experiments and are represented as the Pearson's coefficient of GFP/RFP overlap (±s.d.). A higher Pearson's coefficient of GFP/RFP signal overlap represents a greater number of autophagosomes when compared with autolysosomes.(f) Confocal immunofluorescence microscopy of Tom1-depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and cathepsin D (red). Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images.(g) HeLa cells with stable expression of HttQ72-GFP were transiently transfected with Tom1 siRNA (top right), saponin-extracted, and processed for immunofluorescence microscopy (left) to quantify HttQ72-GFP punctae (bottom right). Immunolabelling against GFP (green) and p62 (red) was performed. Nuclei (blue) were labelled with Hoechst. The results are represented as the number of GFP-expressing cells with greater than 15 GFP-positive spots per cell (±s.d.; n = 3). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


figf8 ( a ) Left , RPE cells with stable expression of GFP - LC3 following mock - or myosin - VI - siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1 / Tom1L2 ( red ) . Nuclei ( blue ) were labelled with Hoechst . The insets show higher magnifications of the areas outlined in the main panels and the arrows highlight areas of complete overlapping co - localization and arrowheads highlight adjacent localization of Tom1 relative to LC3 . Right , quantification was performed on > 75 cells for the relative position ( adjacent or complete overlap ) of Tom1 - positive vesicles in relation to LC3 - positive vesicles . The results are represented as the average number of Tom1 - positive vesicles per cell categorized by their relative position to LC3 ( ± s . d . ; n = 3 ) . ( b ) GFP - LC3 - expressing RPE cells were depleted of myosin VI by siRNA . Cells were pulse labelled with Texas - red - dextran ( red ) for 4 h , before chase into fresh medium containing 1 μM MG132 for 2 h . Cells were processed for immunofluorescence microscopy and immunostained for GFP ( green ) . Nuclei were labelled with Hoechst ( blue ) . The insets represent higher magnifications of the areas outlined in the main panels . ( c ) Mander ' s overlap coefficients for the degree of LC3 signal co - localizing with dextran were calculated using Volocity software . The results represent > 20 cells from n = 3 independent experiments and are illustrated as a box and whisker plot . The box represents the median , 25th and 75th percentiles and whiskers represent the maximum and minimum . ( d ) RPE cells stably expressing GFP - LC3 were depleted of myosin VI and Tom1 by siRNA . Cells were pulse - labelled with Texas - red ( TR ) - dextran for 16 h followed by a chase period of 4 h . Cells were processed for immunoelectron microscopy and labelled with 15 nm gold particles against GFP - LC3 and 5 nm gold particles against Texas - red - dextran . Scale bars , 20 μm ( a , b ) ; 200 nm ( d ) .

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figf8(a) Left, RPE cells with stable expression of GFP-LC3 following mock- or myosin-VI-siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1/Tom1L2 (red). Nuclei (blue) were labelled with Hoechst. The insets show higher magnifications of the areas outlined in the main panels and the arrows highlight areas of complete overlapping co-localization and arrowheads highlight adjacent localization of Tom1 relative to LC3. Right, quantification was performed on >75 cells for the relative position (adjacent or complete overlap) of Tom1-positive vesicles in relation to LC3-positive vesicles. The results are represented as the average number of Tom1-positive vesicles per cell categorized by their relative position to LC3 (±s.d.; n = 3).(b) GFP-LC3-expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labelled with Texas-red-dextran (red) for 4 h, before chase into fresh medium containing 1 μM MG132 for 2 h. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labelled with Hoechst (blue). The insets represent higher magnifications of the areas outlined in the main panels. (c) Mander's overlap coefficients for the degree of LC3 signal co-localizing with dextran were calculated using Volocity software. The results represent >20 cells from n = 3 independent experiments and are illustrated as a box and whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum.(d) RPE cells stably expressing GFP-LC3 were depleted of myosin VI and Tom1 by siRNA. Cells were pulse-labelled with Texas-red (TR)-dextran for 16 h followed by a chase period of 4 h. Cells were processed for immunoelectron microscopy and labelled with 15 nm gold particles against GFP-LC3 and 5 nm gold particles against Texas-red-dextran. Scale bars, 20 μm (a,b); 200 nm (d).


Figure 1B WB showing depletion of PERK in Perk KO astrocytes and neurons . D - I eIF2α phosphorylation in WT and Perk KO astrocytes ( N = 3 ) and neurons ( N = 6 ) ± 20 - 24 hours of TM - induced ER stress . Representative WB ( D , G ) , quantification of absolute phosphorylated eIF2α level ( p - eIF2α / eIF2α ) ( E , H ) and the ER stress - induced p - eIf2α response ( F , I ) . Data information : ( E , H , Data are normalised to untreated WT . ( F , I Data are related to untreated cells of the same genotype . Baseline level ( without ER stress ) is depicted by a dashed line . Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( E , H , Students t - test ( F , I ) J - O Protein synthesis in WT and Perk KO astrocytes ( N = 5 ) and neurons ( N = 5 ) ± 20 - 24 hours of TM - induced ER stress . Representative images obtained by high - content microscopy showing puromycinilated proteins detected by immunofluorescence ( J , M ) , quantification of puromycin intensity as measure for de novo protein synthesis ( K , N ) and the ER stress - induced protein synthesis response ( L , O ) . Astrocytic soma ( GFAP , white ) , dendrites ( MAP2 , white ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesized proteins ( Puromycin , green ) . Scale bar : 25 μm . Data information : K , N ) Data are normalised to untreated WT . , L , O ) Data are related to untreated cells of the same genotype . Baseline level ( without ER stress ) is depicted by a dashed line . Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test K , N ) ; Nested t - test ( L , O ) .

O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B WB showing depletion of PERK in Perk KO astrocytes and neurons.D-I eIF2α phosphorylation in WT and Perk KO astrocytes (N=3) and neurons (N=6) ± 20-24 hours of TM-induced ER stress. Representative WB (D, G), quantification of absolute phosphorylated eIF2α level (p-eIF2α/eIF2α) (E, H) and the ER stress-induced p-eIf2α response (F, I). Data information: (E, H, Data are normalised to untreated WT. (F, I Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: ** P<0.01, *** P<0.001, **** P<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (E, H, Students t-test (F, I)J-O Protein synthesis in WT and Perk KO astrocytes (N=5) and neurons (N=5) ± 20-24 hours of TM-induced ER stress. Representative images obtained by high-content microscopy showing puromycinilated proteins detected by immunofluorescence (J, M), quantification of puromycin intensity as measure for de novo protein synthesis (K, N) and the ER stress-induced protein synthesis response (L, O). Astrocytic soma (GFAP, white), dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green). Scale bar: 25 μm. Data information: K, N) Data are normalised to untreated WT. , L, O) Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: ** P<0.01, *** P<0.001, **** P<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test K , N); Nested t-test (L, O).


Figure 2A - F ATF4 expression in WT and Perk KO astrocytes ( N = 3 ) and neurons ( N = 9 ) ± 20 - 24 hours of TM - induced ER stress . ( A , D ) Representative confocal images of ATF4 immunofluorescence . Astrocytic soma ( GFAP , cyan ) , dendrites ( MAP2 , cyan ) , nuclei ( ( Δ ) Cre , green ) , ATF4 ( magenta ) . Scale bar : 10 μm . ( B , E ) Quantification of absolute nuclear ATF4 intensity . Data are normalised to untreated WT . ( C , F ) Quantification of the ER stress - induced ATF4 response . Data are related to untreated cells of the same genotype . Baseline level ( without ER stress ) is depicted by a dashed line . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant p - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( B , E ) ; Nested t - test ( C , F ) . G - J ( G , I ) Venn diagram showing the number of significantly upregulated ATF4 target genes in WT ( grey and blue ) and Perk KO ( green and blue ) astrocytes ( G , N = 3 ) and neurons ( I , N = 3 ) upon 20 - 24 hours of TM - induced ER stress determined by mRNA - seq . Percentage : fraction of upregulated ATF4 target genes in WT cells that are also upregulated in Perk KO cells . ( H , N = 3 ; J , N = 3 ) Log2 fold change of ATF4 target genes that are significantly upregulated in both WT and Perk KO cells ( blue in Venn diagram ) . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant p - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-F ATF4 expression in WT and Perk KO astrocytes (N=3) and neurons (N=9) ± 20-24 hours of TM-induced ER stress. (A, D) Representative confocal images of ATF4 immunofluorescence. Astrocytic soma (GFAP, cyan), dendrites (MAP2, cyan), nuclei ((Δ)Cre, green), ATF4 (magenta). Scale bar: 10 μm. (B, E) Quantification of absolute nuclear ATF4 intensity. Data are normalised to untreated WT. (C, F) Quantification of the ER stress-induced ATF4 response. Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant p-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E); Nested t-test (C, F).G-J (G, I) Venn diagram showing the number of significantly upregulated ATF4 target genes in WT (grey and blue) and Perk KO (green and blue) astrocytes (G, N=3) and neurons (I, N=3) upon 20-24 hours of TM-induced ER stress determined by mRNA-seq. Percentage: fraction of upregulated ATF4 target genes in WT cells that are also upregulated in Perk KO cells. (H, N=3; J, N=3) Log2 fold change of ATF4 target genes that are significantly upregulated in both WT and Perk KO cells (blue in Venn diagram). Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant p-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant.


Figure 3Protein synthesis and ATF4 expression in mouse ( A - F ) astrocytes ( A - C and neurons ( D - F ± 20 - 24 hours of TM - induced ER stress ± PERK - i ( Pi ) . ( A , D , Representative images obtained by high - content microscopy . Astrocytic soma ( GFAP dendrites ( MAP2 , white ) , nuclei ( ΔCre , red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . ( B , N = 5 ; E , N = 5 ; Quantification of puromycin intensity as measure for de novo protein synthesis . ( C , N = 5 ; F , N = 7 ; Quantification of absolute nuclear ATF4 intensity . Data information : Data are normalised to untreated WT and presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test . Protein synthesis and ATF4 expression in human ( G - L ) astrocytes G - I , ) and neurons J - L ) ± 20 - 24 hours of TM - induced ER stress ± PERK - i ( Pi ) . G , J ) Representative images obtained by high - content microscopy . Astrocytic soma Phalloidin , white ) , dendrites ( MAP2 , white ) , nuclei ( ΔCre , red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . ; H , N = 3 ; K , N = 3 ) Quantification of puromycin intensity as measure for de novo protein synthesis . I , N = 4 ; L , N = 3 ) Quantification of absolute nuclear ATF4 intensity . Data information : Data are normalised to untreated WT and presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Protein synthesis and ATF4 expression in mouse (A-F) astrocytes (A-C and neurons (D-F ± 20-24 hours of TM-induced ER stress ± PERK-i (Pi). (A, D, Representative images obtained by high-content microscopy. Astrocytic soma (GFAP dendrites (MAP2, white), nuclei (ΔCre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. (B, N=5; E, N=5; Quantification of puromycin intensity as measure for de novo protein synthesis. (C, N=5; F, N=7; Quantification of absolute nuclear ATF4 intensity. Data information: Data are normalised to untreated WT and presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test.Protein synthesis and ATF4 expression in human (G-L) astrocytes G-I, ) and neurons J-L) ± 20-24 hours of TM-induced ER stress ± PERK-i (Pi). G, J) Representative images obtained by high-content microscopy. Astrocytic soma Phalloidin, white), dendrites (MAP2, white), nuclei (ΔCre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. ; H, N=3; K, N=3) Quantification of puromycin intensity as measure for de novo protein synthesis. I, N=4; L, N=3) Quantification of absolute nuclear ATF4 intensity. Data information: Data are normalised to untreated WT and presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test.


Figure 4D - G ROS accumulation in WT and Perk KO neurons ( N = 3 ) and astrocytes ( N = 3 ) ± 20 - 24 hours of TM - induced ER stress . ( D , F ) Representative images obtained by high - content microscopy . Dendrites ( MAP2 , white ) , astrocytic soma ( Phalloidin , white ) , nuclei ( ( Δ ) Cre , red ) , ROS ( CellROX green , green ) . Scale bar : 25 μm . ( E , G ) Quantification of CellROX green intensity . Data information : ( E , G , Data are normalised to untreated WT . Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( E , G , H mRNA expression of Eif2ak1 ( HRI ) in HRI KD neurons determined by RT - qPCR , normalised to WT transduced with shRNA control ( represented by a dashed line ) ( N = 3 ) . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis One sample t - test with a hypothetical value of 100 ( HI Quantification of the increase in nuclear ATF4 intensity in WT neurons ± HRI KD ± 20 hours of sodium arsenite ( Ars ) treatment ( N = 3 ) . J The remaining ATF4 response ( % ) upon HRI KD , related to ATF4 response of WT transduced with shRNA control ( 100 % , represented by a dashed line ) ( N = 3 ) . Calculated from data in ( I ) : [ [ ( KD _ Ars + - KD _ Ars - ) / KD _ Ars - ] / [ ( WT _ Ars + - WT _ Ars - ) / WT _ Ars - ] ] * 100 . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test I , One sample t - test with a hypothetical value of 100 J ) K - M eIF2α phosphorylation in HRI KD WT and Perk KO neurons ( N = 3 ) ± 20 - 24 hours of TM - induced ER stress . Representative WB ( K ) , quantification of the ER stress - induced p - eIf2α response ( L ) and absolute phosphorylated eIF2α level ( p - eIF2α / eIF2α ) ( M ) . Data information : M , Data are normalised to untreated WT . ( L , Data are related to untreated cells of the same genotype and normalised to WT without HRI KD . Baseline level ( without ER stress ) is depicted by a dashed line . Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test L , Students t - test ( M ) N - S Protein synthesis ( N = 3 ) and ATF4 expression ( N = 4 ) in HRI KD WT and Perk KO neurons ± 20 - 24 hours of TM - induced ER stress . Representative images obtained by ( N ) confocal and ( Q ) high - content microscopy . Dendrites ( MAP2 , white / cyan ) , nuclei ( ( Δ ) Cre , green / red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . Quantification of the ER stress - induced protein synthesis response ( O ) and puromycin intensity as measure for de novo protein synthesis ( P ) . Quantification of the ER stress - induced ATF4 response ( R ) and absolute nuclear ATF4 intensity ( S ) . Data information : P , S ) Data are normalised to untreated WT . O , R ) Data are related to untreated cells of the same genotype and normalised to WT without HRI KD . Baseline level ( without ER stress ) is depicted by a dashed line . Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test O , R ) ; Nested t - test ( P , S ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4D-G ROS accumulation in WT and Perk KO neurons (N=3) and astrocytes (N=3) ± 20-24 hours of TM-induced ER stress. (D, F) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), astrocytic soma (Phalloidin, white), nuclei ((Δ)Cre, red), ROS (CellROX green, green). Scale bar: 25 μm. (E, G) Quantification of CellROX green intensity. Data information: (E, G, Data are normalised to untreated WT. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (E, G,H mRNA expression of Eif2ak1 (HRI) in HRI KD neurons determined by RT-qPCR, normalised to WT transduced with shRNA control (represented by a dashed line) (N=3). Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis One sample t-test with a hypothetical value of 100 (HI Quantification of the increase in nuclear ATF4 intensity in WT neurons ± HRI KD ± 20 hours of sodium arsenite (Ars) treatment (N=3). J The remaining ATF4 response (%) upon HRI KD, related to ATF4 response of WT transduced with shRNA control (100%, represented by a dashed line) (N=3). Calculated from data in (I): [[(KD_Ars+-KD_Ars-)/KD_Ars-]/[(WT_Ars+-WT_Ars-)/WT_Ars-]]*100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test I, One sample t-test with a hypothetical value of 100 J)K-M eIF2α phosphorylation in HRI KD WT and Perk KO neurons (N=3) ± 20-24 hours of TM-induced ER stress. Representative WB (K), quantification of the ER stress-induced p-eIf2α response (L) and absolute phosphorylated eIF2α level (p-eIF2α/eIF2α) (M). Data information: M, Data are normalised to untreated WT. (L, Data are related to untreated cells of the same genotype and normalised to WT without HRI KD. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test L, Students t-test (M)N-S Protein synthesis (N=3) and ATF4 expression (N=4) in HRI KD WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress. Representative images obtained by (N) confocal and (Q) high-content microscopy. Dendrites (MAP2, white/cyan), nuclei ((Δ)Cre, green/red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis response (O) and puromycin intensity as measure for de novo protein synthesis (P). Quantification of the ER stress-induced ATF4 response (R) and absolute nuclear ATF4 intensity (S). Data information: P, S) Data are normalised to untreated WT. O, R) Data are related to untreated cells of the same genotype and normalised to WT without HRI KD. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test O, R); Nested t-test (P, S).


Figure 5A - F Protein synthesis ( A - C , N = 3 ) and ATF4 expression ( D - F , N = 5 ) in WT and Perk KO neurons overexpressing eIF2αWT or phospho - deficient eIF2αS51A ± 20 - 24 hours of TM - induced ER stress . Representative images obtained by ( A ) confocal and ( D ) high - content microscopy . Dendrites ( MAP2 , white / cyan ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . Quantification of the ER stress - induced protein synthesis ( B ) and ATF4 response ( E ) . Data are normalised to WT eIF2αWT . Quantification showing the percentage change in the ER stress - induced protein synthesis ( C ) and ATF4 ( F ) response induced by eIF2αS51A compared to eIF2αWT . ( C ) Calculated from data in ( B ) : [ ( eIF2αS51A - eIF2αWT ) / eIF2αWT ] * 100 . ( F ) Calculated from data in ( E ) : [ ( eIF2αS51A - eIF2αWT ) / eIF2αWT ] * - 100 . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * * p < 0 . 001 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( B , E , Nested t - test ( C , F ) . Expression of p - eIF2α sensitive ( 5 ' ATF4 - uORFWT ) and p - eIf2α insensitive ( 5 ' ATF4 - uORFMut ) EYFP translational reporters in WT and Perk KO neurons ± 20 - 24 hours of TM - induced ER stress ( N = 3 ) . ( I , K ) Representative confocal images . Dendrites ( MAP2 , cyan ) , nuclei ( ( Δ ) Cre , red ) , endogenous ATF4 ( magenta ) , ATF4 reporter ( green ) . Scale bar : 10 μm . ( J , L ) Quantification of EYFP intensity , normalised to untreated WT . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * * p < 0 . 001 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test J , L )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-F Protein synthesis (A-C, N=3) and ATF4 expression (D-F, N=5) in WT and Perk KO neurons overexpressing eIF2αWT or phospho-deficient eIF2αS51A ± 20-24 hours of TM-induced ER stress. Representative images obtained by (A) confocal and (D) high-content microscopy. Dendrites (MAP2, white/cyan), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (B) and ATF4 response (E). Data are normalised to WT eIF2αWT. Quantification showing the percentage change in the ER stress-induced protein synthesis (C) and ATF4 (F) response induced by eIF2αS51A compared to eIF2αWT. (C) Calculated from data in (B): [(eIF2αS51A-eIF2αWT)/eIF2αWT] *100. (F) Calculated from data in (E): [(eIF2αS51A-eIF2αWT)/eIF2αWT]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E, Nested t-test (C, F).Expression of p-eIF2α sensitive (5'ATF4-uORFWT) and p-eIf2α insensitive (5'ATF4-uORFMut) EYFP translational reporters in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=3). (I, K) Representative confocal images. Dendrites (MAP2, cyan), nuclei ((Δ)Cre, red), endogenous ATF4 (magenta), ATF4 reporter (green). Scale bar: 10 μm. (J, L) Quantification of EYFP intensity, normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test J, L)


Figure 6A - F Protein synthesis and ATF4 expression in ISRIB - treated WT and Perk KO neurons ± 20 - 24 hours of TM - induced ER stress ( N = 4 ) . ( A , D ) Representative images obtained by high - content microscopy . Dendrites ( MAP2 , white ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . Quantification of the ER stress - induced protein synthesis ( B ) and ATF4 ( E ) response . Data are normalised to WT without ISRIB . Baseline level ( without ER stress ) is depicted by dashed line . Quantification showing the percentage change in the ER stress - induced protein synthesis ( C ) and ATF4 ( F ) response induced by ISRIB . ( C ) Calculated from data in ( B ) : [ ( ISRIB + - ISRIB - ) / ISRIB - ] * 100 . ( F ) Calculated from data in ( E ) : [ ( ISRIB + - ISRIB - ) / ISRIB - ] * - 100 . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( B , E ) ; Nested t - test ( C , F ) G - M Protein synthesis and ATF4 expression in 2b5he and 2b5ho astrocytes and neurons ± 20 - 24 hours of TM - induced ER stress in the presence or absence of PERK - i ( Pi ) ( N = 3 ) . ( G , I , K , M ) Representative images obtained by high - content microscopy . Astrocytic soma ( Phalloidin , white ) , dendrites ( MAP2 , white ) , nuclei ( DAPI , red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . ( H , L ) Quantification of puromycin intensity as measure for de novo protein synthesis and ( J , N ) absolute nuclear ATF4 intensity . Data are normalised to untreated WT . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Three - way ANOVA with Tukey ' s post hoc test ( H , J , L , N ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-F Protein synthesis and ATF4 expression in ISRIB-treated WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=4). (A, D) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (B) and ATF4 (E) response. Data are normalised to WT without ISRIB. Baseline level (without ER stress) is depicted by dashed line. Quantification showing the percentage change in the ER stress-induced protein synthesis (C) and ATF4 (F) response induced by ISRIB. (C) Calculated from data in (B): [(ISRIB+-ISRIB-)/ISRIB-]*100. (F) Calculated from data in (E): [(ISRIB+-ISRIB-)/ISRIB-]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E); Nested t-test (C, F)G-M Protein synthesis and ATF4 expression in 2b5he and 2b5ho astrocytes and neurons ± 20-24 hours of TM-induced ER stress in the presence or absence of PERK-i (Pi) (N=3). (G, I, K, M) Representative images obtained by high-content microscopy. Astrocytic soma (Phalloidin, white), dendrites (MAP2, white), nuclei (DAPI, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. (H, L) Quantification of puromycin intensity as measure for de novo protein synthesis and (J, N) absolute nuclear ATF4 intensity. Data are normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Three-way ANOVA with Tukey's post hoc test (H, J, L, N).


Figure 7A Differentially expressed genes ( DEGs ) involved in oxidative stress related processes comparing TM - treated Perk KO and WT neurons . Blue arrow : Ang . B , C Ang mRNA level in WT and Perk KO neurons and astrocytes ± 20 - 24 hours of TM - induced ER stress , determined by mRNA - seq ( N = 3 ) . FPMK : Fragments Per Kilobase of transcript per Million . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( B , C , D , E Representative WB and quantification of ANG protein level ( N = 3 ) . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test E , F , G tRNA unfolding in WT and Perk KO neurons ± 20 - 24 hours of TM - induced ER stress ( N = 4 ) . ( F ) Representative images obtained by high - content microscopy of 1 - methyladenosine ( m1A ) immunofluorescence to determine tRNA unfolding . Dendrites ( MAP2 , white ) , nuclei ( ( Δ ) Cre , red ) , unfolded tRNA ( m1A , green ) . Scale bar : 25 μm . ( G ) Quantification of m1A intensity , normalised to untreated WT . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test G , H - M Abundance of small RNAs in WT and Perk KO astrocytes ( N = 3 ) and neurons ( N = 6 ) ± 20 - 24 hours of TM - induced ER stress , determined by high - resolution automated electrophoresis ( H , K ) Representative gel image . Electropherogram ( I , L ) and zoom ( J , M ) showing quantified fluorescence units ( FU ) of small RNAs , size ranges of tRNAs and tsncRNAs are indicated . Shown is the mean of the biological replicates . N - R Protein synthesis and ATF4 expression in WT and Perk KO neurons ± 20 - 24 hours of TM - induced ER stress in the presence or absence of ANG - I ( N = 3 ) . ( N ) Representative images obtained by high - content microscopy . Dendrites ( MAP2 , white ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesis of proteins ( Puromycin , green ) . Scale bar : 25 μm . Quantification of the ER stress - induced protein synthesis ( O ) and ATF4 response ( Q ) . Data are normalised to WT without ANG - i . Baseline level ( without ER stress ) is depicted by dashed line . Quantification showing the percentage change in the ER stress - induced protein synthesis ( P ) and ATF4 response ( R ) induced by ANG - i . ( P ) Calculated from data in ( O ) : [ ( ANG - i + - ANG - i - ) / ANG - i - ] * 100 . ( R ) Calculated from data in ( Q ) : [ ( ANG - i + - ANG - i - ) / ANG - i - ] * - 100 . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test O , Q ) ; Nested t - test ( P , R ) .

O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Differentially expressed genes (DEGs) involved in oxidative stress related processes comparing TM-treated Perk KO and WT neurons. Blue arrow: Ang.B, C Ang mRNA level in WT and Perk KO neurons and astrocytes ± 20-24 hours of TM-induced ER stress, determined by mRNA-seq (N=3). FPMK: Fragments Per Kilobase of transcript per Million. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, C,D, E Representative WB and quantification of ANG protein level (N=3). Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test E,F, G tRNA unfolding in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=4). (F) Representative images obtained by high-content microscopy of 1-methyladenosine (m1A) immunofluorescence to determine tRNA unfolding. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), unfolded tRNA (m1A, green). Scale bar: 25 μm. (G) Quantification of m1A intensity, normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test G,H-M Abundance of small RNAs in WT and Perk KO astrocytes (N=3) and neurons (N=6) ± 20-24 hours of TM-induced ER stress, determined by high-resolution automated electrophoresis (H, K) Representative gel image. Electropherogram (I, L) and zoom (J, M) showing quantified fluorescence units (FU) of small RNAs, size ranges of tRNAs and tsncRNAs are indicated. Shown is the mean of the biological replicates.N-R Protein synthesis and ATF4 expression in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress in the presence or absence of ANG-I (N=3). (N) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesis of proteins (Puromycin, green). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (O) and ATF4 response (Q). Data are normalised to WT without ANG-i. Baseline level (without ER stress) is depicted by dashed line. Quantification showing the percentage change in the ER stress-induced protein synthesis (P) and ATF4 response (R) induced by ANG-i. (P) Calculated from data in (O): [(ANG-i+-ANG-i-)/ANG-i-]*100. (R) Calculated from data in (Q): [(ANG-i+-ANG-i-)/ANG-i-]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test O, Q); Nested t-test (P, R).


Figure 1B - C . Graphs showing the relative weight ( B ; n = 15 WT , 12 ECko ; curve comparison ) and the survival ( C ; n = 15 WT , 11 ECko ; Log - Rank test ) of mice upon tamoxifen treatment . Data information : Data shown as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant . D . Representative images of the intestine of Casp8WT and Casp8ECko mice , with mild and severe hemorrhages at a late disease stage . E - F . Representative images of H & E staining ( E ) and quantification of intestinal pathology ( F ) of the small intestine at an early disease stage in Casp8WT and Casp8ECko mice ( enteropathy score ; n = 6 WT , 6 ECko ; two tailed unpaired Student ' s t - test ) . Scale bars : 100µm ; insets 50µm . Data information : Data shown as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant . G - H . Quantification of intestinal pathology ( enteropathy score , G ; n = 8 WT , 7 ECko ; two tailed unpaired Student ' s t - test ) and hemorrhages into the mucosa ( H ; n = 4 WT , 5 ECko ; two tailed unpaired Student ' s t - test ) at a late disease stage in Casp8WT and Casp8ECko mice . Data information : Data shown as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant . I . Overview of H & E stained swiss roll preparations of Casp8WT and Casp8ECko mice . Scale bars : 2mm . J . Representative images of H & E staining of the small intestine of Casp8WT ( j ' ) and Casp8ECko mice with mild ( j ' ' ) and more severely ( j ' ' ' ) affected areas at a late disease stage ( white arrows point to hemorrhages , asterisk points to focal erosion of the epithelium ) . Scale bars : 100µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B-C. Graphs showing the relative weight (B; n= 15 WT, 12 ECko; curve comparison) and the survival (C; n= 15 WT, 11 ECko; Log-Rank test) of mice upon tamoxifen treatment. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.D. Representative images of the intestine of Casp8WT and Casp8ECko mice, with mild and severe hemorrhages at a late disease stage.E-F. Representative images of H&E staining (E) and quantification of intestinal pathology (F) of the small intestine at an early disease stage in Casp8WT and Casp8ECko mice (enteropathy score; n= 6 WT, 6 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm; insets 50µm. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.G-H. Quantification of intestinal pathology (enteropathy score, G; n= 8 WT, 7 ECko; two tailed unpaired Student's t-test) and hemorrhages into the mucosa (H; n= 4 WT, 5 ECko; two tailed unpaired Student's t-test) at a late disease stage in Casp8WT and Casp8ECko mice. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.I. Overview of H&E stained swiss roll preparations of Casp8WT and Casp8ECko mice. Scale bars: 2mm.J. Representative images of H&E staining of the small intestine of Casp8WT (j') and Casp8ECko mice with mild (j'') and more severely (j''') affected areas at a late disease stage (white arrows point to hemorrhages, asterisk points to focal erosion of the epithelium). Scale bars: 100µm.


Figure 2A - B . QPCR analysis of genes associated to immune and bacterial response ( A ) , vessel permeability and EC activation ( B ) in small intestine lysates at a late disease timepoint in Casp8WT and Casp8ECko mice ( n = 4 WT , 3 - 4 ECko , biological replicates , multiple t - test with Holm - Sidak method ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the complement cascade ( C ) ( n = 4 WT , 4 ECko , multiple t - test with Holm - Sidak method ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the mucosal healing ( D ) ( n = 4 WT , 4 ECko , multiple t - test with Holm - Sidak method ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Representative images ( E ; area between dotted lines shows crypts ) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice ( n = 5 WT , 6 ECko Scale bars : 100µm . quantification ( F ) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice ( n = 5 WT , 6 ECko two tailed unpaired Student ' s t - test ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . G . QPCR analysis of the proliferation markers Ki67 and c - myc in ileal lysates from late disease stage Casp8WT and Casp8ECko mice ( n = 4 WT , 3 - 4ECko , two - way ANOVA with Sidak method ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . H . Representative images of E - Cadherin staining , showing intact epithelial barrier integrity in Casp8ECko mice in healthy areas ( h ' ) , and disrupted barrier integrity in intestinal lesions ( h ' ' ) at late disease stages compared to Casp8WT mice ( n = 4 WT , 4 ECko ) . Scale bars : 100µm . I . Representative pictures of CD45 and NIMPR14 staining at late disease stages ( n = 5 WT , 5 ECko ) , showing accumulation of CD45 + and NIMPR14 + cells ( black arrowheads ) in Casp8ECko intestine at sites of impaired epithelial barrier integrity and tissue necrosis ( red asterisks ) . Scale bars : 100µm . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-B. QPCR analysis of genes associated to immune and bacterial response (A), vessel permeability and EC activation (B) in small intestine lysates at a late disease timepoint in Casp8WT and Casp8ECko mice (n= 4 WT, 3-4 ECko, biological replicates, multiple t-test with Holm-Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the complement cascade (C) (n= 4 WT, 4 ECko, multiple t-test with Holm- Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the mucosal healing (D) (n= 4 WT, 4 ECko, multiple t-test with Holm- Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.Representative images (E; area between dotted lines shows crypts) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice (n= 5 WT, 6 ECko Scale bars: 100µm.quantification (F) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice (n= 5 WT, 6 ECko two tailed unpaired Student's t-test). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.G. QPCR analysis of the proliferation markers Ki67 and c-myc in ileal lysates from late disease stage Casp8WT and Casp8ECko mice (n= 4 WT, 3-4ECko, two-way ANOVA with Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.H. Representative images of E-Cadherin staining, showing intact epithelial barrier integrity in Casp8ECko mice in healthy areas (h'), and disrupted barrier integrity in intestinal lesions (h'') at late disease stages compared to Casp8WT mice (n= 4 WT, 4 ECko). Scale bars: 100µm.I. Representative pictures of CD45 and NIMPR14 staining at late disease stages (n= 5 WT, 5 ECko), showing accumulation of CD45+ and NIMPR14+ cells (black arrowheads) in Casp8ECko intestine at sites of impaired epithelial barrier integrity and tissue necrosis (red asterisks). Scale bars: 100µm.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.


Figure 3A . Representative pictures showing Peyer ´ s Patches in Casp8WT mice , and different severities of hemorrhage formation in Casp8ECko mice at an early disease stage . Scale bars : 1mm . Quantification of lymphatic vessel density ( B ) in Peyer ' s Patches in Casp8WT and Casp8ECko mice at an early disease stage ( n = 9 WT , 7 - 10 ECko Peyer ' s Patches are pooled from 4 - 6 mice per genotype ; one - way ANOVA with tukey ' s multiple comparison ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant . representative pictures of CD31 ( pan EC ) and Lyve1 ( lymphatic ) staining ( C ) in Peyer ' s Patches in Casp8WT and Casp8ECko mice at an early disease stage ( n = 9 WT , 7 - 10 ECko Peyer ' s Patches are pooled from 4 - 6 mice per genotype Scale bars : 200µm . D - F . Representative images ( D ) and quantifications ( E , F ) of stainings for CD31 , Lyve1 and DAPI ( nuclei ) in small intestinal wholemounts at an early disease stage ( blue arrowheads point to defective lacteals , white arrowheads indicate edema ) in Casp8ECko mice ( E ; n = 7 WT , 6 ECko . F ; n = 7 WT , 10 ECko ; two tailed unpaired Student ' s t - test ) . Scale bars : 100µm . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant . G . Representative images of small intestine sections of mice injected with a 70kDa fluorescently labeled dextran and stained for CD31 at an early disease stage . White arrowheads point to extravasated dextran in Casp8ECko mice ( n = 5 WT , 5 ECko ) . Scale bars : 50µm . H . Quantification of extravasated dextran in small intestine and brain lysates in Casp8WT and Casp8ECko mice at an early disease stage ( n = 4 WT , 4 - 5 ECko ; two - way ANOVA with tukey ' s multiple comparison ) . RFU : relative fluorescent units . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant . I . Representative images of CD31 and VE - Cadherin staining in intestinal wholemounts at an early disease stage ( n = 5 WT , 5 ECko ) in Casp8WT and Casp8ECko mice ( red arrowheads point to VE - Cadherin ' empty ' vessel patches ) . Scale bars : 25µm .

O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Representative pictures showing Peyer´s Patches in Casp8WT mice, and different severities of hemorrhage formation in Casp8ECko mice at an early disease stage. Scale bars: 1mm.Quantification of lymphatic vessel density (B) in Peyer's Patches in Casp8WT and Casp8ECko mice at an early disease stage (n = 9 WT, 7-10 ECko Peyer's Patches are pooled from 4-6 mice per genotype; one-way ANOVA with tukey's multiple comparison). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.representative pictures of CD31 (pan EC) and Lyve1 (lymphatic) staining (C) in Peyer's Patches in Casp8WT and Casp8ECko mice at an early disease stage (n = 9 WT, 7-10 ECko Peyer's Patches are pooled from 4-6 mice per genotype Scale bars: 200µm.D-F. Representative images (D) and quantifications (E, F) of stainings for CD31, Lyve1 and DAPI (nuclei) in small intestinal wholemounts at an early disease stage (blue arrowheads point to defective lacteals, white arrowheads indicate edema) in Casp8ECko mice (E; n= 7 WT, 6 ECko. F; n= 7 WT, 10 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm. Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.G. Representative images of small intestine sections of mice injected with a 70kDa fluorescently labeled dextran and stained for CD31 at an early disease stage. White arrowheads point to extravasated dextran in Casp8ECko mice (n= 5 WT, 5 ECko). Scale bars: 50µm. H. Quantification of extravasated dextran in small intestine and brain lysates in Casp8WT and Casp8ECko mice at an early disease stage (n= 4 WT, 4-5 ECko; two-way ANOVA with tukey's multiple comparison). RFU: relative fluorescent units. Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.I. Representative images of CD31 and VE-Cadherin staining in intestinal wholemounts at an early disease stage (n= 5 WT, 5 ECko) in Casp8WT and Casp8ECko mice (red arrowheads point to VE-Cadherin 'empty' vessel patches). Scale bars: 25µm.


Figure 4A - B . Representative images ( A ) and quantification ( B ) of CD31 , TUNEL and DAPI staining in small intestine sections of Casp8WT and Casp8ECko mice at an early disease stage ( red dotted lines : epithelium ; white dotted lines : endothelium ) in ( n = 5 WT , 5 ECko , multiple t - tests with Holm - Sidak method ) . Scale bars : 100µm . Data information : All data is shown as mean ± SEM . ns : not significant . D - E . Graphs showing survival and relative weight of Casp8WT / MLKLko and Casp8ECko / MLKLko mice upon tamoxifen treatment . ( D ; n = 5 - 8 WT / MLKLko , 5 - 8 ECko / MLKLko ; Log - Rank test , E ; n = 7 WT , 8 ECko ; two tailed unpaired Student ' s t - test ) . F . Representative pictures of intestines of Casp8WT / MLKLko and Casp8ECko / MLKLko mice at 30 days after tamoxifen treatment ( n = 8 WT , 8 ECko ) . G . Representative images of stainings for CD31 , Lyve1 and DAPI in small intestinal wholemounts from Casp8WT / MLKLko and Casp8ECko / MLKLko mice at 30 days after tamoxifen treatment . Scale bars : 100µm . H - K . Quantification of vessel area ( H , I ) and lacteal length ( J , K ) at two different timepoints after tamoxifen treatment in Casp8WT / MLKLko and Casp8ECko / MLKLko mice ( n = 4 - 6 WT / MLKLko , 5 ECko / MLKLko ; two tailed unpaired Student ' s t - test ) . Data information : All data is shown as mean ± SEM . ns : not significant . L . Graph showing quantification of extravasated 70kDa Dextran from small intestine tissue of Casp8WT / MLKLko and Casp8ECko / MLKLko mice at 30 days after tamoxifen treatment ( n = 7 WT / MLKLko , 6 ECko / MLKLko ; two tailed unpaired Student ' s t - test ) . Data information : All data is shown as mean ± SEM . ns : not significant . M - N . Representative pictures ( M ) and quantification ( N ) of lymphatic vessel density in Peyer ' s Patches of Casp8WT / MLKLko and Casp8ECko / MLKLko mice at 140 days after tamoxifen treatment ( n = 8 WT , 7 ECko Peyer ' s Patches are pooled from 4 mice per genotype ; two - tailed unpaired students t - test ) . Scale bars : 200µm . Data information : All data is shown as mean ± SEM . ns : not significant . O . Representative pictures of H & E stainings of small intestine tissue of Casp8WT / MLKLko and Casp8ECko / MLKLko mice ( n = 4 - 6 WT , 4 - 5 ECko ) . Scale bars : 100µm . P - Q . Quantification of intestinal pathology in Casp8WT / MLKLko and Casp8ECko / MLKLko mice at two different timepoints after tamoxifen treatment ( enteropathy score ; n = 4 - 6 WT / MLKLko , 5 ECko / MLKLko ; two tailed unpaired Student ' s t - test ) . Data information : All data is shown as mean ± SEM . ns : not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-B. Representative images (A) and quantification (B) of CD31, TUNEL and DAPI staining in small intestine sections of Casp8WT and Casp8ECko mice at an early disease stage (red dotted lines: epithelium; white dotted lines: endothelium) in (n= 5 WT, 5 ECko, multiple t-tests with Holm-Sidak method). Scale bars: 100µm. Data information: All data is shown as mean ± SEM. ns: not significant.D-E. Graphs showing survival and relative weight of Casp8WT/MLKLko and Casp8ECko/MLKLko mice upon tamoxifen treatment. (D; n= 5-8 WT/ MLKLko, 5-8 ECko/MLKLko; Log-Rank test, E; n= 7 WT, 8 ECko; two tailed unpaired Student's t-test).F. Representative pictures of intestines of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 8 WT, 8 ECko).G. Representative images of stainings for CD31, Lyve1 and DAPI in small intestinal wholemounts from Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment. Scale bars: 100µm.H-K. Quantification of vessel area (H, I) and lacteal length (J, K) at two different timepoints after tamoxifen treatment in Casp8WT/MLKLko and Casp8ECko/MLKLko mice (n= 4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.L. Graph showing quantification of extravasated 70kDa Dextran from small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 7 WT/MLKLko, 6 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.M-N. Representative pictures (M) and quantification (N) of lymphatic vessel density in Peyer's Patches of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 140 days after tamoxifen treatment (n = 8 WT, 7 ECko Peyer's Patches are pooled from 4 mice per genotype; two-tailed unpaired students t-test). Scale bars: 200µm. Data information: All data is shown as mean ± SEM. ns: not significant.O. Representative pictures of H&E stainings of small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice (n= 4-6 WT, 4-5 ECko). Scale bars: 100µm. P-Q. Quantification of intestinal pathology in Casp8WT/MLKLko and Casp8ECko/MLKLko mice at two different timepoints after tamoxifen treatment (enteropathy score; n=4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.


Figure 5A . Representative pictures of TUNEL staining together with CD31 and Lyve - 1 in Peyer ' s Patches of Casp8WT and Casp8ECko mice . TUNEL + LECs , but not BECs could already be seen in non - hemorrhagic Peyer ' s Patches ( a ' ) , and further accumulated in hemorrhagic Peyer ' s Patches ( a ' ' ) . White dotted areas outline location of the lymphatic plexus . White arrowheads point to TUNEL + LECs ( note that necroptotic LECs only weakly express Lyve - 1 ) . Scale bars : 50µm , insets : 10µm . C . Survival ( n = 5 WT , 6 BECko ; Log - Rank test ) of Casp8WT and Casp8ECko mice upon tamoxifen treatment . D . Representative pictures of intestines of Casp8WT and Casp8BECko mice . Representative pictures of H & E staining of small intestine of Casp8WT and Casp8BECko mice ( E ) , enteropathy score ( F ; n = 5 WT , 6 ECko , two tailed unpaired Student ' s t - test ) Scale bars : 100µm , insets : 50µm . Data information : Data shown as mean ± SEM . ns : not significant . mucosal hemorrhages ( G ; n = 5 WT , 6 ECko , two tailed unpaired Student ' s t - test ) . Data information : Data shown as mean ± SEM . ns : not significant . Representative images of stainings for CD31 , Lyve1 and DAPI in small intestinal wholemounts ( H ) and quantification of vessel density ( I ) in Casp8BECko compared to Casp8WT mice ( I ; n = 5 WT , 6 BECko ; two tailed unpaired Student ' s t - test , Scale bars : 100µm . Data information : Data shown as mean ± SEM . ns : not significant . quantification of lacteal length ( J ) in Casp8BECko compared to Casp8WT mice ; n = 5 WT , 5 BECko ; two tailed unpaired Student ' s t - test with Welch ' s correction ) . Data information : Data shown as mean ± SEM . ns : not significant . K - L . Representative pictures ( K ) and quantification ( L ) of lymphatic vessel density Peyer ' s Patches in Casp8WT and Casp8BECko mice ( n = 10 WT , 11 BECko Peyer ' s Patches from 5 - 6 mice per genotype ; two tailed unpaired Student ' s t - test ) . Scale bars : 200µm . Data information : Data shown as mean ± SEM . ns : not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Representative pictures of TUNEL staining together with CD31 and Lyve-1 in Peyer's Patches of Casp8WT and Casp8ECko mice. TUNEL+ LECs, but not BECs could already be seen in non-hemorrhagic Peyer's Patches (a'), and further accumulated in hemorrhagic Peyer's Patches (a''). White dotted areas outline location of the lymphatic plexus. White arrowheads point to TUNEL+ LECs (note that necroptotic LECs only weakly express Lyve-1). Scale bars: 50µm, insets: 10µm.C. Survival (n= 5 WT, 6 BECko; Log-Rank test) of Casp8WT and Casp8ECko mice upon tamoxifen treatment.D. Representative pictures of intestines of Casp8WT and Casp8BECko mice.Representative pictures of H&E staining of small intestine of Casp8WT and Casp8BECko mice (E), enteropathy score (F; n= 5 WT, 6 ECko, two tailed unpaired Student's t-test) Scale bars: 100µm, insets: 50µm. Data information: Data shown as mean ± SEM. ns: not significant.mucosal hemorrhages (G; n= 5 WT, 6 ECko, two tailed unpaired Student's t-test). Data information: Data shown as mean ± SEM. ns: not significant.Representative images of stainings for CD31, Lyve1 and DAPI in small intestinal wholemounts (H) and quantification of vessel density (I) in Casp8BECko compared to Casp8WT mice (I; n= 5 WT, 6 BECko; two tailed unpaired Student's t-test, Scale bars: 100µm. Data information: Data shown as mean ± SEM. ns: not significant.quantification of lacteal length (J) in Casp8BECko compared to Casp8WT mice ; n= 5 WT, 5 BECko; two tailed unpaired Student's t-test with Welch's correction). Data information: Data shown as mean ± SEM. ns: not significant.K-L. Representative pictures (K) and quantification (L) of lymphatic vessel density Peyer's Patches in Casp8WT and Casp8BECko mice (n = 10 WT, 11 BECko Peyer's Patches from 5-6 mice per genotype; two tailed unpaired Student's t-test). Scale bars: 200µm. Data information: Data shown as mean ± SEM. ns: not significant.


Figure 6B . Survival graph after intravenous TNF - α injection of Casp8WT vs . Casp8ECko mice within the first 24h ( n = 11 WT , 11 ECko ; Log - Rank test ) . C . Representative images of the small intestines 24h after TNF - α injection in Casp8WT and Casp8ECko mice ( blue arrowheads point to Peyer ' s Patches ) . D - E . Representative pictures of Peyer ' s Patches ( D ) and quantification of lymphatic vessel density ( E ) in Casp8WT , Casp8ECko and Casp8ECko / MLKLko mice injected with TNF - α ( n = 7 WT , 5 ECko , 15 ECko / MLKLko . Peyer ' s Patches are pooled from 3 - 5 mice per genotype ) . Scale bars : 200µm . Data information : All data is shown as mean ± SEM . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; ns : not significant . F . Representative pictures of TUNEL staining together with CD31 and Lyve - 1 in Peyer ' s Patches of Casp8WT , Casp8ECko and Casp8ECko / MLKLko mice injected with TNF - α . TUNEL + LECs , but not BECs could already be seen in Casp8ECko , but neither in Casp8WT nor in Casp8ECko / MLKLko mice . White dotted areas outline location of the lymphatic plexus . Yellow arrowheads point to TUNEL + ECs ) . Scale bars : 50µm . H . Survival graph of Casp8WT and Casp8ECko mice after Enbrel ® treatment ( n = 7 WT , 5 ECko + IgG , 8 ECko + Enbrel ; Log - Rank test ) . Representative images of small intestines ( I ) of Casp8ECko mice after Enbrel ® treatment Scale bars : 100µm . Representative images of 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 .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B. Survival graph after intravenous TNF-α injection of Casp8WT vs. Casp8ECko mice within the first 24h (n=11 WT, 11 ECko; Log-Rank test).C. Representative images of the small intestines 24h after TNF-α injection in Casp8WT and Casp8ECko mice (blue arrowheads point to Peyer's Patches).D-E. Representative pictures of Peyer's Patches (D) and quantification of lymphatic vessel density (E) in Casp8WT, Casp8ECko and Casp8ECko/MLKLko mice injected with TNF-α (n=7 WT, 5 ECko, 15 ECko/MLKLko. Peyer's Patches are pooled from 3-5 mice per genotype). Scale bars: 200µm. Data information: All data is shown as mean ± SEM. * P<0.05, *** P <0.001, **** P <0.0001; ns: not significant.F. Representative pictures of TUNEL staining together with CD31 and Lyve-1 in Peyer's Patches of Casp8WT, Casp8ECko and Casp8ECko/MLKLko mice injected with TNF-α. TUNEL+ LECs, but not BECs could already be seen in Casp8ECko, but neither in Casp8WT nor in Casp8ECko/MLKLko mice. White dotted areas outline location of the lymphatic plexus. Yellow arrowheads point to TUNEL+ECs). Scale bars: 50µm.H. Survival graph of Casp8WT and Casp8ECko mice after Enbrel® treatment (n=7 WT, 5 ECko + IgG, 8 ECko + Enbrel; Log-Rank test).Representative images of small intestines (I) of Casp8ECko mice after Enbrel® treatment Scale bars: 100µm.Representative images of 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.