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

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

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.


D - G ROS accumulation in WT Perk KO neurons ( N = 3 ) astrocytes ( N = 3 ) ± 20 - 24 hours of TM - induced ER stress . ( D , F ) Representative obtained by high - content microscopy . Dendrites ( MAP2 , ) , astrocytic soma ( Phalloidin , ) , nuclei ( ( Δ ) Cre , red ) , ROS ( CellROX green , green ) . Scale bar : 25 μm . ( E , G ) of CellROX green . Data information : ( E , G , Data are normalised to untreated WT . Data are presented as mean ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ns : not significant . Statistical : Two - way ANOVA with Tukey ' s post hoc ( E , G ,

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

D-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 ) ( N = 3 ) . Data information : Data are presented as mean ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ns : not significant . Statistical One sample t - with a hypothetical value of 100 (

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

H 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 (H


I of increase in ATF4 in WT neurons ± HRI KD ± 20 hours of sodium arsenite ( Ars ) treatment ( N = 3 ) . J remaining ATF4 response ( % ) upon HRI KD , related to ATF4 response of WT transduced with shRNA control ( 100 % , represented by a dashed ) ( 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 ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ns : not significant . Statistical : Two - way ANOVA with Tukey ' s post hoc I , One sample t - with a hypothetical value of 100 J )

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

I 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 Perk KO neurons ( N = 3 ) ± 20 - 24 hours of TM - induced ER stress . Representative WB ( K ) , of ER stress - induced p - eIf2α response ( L ) absolute phosphorylated eIF2α level ( p - eIF2α / eIF2α ) ( M ) . Data information : M , Data are normalised to untreated WT . ( L , Data are related to untreated of same genotype normalised to WT without HRI KD . Baseline level ( without ER stress ) is depicted by a dashed . Data are presented as mean ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ns : not significant . Statistical : Two - way ANOVA with Tukey ' s post hoc L , Students t - ( M )

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

K-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 synthesis ( N = 3 ) ATF4 expression ( N = 4 ) in HRI KD WT Perk KO neurons ± 20 - 24 hours of TM - induced ER stress . Representative obtained by ( N ) confocal ( Q ) high - content microscopy . Dendrites ( MAP2 , / cyan ) , nuclei ( ( Δ ) Cre , green / red ) , de novo synthesized ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . of ER stress - induced synthesis response ( O ) puromycin as measure for de novo synthesis ( P ) . of ER stress - induced ATF4 response ( R ) absolute ATF4 ( S ) . Data information : P , S ) Data are normalised to untreated WT . O , R ) Data are related to untreated of same genotype normalised to WT without HRI KD . Baseline level ( without ER stress ) is depicted by a dashed . Data are presented as mean ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ns : not significant . Statistical : Two - way ANOVA with Tukey ' s post hoc O , R ) ; Nested t - ( P , S ) .

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

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).


A - F synthesis ( A - C , N = 3 ) ATF4 expression ( D - F , N = 5 ) in WT Perk KO neurons overexpressing eIF2αWT or phospho - deficient eIF2αS51A ± 20 - 24 hours of TM - induced ER stress . Representative obtained by ( A ) confocal ( D ) high - content microscopy . Dendrites ( MAP2 , / cyan ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesized ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . of ER stress - induced synthesis ( B ) ATF4 response ( E ) . Data are normalised to WT eIF2αWT . showing change in ER stress - induced synthesis ( C ) 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 ± . N : Biological replicate . Relevant P - values are indicated : * * * p < 0 . 001 , * * * * p < 0 . 0001 ns : not significant . Statistical : Two - way ANOVA with Tukey ' s post hoc ( B , E , Nested t - ( C , F ) .

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

A-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 ) p - eIf2α insensitive ( 5 ' ATF4 - uORFMut ) EYFP translational reporters in WT Perk KO neurons ± 20 - 24 hours of TM - induced ER stress ( N = 3 ) . ( I , K ) Representative confocal . Dendrites ( MAP2 , cyan ) , nuclei ( ( Δ ) Cre , red ) , endogenous ATF4 ( magenta ) , ATF4 reporter ( green ) . Scale bar : 10 μm . ( J , L ) of EYFP , normalised to untreated WT . Data information : Data are presented as mean ± . N : Biological replicate . Relevant P - values are indicated : * * * p < 0 . 001 , * * * * p < 0 . 0001 ns : not significant . Statistical : Two - way ANOVA with Tukey ' s post hoc J , L )

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

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)


A - F synthesis ATF4 expression in ISRIB - treated WT Perk KO neurons ± 20 - 24 hours of TM - induced ER stress ( N = 4 ) . ( A , D ) Representative obtained by high - content microscopy . Dendrites ( MAP2 , ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesized ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . of ER stress - induced synthesis ( B ) ATF4 ( E ) response . Data are normalised to WT without ISRIB . Baseline level ( without ER stress ) is depicted by dashed . showing change in ER stress - induced synthesis ( C ) 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 ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ns : not significant . Statistical : Two - way ANOVA with Tukey ' s post hoc ( B , E ) ; Nested t - ( C , F )

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

A-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 synthesis ATF4 expression in 2b5he 2b5ho astrocytes neurons ± 20 - 24 hours of TM - induced ER stress in presence or absence of PERK - i ( Pi ) ( N = 3 ) . ( G , I , K , M ) Representative obtained by high - content microscopy . Astrocytic soma ( Phalloidin , ) , dendrites ( MAP2 , ) , nuclei ( DAPI , red ) , de novo synthesized ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . ( H , L ) of puromycin as measure for de novo synthesis ( J , N ) absolute ATF4 . Data are normalised to untreated WT . Data information : Data are presented as mean ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ns : not significant . Statistical : Three - way ANOVA with Tukey ' s post hoc ( H , J , L , N ) .

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

G-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).


A Differentially expressed genes ( DEGs ) involved in oxidative stress related processes comparing TM - treated Perk KO WT neurons . Blue arrow : Ang .

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

A 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 Perk KO neurons 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 ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical : Two - way ANOVA with Tukey ' s post hoc ( B , C ,

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

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 of ANG level ( N = 3 ) . Data information : Data are presented as mean ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical : Two - way ANOVA with Tukey ' s post hoc E ,

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

D, E Representative 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 Perk KO neurons ± 20 - 24 hours of TM - induced ER stress ( N = 4 ) . ( F ) Representative obtained by high - content microscopy of 1 - methyladenosine ( m1A ) immunofluorescence to determine tRNA unfolding . Dendrites ( MAP2 , ) , nuclei ( ( Δ ) Cre , red ) , unfolded tRNA ( m1A , green ) . Scale bar : 25 μm . ( G ) of m1A , normalised to untreated WT . Data information : Data are presented as mean ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical : Two - way ANOVA with Tukey ' s post hoc G ,

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

F, 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 Perk KO astrocytes ( N = 3 ) 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 ) zoom ( J , M ) showing quantified units ( FU ) of small RNAs , ranges of tRNAs tsncRNAs are indicated . Shown is mean of biological replicates .

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

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 synthesis ATF4 expression in WT Perk KO neurons ± 20 - 24 hours of TM - induced ER stress in presence or absence of ANG - I ( N = 3 ) . ( N ) Representative obtained by high - content microscopy . Dendrites ( MAP2 , ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesis of ( Puromycin , green ) . Scale bar : 25 μm . of ER stress - induced synthesis ( O ) ATF4 response ( Q ) . Data are normalised to WT without ANG - i . Baseline level ( without ER stress ) is depicted by dashed . showing change in ER stress - induced synthesis ( P ) 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 ± . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical : Two - way ANOVA with Tukey ' s post hoc O , Q ) ; Nested t - ( P , R ) .

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

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).


( B ) U2OS stably expressing FLAG - TIP60 or empty vector ( vec ) were subjected to FLAG - immunoprecipitation from lysates analysed by Western blotting

(B) U2OS stably expressing FLAG-TIP60 or empty vector (vec) were subjected to FLAG- immunoprecipitation from nuclear lysates and analysed by Western blotting


( C ) HEK293T were transfected with pcDNA3 . 1 encoding FLAG - TIP60WT , FLAG - TIP60S90A or empty vector ( vec ) . samples were subjected to FLAG affinity - purification , dephosphorylated with shrimp alkaline phosphatase ( rSAP ) incubated as indicated with CDK9 / cyclinT1 in presence of ATP . Phosphorylation of TIP60 was analysed by a phosphoS90 - specific TIP60 antibody , blot was further probed with antibodies specific for FLAG CDK9

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

(C) HEK293T cells were transfected with pcDNA3.1 encoding FLAG-TIP60WT, FLAG- TIP60S90A or empty vector (vec). The samples were subjected to FLAG affinity-purification, dephosphorylated with shrimp alkaline phosphatase (rSAP) and incubated as indicated with CDK9/cyclinT1 in presence of ATP. Phosphorylation of TIP60 was analysed by a phosphoS90-specific TIP60 antibody, and the blot was further probed with antibodies specific for FLAG and CDK9


( D ) p53 - / - hRasG12V MEF were treated with DMSO or 1µM SNS - 032 ( SNS ) for 1h . lysates were analysed by Western blotting

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

(D) p53-/- hRasG12V MEF were treated with DMSO or 1µM SNS-032 (SNS) for 1h. Nuclear lysates were analysed by Western blotting


( E ) p53 - / - hRasG12V MEF were treated with DMSO , 1µM SNS - 032 ( SNS ) or 50µM DRB followed by addition of 100nM Calyculin A as indicated . lysates were analysed by Western blotting

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

(E) p53-/- hRasG12V MEF were treated with DMSO, 1µM SNS-032 (SNS) or 50µM DRB followed by addition of 100nM Calyculin A as indicated. Nuclear lysates were analysed by Western blotting


( F ) U2OS stably expressing FLAG - TIP60WT were transfected with a pool of four different CDK9 - targeting siRNAs or a control siRNA targeting Luciferase . lysates were analysed by Western blotting

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

(F) U2OS cells stably expressing FLAG-TIP60WT were transfected with a pool of four different CDK9-targeting siRNAs or a control siRNA targeting Luciferase. Nuclear lysates were analysed by Western blotting


( A ) Localization of TIP60 was analysed employing U2OS stably expressing FLAG - TIP60WT , FLAG - TIP60S90A or FLAG - TIP60S86A . Nucleoplasm ( nuc . ) ( chr . ) fractions were generated were subjected to Western blot

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

(A) Localization of TIP60 was analysed employing U2OS cells stably expressing FLAG- TIP60WT, FLAG-TIP60S90A or FLAG-TIP60S86A. Nucleoplasm (nuc.) and chromatin (chr.) fractions were generated and were subjected to Western blot analysis


( B ) experiment as described in ( A ) was repeated twice with U2OS twice with Ba / F3 blots were quantified . Each dot represents ration of FLAG - TiIP60 to H4 in fraction of altogether four experiments as shown in ( A )

O O O O O O O O O O O O O O O O O O O O O O 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) The experiment as described in (A) was repeated twice with U2OS cells and twice with Ba/F3 cells and the blots were quantified. Each dot represents the ration of FLAG-TiIP60 to H4 in the chromatin fraction of altogether four individual experiments as shown in (A)


( C ) extracts of U2OS stably expressing FLAG - tagged TIP60WT or TIP60S90A , or FLAG - tagged chromodomain mutants TIP60F50A , TIP60Y47A , TIP60Y44F or empty vector ( vec ) were subjected to fractionation into nucleoplasm ( nuc . ) ( chr . ) fraction . Both fractions were analysed by Western blotting

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

(C) Nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT or TIP60S90A, or FLAG-tagged chromodomain mutants TIP60F50A, TIP60Y47A, TIP60Y44F or the empty vector (vec) were subjected to fractionation into nucleoplasm (nuc.) and chromatin (chr.) fraction. Both fractions were analysed by Western blotting


( D ) extracts of U2OS stably expressing FLAG - tagged TIP60WT , TIP60S90A , TIP60S90E , TIP60S90D or empty vector ( vec ) were subjected to fractionation into nucleoplasm ( nuc . ) ( chr . ) fraction . Both fractions were analysed by Western

(D) Nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A, TIP60S90E, TIP60S90D or the empty vector (vec) were subjected to fractionation into nucleoplasm (nuc.) and chromatin (chr.) fraction. Both fractions were analysed by Western blotting


( E ) U2OS stably expressing FLAG - tagged TIP60WT , TIP60S90A or empty vector ( vec ) were treated with 2 µM SNS - 032 ( SNS ) for 3 hours as indicated . nucleus was fractionated into nucleoplasm . Both fractions were analysed by Western

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

(E) U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A or empty vector (vec) were treated with 2 µM SNS-032 (SNS) for 3 hours as indicated. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting


( A ) FLAG - immunoprecipitations were performed from extracts of U2OS stably expressing FLAG - tagged TIP60WT , TIP60S90A , TIP60S86A or empty vector ( vec ) . Immunoprecipitated material input lysate were subjected to Western blot ( B ) experiment as shown in ( A ) was repeated three times . graphs error bars represent mean standard deviation of these three experiments ( one - way ANOVA , Fisher " s LSD , WT vs . S86A p = 0 , 5992 , WT vs . S90A p = 0 , 0429 , S90A vs . S86A p = 0 , 0176 ( * p < 0 , 05 ; n . s . not significant , p > 0 , 05 ) )

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

(A) FLAG- immunoprecipitations were performed from nuclear extracts of U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A, TIP60S86A or empty vector (vec). Immunoprecipitated material and input lysate were subjected to Western blot analysis (B) The experiment as shown in (A) was repeated three times. The graphs and error bars represent mean and standard deviation of these three individual experiments (one-way ANOVA, Fisher"s LSD test, WT vs. S86A p=0,5992, WT vs. S90A p=0,0429, S90A vs. S86A p=0,0176 (*p < 0,05; n.s. not significant, p > 0,05))


( C ) HEK293 were transfected with pcDNA3 . 1 encoding FLAG - TIP60WT , FLAG - TIP60S90A or empty vector . FLAG - immunoprecipitations were performed from extracts . Immunoprecipitated material input lysate were subjected to Western blot

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

(C) HEK293 cells were transfected with pcDNA3.1 encoding FLAG-TIP60WT, FLAG-TIP60S90A or empty vector. FLAG-immunoprecipitations were performed from nuclear extracts. Immunoprecipitated material and input lysate were subjected to Western blot analysis


( D ) U2OS stably expressing FLAG - TIP60WT or empty vector were treated with 2µM SNS - 032 ( SNS ) for 2 hours as indicated . FLAG - immunoprecipitations were performed from extracts . This material input lysate were subjected to Western blot

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

(D) U2OS cells stably expressing FLAG-TIP60WT or empty vector were treated with 2µM SNS-032 (SNS) for 2 hours as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. This material and input lysate were subjected to Western blot analysis


( E ) U2OS stably expressing FLAG - TIP60WT , FLAG - TIP60S90A or empty vector ( vec ) were subjected to ChIP using anti - FLAG antibody . Immunoprecipitated input were subjected to quantitative real - time PCR using primers annealing in myc locus about 50bp upstream ( myc + 50 ) or about 500 bp downstream ( myc - 500 ) of transcription start site . Promoter occupancy of FLAG - TIP60WT or FLAG - TIP60S90A was determined as input in relation to FLAG - TIP60WT . graphs error bars represent mean standard deviation of three experiments ( one - way ANOVA , Bonferroni " s multiple comparison , myc + 50 WT vs . S90A p = 0 , 0289 , myc - 500 WT vs . S90A p = 0 , 0155 ( * p < 0 , 05 ) )

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

(E) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A or empty vector (vec) were subjected to ChIP using anti-FLAG antibody. Immunoprecipitated DNA and input were subjected to quantitative real-time PCR using primers annealing in the myc locus about 50bp upstream (myc+50) or about 500 bp downstream (myc-500) of the transcription start site. Promoter occupancy of FLAG-TIP60WT or FLAG-TIP60S90A was determined as percent input in relation to FLAG -TIP60WT. The graphs and error bars represent mean and standard deviation of three individual experiments (one-way ANOVA, Bonferroni"s multiple comparison test, myc+50 WT vs. S90A p=0,0289, myc-500 WT vs. S90A p=0,0155 (*p < 0,05))


( A ) U2OS stably expressing FLAG - TIP60WT , FLAG - TIP60S90A or empty vector ( vec ) were treated with 2µM SNS - 032 ( SNS ) , 2µM JQ1 ( JQ1 ) or solvent DMSO ( d . ) for 2h as indicated . FLAG - immunoprecipitations were performed from extracts . Immunoprecipitated material input lysate were subjected to Western blot

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

(A) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A or empty vector (vec) were treated with 2µM SNS-032 (SNS), 2µM JQ1 (JQ1) or the solvent DMSO (d.) for 2h as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. Immunoprecipitated material and input lysate were subjected to Western blot analysis


( B ) U2OS stably expressing FLAG - TIP60WT were treated with 2µM JQ1 ( JQ1 ) for 2h . nucleus was fractionated into nucleoplasm . Both fractions were analysed by Western blotting

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

(B) U2OS cells stably expressing FLAG-TIP60WT were treated with 2µM JQ1 (JQ1) for 2h. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting


( C ) HEK293 were transiently transfected with pcDNA4 encoding Brd4 or GFP . nucleus was fractionated into nucleoplasm . Both fractions were analysed by Western blotting

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

(C) HEK293 cells were transiently transfected with pcDNA4 encoding Brd4 or GFP. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting


( A ) U2OS were transfected with a siRNA pool targeting human TIP60 ( Dharmacon ) or luciferase ( Dharmacon ) . lysates were analysed by Western blotting 24h after transfection

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

(A) U2OS cells were transfected with a siRNA pool targeting human TIP60 (Dharmacon) or luciferase (Dharmacon). The nuclear lysates were analysed by Western blotting 24h after transfection


( B ) U2OS stably expressing FLAG - TIP60WT , FLAG - TIP60S90A , FLAG - TIP60S86A or empty vector ( vec ) were analysed by Western blotting 60 hours after viral transduction

O O O O O O O O O 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) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A, FLAG-TIP60S86A or empty vector (vec) were analysed by Western blotting 60 hours after viral transduction


( A ) Confocal live imaging of HeLa overexpressing GFP - Bax ( green ) mitochondria marked with TMRE 100 nM ( magenta ) show GFP - Bax distribution healthy mitochondria . Lower panels are zoomed corresponding to rectangle in upper panel represent merged channels . Time shows minutes after apoptosis induction with Staurosporine ( STS ) 1 µM . Scale bars , 5µm ( overview ) 2µm ( zooms ) ( B ) HeLa shown in A at longer times after STS treatment . show sequential GFP - Bax translocation ( green ) , which correlates with mitochondria depolarization ( TMRE , magenta ) . Scale bars , 5 µm ( C ) HeLa overexpressing GFP - Bax mutant ( 1 - 2 / L - 6 ) ( green ) mitochondria marked with TMRE 100 nM ( magenta ) . show constitutive GFP - Bax localization to healthy mitochondria without apoptosis induction . Right panels are zoomed corresponding to rectangle in left panel represent overlaid channels . Scale bars , 5µm ( overview ) 2 µm ( zooms ) ( D ) Standard deviation of of GFP - Bax blue ) versus corrected total ( CTCF ) of TMRE ( purple ) in ( N = 4 ) . CTCF = Integrated Density - ( of selected X Mean of 3 background readings ) . Time 0 corresponds to normalized time when both events cross in each . Black represents average of .

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

(A) Confocal live cell imaging of HeLa cells overexpressing GFP-Bax (green) and mitochondria marked with TMRE 100 nM (magenta) show cytosolic GFP-Bax distribution and healthy mitochondria. Lower panels are zoomed images corresponding to the white rectangle in the upper panel and represent the individual and merged channels. Time shows the minutes after apoptosis induction with Staurosporine (STS) 1 µM. Scale bars, 5µm (overview) and 2µm (zooms)(B) HeLa cells shown in A at longer times after STS treatment. Images show sequential GFP-Bax translocation (green), which correlates with mitochondria depolarization (TMRE, magenta). Scale bars, 5 µm(C) HeLa cells overexpressing GFP-Bax mutant (1-2/L-6) (green) and mitochondria marked with TMRE 100 nM (magenta). Images show constitutive GFP-Bax localization to healthy mitochondria without apoptosis induction. Right panels are zoomed images corresponding to the white rectangle in the left panel and represent the individual and overlaid channels. Scale bars, 5µm (overview) and 2 µm (zooms)(D) Standard deviation of the fluorescence intensity of GFP-Bax blue) versus the corrected total cell fluorescence (CTCF) of TMRE (purple) in individual cells (N=4). CTCF= Integrated Density - (Area of selected cell X Mean fluorescence of 3 background readings). Time 0 corresponds to the normalized time when both events cross in each cell. Black line represents the average of the individual cells.


( A ) Overview of a reconstructed superresolution image of GFP - Bax overexpressing HeLa stained with AF647 - anti - GFP - nanobodies . Image was acquired on fixed 3 h after apoptosis induction with STS . Dotted shows shape ( see Fig EV3 ) . Scale bar , 5 µm . ( B ) Magnified reconstructed superresolution image corresponding to rectangle in A showing shapes of GFP - Bax WT structures ( arrows ) . Scale bar , 500 nm . ( C ) Gallery of typical GFP - Bax WT structures during apoptosis found in analyzed . Scale bars , 100 nm . ( D ) Overview ( left ) 3 enlarged insets ( right ) of HeLa overexpressing GFP - Bax 1 - 2 / L - 6 stained with AF647 - anti - GFP - nanobodies . Figure shows reconstructed superresolution acquired without apoptosis induction . Scale bars , 5 µm ( overview ) 500 nm ( insets ) .

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

(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HeLa cells stained with AF647-anti-GFP-nanobodies. Image was acquired on fixed cells 3 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Magnified reconstructed superresolution image corresponding to the white rectangle in A showing the shapes of GFP-Bax WT structures (white arrows). Scale bar, 500 nm.(C) Gallery of typical GFP-Bax WT structures during apoptosis found in all the analyzed cells. Scale bars, 100 nm.(D) Overview (left) and 3 enlarged insets (right) of HeLa cells overexpressing GFP-Bax 1-2/L-6 stained with AF647-anti-GFP-nanobodies. Figure shows reconstructed superresolution images acquired without apoptosis induction. Scale bars, 5 µm (overview) and 500 nm (insets).


( A ) of structures found on Bax wild type ( purple ) on Bax 1 - 2 / L - 6 ( grey ) overexpressing . Data show total number of structures in measurements . n ( Bax wild type ) = 13 , n ( Bax 1 - 2 / L - 6 ) = 11 . ( B ) Cumulative distribution plots of GFP - Bax wild type ( purple ) GFP - Bax 1 - 2 / L - 6 ( grey ) showing differences in amplitude frequency . Each plot represents a single . Thick lines represent average cumulative distributions for single transfected with GFP - Bax WT or mutant GFP - Bax 1 - 2 / L - 6 .

(A) Quantification of the structures found on Bax wild type (purple) and on Bax 1-2/L-6 (grey) overexpressing cells. Data show the total number of structures in all the measurements. n (Bax wild type)= 13, n (Bax 1-2/L-6)=11.(B) Cumulative distribution plots of GFP-Bax wild type (purple) and GFP-Bax 1-2/L-6 (grey) showing differences in amplitude and frequency. Each plot represents a single cell. Thick lines represent average cumulative distributions for all the single cells transfected with GFP-Bax WT or mutant GFP-Bax 1-2/L-6.


( A ) Overview of a reconstructed superresolution image of GFP - Bax overexpressing HCT116Bax / Bak - / - stained with AF647 - anti - GFP - nanobodies Image was acquired on fixed 7 h after apoptosis induction with STS . Dotted shows shape ( see Fig EV3 ) . Scale bar , 5 µm . ( B ) Gallery of typical GFP - Bax WT non - random structures during apoptosis on HCT116Bax / Bak - / - . Scale bars , 100 nm .

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

(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HCT116Bax/Bak -/- cells stained with AF647-anti-GFP-nanobodies Image was acquired on fixed cells 7 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Gallery of typical GFP-Bax WT non-random structures during apoptosis on HCT116Bax/Bak -/- cells. Scale bars, 100 nm.


( C ) Comparison of confocal ( left ) STED ( right ) of GFP - Bax overexpressing HeLa 2h after STS treatment . STED reveals a notable increase in resolution . Scale bars , 500 nm . ( D ) Representative GFP - Bax non - random structures found with STED ( lines , arcs , rings , double lines ) . Scale bars , 100 nm .

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

(C) Comparison of confocal (left) and STED (right) images of GFP-Bax overexpressing HeLa cells 2h after STS treatment. STED reveals a notable increase in resolution. Scale bars, 500 nm.(D) Representative GFP-Bax non-random structures found with STED (lines, arcs, rings, double lines). Scale bars, 100 nm.


( A ) Representative pictures of HeLa wild - type . Bottom panels show healthy mitochondria . Scale bars , 500nm ( overview ) 200 nm ( zooms ) . ( B ) Representative pictures of apoptotic HeLa wild - type 4h after STS treatment ( 1µM ) . Black arrowheads point to defects on mitochondrial outer . Scale bars , 500nm ( overview ) 200 nm ( zooms ) ( C ) Representative pictures of apoptotic HeLaGFP - Bax transfected 4h after STS treatment ( 1µM ) . Black arrowheads point to defects on mitochondrial outer . Scale bars , 500nm ( overview ) 200 nm ( zooms ) .

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

(A) Representative TEM pictures of HeLa wild-type cells. Bottom panels show healthy mitochondria. Scale bars, 500nm (overview) and 200 nm (zooms).(B) Representative TEM pictures of apoptotic HeLa wild-type cells 4h after STS treatment (1µM). Black arrowheads point to defects on the mitochondrial outer membrane. Scale bars, 500nm (overview) and 200 nm (zooms)(C) Representative TEM pictures of apoptotic HeLaGFP-Bax transfected cells 4h after STS treatment (1µM). Black arrowheads point to defects on the mitochondrial outer membrane. Scale bars, 500nm (overview) and 200 nm (zooms).


( A ) Left panel shows channels overlaid of confocal live imaging of HeLa overexpressing GFP - Bax ( green ) mito - dsRed ( magenta ) at 0 85 minutes after apoptosis induction with STS 1µM . Right panel shows zoomed overlaid of HeLa following GFP - Bax translocation ( green ) mitochondrial fragmentation ( magenta ) during apoptosis induction with STS 1µM . Scale bars , 10 µm ( left ) 2 µm ( right ) ( B ) Reconstructed dual - color superresolution image of GFP - Bax overexpressing HeLa . GFP - Bax was imaged by immunosstaining with AF647 - anti - GFP - nanobodies mitochondria were immunostained with CoxIV ( ) cf680 ( secondary ) antibodies . Image was acquired 3h after STS treatment . Left panel shows overview ( scale bar , 5 µm ) , right panel up shows overview inset ( scale bar , 500nm ) right panel down shows a non - random linear GFP - Bax assembly at mitochondria ( scale bar , 100nm ) . ( C ) Reconstructed dual - color superresolution image of HeLa co - overexpressing mMaple - mito GFP - Bax stained with AF647 - anti - GFP - nanobodies . Image was acquired 3h after STS treatment . Lower panel shows enlarged insets corresponding to rectangles in upper panel . Scale bars , 1 µm ( overview ) 100 nm ( insets )

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

(A) Left panel shows the individual channels and overlaid images of confocal live cell imaging of HeLa cells overexpressing GFP-Bax (green) and mito-dsRed (magenta) at 0 and 85 minutes after apoptosis induction with STS 1µM. Right panel shows zoomed overlaid images of HeLa cells following GFP-Bax translocation (green) and mitochondrial fragmentation (magenta) during apoptosis induction with STS 1µM. Scale bars, 10 µm (left) and 2 µm (right)(B) Reconstructed dual-color superresolution image of GFP-Bax overexpressing HeLa cells. GFP-Bax was imaged by immunosstaining with AF647-anti-GFP-nanobodies and mitochondria were immunostained with CoxIV (primary) and cf680 (secondary) antibodies. Image was acquired 3h after STS treatment. Left panel shows the cell overview (scale bar, 5 µm), right panel up shows the overview inset (scale bar, 500nm) and right panel down shows a non-random linear GFP-Bax assembly at mitochondria (scale bar, 100nm).(C) Reconstructed dual-color superresolution image of HeLa cells co-overexpressing mMaple-mito and GFP-Bax and stained with AF647-anti-GFP-nanobodies. Image was acquired 3h after STS treatment. Lower panel shows enlarged insets corresponding to the white rectangles in the upper panel. Scale bars, 1 µm (overview) and 100 nm (insets)


( A ) AFM image of a control SLB prepared from LUVs with a mitochondrial - like lipid composition . bilayer looks flat , without or defects . Scale bar , 1 µm . ( B ) AFM image of a SLB prepared from LUVs with a mitochondrial - like lipid composition pre - incubated with heat - activated Bax . green arrows indicate presence of pores , which are heterogeneous in shape . edges of pores present protrusions corresponding to Bax clusters . Scale bar , 1 µm .

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

(A) AFM image of a control SLB prepared from LUVs with a mitochondrial-like lipid composition. The bilayer looks flat, without aggregates or defects. Scale bar, 1 µm.(B) AFM image of a SLB prepared from LUVs with a mitochondrial-like lipid composition pre-incubated with heat-activated Bax. The green arrows indicate the presence of membrane pores, which are heterogeneous in size and shape. The edges of the pores present protrusions corresponding to Bax clusters. Scale bar, 1 µm.


( C ) 3D AFM topography of a Bax arc ( left ) ring ( right ) . Both reveal a circular hole that spans lipid ( green ) . Bax molecules around pore rim ( magenta ) protrude 3 , 97 ± 1 , 02 nm above plane , as confirmed by height profiles shown below each image ( corresponding to in 2D image insets ) . topography of arc structure reveals a pore only partially surrounded by Bax molecules , while lipids alone form of pore rim . are shown in a 42 ° tilted representation .

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

(C) 3D AFM topography of a Bax arc (left) and ring (right). Both images reveal a circular dark hole that spans the lipid membrane (green). Bax molecules around the pore rim (magenta and white) protrude 3,97 ± 1,02 nm above the membrane plane, as confirmed by the height profiles shown below each image (corresponding to the white line in the 2D image insets). The topography of the arc structure reveals a pore only partially surrounded by Bax molecules, while lipids alone form the rest of the pore rim. Images are shown in a 42° tilted representation.


( D ) Quantitative of distinct structures found for activated Bax on SLBs . Data show total number of structures in measurements .

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

(D) Quantitative analysis of the distinct structures found for activated Bax on SLBs. Data show the total number of structures in all the measurements.


( C - D ) Snapshots from confocal live imaging movies of a representative ( N = 7 ) during cellularisation co - expressing opto - Notch , Mito - Zdk , MCP : : GFP sim - MS2 . Continuous photo - activation from onset of cellularisation with a stack of z = 25μm ( λ = 488 nm ) was alternated with mCherry excitation ( λ = 561 nm ) at 1 min intervals . Single confocal z - slices of mCherry channel are shown to record opto - Notch localization before , 10 minutes after sustained photo - activation . Magnified insets show opto - Notch bound to mitochondrial anchor in ( C ) in nucleus upon photo - activation ( D ) . Scale bar , 10 μm .

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

(C-D) Snapshots from confocal live imaging movies of a representative embryo (N=7) during cellularisation co-expressing opto-Notch, Mito-Zdk, MCP::GFP and sim-MS2. Continuous photo-activation from the onset of cellularisation with a stack size of z= 25μm (λ=488 nm) was alternated with mCherry excitation (λ=561 nm) at 1 min intervals. Single confocal z-slices of the mCherry channel are shown to record opto-Notch localization before, and 10 minutes after sustained photo-activation. Magnified insets show opto-Notch bound to the mitochondrial anchor in the dark (C) and in the nucleus upon photo-activation (D). Scale bar, 10 μm.


( F - H ) Maximum z - projections ( z = 25 μm ) of same simultaneously photoactivated imaged in GFP channel ( λ = 488 nm ) to record sim expression at a time resolution of 30 s . Shown are first cycle of photo - activation , which approximates state ( F ) , 15 min ( G ) , 35 min ( H ) after continuous photo - activation . Magnified insets show that sim is not expressed in at this stage ( F ) , but it is expressed in lateral ectodermal after 15 min of photo - activation ( G ) , appeared to be shut off in most nuclei after 35 mins ( H ) . Scale bar , 10 μm .

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

(F-H) Maximum intensity z-projections (z = 25 μm) of the same embryo simultaneously photoactivated and imaged in the GFP channel (λ=488 nm) to record sim expression at a time resolution of 30 s. Shown are the first cycle of photo-activation, which approximates the dark state (F), 15 min (G), and 35 min (H) after continuous photo-activation. Magnified insets show that sim is not expressed in the dark at this stage (F), but it is expressed in lateral ectodermal cells after 15 min of photo-activation (G), and appeared to be shut off in most nuclei after 35 mins (H). Scale bar, 10 μm.


number of sim - expressing nuclei as counted by automated spot detection over time ( A ) Continuous opto - Notch activation initially induces sim expression across a large number of nuclei . Over time , majority of nuclei adapt to input terminate expression .

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

The number of sim-expressing nuclei as counted by automated spot detection over time (A) Continuous opto-Notch activation initially induces sim expression across a large number of nuclei. Over time, the majority of nuclei adapt to the input and terminate expression.


( D - E ) of localisation of mCherry : : NICD during opto - Notch photo - activation . Raw data was bleaching - corrected with an exponential fit shown is / total , which further normalises bleaching - to - variability . NICD displays persistent enrichment during continuous activation ( D ) shuttles back forth between nucleus cytoplasm under pulsatile protocol ( E ) .

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

(D-E) Quantification of nuclear localisation of mCherry::NICD during opto-Notch photo-activation. Raw data was bleaching-corrected with an exponential fit and shown is the nuclear/total ratio, which further normalises bleaching and embryo-to-embryo variability. NICD displays persistent nuclear enrichment during continuous activation (D) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol (E).


( D - E ) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant ( Hairless H2 ) under overexpression of Twist ( Twist OE ) observed outcomes are most consistent with predictions from state - dependent inactivation model .

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

(D-E) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant (Hairless H2) and under overexpression of Twist (Twist OE) The observed outcomes are most consistent with the predictions from the state-dependent inactivation model.


( A ) In vitro pull - downs between strep ( II ) - tagged DDB1 - DCAF12 untagged wild type ( WT ) CCT5 or CCT5 ( 1 - 529 ) ( Δ ( Ct ) ) seen on a Coomassie - stained SDS - PAGE gel .

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

(A) In vitro pull-downs between strep(II)-tagged DDB1-DCAF12 and untagged wild type (WT) CCT5 or CCT5 (1-529) (Δ(Ct)) seen on a Coomassie-stained SDS-PAGE gel.


( B ) Titration curves between a ATTO488CCT520 degron peptide TbDDB1 - DCAF12 or terbium - coupled streptavidin ( Tb - SA ) ( n = 3 ) . Signal originating in absence of TbDDB1 - DCAF12 is unspecific was subtracted in subsequent experiments .

(B) Titration curves between a fluorescent ATTO488CCT520 degron peptide and TbDDB1-DCAF12 or terbium-coupled streptavidin (Tb-SA) (n=3). Signal originating in the absence of TbDDB1-DCAF12 is unspecific and was subtracted in subsequent experiments.


( C ) TR - FRET counter - titration of unlabeled wild type ( WT ) CCT5 or an unlabeled CCT520 peptide into pre - assembled TbDDB1 - DCAF12488 ( n = 3 ) .

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

(C) TR-FRET counter-titration of unlabeled wild type (WT) CCT5 or an unlabeled CCT520 peptide into pre-assembled TbDDB1-DCAF12488 (n=3).


( D ) TR - FRET counter - titrations of label - free CCT5 C - terminal peptides into TbDDB1 - DCAF12488 ( n = 3 ) . Sequences of peptides IC50 values for titrations are listed on table . Peptides are labeled for their degron position corresponding CCT5 acid number . ( E ) Counter - titration of unlabeled CCT510 mutant peptides into TbDDB1 - DCAF12488 ( n = 3 ) . Sequences of peptides IC50 values for titrations are listed on table . Peptides are labeled for their degron position .

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

(D) TR-FRET counter-titrations of label-free CCT5 C-terminal peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position and corresponding CCT5 amino acid number. (E) Counter-titration of unlabeled CCT510 mutant peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position. D


( D ) Titration curves between a ATTO488CCT520 degron peptide wild type ( WT ) or mutant TbDDB1 - DCAF12 complexes ( n = 3 ) . Unspecific signal arising from Tb - SA label was subtracted .

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

(D) Titration curves between a fluorescent ATTO488CCT520 degron peptide and wild type (WT) or mutant TbDDB1-DCAF12 complexes (n=3). Unspecific signal arising from the Tb-SA label was subtracted.


( E ) In vitro ubiquitination of CCT5 by wild type ( WT ) or mutant CRL4DCAF12 complexes in presence of ubiquitin , ATP , E1 E2 enzymes .

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

(E) In vitro ubiquitination of CCT5 by wild type (WT) or mutant CRL4DCAF12 complexes in the presence of ubiquitin, ATP, E1 and E2 enzymes.


( A ) Counter - titration of unlabeled CCT5 or unlabeled TRiC into pre - assembled TbDDB1 - DCAF12488 ( n = 3 ) .

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

(A) Counter-titration of unlabeled CCT5 or unlabeled TRiC into pre-assembled TbDDB1-DCAF12488 (n=3).


( B ) In vitro ubiquitination of monomeric wild type CCT5 or TRiC by CRL4DCAF12 in presence of ubiquitin , ATP , E1 E2 enzymes . CCT5 , but not TRiC , is modified with poly - ubiquitin ( Ubn ) chains by CRL4DCAF12 . Free CCT5 carries an N - terminal strep ( II ) tag differs in electrophoretic mobility from untagged CCT5 purified in with TRiC .

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

(B) In vitro ubiquitination of monomeric wild type CCT5 or TRiC by CRL4DCAF12 in the presence of ubiquitin, ATP, E1 and E2 enzymes. CCT5, but not TRiC, is modified with poly-ubiquitin (Ubn) chains by CRL4DCAF12. Free CCT5 protein carries an N-terminal strep(II) tag and differs in electrophoretic mobility from untagged CCT5 purified in complex with TRiC.


Representative false colour of leaf surface captured by a thermal camera from WT , stp1 - 1 , stp4 - 1 , stp1stp4 , stp13 , stp1stp13 stp4stp13 .

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

Representative false colour images of leaf surface temperature captured by a thermal camera from WT, stp1-1, stp4-1, stp1stp4, stp13, stp1stp13 and stp4stp13 plants.


Normalized leaf surface over phenotyping period . Data shown are means ± ; n = 10 per genotype time point .

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

Normalized leaf surface temperature over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Whole - recordings of changes in stomatal conductance ( gs ) from WT , stp1 - 1 , stp4 - 1 stp1stp4 . Data shown are means ± ; n ≥ 3 per genotype .

Whole-plant recordings of changes in stomatal conductance (gs) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.


Whole - recordings of changes in stomatal conductance ( gs ) from self - grafted donor lines ( WT / WT , stp1stp4 / stp1stp4 ) reciprocal grafting of shoot / root ( WT / stp1stp4 , stp1stp4 / WT ) . Data shown are means ± ; n = 3 per genotype .

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

Whole-plant recordings of changes in stomatal conductance (gs) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.


Content of soluble sugars in guard - enriched epidermal peels of WT stp1stp4 at end of night ( EoN ) after 40 min of illumination with at 150 µmol m - 2 s - 1 following EoN . Data for two independent experiments are shown ; means ± ; n ≥ 7 per genotype time point .

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

Content of soluble sugars in guard cell-enriched epidermal peels of WT and stp1stp4 plants at the end of the night (EoN) and after 40 min of illumination with white light at 150 µmol m-2 s-1 following the EoN. Data for two independent experiments are shown; means ± SEM; n ≥ 7 per genotype and time point.


Representative confocal microscopy of propidium iodide - stained guard starch granules of intact leaves of WT , stp1 - 1 , stp4 - 1 , stp1stp4 , suc1 suc3 . Scale bar , 10 µm .

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

Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants. Scale bar, 10 µm.


Starch dynamics in guard of intact leaves of WT , stp1 - 1 , stp4 - 1 , stp1stp4 , suc1 suc3 at end of night ( EoN ) after one three hours of illumination with 150 µmol m - 2 s - 1 of . Data for three independent experiments are shown ; means ± ; n = 120 guard per genotype time point .

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

Starch dynamics in guard cells of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for three independent experiments are shown; means ± SEM; n = 120 individual guard cells per genotype and time point.


Representative false colour of photosystem II ( PSII ) operating efficiency ( ΦPSII ) captured by chlorophyll imaging from WT , stp1 - 1 , stp4 - 1 stp1stp4 . ΦPSII was measured at a photosynthetically active radiation ( PAR ) of 440 µmol m - 2 s - 1 .

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

Representative false colour images of photosystem II (PSII) operating efficiency (ΦPSII) captured by chlorophyll fluorescence imaging from WT, stp1-1, stp4-1 and stp1stp4 plants. ΦPSII was measured at a photosynthetically active radiation (PAR) of 440 µmol m-2 s-1.


ΦPSII quantified over phenotyping period . Data shown are means ± ; n = 10 per genotype time point .

ΦPSII quantified over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Whole - recordings of changes in CO2 assimilation ( A ) from WT , stp1 - 1 , stp4 - 1 stp1stp4 . Data shown are means ± ; n ≥ 3 per genotype .

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

Whole-plant recordings of changes in CO2 assimilation (A) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.


Whole - recordings of changes in CO2 assimilation ( A ) from self - grafted donor lines ( WT / WT , stp1stp4 / stp1stp4 ) reciprocal grafting of shoot / root ( WT / stp1stp4 , stp1stp4 / WT ) . Data shown are means ± ; n = 3 per genotype .

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

Whole-plant recordings of changes in CO2 assimilation (A) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.


of leaf glucose ( Glc ) . Data shown are means ± ; n = 8 per genotype time point . of leaf fructose ( Fru ) . Data shown are means ± ; n = 8 per genotype time point . of leaf sucrose ( Suc ) . Data shown are means ± ; n = 8 per genotype time point .

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

Quantification of leaf glucose (Glc). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf fructose (Fru). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf sucrose (Suc). Data shown are means ± SEM; n = 8 per genotype and time point.


of leaf starch . Data shown are means ± ; n = 8 per genotype time point .

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

Quantification of leaf starch. Data shown are means ± SEM; n = 8 per genotype and time point.


Representative Red Green Blue ( RGB ) of 3 - ( day 0 ) 4 - week - old ( day 7 ) WT , stp1 - 1 , stp4 - 1 stp1stp4 .

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

Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 7) WT, stp1-1, stp4-1 and stp1stp4 plants.


Projected rosette over phenotyping period . Data shown are means ± ; n = 10 per genotype time point .

Projected rosette area over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Photosynthetic CO2 assimilation ( A ) in - adapted WT stp1stp4 in response to a step increase in CO2 concentrations from 0 to 1000 ppm . Data shown are means ± ; n = 3 per genotype .

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

Photosynthetic CO2 assimilation (A) in dark-adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm. Data shown are means ± SEM; n=3 per genotype.


of leaf glucose ( Glc ) . Data for two independent experiments are shown ; means ± ; n ≥ 17 per genotype time point .

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

Quantification of leaf glucose (Glc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.


of leaf fructose ( Fru ) . Data for two independent experiments are shown ; means ± ; n ≥ 17 per genotype time point . of leaf sucrose ( Suc ) . Data for two independent experiments are shown ; means ± ; n ≥ 17 per genotype time point . of leaf starch . Data for two independent experiments are shown ; means ± ; n ≥ 17 per genotype time point .

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

Quantification of leaf fructose (Fru). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf sucrose (Suc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf starch. Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.


Representative Red Green Blue ( RGB ) of 3 - ( day 0 ) 4 - week - old ( day 6 ) WT stp1stp4 grown under 600 ppm CO2 .

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

Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 6) WT and stp1stp4 plants grown under 600 ppm CO2.


Projected rosette over phenotyping period . Data shown are from two independent experiments ; means ± ; n ≥ 24 per genotype time point .

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

Projected rosette area over the phenotyping period. Data shown are from two independent experiments; means ± SEM; n ≥ 24 per genotype and time point.


Representative confocal microscopy of propidium iodide - stained guard starch granules of intact leaves of WT stp1stp4 grown under 600 ppm CO2 . Scale bar , 10 µm .

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

Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2. Scale bar, 10 µm.


Starch dynamics in guard of intact leaves of WT stp1stp4 grown under 600 ppm CO2 at end of night ( EoN ) after one three hours of illumination with 150 µmol m - 2 s - 1 of . Data for two independent experiments are shown ; means ± ; n = 80 guard per genotype time point .

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

Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for two independent experiments are shown; means ± SEM; n = 80 individual guard cells per genotype and time point.


( A ) RNAscope in - situ hybridization of Ch25h transcripts ( red ) in spinal cord of non - immunized mice ( NI ) ( left panels ) mice 17 days after EAE immunization ( peak disease ) ( 2 right panels ) shown in endothelial ( Isolectin B4 ( IsoB4 ) green top panels ) activated macrophages / microglia ( Ionized calcium biding adaptor molecule 1 ( IBA1 ) green lower panels ) . Nuclei are shown in blue . ( B ) Quantitative of Ch25h mRNA expression in spinal cord of NI EAE mice comparing expression in macrophages / microglia ( MG ) endothelial ( EC ) , n = 6 biological replicates / group .

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

(A) RNAscope fluorescence in-situ hybridization of Ch25h transcripts (red) in the spinal cord of non-immunized mice (NI) (left panels) and mice 17 days after EAE immunization (peak disease) (2 right panels) shown in endothelial cells (Isolectin B4 (IsoB4) green top panels) and activated macrophages/microglia (Ionized calcium biding adaptor molecule 1 (IBA1) green lower panels). Nuclei are shown in blue. (B) Quantitative analysis of Ch25h mRNA expression in the spinal cord of NI and EAE mice comparing expression in macrophages/microglia (MG) and endothelial cells (EC), n = 6 biological replicates/group.


( E ) of eGFP + CNS ECs in same condition as in ( D ) . Symbols indicate mice bars indicate mean ± SD . Ch25hfl / fl NI : n = 4 biological replicates , Ch25hfl / fl EAE : n = 5 biological replicates , Ch25hECKO NI : n = 4 biological replicates , Ch25hECKO EAE : n = 4 biological replicates . Representative results of 1 experiment .

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

(E) Percentage of eGFP+ CNS ECs in the same condition as in (D). Symbols indicate individual mice and bars indicate mean ± SD. Ch25hfl/fl NI: n = 4 biological replicates, Ch25hfl/fl EAE: n = 5 biological replicates, Ch25hECKO NI: n = 4 biological replicates, Ch25hECKO EAE: n = 4 biological replicates. Representative results of 1 experiment.


( A ) EAE disease course in Ch25hECKO Cre negative ( Ch25hfl / fl ) . Top panel : EAE clinical score . Bars indicate mean ± . Bottom panel : Survival of EAE , depicting disease incidence . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on graph . ( B ) As in ( A ) except that Ch25h fl / fl - Pdgfb - iCreERT2 mice that express Cre recombinase in blood endothelial ( Ch25hBECKO ) are shown . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on graph . ( C ) As in ( A ) except that Ch25hfl / fl - Prox1 - CreERT2 mice that express Cre recombinase in lymphatic endothelial ( Ch25hLECKO ) are shown . Representative results of 2 experiments with a minimum of n = 5 biological replicates / group as indicated on graph .

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

(A) EAE disease course in Ch25hECKO and Cre negative littermates (Ch25hfl/fl). Top panel: EAE clinical score. Bars indicate mean ± SEM. Bottom panel: Survival analysis of EAE, depicting disease incidence. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph. (B) As in (A) except that Ch25h fl/fl- Pdgfb-iCreERT2 mice that express Cre recombinase in blood endothelial cells (Ch25hBECKO) are shown. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph. (C) As in (A) except that Ch25hfl/fl- Prox1-CreERT2 mice that express Cre recombinase in lymphatic endothelial cells (Ch25hLECKO) are shown. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph.


( D , E ) Histopathological quantifications ( D ) representatives ( E ) of spinal cord sections of immunized Ch25hfl / fl mice ( n = 7 biological replicates ) Ch25hECKO mice ( n = 10 biological replicates ) at day 21 post - immunization for infiltration ( HE ) . Five sections per mouse were quantified . Scale bars 200 µm ( top panels ) , 100 µm ( bottom panels ) .

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

(D, E) Histopathological quantifications (D) and representatives staining (E) of spinal cord sections of immunized Ch25hfl/fl mice (n = 7 biological replicates) and Ch25hECKO mice (n = 10 biological replicates) at day 21 post-immunization for cellular infiltration (HE). Five sections per mouse were quantified. Scale bars 200 µm (top panels), 100 µm (bottom panels).


( A ) RT - qPCR of Ch25h expression in mouse brain microvascular endothelial ( pMBMECs ) isolated from Ch25hfl / fl Ch25hECKO mice . were left unstimulated ( NS ) or stimulated with IL - 1β . n = 5 biological replicates / group . experiment was performed 3 times .

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

(A) RT-qPCR analysis of Ch25h expression in primary mouse brain microvascular endothelial cells (pMBMECs) isolated from Ch25hfl/fl and Ch25hECKO mice. Cells were left unstimulated (NS) or stimulated with IL-1β. n = 5 biological replicates/group. The experiment was performed 3 times.


( F ) RT - qPCR of pMBMECs isolated from Ch25hECKO Ch25hfl / fl mice stimulated with IL - 1β . n = 4 biological replicates / group . experiment was realized once .

(F) RT-qPCR of pMBMECs isolated from Ch25hECKO and Ch25hfl/fl mice and stimulated with IL-1β. n = 4 biological replicates/group. The experiment was realized once.


( G ) RT - qPCR of CNS endothelial sorted from Ch25hECKO Ch25hfl / fl mice during EAE ( day 10 after immunization ) . Ch25hECKO n = 3 biological replicates , Ch25hfl / fl n = 4 biological replicates . experiment was performed once .

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

(G) RT-qPCR of CNS endothelial cells sorted from Ch25hECKO and Ch25hfl/fl mice during EAE (day 10 after immunization). Ch25hECKO n = 3 biological replicates, Ch25hfl/fl n = 4 biological replicates. The experiment was performed once.


( C ) 25 - OHC , 7 - keto - 25 - OHC , 24 ( S ) - OHC in same conditions as in ( B ) . Bars represent mean ± SD . n = 6 biological replicates / group except for Ch25fl / fl IL - 1β were n = 5 biological replicates . experiment was performed once . ( D ) 25 - OHC , 7 - keto - 25 - OHC , 24 ( S ) - OHC in spinal cord tissue extracted from Ch25hfl / fl ( n = 8 ) , Ch25hECKO ( n = 3 ) Ch25hBBBKO ( n = 6 ) at peak of EAE .

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

(C) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC concentration in the same conditions as in (B). Bars represent mean ± SD. n = 6 biological replicates/group except for Ch25fl/fl IL-1β were n = 5 biological replicates. The experiment was performed once. (D) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC levels in spinal cord tissue extracted from Ch25hfl/fl (n = 8), Ch25hECKO (n = 3) and Ch25hBBBKO (n = 6) at the peak of EAE.


( G ) Prostaglandin ( nM ) in supernatant of pMBMECs comparing conditions mentioned in ( A ) . n = 5 biological replicates / group bars represent mean ± SD .

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

(G) Prostaglandin concentration (nM) in the supernatant of pMBMECs comparing conditions mentioned in (A). n = 5 biological replicates/group and bars represent mean ± SD.


( C ) Impact of CNS PMN - MDSC BM - PMN on CD4 + T proliferation assessed by CFSE dilution using flow cytometry . PMN - MDSC were FACS - sorted from CNS ( right panel ) BM ( left panel ) of WT mice at peak of EAE . NS = non stimulated . n = 4 biological replicates / group . Bars indicate mean ± SD . CNS PMN - MDSC / CD4 + T : representative results of 2 independent experiments ; BM - PMN : 1 experiment .

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

(C) Impact of CNS PMN-MDSC and BM-PMN on CD4+ T cell proliferation assessed by CFSE dilution using flow cytometry. PMN-MDSC were FACS-sorted from the CNS (right panel) and BM (left panel) of WT mice at the peak of EAE. NS = non stimulated. n = 4 biological replicates/group. Bars indicate mean ± SD. CNS PMN-MDSC/CD4+ T cells: representative results of 2 independent experiments; BM-PMN: 1 experiment.


( H ) ( upper left panel ) absolute number ( lower left panel ) of CNS live CD45 + CD11b + Ly6CintLy6G + PMN - MDSC ; of live CD4 + CD44 + Ki67 + ( upper right panel ) ( lower right panel ) of absolute number of PMN - MDSC proliferating CD4 + T in CNS at peak of disease of Ch25hfl / fl ( n = 9 ) Ch25hECKO mice ( n = 8 ) . Symbols depict mice bars indicate mean ± SD . Combined results of 2 independent experiments .

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

(H) Percentage (upper left panel) and absolute number (lower left panel) of CNS live cells CD45+CD11b+Ly6CintLy6G+ PMN-MDSC; Percentage of live CD4+CD44+Ki67+ cells (upper right panel) and ratio (lower right panel) of absolute number of PMN-MDSC and proliferating CD4+ T cells in the CNS at the peak of the disease of Ch25hfl/fl (n = 9) and Ch25hECKO mice (n = 8). Symbols depict individual mice and bars indicate mean ± SD. Combined results of 2 independent experiments.


( I ) EAE disease course in Ch25hBBBKO mice ( n = 11 ) where Cre - recombinase is expressed in endothelial of CNS Cre negative ( Ch25hfl / fl : n = 7 Ch25hfl / wt : n = 2 ) . Bars indicate mean ± . Representative results of 2 independent experiments .

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

(I) EAE disease course in Ch25hBBBKO mice (n = 11) where the Cre-recombinase is expressed in endothelial cells of the CNS and Cre negative littermates (Ch25hfl/fl: n = 7 and Ch25hfl/wt : n = 2). Bars indicate mean ± SEM. Representative results of 2 independent experiments.


( B ) Survival in same conditions as in ( A ) . Ch25hfl / fl isotype : n = 7 , Ch25hECKO isotype : n = 8 , Ch25hfl / fl Combo : n = 8 , Ch25hECKO Combo : n = 9 .

O O O O O O O O O O O O O O O O O O O O O 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) Survival analysis in the same conditions as in (A). Ch25hfl/fl isotype: n = 7, Ch25hECKO isotype: n = 8, Ch25hfl/fl Combo: n = 8, Ch25hECKO Combo: n = 9.


( C ) Representative contour plots of PMN - MDSC in CNS at day 22 of EAE assessed by flow cytometry in same conditions as in ( A ) . ( D ) As in ( C ) except that statistical of PMN - MDSC in live are shown . Symbols depict mice bars mean ± SD . Ch25hfl / fl Isotype n = 4 , Ch25hECKO isotype n = 4 , Ch25hfl / fl Combo , n = 3 , Ch25hECKO Combo n = 4 . (

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

(C) Representative contour plots of PMN-MDSC in the CNS at day 22 of EAE assessed by flow cytometry in the same conditions as in (A). (D) As in (C) except that statistical analysis and percentage of PMN-MDSC in live cells are shown. Symbols depict individual mice and bars mean ± SD. Ch25hfl/fl Isotype n = 4, Ch25hECKO isotype n = 4, Ch25hfl/fl Combo, n = 3, Ch25hECKO Combo n = 4. (


( E ) Representative contour plots of CD101 representative histograms of Ly6G expression in blood neutrophils at day 16 of EAE assessed by flow cytometry . Ch25hECKO Ch25hfl / fl mice were treated with isotype or Combo treatment which was initiated at first symptoms of EAE ( day 14 after immunization ) . ( F ) As in ( E ) except that statistical is shown . Symbols depict mice , bars mean ± SD . Ch25hfl / lfl isotype n = 7 , Ch25hECKO isotype n = 5 , Ch25hfl / lfl Combo n = 6 , Ch25hECKO Combo n = 5 . experiment was performed once . (

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

(E) Representative contour plots of CD101 and representative histograms of Ly6G expression in blood neutrophils at day 16 of EAE assessed by flow cytometry. Ch25hECKO and Ch25hfl/fl mice were treated with isotype or Combo treatment which was initiated at the first symptoms of EAE (day 14 after immunization). (F) As in (E) except that statistical analysis is shown. Symbols depict individual mice, bars mean ± SD. Ch25hfl/lfl isotype n = 7, Ch25hECKO isotype n = 5, Ch25hfl/lfl Combo n = 6, Ch25hECKO Combo n = 5. The experiment was performed once. (


Reverse Transcriptase PCR showing that bd1971 , control gene dnaK , are expressed throughout predatory cycle of Bdellovibrio bacteriovorus . was isolated at time points indicated across top of gel during one round of synchronous Bdellovibrio infection of E . coli . Primers were designed to anneal specifically to gene of interest . L = 100bp ladder , AP = Attack Phase , 15 - 45 = 15 - 45 minutes respectively since infection , 1h - 4h = hours respectively since infection . Ec = E . coli S17 - 1 ( negative control : no Bdellovibrio ) ; NT = control with no template ; gen = B . bacteriovorus HD100 genomic ( positive control ) . cartoon above represents each stage in predatory cycle .

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

Reverse Transcriptase PCR showing that bd1971, and control gene dnaK, are expressed throughout the predatory cycle of Bdellovibrio bacteriovorus. RNA was isolated at the time points indicated across the top of the gel during one round of synchronous Bdellovibrio infection of E. coli cells. Primers were designed to anneal specifically to the gene of interest. L = 100bp DNA ladder, AP = Attack Phase cells, 15-45 = 15-45 minutes respectively since infection, 1h-4h = hours respectively since infection. Ec = E. coli strain S17-1 RNA (negative control: no Bdellovibrio RNA); NT = control with no template RNA; gen = B. bacteriovorus HD100 genomic DNA (positive control). The cartoon above represents each stage in the predatory cycle.


Epifluorescent of E . coli invaded by B . bacteriovorus HD100 with mCherry tagged Bd1971 at times indicated throughout invasive process during one round of synchronous infection . was acquired with a two second exposure maximum sensitivity gain but different balance contrast in channel were used to produce these ( see methods ) as was fainter at later timepoints but we wished to show whether or not localization within Bdellovibrio altered during predation . merges with phase contrast are shown . Scale bars are 2 µm . are representative of three independent experiments .

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

Epifluorescent images of E. coli invaded by B. bacteriovorus HD100 with mCherry tagged Bd1971 at the times indicated throughout the invasive process during one round of synchronous infection. Fluorescence was acquired with a two second exposure and maximum sensitivity gain but different balance and contrast levels in the fluorescence channel were used to produce these images (see methods) as the fluorescence was fainter at later timepoints but we wished to show whether or not the cellular localization within the Bdellovibrio altered during predation. Fluorescence images and merges with phase contrast images are shown. Scale bars are 2 µm. Images are representative of three independent experiments.


Relative of c - di - GMP in matched biomass host - independent ( HI ) cultures of bd1971 : : kan compared with fliC1 merodiploid KnR HI control ( Wild - Type for bd1971 ) . bd1971 mutant contains significantly more c - di - GMP than control . Data are from 3 independent experiments .

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

Relative levels of c-di-GMP in matched cell biomass host-independent (HI) cultures of bd1971::kan compared with fliC1 merodiploid KnR HI control strain (Wild-Type for bd1971). The bd1971 mutant strain contains significantly more c-di-GMP than the control. Data are from 3 independent experiments.


Relative of c - di - GMP in matched biomass wild - type predatory cultures of HD100 , silent Bd1971 deletion ( Δ1971 ) , Bd1971D306307A ( bd1971DD ) Bd1971R67D ( bd1971R ) . mutant strains had higher of c - di - GMP compared to wild type . Error bars show . Data are from six independent experiments for HD100 ΔBd1971 from four independent experiments for Bd1971DD Bd1971R mutants .

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

Relative levels of c-di-GMP in matched cell biomass wild-type predatory cultures of HD100 strain, silent Bd1971 deletion strain (Δ1971), Bd1971D306307A strain (bd1971DD) and Bd1971R67D strain (bd1971R). All mutant strains had higher levels of c-di-GMP compared to the wild type strain. Error bars show SEM. Data are from six independent experiments for HD100 and ΔBd1971 and from four independent experiments for Bd1971DD and Bd1971R mutants.


Apo - Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations ( 2 . 5 mM ) of cAMP , cGMP or no nucleotide phosphodiesterase activity with respect to generic substrate PNPP ( 5 mM ) was measured spectrophotometrically . Values are average of three repeats , error bars represent standard deviation .

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

Apo-Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations (2.5 mM) of cAMP, cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP (5 mM) was measured spectrophotometrically. Values are the average of three repeats, error bars represent standard deviation.


HPLC traces of reaction products generated from 100 µM c - di - GMP incubated with apo - Bd1971 ( Control ) , or supplemented with either 100 µM cAMP or cGMP . Peaks were identified by reference to standards also resolved by HPLC are labeled in figure .

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

HPLC traces of reaction products generated from 100 µM c-di-GMP incubated with apo-Bd1971 (Control), or protein supplemented with either 100 µM cAMP or cGMP. Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure.


ITC titration curves relative heats of binding for ( A ) cAMP binding to Bd1971ΔEAL . Successive injections of 6 µl of cNMP ( 0 . 5 - 1 . 6 mM ) were made into 1 . 4 ml of apo - Bd1971ΔEAL ( 50 - 150 µM ) heat changes measured . top panels show ITC titrations bottom panels show binding isotherm associated with each ITC experiment .

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

ITC titration curves and relative heats of binding for (A) cAMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.


ITC titration curves relative heats of binding for cGMP binding to Bd1971ΔEAL . Successive injections of 6 µl of cNMP ( 0 . 5 - 1 . 6 mM ) were made into 1 . 4 ml of apo - Bd1971ΔEAL ( 50 - 150 µM ) heat changes measured . top panels show ITC titrations bottom panels show binding isotherm associated with each ITC experiment .

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

ITC titration curves and relative heats of binding for cGMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.


Data were fitted with an allosteric sigmoidal model in GraphPad Prism software used to determine kinetic parameters associated with cNMP dependent phosphodiesterase activity . Data points for both sets of results are average of three experimental replicates with standard deviation shown by intersecting bars . Results are representative of at least three independent experiments .

Data were fitted with an allosteric sigmoidal model in GraphPad Prism software and used to determine the kinetic parameters associated with cNMP dependent phosphodiesterase activity.Data points for both sets of results are the average of three experimental replicates with the standard deviation shown by intersecting bars. Results are representative of at least three independent experiments.