Representative of vacuolar of late meristematic atrichoblast . PI ( green ) MDY - 64 ( yellow ) depicts wall vacuolar , respectively . ( D ) Col - 0 ( n = 28 ) fer - 4 ( n = 28 ) mutant seedling roots were grown on surface or into matrix of 2 % agar - containing solid medium . Kruskal Wallis followed by Dunn ´ s multiple comparison ( b : p < 0 . 05 , c : p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (D) Col-0 (n=28) and fer-4 (n=28) mutant seedling roots were grown on the surface or into the matrix of 2 % agar-containing solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . PI ( green ) MDY - 64 ( yellow ) depicts wall vacuolar , respectively . ( A ) Vacuolar of Col - 0 control ( n = 52 ) lrx3 / 4 / 5 triple mutants ( n = 48 ) . Mann Whitney U ( * * * p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (A) Vacuolar morphology of Col-0 control (n=52) and lrx3/4/5 triple mutants (n=48). Mann Whitney U test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . ( B ) 3 - D reconstructions of PI - stained wall ( red ) BCECF - stained vacuole ( green ) of late meristematic . Boxplot depicts vacuolar occupancy of in Col - 0 control ( n = 11 ) lrx3 / 4 / 5 ( n = 10 ) . Student ´ s t - ( * * * p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. (B) 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Boxplot depicts vacuolar occupancy of the cell in Col-0 control (n=11) and lrx3/4/5 (n=10). Student´s t-test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . PI ( green ) MDY - 64 ( yellow ) depicts wall vacuolar , respectively . ( C ) Col - 0 ( n = 40 - 44 ) lrx3 / 4 / 5 ( n = 36 ) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium . Kruskal Wallis followed by Dunn ´ s multiple comparison ( b : p < 0 . 01 , c : p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (C) Col-0 (n=40-44) and lrx3/4/5 (n=36) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.01, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . PI ( green ) MDY - 64 ( yellow ) depicts wall vacuolar , respectively . ( D ) Col - 0 ( n = 40 - 48 ) fer - 4 ( n = 28 - 32 ) seedlings were grown on surface or into matrix of 2 % agar - containing solid medium . Kruskal Wallis followed by Dunn ´ s multiple comparison ( b : p < 0 . 05 , c : p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (D) Col-0 (n=40-48) and fer-4 (n=28-32) seedlings were grown on the surface or into the matrix of 2 % agar-containing solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


A Rosette phenotype of 3 - - old Col - 0 , fer - 4 , lrx3 / 4 / 5 fer - 4 / lrx3 / 4 / 5 ( upper panel ) . Vacuolar ( MDY - 64 - stained ) of late meristematic atrichoblast of Col - 0 , fer - 4 , lrx3 / 4 / 5 fer - 4 / lrx3 / 4 / 5 ( lower panel ) . Scale bars : 1 cm ( upper row ) 5 µm ( lower row ) .

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A Rosette phenotype of 3-weeks-old Col-0, fer-4, lrx3/4/5 and fer-4/lrx3/4/5 (upper panel). Vacuolar morphology (MDY-64-stained) of late meristematic atrichoblast cells of Col-0, fer-4, lrx3/4/5 and fer-4/lrx3/4/5 (lower panel). Scale bars: 1 cm (upper row) and 5 µm (lower row).


B LRR4 - HA RALF1 - FLAG were transiently expressed ( as indicated by + - ) in Nicotiana benthamiana Immunoprecipitation subsequent detection of by western blotting was done as labelled .

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B LRR4-HA and RALF1-FLAG were transiently expressed (as indicated by + and -) in Nicotiana benthamiana Immunoprecipitation and subsequent detection of the proteins by western blotting was done as labelled.


C Relative root [ % of control ] of Col - 0 ( n = 11 - 13 ) , lrx3 / 4 / 5 ( n = 9 - 13 ) fer - 4 ( n = 11 - 12 ) after 3 days of RALF1 treatment . Statistical analyses were performed for each using one - way ANOVA followed by Bonferroni post ( 1 µM 1 . 25 µM b : p < 0 . 001 ; 1 . 5 µM b : p < 0 . 05 , c : p < 0 . 001 ) . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values . Representative experiment is shown .

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C Relative root length [% of control] of Col-0 (n=11-13 ), lrx3/4/5 (n=9-13 ) and fer-4 (n=11-12) after 3 days of RALF1 treatment. Statistical analyses were performed for each concentration using one-way ANOVA followed by Bonferroni post test (1 µM and 1.25 µM b: p < 0.001; 1.5 µM b: p < 0.05, c: p < 0.001). Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values. Representative experiment is shown.


D Nicotiana benthamiana was transiently transformed with LRR4 - HA NtermFER - FLAG . Immunoprecipitation subsequent detection of by western blotting was done as labelled .

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D Nicotiana benthamiana was transiently transformed with LRR4-HA and NtermFER-FLAG. Immunoprecipitation and subsequent detection of the proteins by western blotting was done as labelled.


E Yeast was transformed with either empty plasmids ( e / e ) , NtermFER - FLAG or LRR4 - HA with empty plasmid , or both NtermFER - FLAG LRR4 - HA . Growth of yeast on quadruple drop - out medium was only observed in transformed with NtermFER - FLAG LRR4 - HA .

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E Yeast was transformed with either empty plasmids (e / e), NtermFER-FLAG or LRR4-HA with the empty plasmid, or both NtermFER-FLAG and LRR4-HA. Growth of yeast on quadruple drop-out medium was only observed in cells transformed with NtermFER-FLAG and LRR4-HA.


A Rosette phenotype of 3 - - old Col - 0 , lrx3 / 4 / 5 35S : : LRR4 - cit . Data information : Scale bars : 1

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A Rosette phenotype of 3-weeks-old Col-0, lrx3/4/5 and 35S::LRR4-cit. Data information: Scale bars: 1 cm


B Representative of vacuolar of late meristematic of Col - 0 35S : : LRR4 - cit . PI ( green ) MDY - 64 ( yellow ) depict wall vacuolar , respectively . Col - 0 ( n = 36 ) 35S : : LRR4 - cit ( n = 32 - 36 ) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium . Kruskal Wallis followed by Dunn ´ s multiple comparison ( b : p < 0 . 05 , c : p < 0 . 001 ) . Data information : 5 µm ( B ) . Boxplots : Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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B Representative images and quantification of vacuolar morphology of late meristematic cells of Col-0 and 35S::LRR4-cit. PI (green) and MDY-64 (yellow) staining depict cell wall and vacuolar membrane, respectively. Col-0 (n=36) and 35S::LRR4-cit (n=32-36) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). Data information: 5 µm (B). Boxplots: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


C Relative root of Col - 0 ( n = 11 - 13 ) 35S : : LRR4 - cit ( n = 11 - 14 ) after 3 days of RALF1 treatment . Statistical analyses were performed for each using Student ´ s t - ( * * * p < 0 . 001 ) . Boxplots : Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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C Relative root length of Col-0 (n=11-13) and 35S::LRR4-cit (n=11-14) after 3 days of RALF1 treatment. Statistical analyses were performed for each concentration using Student´s t-test (***p < 0.001). Boxplots: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Heat map showing absolute of unique peptides for S . indica apoplastic present at three time points in culture filtrate . S1 / S2 / S3 = biologically independent sample 1 , 2 3 ; d = deglycosylated .

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Heat map showing absolute counts of unique peptides for S. indica apoplastic proteins present at all three time points and in culture filtrate. S1/S2/S3 = biologically independent sample 1, 2 and 3; d = deglycosylated.


eATP in APF from S . indica inoculated mock - treated barley root samples collected at predominantly biotrophic phase ( 3 5 dpi ) as well as predominantly associated phase ( 7 10 dpi ) . Error bars show standard error of mean obtained from three independent biological replicates at p < 0 . 05 ( * ) , 0 . 01 ( * * ) analyzed by Student ' s t - .

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eATP levels in APF from S. indica inoculated and mock-treated barley root samples collected at the predominantly biotrophic phase (3 and 5 dpi) as well as the predominantly cell death associated phase (7 and 10 dpi). Error bars show the standard error of the mean obtained from three independent biological replicates at p< 0.05 (*), 0.01 (**) analyzed by Student's t-test.


eATP measured from culture medium collected from mock treated S . indica colonized Arabidopsis seedlings at 3 , 5 , 7 10 dpi . Error bars represent ± SE of mean from three independent biological replicates . RLU : relative units . Asterisks indicate significance at p < 0 . 05 ( * ) , 0 . 01 ( * * ) analyzed by Student ' s t - .

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eATP levels measured from culture medium collected from mock treated and S. indica colonized Arabidopsis seedlings at 3, 5, 7 and 10 dpi. Error bars represent ±SE of the mean from three independent biological replicates. RLU: relative light units. Asterisks indicate significance at p< 0.05 (*), 0.01 (**) analyzed by Student's t-test.


S . indica colonization of Arabidopsis dorn1 - 3 mutant parental Col - 0aq lines quantified by qPCR at 3 , 5 , 7 10 dpi . of fungal ( SiTEF ) to ( AtUBI ) amplicons representing fungal colonization in root tissue was calculated using gDNA as template 2 - ΔCT method . Error bars represent standard error of mean of three technical replicates . experiment was repeated 3 times for 3 , 5 7 dpi with similar outcomes .

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S. indica colonization of Arabidopsis dorn1-3 mutant and the parental Col-0aq lines quantified by qPCR at 3, 5, 7 and 10 dpi. The ratio of fungal (SiTEF) to plant (AtUBI) amplicons representing fungal colonization levels in plant root tissue was calculated using gDNA as template and the 2-ΔCT method. Error bars represent standard error of the mean of three technical replicates. The experiment was repeated 3 times for 3, 5 and 7 dpi with similar outcomes.


Transcript of S . indica E5 ′ NT during colonization of barley Arabidopsis at different symbiotic stages in axenic culture . Error bars represent standard error of mean of three independent biological replicates . CM = medium .

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Transcript levels of S. indica E5′NT during colonization of barley and Arabidopsis at different symbiotic stages and in axenic culture. Error bars represent standard error of the mean of three independent biological replicates. CM = complex medium.


Ecto - 5 ′ - nucleotidase activity measured in preparations of Arabidopsis expressing Pro35S : : E5 ′ NT ( # 303 ) , Pro35S : : SPE5 ′ NT : mCherry : E5 ′ NTwoSP ( # 304 ) or Pro35S : : mCherry ( # 305 ) . E5 ′ NT activity was measured after incubation with 100 µM of either ATP , ADP or AMP . In preparations from Pro35S : : E5 ′ NT ( # 303 ) lines phosphate release was specifically increased upon incubation with purines . Error bars represent standard error of mean from three technical repetitions . coomassie stained SDS - PAGE shows pattern of fractions for transgenic lines . Equal volumes were loaded . experiment was repeated two times with similar results .

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

Ecto-5′-nucleotidase activity measured in membrane protein preparations of Arabidopsis plants expressing Pro35S::E5′NT (#303), Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) or Pro35S::mCherry (#305). E5′NT activity was measured after incubation with 100 µM of either ATP, ADP or AMP. In the membrane protein preparations from Pro35S::E5′NT (#303) lines phosphate release was specifically increased upon incubation with purines. Error bars represent the standard error of the mean from three technical repetitions. The coomassie stained SDS-PAGE shows the protein pattern of the membrane fractions for the individual transgenic lines. Equal volumes were loaded. The experiment was repeated two times with similar results.


Confocal microscopy of Arabidopsis roots expressing either mCherry ( # 305 ) or Pro35S : : SPE5 ′ NT : mCherry : E5 ′ NTwoSP ( # 304 ) showing secretion of E5 ′ NT fusion . mCherry show z - stacks of 14 image planes of 1 µm each . Scale bar = 20 µm .

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Confocal microscopy images of Arabidopsis roots expressing either cytosolic mCherry (#305) or Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) showing secretion of the E5′NT fusion protein. mCherry images show z-stacks of 14 image planes of 1 µm each. Scale bar=20 µm.


transgenic Arabidopsis Pro35S : : E5 ′ NT ( # 303 ) expressing untagged full SiE5 ′ NT was better colonized by S . indica . Error bars of qPCR data represent ± SE of mean from three independent biological replicates . Asterisks indicate significance ( Student ' s t - , * p < 0 . 05 ) .

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The transgenic Arabidopsis line Pro35S::E5′NT (#303) expressing untagged full length SiE5′NT was better colonized by S. indica. Error bars of the qPCR data represent ±SE of the mean from three independent biological replicates. Asterisks indicate significance (Student's t-test, * p< 0.05).


S . indica induced eATP release in different Arabidopsis transgenic lines . Culture medium was collected from mock treated or S . indica inoculated seedlings growing in liquid medium at 5 dpi released eATP was measured . RLU : relative units . Error bars represent ± SE of mean from three biological replicates . Asterisks indicate significance to other samples within same treatment group ( ANOVA , p < 0 . 05 ) .

S. indica induced eATP release in different Arabidopsis transgenic lines. Culture medium was collected from mock treated or S. indica inoculated seedlings growing in liquid medium at 5 dpi and released eATP was measured. RLU: relative light units. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05).


S . indica colonization of transgenic lines at 5 dpi . Error bars represent ± SE of mean from three biological replicates . Asterisks indicate significance to other samples within same treatment group ( ANOVA , p < 0 . 05 ) .

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S. indica colonization of transgenic lines at 5 dpi. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05).


Expression of eATP responsive gene At1g58420 measured by qRT - PCR . Error bars represent ± SE of mean from three independent biological replicates ( independent from those shown in Fig . 2C ) . Letters indicate significant groups ( ANOVA , p < 0 . 01 , for 305 p < 0 . 05 ) .

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Expression analysis of the eATP responsive gene At1g58420 measured by qRT-PCR. Error bars represent ±SE of the mean from three independent biological replicates (independent from those shown in Fig. 2C). Letters indicate significant groups (ANOVA, p<0.01, for the line 305 p<0.05).


Relative fluxes of phosphates ( Pi ) sugars ( C ) across fungal plasma . Without additional Pi - source in apoplast ( value 0 at x - axis , black dots ) there is a constant flux of phosphate via proton - coupled phosphate ( H / P ) transporter from fungus to apoplast a constant flux of sugar from apoplast to fungus . For better comparison these fluxes were normalized to 1 - 1 , respectively , other fluxes were calculated relative to these control values . With increasing ATP - release Pi - production Pi - efflux gets smaller is zero at a relative Pi - production rate of 1 ( grey triangles ) . At higher ATP - release Pi - production rates fungus imports Pi , i . e . transport direction of phosphate has been inverted in comparison to control condition ( square ) . C - flux via proton - coupled sugar ( H / C ) transporter is not affected by additional Pi - source ( horizontal grey ) .

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Relative fluxes of phosphates (Pi) and sugars (C) across the fungal plasma membrane. Without additional Pi-source in the apoplast (value 0 at the x-axis, black dots) there is a constant flux of phosphate via the proton-coupled phosphate (H/P) transporter from the fungus to the apoplast and a constant flux of sugar from the apoplast to the fungus. For better comparison these fluxes were normalized to 1 and -1, respectively, and all other fluxes were calculated relative to these control values. With increasing ATP-release and Pi-production the Pi-efflux gets smaller and is zero at a relative Pi-production rate of 1 (grey triangles). At higher ATP-release and Pi-production rates the fungus imports Pi, i.e. the transport direction of phosphate has been inverted in comparison to the control condition (white square). The C-flux via the proton-coupled sugar (H/C) transporter is not affected by the additional Pi-source (horizontal grey line).


Relative fluxes of Pi C across plasma . Without additional Pi - source in apoplast ( value 0 at x - axis , black dots ) there is a constant flux of sugar via H / C transporter from to apoplast ( thereafter to fungus ) a constant flux of phosphate ( coming from fungus ) via H / P transporter from apoplast to . With increasing ATP - release Pi - production Pi - influx via H / P transporter increases while C - flux via H / C transporter is unaffected by additional Pi - source ( horizontal grey ) .

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Relative fluxes of Pi and C across the plant plasma membrane. Without additional Pi-source in the apoplast (value 0 at the x-axis, black dots) there is a constant flux of sugar via the H/C transporter from the plant to the apoplast (and thereafter to the fungus) and a constant flux of phosphate (coming from the fungus) via the H/P transporter from the apoplast to the plant. With increasing ATP-release and Pi-production the Pi-influx via the H/P transporter increases while the C-flux via the H/C transporter is unaffected by the additional Pi-source (horizontal grey line).


Phosphate fluxes across plasma . Besides Pi - uptake via H / P transporter ( B , black , for clarity not shown in C ) , loses Pi due to ATP - release ( grey ) . difference between Pi - uptake Pi - loss is net Pi - balance of ( grey ) . Without ATP - release ( value 0 at x - axis , black dots ) gains Pi . At a relative ATP - release Pi - production of 1 ( grey triangles ) release as much phosphate as it takes up , while at higher ATP - release values , loses Pi .

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Phosphate fluxes across the plant plasma membrane. Besides the Pi-uptake via the H/P transporter (B, black line, for clarity not shown in C), the plant loses Pi due to the ATP-release (light grey line). The difference between Pi-uptake and Pi-loss is the net Pi-balance of the plant (dark grey line). Without ATP-release (value 0 at the x-axis, black dots) the plant gains Pi. At a relative ATP-release and Pi-production of 1 (grey triangles) the plant release as much phosphate as it takes up, while at higher ATP-release values, the plant loses Pi.


( a ) Immunoblot of caspase - 1 IL - 1β in lysates ( Lys ) supernatants ( Sup ) of peritoneal macrophages obtained from Map1lc3b - / - mice Becn1 + / - mice corresponding wild - type littermate mice , then left unstimulated ( - ) or stimulated with LPS alone ( LPS ) or LPS followed by 30 min of ATP treatment ( L + A ) . p10 , mature form of caspase - 1 . Right margin , molecular in kilodaltons ( kDa ) .

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(a) Immunoblot analysis of caspase-1 and IL-1β in lysates (Lys) and supernatants (Sup) of peritoneal macrophages obtained from Map1lc3b-/- mice and Becn1+/- mice and the corresponding wild-type littermate mice, then left unstimulated (-) or stimulated with LPS alone (LPS) or LPS followed by 30 min of ATP treatment (L + A). p10, mature form of caspase-1. Right margin, molecular size in kilodaltons (kDa).


( b ) Enzyme - linked immunosorbent ( ELISA ) of cytokine secretion by peritoneal macrophages stimulated with LPS , followed by treatment for 1 h with ATP .

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(b) Enzyme-linked immunosorbent assay (ELISA) of cytokine secretion by peritoneal macrophages stimulated with LPS, followed by treatment for 1 h with ATP.


( c ) Immunoblot of caspase - 1 IL - 1β in lysates ( left ) ELISA of cytokines in supernatants ( right ) of Map1lc3b + / + Map1lc3b - / - BMDMs incubated with LPS , followed by 30 min ( immunoblot ) or 1 h ( ELISA ) of ATP treatment .

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(c) Immunoblot analysis of caspase-1 and IL-1β in lysates (left) and ELISA of cytokines in supernatants (right) of Map1lc3b+/+ and Map1lc3b-/- BMDMs incubated with LPS, followed by 30 min (immunoblot) or 1 h (ELISA) of ATP treatment.


( d ) ELISA of IL - 1β secretion by LPS - primed Map1lc3b - / - mice Becn1 + / - BMDMs incubated for 1 h with z - YVAD - fmk ( z - YVAD ; 10 μM ) or vehicle ( dimethylsulfoxide ( DMSO ) ) , followed by stimulation for 1 h with ATP . * P 0 . 05 ( Student ' s t - ) . Data are representative of three or four experiments ( mean s . d . in b - d ) .

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(d) ELISA of IL-1β secretion by LPS-primed Map1lc3b-/- mice and Becn1+/- BMDMs incubated for 1 h with z-YVAD-fmk (z-YVAD; 10 μM) or vehicle (dimethylsulfoxide (DMSO)), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three or four experiments (mean and s.d. in b-d).


( a , b ) Transmission electron microscopy of morphological changes in mitochondria ( arrows ) in Map1lc3b − / − BMDMs ( a ) or Becn1 + / − BMDMs ( b ) left untreated or incubated for 6 h with LPS ( 10 ng / ml ) then stimulated for 30 min with ATP ( 1 mM ) . Outlined areas ( middle ) enlarged at right . Scale bars , 500 nm .

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(a,b) Transmission electron microscopy of morphological changes in mitochondria (arrows) in Map1lc3b−/− BMDMs (a) or Becn1+/− BMDMs (b) left untreated or incubated for 6 h with LPS (10 ng/ml) and then stimulated for 30 min with ATP (1 mM). Outlined areas (middle) enlarged at right. Scale bars, 500 nm.


( c ) Flow cytometry of Map1lc3b − / − Becn1 + / − macrophages left unstained or labeled with MitoSOX .

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(c) Flow cytometry of Map1lc3b−/− and Becn1+/− macrophages left unstained or labeled with MitoSOX.


( d ) Flow cytometry of macrophages left untreated or stimulated with LPS ATP ; were stained for 15 min with MitoTracker Deep Red MitoTracker Green before ATP treatment . Data are representative of three experiments .

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(d) Flow cytometry of macrophages left untreated or stimulated with LPS and ATP; cells were stained for 15 min with MitoTracker Deep Red and MitoTracker Green before ATP treatment. Data are representative of three experiments.


( a ) ELISA of cytokines in supernatants ( left ) immunoblot of caspase - 1 IL - 1β in lysates ( right ) of J774A . 1 macrophages exposed to EtBr ( 0 , 10 , 100 or 500 ng / ml ; wedges ) left unstimulated or stimulated with LPS alone or with LPS ATP .

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(a) ELISA of cytokines in supernatants (left) and immunoblot analysis of caspase-1 and IL-1β in lysates (right) of J774A.1 macrophages exposed to EtBr (0, 10, 100 or 500 ng/ml; wedges) and left unstimulated or stimulated with LPS alone or with LPS and ATP.


( b ) Immunoblot of caspase - 1 ASC in lysates of J774A . 1 or ρ0 J774A . 1 macrophages ( treated with 100 ng / ml of EtBr ) incubated with LPS , followed by stimulation for 0 , 15 or 30 min with ATP .

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(b) Immunoblot analysis of caspase-1 and ASC in lysates of J774A.1 or ρ0 J774A.1 macrophages (treated with 100 ng/ml of EtBr) incubated with LPS, followed by stimulation for 0, 15 or 30 min with ATP.


( c ) Cytokine secretion by J774A . 1 or ρ0 J774A . 1 macrophages incubated with various combinations of LPS ATP ( below graph ) .

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(c) Cytokine secretion by J774A.1 or ρ0 J774A.1 macrophages incubated with various combinations of LPS and ATP (below graph).


( d ) Cytokine secretion ( right ) immunoblot of caspase - 1 IL - 1β in lysates ( left ) of LPS - primed macrophages incubated for 1 h with rotenone ( + ; 5 μM ) or DMSO ( − ) before ATP treatment .

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(d) Cytokine secretion (right) and immunoblot analysis of caspase-1 and IL-1β in lysates (left) of LPS-primed macrophages incubated for 1 h with rotenone (+; 5 μM) or DMSO (−) before ATP treatment.


( e ) ELISA of IL - 1β secretion by J774A . 1 or ρ0 J774A . 1 macrophages incubated with LPS rotenone ( or DMSO ) , followed by ATP .

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(e) ELISA of IL-1β secretion by J774A.1 or ρ0 J774A.1 macrophages incubated with LPS and rotenone (or DMSO), followed by ATP.


( f ) Cytokine secretion by peritoneal macrophages preincubated for 1 h with Mito - TEMPO then stimulated with various combinations of LPS ATP .

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(f) Cytokine secretion by peritoneal macrophages preincubated for 1 h with Mito-TEMPO and then stimulated with various combinations of LPS and ATP.


( g , h ) ELISA of IL - 1β secretion ( left ) immunoblot for caspase - 1 in lysates ( right ) of Map1lc3b + / + Map1lc3b − / − ( g ) or Becn1 + / + Becn1 + / − ( h ) peritoneal macrophages incubated for 1 h with Mito - TEMPO ( 500 μM ) , followed by LPS ATP . * P 0 . 05 ( Student ' s t - ) . Data represent three experiments ( mean s . d . in a , c , d - h ) .

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(g,h) ELISA of IL-1β secretion (left) and immunoblot analysis for caspase-1 in lysates (right) of Map1lc3b+/+ and Map1lc3b−/− (g) or Becn1+/+ and Becn1+/− (h) peritoneal macrophages incubated for 1 h with Mito-TEMPO (500 μM), followed by LPS and ATP. *P 0.05 (Student's t-test). Data represent three experiments (mean and s.d. in a,c,d-h).


( a ) ELISA of cytokine secretion by LPS - primed Map1lc3b + / + Map1lc3b − / − BMDMs incubated with cyclosporine A or DMSO , followed by stimulation for 1 h with ATP .

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(a) ELISA of cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− BMDMs incubated with cyclosporine A or DMSO, followed by stimulation for 1 h with ATP.


( b ) ELISA of cytokine secretion by LPS - primed Becn1 + / + or Becn1 + / − BMDMs incubated with cyclosporine A ( 10 μM ) or DMSO , followed by ATP stimulation .

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(b) ELISA of cytokine secretion by LPS-primed Becn1+/+ or Becn1+/− BMDMs incubated with cyclosporine A (10 μM) or DMSO, followed by ATP stimulation.


( c ) Quantitative PCR of mtDNA in LPS - primed BMDMs incubated for 1 h with rotenone ( 5 μM ) or vehicle ( DMSO ) before ATP stimulation .

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(c) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed BMDMs incubated for 1 h with rotenone (5 μM) or vehicle (DMSO) before ATP stimulation.


( d ) Quantitative PCR of mtDNA in macrophages preincubated for 1 h with Mito - TEMPO ( 500 μM ) then incubated with LPS , followed by ATP stimulation .

(d) Quantitative PCR analysis of cytosolic mtDNA in macrophages preincubated for 1 h with Mito-TEMPO (500 μM) and then incubated with LPS, followed by ATP stimulation.


( e ) Quantitative PCR of mtDNA in LPS - primed BMDM incubated with cyclosporine A ( 10 μM ) , plus either rotenone ( 5 μM ) or DMSO , followed by ATP stimulation .

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(e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed BMDM incubated with cyclosporine A (10 μM), plus either rotenone (5 μM) or DMSO, followed by ATP stimulation.


( f ) Quantitative PCR of mtDNA in LPS - primed Map1lc3b − / − Becn1 + / − BMDMs stimulated for 30 min with ATP . amount of mtDNA is presented relative to amount in untreated control , set as 1 . * P 0 . 05 ( Student ' s t - ) . Data are representative of three experiments ( mean s . d . ) .

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(f) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed Map1lc3b−/− and Becn1+/− BMDMs stimulated for 30 min with ATP. The amount of cytosolic mtDNA is presented relative to the amount in untreated control cells, set as 1. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).


( a ) ELISA of cytokines in supernatants of BMDMs incubated for 4 h after delivery of 3 μg DNase I , lactate dehydrogenase LDH ( Control ) or heat - inactivated DNase I ( HI DNase I ) then stimulated with LPS , followed by stimulation for 1 h with ATP .

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(a) ELISA of cytokines in supernatants of BMDMs incubated for 4 h after delivery of 3 μg DNase I, lactate dehydrogenase LDH (Control) or heat-inactivated DNase I (HI DNase I) and then stimulated with LPS, followed by stimulation for 1 h with ATP.


( b ) Quantitative PCR of mtDNA in BMDMs transfected as in a then stimulated with LPS , followed by stimulation for 15 min with ATP .

(b) Quantitative PCR analysis of cytosolic mtDNA in BMDMs transfected as in a and then stimulated with LPS, followed by stimulation for 15 min with ATP.


( c ) ELISA of IL - 1β secretion by Aim2 + / + Aim2 − / − BMDMs primed for 4 h with LPS ( 200 ng / ml ) , then transfected for 6 h with 1 μg mtDNA ( through use of liposomes as vehicle ) , followed by stimulation for 1 h with ATP . * P 0 . 05 , versus Aim2 − / − treated with LPS ATP ( Student ' s t - ) .

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(c) ELISA of IL-1β secretion by Aim2+/+ and Aim2−/− BMDMs primed for 4 h with LPS (200 ng/ml), then transfected for 6 h with 1 μg mtDNA (through the use of liposomes as the vehicle), followed by stimulation for 1 h with ATP. *P 0.05, versus Aim2−/− cells treated with LPS and ATP (Student's t-test).


( d ) ELISA of cytokine secretion by LPS - primed Aim2 − / − BMDMs transfected for 6 h with mtDNA , poly ( dA : dT ) or mtDNA predigested by DNase I ( + DNase ; far left ) , followed by stimulation for 1 h with ATP . * P 0 . 05 ( Student ' s t - ) . Data are representative of three experiments ( mean s . d . ) .

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(d) ELISA of cytokine secretion by LPS-primed Aim2−/− BMDMs transfected for 6 h with mtDNA, poly(dA:dT) or mtDNA predigested by DNase I (+ DNase; far left), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).


( a ) Cytokine secretion by LPS - primed Map1lc3b + / + Map1lc3b − / − ( a ) or Becn1 + / + Becn1 + / − ( b ) peritoneal macrophages incubated for 15 min with glybenclamide ( Glyb ; 100 μM ( a ) or 50 - 100 μM ( b ) ) , Bay 11 - 7082 ( Bay ; 12 μM ) or DMSO ( - ) , followed by ATP .

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(a) Cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− (a) or Becn1+/+ and Becn1+/− (b) peritoneal macrophages incubated for 15 min with glybenclamide (Glyb; 100 μM (a) or 50-100 μM (b)), Bay 11-7082 (Bay; 12 μM) or DMSO (-), followed by ATP.


( c ) Cytokine secretion by LPS - primed wild - type ( WT ) NALP3 - deficient ( NALP3 - KO ) macrophages incubated with rotenone , followed by stimulation for 1 h with ATP .

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(c) Cytokine secretion by LPS-primed wild-type (WT) and NALP3-deficient (NALP3-KO) macrophages incubated with rotenone, followed by stimulation for 1 h with ATP.


( d ) Quantitative PCR of mtDNA in wild - type NALP3 - deficient BMDMs left unstimulated ( - ) or stimulated with LPS , followed by treatment for 15 or 30 min with ATP ( left ) , immunoblot of cytochrome c activation of caspase - 1 in at left ( right ) .

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(d) Quantitative PCR analysis of cytosolic mtDNA in wild-type and NALP3-deficient BMDMs left unstimulated (-) or stimulated with LPS, followed by treatment for 15 or 30 min with ATP (left), and immunoblot analysis of cytosolic cytochrome c and activation of caspase-1 in the cells at left (right).


( e ) Quantitative PCR of mtDNA in LPS - primed wild - type , NALP3 - deficient or ASC - deficient ( ASC - KO ) BMDMs stimulated for 15 min with ATP . * P 0 . 05 , NALP3 - deficient or ASC - deficient versus wild - type treated with LPS ATP ( Student ' s t - ) .

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(e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed wild-type, NALP3-deficient or ASC-deficient (ASC-KO) BMDMs stimulated for 15 min with ATP. *P 0.05, NALP3-deficient or ASC-deficient versus wild-type treated with LPS and ATP (Student's t-test).


( f ) Flow cytometry of untreated macrophages ( control ) or of LPS - primed macrophages left unstained or stained with MitoSOX , then stimulated for 15 min with ATP .

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(f) Flow cytometry of untreated macrophages (control) or of LPS-primed macrophages left unstained or stained with MitoSOX, then stimulated for 15 min with ATP.


( g ) Flow cytometry of untreated macrophages ( control ) or of macrophages stimulated with LPS ATP , stained with MitoTracker Deep Red . Numbers above bracketed lines indicate with loss of mitochondrial potential .

(g) Flow cytometry of untreated macrophages (control) or of macrophages stimulated with LPS and ATP, stained with MitoTracker Deep Red. Numbers above bracketed lines indicate percent cells with loss of mitochondrial membrane potential.


( h ) Flow cytometry of LPS - primed wild - type , ASC - deficient NALP3 - deficient peritoneal macrophages stimulated for 30 min with ATP stained with annexin V . Data represent three experiments ( mean s . d . in a - e ) .

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(h) Flow cytometry of LPS-primed wild-type, ASC-deficient and NALP3-deficient peritoneal macrophages stimulated for 30 min with ATP and stained with annexin V. Data represent three experiments (mean and s.d. in a-e).


( a ) ELISA of serum IL - 1β IL - 18 at 24 h after intraperitoneal injection of LPS ( 12 mg per kg ) into Map1lc3b + / + Map1lc3b − / − mice .

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(a) ELISA of serum IL-1β and IL-18 at 24 h after intraperitoneal injection of LPS (12 mg per kg body weight) into male Map1lc3b+/+ and Map1lc3b−/− mice.


( b ) Survival of Map1lc3b + / + mice ( n = 11 ) Map1lc3b − / − mice ( n = 10 ) after LPS challenge ( 100 mg per kg ) , assessed over a period of 2 .

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(b) Survival of Map1lc3b+/+ mice (n = 11) and Map1lc3b−/− mice (n = 10) after LPS challenge (100 mg per kg body weight), assessed over a period of 2 weeks.


( c , d ) ELISA of serum IL - 1β IL - 18 in Map1lc3b + / + Map1lc3b − / − mice ( c ) Becn1 + / + Becn1 + / − mice ( d ) at 24 h after CLP or sham laparotomy . In a , c , d , each symbol represents an mouse ; small horizontal lines indicate mean . P values , unpaired two - Student ' s t - ( a , c , d ) or log - rank ( b ) . Data are representative of two experiments .

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(c,d) ELISA of serum IL-1β and IL-18 in male Map1lc3b+/+ and Map1lc3b−/− mice (c) and Becn1+/+ and Becn1+/− mice (d) at 24 h after CLP or sham laparotomy. In a,c,d, each symbol represents an individual mouse; small horizontal lines indicate the mean. P values, unpaired two-tailed Student's t-test (a,c,d) or log-rank test (b). Data are representative of two experiments.


( A ) features mapped here displayed differences between CLL ( CLL1 ) an NBC donor ( H7 ) as shown for TCF4 locus , which encodes for a transcription factor from E family . on increased H3K4me1 , H3K27ac ATAC signal , two predicted enhancer loci were marked that became active in CLL . Note that y - axis for - seq is scaled differently for CLL ( 8000 ) NBCs ( 100 ) to visualize that TCF4 gene was not completely silenced but lowly expressed also in NBCs as evident also from H3K36me3 mark . grey depicts active region grey confined enhancer locus coinciding with an open region .

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(A) Chromatin features mapped here displayed differences between CLL (patient CLL1) and an NBC donor (H7) as shown for the TCF4 locus, which encodes for a transcription factor from the E protein family. Based on the increased H3K4me1, H3K27ac and ATAC signal, two predicted enhancer loci were marked that became active in CLL. Note that the y-axis for RNA-seq is scaled differently for CLL (8000) and NBCs (100) to visualize that the TCF4 gene was not completely silenced but lowly expressed also in NBCs as evident also from the H3K36me3 mark. Light grey depicts active chromatin region and dark grey the confined enhancer locus coinciding with an open chromatin region.


( D ) Distribution of ~ 24 , 400 annotated differentially accessible regions ( ATAC - seq ) in NBC CLL samples according to state annotation . In total 7 , 605 annotated regions gained an ATAC - seq signal in CLL , while it was lost at 16 , 790 loci .

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(D) Distribution of ~24,400 annotated differentially accessible regions (ATAC-seq) in NBC and CLL samples according to the chromatin state annotation. In total 7,605 annotated regions gained an ATAC-seq signal in CLL, while it was lost at 16,790 loci.


( A ) Left , example of a large PMD on chromosome 2 derived from a consensus of CLL samples ( n = 11 ) . Right , genome - wide of PMDs across CLL samples ( n = 11 ) NBCs ( n = 6 ) . Red , methylated ; blue , unmethylated .

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(A) Left, example of a large PMD on chromosome 2 derived from a consensus of CLL samples (n=11). Right, genome-wide quantification of PMDs across CLL samples (n=11) and NBCs (n=6). Red, methylated DNA; blue, unmethylated DNA.


( B ) Expression of genes located inside ( blue ) outside ( grey ) PMD regions . ( C ) Fraction of differentially expressed genes inside or outside PMD regions . Up - downregulated genes are shown in red green , respectively .

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(B) Expression of genes located inside (blue) and outside (grey) the PMD regions. (C) Fraction of differentially expressed genes inside or outside PMD regions. Up- and downregulated genes are shown in red and green, respectively.


( D ) Upper panel : Average signal of histone modification marks normalized to H3 standard deviation in 5 - kb windows around ± 50 kb flanking regions of PMD boundaries . Normalized fold changes were calculated by dividing to average signal flanking outside PMD boundaries . Blue box , within PMDs ; thin , outside PMDs , nor . - normalized . Lower panel : Distribution of bound CTCF sites in CLL as determined by ChIP - seq ( blue ) around ± 50 kb flanking regions of PMD boundaries in 5 kb windows . height of curves gives sum of next nearest CTCF peak at given to PMD boundary .

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(D) Upper panel: Average signal of histone modification marks normalized to H3 and standard deviation in 5-kb windows around the ±50 kb flanking regions of PMD boundaries. Normalized fold changes were calculated by dividing to the average signal flanking outside the PMD boundaries. Blue box, within PMDs; thin line, outside PMDs, nor. - normalized. Lower panel: Distribution of bound CTCF sites in CLL cells as determined by ChIP-seq (blue line) around the ±50 kb flanking regions of PMD boundaries in 5 kb windows. The height of the curves gives the sum of the next nearest CTCF peak at the given distance to the PMD boundary.


( A ) Correlation function of H3K4me3 ChIP - seq reads . A broadening of H3K4me3 domains in CLL by 1 - 2 nucleosomes was detected . Error bars represent .

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(A) Correlation function of H3K4me3 ChIP-seq reads. A broadening of H3K4me3 domains in CLL by 1-2 nucleosomes was detected. Error bars represent the SEM.


( B ) H3K4me3 peak width distribution at common promoters in CLL NBC controls .

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(B) H3K4me3 peak width distribution at common promoters in CLL and NBC controls.


( C ) Distribution of nucleosome occupancy calculated from histone H3 ChIP - seq averaged over a 1000 bp window within promoters . H3K4me3 regions displayed higher nucleosome density for CLL as compared to NBC samples .

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(C) Distribution of nucleosome occupancy calculated from histone H3 ChIP-seq averaged over a 1000 bp window within promoters. H3K4me3 regions displayed higher nucleosome density for CLL as compared to NBC samples.


( D ) Exemplary region at TAF13 promoter showing higher H3K4me3 upstream of TSS with lost ATAC signal ( grey bar ) as compared to NBC control .

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(D) Exemplary region at the TAF13 promoter showing higher H3K4me3 levels upstream of the TSS with lost ATAC signal (grey bar) as compared to the NBC control.


( E ) TF motif of ATAC signal lost at CLL promoters with broadened H3K4me3 regions . An enrichment for NFY TF motif was detected .

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(E) TF motif analysis of ATAC signal lost at CLL promoters with broadened H3K4me3 regions. An enrichment for the NFY TF motif was detected.


( F ) of genome - wide histone modification patterns at promoters within - 2 to 5 kb around TSS ( x - axis ) for an NBC ( H3 ) a ( CLL1 ) sample . Each on y - axis corresponds to one promoter . clustering revealed one cluster with loss of H3K4me3 at bivalent promoters in CLL .

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(F) Heatmap of genome-wide histone modification patterns at promoters within -2 to 5 kb around the TSS (x-axis) for an NBC (H3) and a patient (CLL1) sample. Each line on the y-axis corresponds to one promoter. The clustering revealed one cluster with loss of H3K4me3 at bivalent promoters in CLL.


( D ) Clustering of samples via expression of bidirectionally regions that are differential between NBCs CLL quantified using DESeq2 .

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(D) Clustering of samples via expression of bidirectionally regions that are differential between NBCs and CLL and quantified using DESeq2.


( E ) Volcano plot of differential super - enhancers targeting known leukemia cancer genes . Examples include SE loss at CDKN1A , PI3KC2B KMT2B ( MLL2 ) SE gain at FMOD , CREB3L2 , CTLA4 , TCF4 , LEF1 BCL2 . Points represent non - differential SEs ( grey ) , differential SEs ( FDR < 0 . 01 ) with fold change greater than 1 ( orange ) .

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(E) Volcano plot of differential super-enhancers targeting known leukemia and cancer genes. Examples include SE loss at CDKN1A, PI3KC2B and KMT2B (MLL2) and SE gain at FMOD, CREB3L2, CTLA4, TCF4, LEF1 and BCL2. Points represent non-differential SEs (grey), and differential SEs (FDR < 0.01) with fold change greater than 1 (orange).


( F ) expression changes of selected genes associated with differential SEs . Top : genes significantly ( FDR < 0 . 05 ) up - regulated by SEs in CLL . Bottom : Genes significantly down - regulated by SEs in CLL .

(F) RNA expression changes of selected genes associated with differential SEs. Top: genes significantly (FDR < 0.05) up-regulated by SEs in CLL. Bottom: Genes significantly down-regulated by SEs in CLL.


( G ) Comparison of normalized gene expression of histone deacetylases between CLL NBCs . Histone deacetylases significantly up - regulated in CLL are shown in bold .

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(G) Comparison of normalized gene expression of histone deacetylases between CLL and NBCs. Histone deacetylases significantly up-regulated in CLL are shown in bold.


( H ) HDAC activity its inhibition by panobinostat in B from CLL ( red ) in comparison to healthy donors ( grey ) . Error bars indicate standard deviation measured in four biological replicates .

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(H) HDAC activity and its inhibition by panobinostat in B cells from CLL patients (red) in comparison to healthy donors (grey). Error bars indicate standard deviation measured in four biological replicates.


( I ) Genome browser view of H3K27ac tracks ( in grey ) at exemplary genes for NBCs CLL 24 hours after mock after panobinostat treatment . At genes such as CDKN1A ( cycle control ) KLF13 , reduced H3K27ac signal in CLL was increased upon HDAC inhibition to level found in NBCs . WNT11 is shown as an example of a de novo gain of an active enhancer due to treatment with panobinostat .

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(I) Genome browser view of H3K27ac tracks (in grey) at exemplary genes for NBCs and CLL cells 24 hours after mock and after panobinostat treatment. At genes such as CDKN1A (cell cycle control) and KLF13, reduced H3K27ac signal in CLL was increased upon HDAC inhibition to the level found in NBCs. WNT11 is shown as an example of a de novo gain of an active enhancer due to treatment with panobinostat.


( J ) displaying changes in H3K27ac read occupancy in CLL upon panobinostat treatment for 24 h . A general gain of H3K27ac in enhancers upon panobinostat treatment was observed .

(J) Heatmap displaying changes in H3K27ac read occupancy in CLL upon panobinostat treatment for 24 h. A general gain of H3K27ac in enhancers upon panobinostat treatment was observed.


( B ) H3K27ac ( left ) methylation ( right ) at NFAT binding sites . CLL showed both an H3K27ac enrichment - hypomethylation at NFAT target sites , suggesting a higher activity of TFs from NFAT family in CLL .

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(B) H3K27ac (left) and DNA methylation (right) at NFAT binding sites. CLL cells showed both an H3K27ac enrichment and DNA-hypomethylation at NFAT target sites, suggesting a higher activity of TFs from the NFAT family in CLL.


( D ) ATAC footprints for E2A , EBF CTCF motifs from Homer . E2A motif footprint ( binding site of E family TFs like TCF4 ) displayed an increased binding signal in CLL while sites with EBF CTCF motif lost ATAC signal .

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(D) ATAC footprints for E2A, EBF and CTCF motifs from the Homer analysis. The E2A motif footprint (binding site of E protein family TFs like TCF4) displayed an increased binding signal in CLL while sites with the EBF and CTCF motif lost the ATAC signal.


( E ) Expression of genes nearest to target enhancers with constitutively bound ( " stable " ) CTCF vs . enhancers that lost CTCF in CLL . Loss of CTCF binding correlated with reduced gene expression .

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(E) Expression of the genes nearest to target enhancers with constitutively bound ("stable") CTCF vs. enhancers that lost CTCF in CLL. Loss of CTCF binding correlated with reduced gene expression.


( G ) Enhancer - promoter rewiring at NFKB2 locus . A switch of interactions between NFKB2 promoter two different enhancers in CLL ( red ) vs . NBCs ( grey ) was observed . on CTCF ChIP - seq both intronic enhancers at NFKB2 FBXL15 gene show ( nearby ) constitutively bound CTCF , even though targets of two enhancers are switched .

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(G) Enhancer-promoter rewiring at the NFKB2 locus. A switch of interactions between the NFKB2 promoter and two different enhancers in CLL (red line) vs. NBCs (grey line) was observed. Based on the CTCF ChIP-seq analysis both intronic enhancers at the NFKB2 and the FBXL15 gene show (nearby) constitutively bound CTCF, even though the targets of the two enhancers are switched.


( E ) of regulation of SNX22 by intronic H464 enhancer shown in panel B . This enhancer lost its ATAC signal at predicted EBF1 binding site . EBF1 ChIP - seq validated that EBF1 is indeed lost at this site . Although H3K27ac at locus was only slightly reduced , transcription of SNX22 was largely reduced . These findings are consistent with a mechanism where EBF1 binding drives gene expression of SNX22 by binding to H464 .

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(E) Analysis of regulation of SNX22 by the intronic H464 enhancer shown in panel B. This enhancer lost its ATAC signal at the predicted EBF1 binding site. EBF1 ChIP-seq analysis validated that EBF1 is indeed lost at this site. Although H3K27ac at the locus was only slightly reduced, transcription of SNX22 was largely reduced. These findings are consistent with a mechanism where EBF1 binding drives gene expression of SNX22 by binding to H464.


B Silver stain of GST ‐ PTB RRM2pull ‐ down of wild ‐ type RRM2 Y247Q mutant . Five microlitre of pull ‐ down was run on a 15 % SDS - PAGE silver ‐ stained . Three strong bands can be seen which are due to recombinant beads used for pull ‐ down . region encompassing 50 kDa to top of gel was sliced subjected to in ‐ gel digestion mass spectrometry . two strongest bands visible in this region are labelled as Matrin3 Raver1 .

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B Silver stain of the GST‐PTB RRM2pull‐down of the wild‐type RRM2 and the Y247Q mutant. Five microlitre of the pull‐down was run on a 15% SDS-PAGE and silver‐stained. Three strong bands can be seen which are due to the recombinant protein and the beads used for the pull‐down. The region encompassing 50 kDa to the top of the gel was sliced and subjected to in‐gel digestion and mass spectrometry. The two strongest bands visible in this region are labelled as Matrin3 and Raver1.


C identified in GST ‐ PTB RRM2 pull ‐ down ranked by their unique peptide number . table shows different we found binding to RRM2 , as well as when present , of PRI motif . indicated function is only a guideline as many have more functions than shown .

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C Proteins identified in the GST‐PTB RRM2 pull‐down ranked by their unique peptide number. The table shows the different proteins we found binding to RRM2, as well as when present, the sequence of the PRI motif. The indicated function is only a guideline as many have more functions than shown.


D Western blot of GSTpull ‐ down using antibodies against Matrin3 Raver1 . Lanes 1 2 show 5 10 % of input , respectively , lanes 3 4 show GST ‐ PTB full ‐ pull ‐ down of wild ‐ type Y247Q mutant , respectively , lanes 5 6 show GST ‐ PTB RRM2 pull ‐ down of wild ‐ type Y247Q mutant , respectively , lane 7 with pull ‐ down using GST alone .

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D Western blot of the GSTpull‐down using antibodies against Matrin3 and Raver1. Lanes 1 and 2 show 5 and 10% of input, respectively, lanes 3 and 4 show GST‐PTB full‐length pull‐down of wild‐type and Y247Q mutant, respectively, and lanes 5 and 6 show GST‐PTB RRM2 pull‐down of wild‐type and Y247Q mutant, respectively, and lane 7 with pull‐down using GST alone.


B FLAGimmunoprecipitation of Matrin3 Raver1 , both with wild ‐ type with PRI mutated , FLAG ‐ MS2 as a negative control . immunoprecipitated was separated in a SDS - PAGE subjected to Western blot using antibody against PTB which showed interaction to wild ‐ type Matrin3 ( lane 2 ) Raver1 ( lane 4 ) . input was also analysed by Western blot with antibodies against PTB as a loading control against FLAG tag to ensure equal expression of .

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B FLAGimmunoprecipitation of Matrin3 and Raver1, both with wild‐type and with PRI mutated, and FLAG‐MS2 as a negative control. The immunoprecipitated complex was separated in a SDS-PAGE and subjected to Western blot using antibody against PTB which showed interaction to wild‐type Matrin3 (lane 2) and Raver1 (lane 4). The input was also analysed by Western blot with antibodies against PTB as a loading control and against the FLAG tag to ensure equal expression of proteins.


C 20 ‐ residue Raver1 491 - 511 ( lane 1 ) Matrin3 346 - 365 ( lane 3 ) peptide fused to MS2 were transcribed translated in vitro ( Input ) then pulled down with GST ‐ PTB or with GST ‐ SXL as a control . Effects of single mutation of Matrin3 PRI GILPPP to GIAPPP were also tested ( lane 4 ) .

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C The 20‐residue Raver1 491-511 (lane 1) and the Matrin3 346-365 (lane 3) peptide fused to MS2 were transcribed and translated in vitro (Input) and then pulled down with GST‐PTB or with GST‐SXL as a control. Effects of single mutation of the Matrin3 PRI GILPPP to GIAPPP were also tested (lane 4).


A Western blot probed for Matrin3 ( top panel ) , PTB ( middle panel ) actin ( lower panel ) . Lanes 1 - 4 contain a twofold dilution of control C2 sample ( lane 1 - 12 . 5 % , lane 2 - 25 % , lane 3 - 50 % , lane 4 - 100 % ) . Lanes 4 - 7 contain equal amount of , as can be confirmed by anti ‐ actin ( lower panel ) , of control sample ( lane 4 ) , double ‐ PTB nPTB siRNA ‐ treated sample ( lane 5 ) , Matrin3 siRNA ‐ treated sample ( lane 6 ) triple knockdown of Matrin3 , PTB nPTB ( lane 7 ) . Lane 7 is from same gel exposure , but some lanes present in original gel were cropped for clarity , a black to indicate cropping was placed .

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A Western blot probed for Matrin3 (top panel), PTB (middle panel) and actin (lower panel). Lanes 1-4 contain a twofold dilution of the control C2 sample (lane 1-12.5%, lane 2-25%, lane 3-50%, lane 4-100%). Lanes 4-7 contain equal amount of proteins, as can be confirmed by the anti‐actin (lower panel), of control sample (lane 4), double‐PTB and nPTB siRNA‐treated sample (lane 5), Matrin3 siRNA‐treated sample (lane 6) and triple knockdown of Matrin3, PTB and nPTB (lane 7). Lane 7 is from the same gel and exposure, but some lanes present in the original gel were cropped for clarity, and a black line to indicate cropping was placed.


F RT - PCR validation of Matrin3 ‐ regulated alternative spicing events in ST7 , ACSL3 , PLEKHA3 , TCF12 , VWA5A , PTBP2 , PTBP3 , C3orf17 , ZMYND8 , VEZT , PIGX DMD genes . In each case , triplicates for each condition ( C - control , M - Matrin3 , PTB / nPTB Matrin3 / PTB / nPTB siRNA transfection samples ) were analysed exon inclusion ( EI ) is shown beneath corresponding lane , along with standard deviation ( s . d . ) .

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F RT-PCR validation of Matrin3‐regulated alternative spicing events in the ST7, ACSL3, PLEKHA3, TCF12, VWA5A, PTBP2, PTBP3, C3orf17, ZMYND8, VEZT, PIGX and DMD genes. In each case, triplicates for each condition (C-control, M-Matrin3, PTB/nPTB and Matrin3/PTB/nPTB siRNA transfection samples) were analysed and exon inclusion (EI) percentage is shown beneath the corresponding lane, along with the standard deviation (s.d.).


A Matrin3 crosslinking in Matrin3 ‐ regulated pre ‐ mRNAs where position of crosslinked nucleotides was mapped onto regulated exon 500 nucleotides upstream downstream of its 3 ′ ss 5 ′ ss , respectively , upstream flanking exon 500 nucleotides downstream of its 5 ′ ss downstream flanking exon with 500 nucleotides upstream of its 5 ′ ss . iCLIP tags were mapped onto silenced ASE ( blue , n = 421 ) , enhanced ASE ( red , n = 187 ) to control ASE ( grey , n = 16 , 242 ) , of occupancy is plotted .

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A Matrin3 crosslinking in Matrin3‐regulated pre‐mRNAs where position of crosslinked nucleotides was mapped onto the regulated exon and the 500 nucleotides upstream and downstream of its 3′ss and 5′ss, respectively, the upstream flanking exon and 500 nucleotides downstream of its 5′ss and the downstream flanking exon with 500 nucleotides upstream of its 5′ss. The iCLIP tags were mapped onto silenced ASE (blue, n = 421), enhanced ASE (red, n = 187) and to control ASE (grey, n = 16,242), and percentage of occupancy is plotted.


B Same as in ( A ) but using iCLIP tags obtained from PTBiCLIP mapped onto PTB / nPTB ‐ regulated ASE , silenced ASE ( blue , n = 729 ) , enhanced ( red , n = 820 ) control ASE ( grey , n = 14 , 599 ) .

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B Same as in (A) but using iCLIP tags obtained from PTBiCLIP and mapped onto PTB/nPTB‐regulated ASE, silenced ASE (blue, n = 729), enhanced (red, n = 820) and control ASE (grey, n = 14,599).


C Matrin3 crosslinked nucleotides mapped onto PTB ‐ regulated ASE .

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C Matrin3 crosslinked nucleotides mapped onto PTB‐regulated ASE.


D PTB crosslinked nucleotides mapped onto Matrin3 ‐ regulated ASE .

D PTB crosslinked nucleotides mapped onto Matrin3‐regulated ASE.


B Western blot of overexpressed Matrin3 its mutants probed with anti ‐ FLAG anti ‐ actin antibodies .

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B Western blot of the overexpressed Matrin3 and its mutants probed with anti‐FLAG and anti‐actin antibodies.


C , D RT - PCR using primers specific to ST7 exon 11 ( C ) ABI2 exon 8 ( D ) splicing reporters , respectively , of samples where FLAG ‐ tagged Matrin3 its mutants have been overexpressed . of at least three replicates for each condition is shown as a histogram of of exon inclusion . * P 0 . 01 compared with control sample ( − ) .

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C, D RT-PCR analysis using primers specific to the ST7 exon 11 (C) and ABI2 exon 8 (D) splicing reporters, respectively, of samples where FLAG‐tagged Matrin3 and its mutants have been overexpressed. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 compared with control sample (−).


E Western blot of overexpressed FLAG ‐ tagged Matrin3 PTB , in control siRNA PTB / nPTB siRNA ‐ transfected , probed with anti ‐ FLAG to detect overexpressed , anti ‐ PTB to detect knockdown anti ‐ actin to confirm loading . A titration of control sample ( lane 1 - 12 . 5 % , lane 2 - 25 % , lane 3 - 50 % lane 4 - 100 % ) is also included to assess of knockdown . band present above PTB doublet in lane 6 is FLAG ‐ tagged PTB .

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E Western blot of overexpressed FLAG‐tagged Matrin3 and PTB, in control siRNA and PTB/nPTB siRNA‐transfected cells, probed with anti‐FLAG to detect overexpressed proteins, anti‐PTB to detect the knockdown levels and anti‐actin to confirm protein loading. A titration of control sample (lane 1-12.5%, lane 2-25%, lane 3-50% and lane 4-100%) is also included to assess the levels of knockdown. The band present above the PTB doublet in lane 6 is FLAG‐tagged PTB.


F , G RT - PCR using primers specific to ST7 exon 11 ( F ) ABI2 exon 8 ( G ) splicing reporters , respectively , of samples where Matrin3 or PTB expression vectors were co ‐ transfected , in control siRNA PTB / nPTB siRNA ‐ transfected . of at least three replicates for each condition is shown as a histogram of of exon inclusion . * P 0 . 01 when compared with control sample in each condition ( ‐ ) .

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F, G RT-PCR analysis using primers specific to the ST7 exon 11 (F) and ABI2 exon 8 (G) splicing reporters, respectively, of samples where Matrin3 or PTB expression vectors were co‐transfected, in control siRNA and PTB/nPTB siRNA‐transfected cells. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 when compared with control sample in each condition (‐).


Western blot of whole lysates from HEK 293 WT NEDP1 KO reveals a loss of free NEDD8 ( indicated by asterisk ) an accumulation of NEDD8 reactive species in NEDP1 KO lysate . predicted molecular sizes of putative , unanchored , poly - NEDD8 chains are denoted by N2 through to N5 . Unconjugated NEDD8 is denoted by N1 .

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Western blot analysis of whole cell lysates from HEK 293 WT and NEDP1 KO cells reveals a loss of free NEDD8 (indicated by asterisk) and an accumulation of NEDD8 reactive species in the NEDP1 KO lysate. The predicted molecular weight sizes of putative, unanchored, poly-NEDD8 chains are denoted by N2 through to N5. Unconjugated NEDD8 is denoted by N1.


NEDD8 affinity resin shows enrichment of endogenous neddylated in WT NEDP1 KO . Recombinant HALO - NEDP1 C163A ( CA ) conjugated to HALO - Link beads was used as an affinity resin to enrich for neddylated in lysates from HEK 293 WT NEDP1 KO . Enriched were resolved by SDS - PAGE processed for Western blot with NEDD8 or Ubiquitin antibodies . HALO - NEDP1 CA specifically enriches for NEDD8 - reactive in both WT NEDP1 KO , but does not enrich for Ubiquitin - modified in either .

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NEDD8 affinity resin shows enrichment of endogenous neddylated proteins in WT and NEDP1 KO cells. Recombinant HALO-NEDP1 C163A (CA) conjugated to HALO-Link beads was used as an affinity resin to enrich for neddylated proteins in lysates from HEK 293 WT and NEDP1 KO cells. Enriched proteins were resolved by SDS-PAGE and processed for Western blot analysis with NEDD8 or Ubiquitin antibodies. HALO-NEDP1 CA specifically enriches for NEDD8-reactive proteins in both WT and NEDP1 KO cells, but does not enrich for Ubiquitin-modified proteins in either cell line.


Components of NEDD8 conjugation machinery are enriched in HALO - NEDP1 pulldowns from NEDP1 KO lysates . Neddylated from HEK293 KO were enriched by HALO - NEDP1 CA pulldown , as in ( B ) but not by NEDD8 nonbinder mutant , HALO - NEDP1 DAGC ( D29W A98K G99K C163A ) . NEDD8 E1s , UBA3 ULA1 , are modified in NEDP1 KO , as well as E2 UBE2M , co - E3s DCNL1 DCNL2 . Cul2 Cul3 are hyper - neddylated in NEDP1 KO . CSN components , CSN5 CSN8 , also co - precipitate in HALO - NEDP1 CA pulldowns .

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Components of the NEDD8 conjugation machinery are enriched in HALO-NEDP1 pulldowns from NEDP1 KO lysates. Neddylated proteins from HEK293 KO cells were enriched by HALO-NEDP1 CA pulldown, as in (B) but not by the NEDD8 nonbinder mutant, HALO-NEDP1 DAGC (D29W A98K G99K C163A). The NEDD8 E1s, UBA3 and ULA1, are modified in NEDP1 KO cells, as well as E2 UBE2M, and co-E3s DCNL1 and DCNL2. Cul2 and Cul3 are hyper-neddylated in NEDP1 KO cells. CSN components, CSN5 and CSN8, also co-precipitate in HALO-NEDP1 CA pulldowns.


Western blot from HEK 293 WT NEDP1 KO of components of NEDD8 conjugation / de - conjugation pathway shows that similar of NEDD8 pathway components are present in both WT NEDP1 KO . Apart from UBA3 , there is no detectable amount of NEDD8 - modified enzymes in whole - lysates from NEDP1 KO .

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Western blot analysis from HEK 293 WT and NEDP1 KO cells of the components of the NEDD8 conjugation/de-conjugation pathway shows that similar levels of NEDD8 pathway components are present in both WT and NEDP1 KO cells. Apart from UBA3, there is no detectable amount of NEDD8-modified enzymes in whole-cell lysates from NEDP1 KO cells.


Poly - NEDD8 chains can be generated by in vitro reactions ( Rxn ) . NAE ( 0 . 15 μM ) , UBE2M , NEDD8 ( 20 μM ) were incubated on ice or incubated at 30 ° C for 3 hours reactions were stopped by addition of LDS loading buffer . Reactions were resolved by SDS - PAGE stained with colloidal Coomaisse . Indicated bands were excised from gel processed for in gel trypsin digestion mass spectrometry . predicted molecular sizes for a theoretical unanchored NEDD8 chain are denoted by N2 - N4 . Unconjugated NEDD8 is indicated by N1 . UBE2M modified by NEDD8 is indicated with an asterisk .

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Poly-NEDD8 chains can be generated by in vitro reactions (Rxn). NAE (0.15 μM), UBE2M, and NEDD8 (20 μM) were incubated on ice or incubated at 30°C for 3 hours and reactions were stopped by addition of LDS loading buffer. Reactions were resolved by SDS-PAGE and stained with colloidal Coomaisse. Indicated bands were excised from the gel and processed for in gel trypsin digestion and mass spectrometry analysis. The predicted molecular weight sizes for a theoretical unanchored NEDD8 chain are denoted by N2-N4. Unconjugated NEDD8 is indicated by N1. UBE2M modified by NEDD8 is indicated with an asterisk.


Neddylated species are NEDD8 E1 dependent . WT NEDP1 KO HEK 293 were treated with NAE inhibitor MLN4924 at 3 μM for indicated time . Lysed were then processed for Western blot . NEDD8 E1 inhibition results in a time - dependent decrease in amount of Cullin non - Cullin NEDD8 reactive bands .

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Neddylated species are NEDD8 E1 dependent. WT and NEDP1 KO HEK 293 cells were treated with NAE inhibitor MLN4924 at 3 μM for the indicated time. Lysed cells were then processed for Western blot analysis. NEDD8 E1 inhibition results in a time-dependent decrease in the amount of Cullin and non-Cullin NEDD8 reactive bands.


Neddylated species are UBE2M dependent . WT NEDP1 KO HEK 293 were left untreated or treated with indicated siRNA for 48 hours . Lysed were then processed for Western blot . Neddylated species are reduced when NEDD8 E2 UBE2M was depleted after siRNA treatment ( si2M ) , but not with control siRNA ( siCTRL ) or when NEDD8 E2 UBE2F was depleted ( si2F ) . double knockdown ( si2M / 2F ) does not further reduce NEDD8 modified .

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Neddylated species are UBE2M dependent. WT and NEDP1 KO HEK 293 cells were left untreated or treated with the indicated siRNA for 48 hours. Lysed cells were then processed for Western blot analysis. Neddylated species are reduced when NEDD8 E2 UBE2M was depleted after siRNA treatment (si2M), but not with control siRNA (siCTRL) or when NEDD8 E2 UBE2F was depleted (si2F). The double knockdown (si2M/2F) does not further reduce NEDD8 modified proteins.


( Left ) Western blot of large scale HALO - NEDP1 pulldowns from HEK 293 NEDP1 KO lysates . ( Right ) Diagram of mass spectrometry sample preparation .

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(Left) Western blot analysis of large scale HALO-NEDP1 pulldowns from HEK 293 NEDP1 KO lysates. (Right) Diagram of mass spectrometry sample preparation.


Scatterplot of identified by mass spectrometry identifies NEDD8 components of NEDD8 conjugation pathway as being most abundant in HEK 293 NEDP1 KO lysates following NEDP1 - CA pulldown . iBAQ of identified in A ) are plotted as log2 value of enrichment ( mass spectrometry of HALO - NEDP1 CA pulldown over HALO - NEDP1 DAGC pulldown ) versus log10 value of iBAQ from HALO - NEDP1 CA pulldown . Blue markers indicate known components of NEDD8 pathway red markers indicate that have been identified as substrates of PARP - 1 in database of ADP - ribosylated , ADPriboDB ( Vivelo et al . , 2017 ) .

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Scatterplot of proteins identified by mass spectrometry analysis identifies NEDD8 and components of the NEDD8 conjugation pathway as being the most abundant proteins in HEK 293 NEDP1 KO lysates following NEDP1-CA pulldown. iBAQ analysis of proteins identified in A) are plotted as the log2 value of the enrichment ratio (mass spectrometry intensity of the HALO-NEDP1 CA pulldown over HALO-NEDP1 DAGC pulldown) versus the log10 value of the iBAQ intensity from the HALO-NEDP1 CA pulldown. Blue markers indicate known components of the NEDD8 pathway and red markers indicate proteins that have been identified as substrates of PARP-1 in the database of ADP-ribosylated proteins, ADPriboDB (Vivelo et al., 2017).


Non - cullin neddylated species accumulate after oxidative stress in WT U2OS with most highly induced species detected at 25 kDa ( as indicated by arrow ) . WT U2OS were treated with indicated of H2O2 for 30 minutes then lysed in LDS sample loading buffer processed for Western blot .

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Non-cullin neddylated species accumulate after oxidative stress in WT U2OS cells with the most highly induced species detected at 25 kDa (as indicated by the arrow). WT U2OS cells were treated with the indicated concentration of H2O2 for 30 minutes and then lysed in LDS sample loading buffer and processed for Western blot analysis.


( Left ) 25 - kDa neddylated species induced by oxidative stress increases with time in WT U2OS . U2OS WT or NEDP1 KO were treated with 600 μM H2O2 for indicated time then lysed in LDS sample loading buffer processed for Western blot . ( Right ) same 25 kDa NEDD8 species , which is strongly induced in WT , is present in untreated NEDP1 KO does not further increase after oxidative stress induced by H2O2 ( as indicated by arrow ) .

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(Left) The 25-kDa neddylated species induced by oxidative stress increases with time in WT U2OS cells. U2OS WT or NEDP1 KO cells were treated with 600 μM H2O2 for the indicated time and then lysed in LDS sample loading buffer and processed for Western blot analysis. (Right) The same 25 kDa NEDD8 species, which is strongly induced in WT cells, is present in untreated NEDP1 KO cells and does not further increase after oxidative stress induced by H2O2 (as indicated by the arrow).


U2OS NEDP1 KO are resistant to PARP - 1 inducer , H2O2 . WT NEDP1 U2OS were plated in 96 - well plates after 24 hours were treated with indicated of H2O2 . They were assessed for viability 24 hours later by CellTiter - Glo . Graphs represent mean ± S . E . M . of survival compared to untreated .

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U2OS NEDP1 KO cells are resistant to the PARP-1 inducer, H2O2. WT and NEDP1 U2OS cells were plated in 96-well plates and after 24 hours were treated with the indicated concentration of H2O2. They were assessed for viability 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


NEDP1 KO are re - sensitized to H2O2 , following re - expression of NEDP1 by transient transfection . U2OS were plated in 96 well plates reversed transfected with NEDP1 or an empty vector . 48 hours after plating , were challenged with indicated amount of H2O2 viability was assessed 24 hours later by CellTiter - Glo . Graphs represent mean ± S . E . M . of survival compared to untreated .

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NEDP1 KO cells are re-sensitized to H2O2, following re-expression of NEDP1 by transient transfection. U2OS cells were plated in 96 well plates and reversed transfected with NEDP1 or an empty vector. 48 hours after plating, cells were challenged with the indicated amount of H2O2 and cell viability was assessed 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


Neddylation of cullins is decreased in NEDP1 KO is rescued by re - expression of NEDP1 by transient transfection . Western blot from HEK 293 WT , NEDP1 KO NEDP1 KO rescued with transient transfection of NEDP1 reveals that upon NEDP1 re - expression , of NEDD8 reactive bands is reduced of free NEDD8 are increased . NEDP1 re - expression also increased neddylation of each Cullin . neddylated band of each Cullin is denoted with a star . graph of mean ± S . E . M . of neddylation of each Cullin in ( E ) .

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Neddylation of cullins is decreased in NEDP1 KO cells and is rescued by re-expression of NEDP1 by transient transfection. Western blot analysis from HEK 293 WT, NEDP1 KO cells and NEDP1 KO cells rescued with transient transfection of NEDP1 reveals that upon NEDP1 re-expression, the intensity of the NEDD8 reactive bands is reduced and the levels of free NEDD8 are increased. NEDP1 re-expression also increased the neddylation of each Cullin. The neddylated band of each Cullin is denoted with a star. Quantification and graph of the mean ± S.E.M. of the percentage neddylation of each Cullin in (E).


Deletion of Nedp1 does not lead to induction Nrf2 response genes . was harvested from WT NEDP1 KO U2OS , reversed transcribed to cDNA , analysed by qPCR for Nrf2 NQO1 expression .

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Deletion of Nedp1 does not lead to induction Nrf2 response genes. RNA was harvested from WT and NEDP1 KO U2OS cells, reversed transcribed to cDNA, and analysed by qPCR for Nrf2 and NQO1 expression.


Deletion of Nedp1 but not of Cand1 in HEK 293 results in resistance to from H2O2 . WT , NEDP1 KO , CAND1 KO were plated in 96 wells plates treated with indicated amount of H2O2 . 24 hours after treatment , viability was measured using CellTiter - Glo . Graphs represent mean ± S . E . M . of survival compared to untreated .

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Deletion of Nedp1 but not of Cand1 in HEK 293 cells results in resistance to cell death from H2O2. WT, NEDP1 KO, and CAND1 KO cells were plated in 96 wells plates and treated with the indicated amount of H2O2. 24 hours after treatment, cell viability was measured using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


PARP - 1 activity is reduced in NEDP1 KO . Western blot of whole lysates from WT NEDP1 KO U2OS after treatment with 600 μM H2O2 for indicated amount of time . PAR polymer generation is induced in WT after H2O2 treatment but induction is reduced in NEDP1 KO .

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PARP-1 activity is reduced in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells after treatment with 600 μM H2O2 for the indicated amount of time. PAR polymer generation is induced in WT cells after H2O2 treatment but induction is reduced in NEDP1 KO cells.


PARP - 1 activity is rescued by re - expression of NEDP1 in NEDP1 KO . Western blot of whole lysates from WT NEDP1 KO U2OS transfected with indicated constructs . 72 hours after transfection , were treated with indicated amount of H2O2 processed for Western blot . PAR polymer generation is reduced in NEDP1 KO but can be rescued by transient re - expression of NEDP1 .

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PARP-1 activity is rescued by re-expression of NEDP1 in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells transfected with the indicated constructs. 72 hours after transfection, cells were treated with the indicated amount of H2O2 and processed for Western blot analysis. PAR polymer generation is reduced in NEDP1 KO cells but can be rescued by the transient re-expression of NEDP1.


PARP - 1 inhibitor Olaparib protects WT from H2O2 treatment to same extent as NEDP1 KO . U2OS were plated in 96 well plates 24 hours later pre - treated with Olaparib ( 10 μM ) for 1 hour before treatment with indicated of H2O2 . 24 hours later , viability was measured using CellTiterGlo . NEDP1 deletion protects from H2O2 treatment to same extent as PARP - 1 inhibition in WT . Olaparib does not provide additional protection to NEDP1 from H2O2 treatment .

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PARP-1 inhibitor Olaparib protects WT cells from H2O2 treatment to the same extent as NEDP1 KO. U2OS cells were plated in 96 well plates and 24 hours later pre-treated with Olaparib (10 μM) for 1 hour before treatment with the indicated concentration of H2O2. 24 hours later, cell viability was measured using the CellTiterGlo assay. NEDP1 deletion protects cells from H2O2 treatment to the same extent as PARP-1 inhibition in WT cells. Olaparib does not provide additional protection to NEDP1 cells from H2O2 treatment.


AIF translocation to nucleus is impaired in NEDP1 KO . WT NEDP1 KO were plated on coverslips 24 hours later were treated with H2O2 ( 600 μM ) for indicated amount of time then processed for immunofluorescence using α - AIF antibodies DAPI . PARP - 1 dependent is induced in WT U2OS , as indicated by translocation of AIF from mitochondria to nucleus ( Scale bar 10 μm ) .

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AIF translocation to the nucleus is impaired in NEDP1 KO cells. WT and NEDP1 KO cells were plated on coverslips and 24 hours later were treated with H2O2 (600 μM) for the indicated amount of time and then processed for immunofluorescence analysis using α-AIF antibodies and DAPI staining. PARP-1 dependent cell death is induced in WT U2OS cells, as indicated by translocation of AIF from the mitochondria to the nucleus (Scale bar 10 μm).


Total AIF from ( D ) was measured with ImageJ plotted as a vertical scatter plot with group mean indicated with a black bar . AIF has translocated to nucleus after WT were exposed to H2O2 but not in NEDP1 KO .

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Total nuclear AIF intensity from (D) was measured with ImageJ and plotted as a vertical scatter plot with the group mean intensity indicated with a black bar. AIF has translocated to the nucleus after WT cells were exposed to H2O2 but not in NEDP1 KO cells.


NEDP1 KO express normal of PARP - 1 AIF . Western blot of whole lysates from WT NEDP1 KO U2OS shows that necessary for induction of PARP - 1 dependent are present at equal in both lines .

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NEDP1 KO cells express normal levels of PARP-1 and AIF. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells shows that the proteins necessary for the induction of PARP-1 dependent cell death are present at equal levels in both cell lines.


NEDP1 KO U2OS are not resistant to induction of apoptosis from combined treatment with TNF - α cycloheximide . U2OS were plated in 96 well plates incubated with cycloheximide ( 10 μg / mL ) before addition of indicated amount of TNF - α . survival was determined 24 hours later using CellTiter - Glo . Graphs represent mean ± S . E . M . of survival compared to untreated .

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NEDP1 KO U2OS cells are not resistant to induction of apoptosis from combined treatment with TNF-α and cycloheximide. U2OS cells were plated in 96 well plates and incubated with cycloheximide (10 μg/mL) before addition of the indicated amount of TNF-α. Cell survival was determined 24 hours later using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


NEDP1 KO U2OS are not resistant to treatment with camptothecin ( CPT ) . WT U2OS NEDP1 KO were plated in 96 - well plates treated with indicated amount of CPT . 48 hours after exposure survival was measured using CellTiterGlo . Graphs represent mean ± S . E . M . of survival compared to untreated .

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NEDP1 KO U2OS cells are not resistant to treatment with camptothecin (CPT). WT U2OS and NEDP1 KO cells were plated in 96-well plates and treated with the indicated amount of CPT. 48 hours after exposure cell survival was measured using the CellTiterGlo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


NEDD8 trimers co - immunoprecipitate with endogenous PARP - 1 in NEDP1 KO . Lysates were prepared from U2OS NEDP1 KO immunoprecipitation was performed with PARP - 1 - Trap or GFP - Trap as a negative control . Bound were resolved by SDS - PAGE processed for Western blot with indicated antibodies .

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NEDD8 trimers co-immunoprecipitate with endogenous PARP-1 in NEDP1 KO cells. Lysates were prepared from U2OS NEDP1 KO cells and immunoprecipitation was performed with PARP-1-Trap or GFP-Trap as a negative control. Bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.