Figure 6A 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 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 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 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 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 .

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

Figure 6A 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 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 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 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 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.


Figure 7A Rosette phenotype of 3 - weeks - old Col - 0 , lrx3 / 4 / 5 and 35S : : LRR4 - cit . Data information : Scale bars : 1 cmB 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 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 .

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

Figure 7A Rosette phenotype of 3-weeks-old Col-0, lrx3/4/5 and 35S::LRR4-cit. Data information: Scale bars: 1 cmB 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 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.


Figure 1Heat 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 .

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Figure 1Heat 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.


Figure 2eATP 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 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 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 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 .

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Figure 2eATP 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 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 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 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.


Figure 4Ecto - 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 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 . 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 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 the mean from three biological replicates . Asterisks indicate significance to all other samples within the same treatment group ( ANOVA , p < 0 . 05 ) . 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 ) .

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Figure 4Ecto-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 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.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 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 the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05).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).


Figure 5Relative 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 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 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 .

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Figure 5Relative 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 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 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.


figf1 ( 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 assay ( ELISA ) of cytokine secretion by peritoneal macrophages stimulated with LPS , followed by treatment for 1 h with ATP . ( 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 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 ) .

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figf1(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 assay (ELISA) of cytokine secretion by peritoneal macrophages stimulated with LPS, followed by treatment for 1 h with ATP.(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 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).


figf2 ( 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 − / − and Becn1 + / − macrophages left unstained or labeled with MitoSOX . ( 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 .

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figf2(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−/− and Becn1+/− macrophages left unstained or labeled with MitoSOX.(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.


figf3 ( 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 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 and ATP ( below graph ) . ( 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 and rotenone ( or DMSO ) , followed by ATP . ( 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 ) 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 ) .

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

figf3(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 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 and ATP (below graph).(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 and rotenone (or DMSO), followed by ATP.(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) 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).


figf4 ( 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 . ( 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 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 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 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 . ) .

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

figf4(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.(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 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 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 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.).


figf5 ( 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 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 + / + 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 - test ) . Data are representative of three experiments ( mean and s . d . ) .

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

figf5(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 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+/+ 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-test). Data are representative of three experiments (mean and s.d.).


figf6 ( 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 ) and NALP3 - deficient ( NALP3 - KO ) macrophages incubated with rotenone , followed by stimulation for 1 h with ATP . ( 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 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 . ( 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 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 ) .

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

figf6(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) and NALP3-deficient (NALP3-KO) macrophages incubated with rotenone, followed by stimulation for 1 h with ATP.(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 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.(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 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).


figf7 ( 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 ) 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 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 .

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

figf7(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) 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 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.


Figure 1 ( 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 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 .

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

Figure 1(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 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.


Figure 2 ( 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 ) 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 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 .

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

Figure 2(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) 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 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.


Figure 3 ( 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 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 . ( 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 analysis of ATAC signal lost at CLL promoters with broadened H3K4me3 regions . An enrichment for the NFY TF motif was detected . ( 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 .

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

Figure 3(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 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.(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 analysis of ATAC signal lost at CLL promoters with broadened H3K4me3 regions. An enrichment for the NFY TF motif was detected.(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.


Figure 4 ( 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 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 ) 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 and NBCs . Histone deacetylases significantly up - regulated in CLL are shown in bold . ( 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 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 ) 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 .

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

Figure 4(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 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) 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 and NBCs. Histone deacetylases significantly up-regulated in CLL are shown in bold.(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 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) 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.


Figure 5 ( 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 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 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 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 .

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Figure 5(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 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 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 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.


Figure 6 ( 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 .

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Figure 6(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.


Figure 1B 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 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 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 .

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Figure 1B 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 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 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.


Figure 2B 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 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 ) .

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Figure 2B 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 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).


Figure 3A 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 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 . ) .

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Figure 3A 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 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.).


Figure 5A 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 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 . D PTB crosslinked nucleotides mapped onto Matrin3 ‐ regulated ASE .

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Figure 5A 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 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.D PTB crosslinked nucleotides mapped onto Matrin3‐regulated ASE.


Figure 6B Western blot of the overexpressed Matrin3 and its mutants probed with anti ‐ FLAG and anti ‐ actin antibodies . 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 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 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 ( ‐ ) .

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Figure 6B Western blot of the overexpressed Matrin3 and its mutants probed with anti‐FLAG and anti‐actin antibodies.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 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 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 (‐).


Figure 1Western 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 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 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 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 , 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 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 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 .

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Figure 1Western 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 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 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 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, 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 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 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.


Figure 2 ( 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 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 ) .

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Figure 2(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 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).


Figure 3Non - 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 ) 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 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 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 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 . 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 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 .

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Figure 3Non-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) 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 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 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 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. 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 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.


Figure 4PARP - 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 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 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 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 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 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 .

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Figure 4PARP-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 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 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 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 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 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.