G of Nox4 - depleted control with depolarized mitochondria , quantified by flow cytometry . n = 3 preparations / group ( 100 , 000 per experiment ) .

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G Percentage of Nox4-depleted and control cells with depolarized mitochondria, quantified by flow cytometry. n=3 cell preparations/group (100,000 cells per experiment).


H Effect of cyclosporin A ( CsA , 1 μmol / L ) on of with depolarized mitochondria . n = 6 - 8 / group .

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H Effect of cyclosporin A (CsA, 1 μmol/L) on the percentage of cells with depolarized mitochondria. n=6-8/group.


I Effect of cyclosporin A ( CsA , 1 μmol / L ) on in serum - starved cardiomyocytes . n = 4 / group .

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I Effect of cyclosporin A (CsA, 1 μmol/L) on cell death in serum-starved cardiomyocytes. n=4/group.


J in serum starved Nox4 knockout MEFs ( Nox4KO ) wild - type MEFs ( WT ) . Cat = PEG - catalase ( 500 U / mL ) . Nox4KO MEFs were transfected either with active Nox4 or a catalytic inactive Nox4 mutant , Nox4P437H ( Mut ) . n = 6 - 12 / group .

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J Cell death in serum starved Nox4 knockout MEFs (Nox4KO) and wild-type MEFs (WT). Cat = PEG-catalase (500 U/mL). Nox4KO MEFs were transfected either with active Nox4 or a catalytic inactive Nox4 mutant, Nox4P437H (Mut). n=6-12/group.


K HMGB1 in supernatants of serum - starved MEFs in similar experiments to those in ( J ) . n = 4 / group . A representative immunoblot is shown to top .

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K HMGB1 levels in the supernatants of serum-starved MEFs in similar experiments to those in (J). n=4/group. A representative immunoblot is shown to the top.


L of MEFs with depolarized mitochondria . n = 6 / group .

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L Percentage of MEFs with depolarized mitochondria. n=6/group.


A Basal mitochondrial calcium assessed in serum - starved rat cardiomyocytes using a mitochondrial - targeted cameleon CFP / YFP FRET probe . Nox4 was depleted with silencing RNAs ( siRNAs ) or were transfected with a control scrambled siRNA ( siScr ) . Representative photomicrographs are shown at top . spectrum color scale represents of emitted ( YFP / CFP ) . mean changes in Nox4 - depleted as compared to scrambled control ( dotted ) at bottom . representative immunoblot shows depletion of Nox4 . Scale bars : 10 µm . n = 3 / group ( with > 50 per experiment ) .

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A Basal mitochondrial calcium levels assessed in serum-starved rat cardiomyocytes using a mitochondrial-targeted cameleon CFP/YFP FRET probe. Nox4 was depleted with silencing RNAs (siRNAs) or cells were transfected with a control scrambled siRNA (siScr). Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). The mean changes in Nox4-depleted cells as compared to scrambled control (dotted line) at the bottom. The representative immunoblot shows depletion of Nox4 protein levels. Scale bars: 10 µm. n=3/group (with >50 cells per individual experiment).


B Basal mitochondrial calcium in WT Nox4KO MEFs after serum starvation . Representative photomicrographs are shown at top . spectrum color scale represents of emitted ( YFP / CFP ) . Mean data at bottom . Scale bars : 10 µm . n = 3 / group ( with > 100 per experiment ) .

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B Basal mitochondrial calcium levels in WT and Nox4KO MEFs after serum starvation. Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). Mean data at the bottom. Scale bars: 10 µm. n=3/group (with >100 cells per individual experiment).


Changes in mitochondrial calcium in serum - starved WT Nox4KO MEFs after addition of histamine ( 100 μmol / L , C ) n = 3 / group ( with > 30 per experiment ) .

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Changes in mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/L, C) n=3/group (with >30 cells per individual experiment).


D Changes in mitochondrial calcium in serum - starved WT Nox4KO MEFs after addition of ATP ( 100 μmol / L , D ) . n = 3 / group ( with > 30 per experiment ) .

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D Changes in mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs after the addition of ATP (100 μmol/L, D). n=3/group (with >30 cells per individual experiment).


E , Changes in ER calcium measured with an ER - targeted cameleon probe in serum - starved WT Nox4KO MEFs after addition of histamine ( 100 μmol / L , E ) n = 3 / group ( with > 30 per experiment ) .

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E, Changes in ER calcium levels measured with an ER-targeted cameleon probe in serum-starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/L, E) n=3/group (with >30 cells per individual experiment).


F Changes in ER calcium measured with an ER - targeted cameleon probe in serum - starved WT Nox4KO MEFs after addition of ATP ( 100 μmol / L , F ) . n = 3 / group ( with > 30 per experiment ) .

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F Changes in ER calcium levels measured with an ER-targeted cameleon probe in serum-starved WT and Nox4KO MEFs after the addition of ATP (100 μmol/L, F). n=3/group (with >30 cells per individual experiment).


G Peak increase in mitochondrial calcium response in response to histamine ( 100 μmol / L ) in WT MEFs with or without treatment with PEG - catalase ( Cat ) , in Nox4KO MEFs with or without transfection with active Nox4 or a catalytically inactive Nox4P437H mutant ( Mut ) . n = 3 / group ( with > 30 per experiment ) .

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G Peak increase in mitochondrial calcium levels response in response to histamine (100 μmol/L) in WT MEFs with or without treatment with PEG-catalase (Cat), and in Nox4KO MEFs with or without transfection with active Nox4 or a catalytically inactive Nox4P437H mutant (Mut). n=3/group (with >30 cells per individual experiment).


H Representative time - course of histamine - induced changes in mitochondrial calcium for experiments shown in ( I ) .

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H Representative time-course of histamine-induced changes in mitochondrial calcium for the experiments shown in (I).


A Immunoblotting of subcellular fractions from serum - starved rat cardiomyoblasts ( H9c2 ) . Input : homogenate before fractionation ; Cyto : cytosol ; ER : endoplasmic reticulum ; Crude M : crude mitochondrial fraction , also containing MAM ; Pure Mito : mitochondria ; MAM : mitochondria associated - ER membranes . Cytochrome c ( Cyt c ) was used as a mitochondrial marker , VDAC as an outer mitochondrial marker , calnexin as an ER marker , Sigma1R FACL4 as MAM markers , tubulin as a marker . B Immunoblotting of subcellular fractions from rat heart . C Immunoblotting of subcellular fractions from rat kidney .

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A Immunoblotting of subcellular fractions from serum-starved rat cardiomyoblasts (H9c2 cells). Input: homogenate before fractionation; Cyto: cytosol; ER: endoplasmic reticulum; Crude M: crude mitochondrial fraction, also containing MAM; Pure Mito: mitochondria; MAM: mitochondria associated-ER membranes. Cytochrome c (Cyt c) was used as a mitochondrial marker, VDAC as an outer mitochondrial membrane marker, calnexin as an ER marker, Sigma1R and FACL4 as MAM markers, and tubulin as a cytosolic marker. B Immunoblotting of subcellular fractions from rat heart. C Immunoblotting of subcellular fractions from rat kidney.


D Electron showing subcellular localization of Nox4 in wild - type ( WT ) Nox4KO mouse hearts , human induced pluripotent - derived cardiomyocytes ( hiPSC - CM ) . hiPSC - CM were depleted of Nox4 using a lentiviral shRNA ( Nox4KD ) or infected with a control lentivirus . FACL4 was used as a MAM marker . Arrow heads show peri - mitochondrial localization of Nox4 FACL4 , in proximity of ER cisternae ribosomes . M = mitochondria , ER = endoplasmic reticulum , T = T - tubule . Scale bars : 100 nm .

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D Electron micrographs showing subcellular localization of Nox4 in wild-type (WT) and Nox4KO mouse hearts, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). hiPSC-CM were depleted of Nox4 using a lentiviral shRNA (Nox4KD) or infected with a control lentivirus. FACL4 was used as a MAM marker. Arrow heads show peri-mitochondrial localization of Nox4 and FACL4, in the proximity of ER cisternae and ribosomes. M=mitochondria, ER=endoplasmic reticulum, T=T-tubule. Scale bars: 100 nm.


A Simplified photomicrographs of proximity ligation studies in WT Nox4KO MEFs , showing borders , nuclei ( blue ) yellow dots corresponding to co - localization of . of number of dots / in each condition is shown to right . Proximity was tested for following couples : FACL4 / Nox4 , InsP3R / Nox4 , FACL4 / InsP3R . Scale bars : 10 µm . n = 3 / group ( with > 30 / experiment ) .

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A Simplified photomicrographs of proximity ligation studies in WT and Nox4KO MEFs, showing cell borders, nuclei (blue) and yellow dots corresponding to co-localization of proteins. Quantification of the number of dots/cell in each condition is shown to the right. Proximity was tested for the following protein couples: FACL4/Nox4, InsP3R/Nox4, and FACL4/InsP3R. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).


B Simplified photomicrographs of proximity ligation in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control ( siScr ) , with as in ( A ) . Proximity was tested for FACL4 / Nox4 couple . Scale bars : 10 µm . n = 3 / group ( with > 30 / experiment ) .

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B Simplified photomicrographs of proximity ligation staining in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control (siScr), with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).


C Simplified photomicrographs of proximity ligation in hiPSC - CM ( human cardiomyocytes ) transduced with Nox4 shRNA or scrambled control , with as in ( A ) . Proximity was tested for FACL4 / Nox4 couple . Scale bars : 10 µm . n = 3 / group ( with > 30 / experiment ) .

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C Simplified photomicrographs of proximity ligation staining in hiPSC-CM (human cardiomyocytes) transduced with Nox4 shRNA or scrambled control, with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).


A Immunoblotting of subcellular fractions from serum - starved cardiomyoblasts for InsP3R , Akt PP2a , as in Fig 3A .

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A Immunoblotting of subcellular fractions from serum-starved cardiomyoblasts for InsP3R, Akt and PP2a, as in Fig 3A.


B Fractionation of crude mitochondrial fractions ( containing mitochondria MAM ) from WT MEFs on a sucrose gradient ( 5 - 60 % ) immunoblotting for relevant . Fraction : 200 µL . Representative of 3 independent experiments .

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B Fractionation of crude mitochondrial fractions (containing mitochondria and MAM) from WT MEFs on a sucrose gradient (5-60%) and immunoblotting for relevant proteins. Fraction size: 200 µL. Representative of 3 independent experiments.


C Immunoblotting for phosphorylated total Akt or InsP3R in crude mitochondrial fractions from serum - starved WT Nox4KO MEFs . FACL4 was used as a loading control for MAM . InsP3R was first immunopreciptated then precipitate was immunoblotted for total InsP3R for phosphorylated Akt - substrate motif RXRXX ( pS / T ) ( p - InsP3R ) . Representative immunoblots are shown at top mean data at bottom . Cat = PEG - catalase . Nox4KO MEFs were transfected either with active Nox4 or a Nox4P437H mutant ( Mut ) . n = 3 / group .

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C Immunoblotting for phosphorylated and total Akt or InsP3R protein in crude mitochondrial fractions from serum-starved WT and Nox4KO MEFs. FACL4 was used as a loading control for the MAM. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Representative immunoblots are shown at the top and mean data at the bottom. Cat = PEG-catalase. Nox4KO MEFs were transfected either with active Nox4 or a Nox4P437H mutant (Mut). n=3/group.


D ROS indexed in MEFs using H2O2 specific HyPer probes targeted to MAM ( FACL4 HyPer ) or cytosol ( Cyto HyPer ) . Imaging was performed after serum starvation for 12h . Representative photomicrographs of are shown to right mean data for changes in presented to left . signal obtained using corresponding ROS - insensitive SypHer probes was used to correct for any pH - induced changes in ; change in between HyPer corresponding SypHer probe ( ΔR ) for each condition is reported . n = 5 independent preparations / group , with at least 20 imaged / preparation . Scale bars : 10 µm .

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D ROS levels indexed in MEFs using H2O2 specific HyPer probes targeted to the MAM (FACL4 HyPer) or the cytosol (Cyto HyPer). Imaging was performed after serum starvation for 12h. Representative photomicrographs of the fluorescence ratio are shown to the right and mean data for changes in fluorescence ratio presented to the left. The signal obtained using corresponding ROS-insensitive SypHer probes was used to correct for any pH-induced changes in fluorescence; the change in fluorescence ratio between HyPer and corresponding SypHer probe (ΔR) for each condition is reported. n=5 independent cell preparations/group, with at least 20 cells imaged/preparation. Scale bars: 10 µm.


B Basal mitochondrial calcium in serum - starved WT Nox4KO MEFs in presence or absence of Akti ( 1 µmol / L ) normalized versus WT MEF control . n = 6 / group with > 20 imaged per experiment .

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B Basal mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs in the presence or absence of Akti (1 µmol/L) normalized versus WT MEF control. n=6/group with >20 cells imaged per experiment.


C Effect of Akti ( 1 µmol / L ) or XeC ( 1 µmol / L ) on histamine - induced changes in mitochondrial calcium in MEFs . time course of changes in calcium is shown on left mean data for peak mitochondrial calcium on right . n = 3 / group with > 40 imaged per experiment .

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C Effect of Akti (1 µmol/L) or XeC (1 µmol/L) on histamine-induced changes in mitochondrial calcium levels in MEFs. The time course of changes in calcium levels is shown on the left and the mean data for peak mitochondrial calcium levels on the right. n=3/group with >40 cells imaged per experiment.


D of in serum - starved WT Nox4KO MEFs in absence or presence of Akti or XeC . n = 6 / group .

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D Quantification of cell death in serum-starved WT and Nox4KO MEFs in the absence or presence of Akti or XeC. n=6/group.


E Cardiac troponin I ( cTnI ) as a marker of cardiomyocyte necrosis in perfusate of isolated WT Nox4KO hearts subjected to ischemia - reperfusion ( I / R ) . XeC was added at a final of 2 µmol / L prior to ischemia . n = 6 - 7 / group .

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E Cardiac troponin I (cTnI) levels as a marker of cardiomyocyte necrosis in the perfusate of isolated WT and Nox4KO hearts subjected to ischemia-reperfusion (I/R). XeC was added at a final concentration of 2 µmol/L prior to ischemia. n=6-7/group.


F Immunoblotting for phosphorylation of Akt InsP3R in crude mitochondrial fractions from WT Nox4KO hearts after I / R . InsP3R was first immunopreciptated then precipitate was immunoblotted for total InsP3R for phosphorylated Akt - substrate motif RXRXX ( pS / T ) ( p - InsP3R ) . Mean data shown to right . n = 3 / group .

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F Immunoblotting for the phosphorylation levels of Akt and InsP3R in crude mitochondrial fractions from WT and Nox4KO hearts after I/R. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Mean data shown to the right. n=3/group.


G Cardiac left ventricular contractile function in isolated Langendorff - perfused WT Nox4KO hearts at baseline ( Basal ) then after I / R . Hearts were treated with XeC ( 1 μmol / L ) or vehicle control for 20 min prior to ischemia . RPP , heart rate x left ventricular pressure product ; DEVP , left ventricular developed pressure . n = 6 - 7 / group .

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G Cardiac left ventricular contractile function in isolated Langendorff-perfused WT and Nox4KO hearts at baseline (Basal) and then after I/R. Hearts were treated with XeC (1 μmol/L) or vehicle control for 20 min prior to ischemia. RPP, heart rate x left ventricular pressure product; DEVP, left ventricular developed pressure. n=6-7/group.


A of MNase - digested fragments from wild - type ( WT , sizes indicated in black ) nap1Δ ( N , sizes indicated in red ) by agarose gel electrophoresis . Diagram illustrating reduction of array in nap1Δ .

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A Analysis of MNase-digested chromatin DNA fragments from wild-type (WT, sizes indicated in black) and nap1Δ (N, sizes indicated in red) cells by agarose gel electrophoresis. Diagram illustrating reduction of array length in nap1Δ cells.


B Frequency distribution of nucleosomal ( WT in grey fill with nap1Δ in blue traces ) from H3 ChIP - seq .

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B Frequency distribution of nucleosomal DNA size (WT in grey fill with nap1Δ in blue traces) from H3 ChIP-seq.


C Calculation of nucleosome fuzziness as standard deviation of nucleosome ( or tag ) positions . See also Appendix Fig S1 .

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C Calculation of nucleosome fuzziness as the standard deviation of nucleosome (or tag) positions. See also Appendix Fig S1.


B Ribbon view of asymmetric crystallographic unit containing a ( yNap1c2 - H2AΔ14 - H2BΔ28 ) 6 assembly . For clarity only layer 1 of is shown . Layer 1 contains three protomers A - C that are related by two perpendicular non - crystallographic dyads ( ) . C Side - view of . layer 2 omitted from panel B is shown in grey . yNap1 dimer is shown in green yellow , H2A in red H2B in blue . See also Fig EV1 . D View of yNap1 - H2A - H2B protomer with labeling of secondary structure elements .

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B Ribbon view of the asymmetric crystallographic unit containing a (yNap1c2-H2AΔ14-H2BΔ28)6 assembly. For clarity only layer 1 of the complex is shown. Layer 1 contains three protomers A-C that are related by two perpendicular non-crystallographic dyads ().C Side-view of the complex. The layer 2 omitted from panel B is shown in grey. The yNap1 dimer is shown in green and yellow, H2A in red and H2B in blue. See also Fig EV1.D View of the yNap1-H2A-H2B protomer complex with labeling of secondary structure elements.


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A Overview of the yNap1c2-H2AΔ14-H2BΔ28 complex.B Residues R17, R20, R29, R32, R35 and K36 of H2A make contacts with the acidic HBR1 in yNap1 comprising residues D201, D205 and E310.C Residues K75, R77 and R81 of H2A make polar contacts with the HBR2 comprising residues E332, D333 and E339 in α8 of yNap1.


D In vitro GST - pulldown showing role of residues in HBR interfaces in H2A - H2B binding . mutant labeled HBR1 ( lane 6 ) contains triple mutation D201R , D205R , E310R HBR2 ( lane 12 ) triple mutations E332 , D333 , E336 . HBR1 + 2 ( lane 13 ) contains six mutations . mutant labeled HBR1 + 2A ( lane 14 ) has Alanine replacements in six HBR residues . Top panel : Bound . Bottom panel : Input .

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D In vitro GST-pulldown assay showing the role of residues in the HBR interfaces in H2A-H2B binding. The mutant labeled HBR1 (lane 6) contains the triple mutation D201R, D205R, E310R and HBR2 (lane 12) the triple mutations E332, D333, E336. HBR1+2 (lane 13) contains all six mutations. The mutant labeled HBR1+2A (lane 14) has Alanine replacements in the six HBR residues. Top panel: Bound. Bottom panel: Input.


E Disruption of Nap1 - histone interaction by H2A mutation . Bottom panel : Inputs of coexpressed ( soluble extract ) top panel material bound to glutathione affinity column were analyzed by SDS - PAGE Coomassie .

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E Disruption of the Nap1-histone interaction by H2A mutation. Bottom panel: Inputs of the coexpressed proteins (soluble extract) and top panel the material bound to the glutathione affinity column were analyzed by SDS-PAGE and Coomassie staining.


F Controls showing input ( bottom ) bound ( top ) fractions . See also Fig EV2 .

F Controls showing input (bottom) and bound (top) fractions. See also Fig EV2.


A EM of yNap1c2 - H2AΔ14 - H2BΔ28 . Representative rawimage of negatively - stained particles . B Representative particles ( 1st row ) , class averages coming from projection matching procedure ( 2nd row ) comparison of class averages with projections from a final model generated after four projection matching cycles ( 3rd row ) .

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A EM analysis of the yNap1c2-H2AΔ14-H2BΔ28 complex. Representative rawimage of negatively-stained particles.B Representative particles (1st row), class averages coming from the projection matching procedure (2nd row) and comparison of the class averages with projections from a final model generated after four projection matching cycles (3rd row).


C From left to right : Crystal structure of ( yNap1c2 - H2A - H2B ) 6 in two different orientations ( brown ) ; 60 Å - filtered structure of ( magenta ) ; 3D reconstruction of from negatively stained particles ( green ) fit of crystal structure into EM map .

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C From left to right: Crystal structure of the (yNap1c2-H2A-H2B)6 complex in two different orientations (brown); 60 Å-filtered structure of the complex (magenta); 3D reconstruction of the complex from negatively stained particles (green) and fit of the crystal structure into the EM map.


D - I Mass spectra of yNap1 variants in absence presence of H2A - H2B . yNap1 in in absence ( D ) or presence of H2A - H2B ( E ) . yNap1c in absence ( F ) or bound to H2A - H2B ( G ) . oligomerization mutant yNap1 _ IF1 alone ( H ) or in with H2A _ IF2Δ14 - H2BΔ28 ( I ) . yNap1 is shown in green circles H2A - H2B as red blue asterisks .

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D-I Mass spectra of yNap1 variants in the absence and presence of H2A-H2B. yNap1 in complex in the absence (D) or presence of H2A-H2B (E). yNap1c in the absence (F) or bound to H2A-H2B (G). The oligomerization mutant yNap1_IF1 alone (H) or in complex with H2A_IF2Δ14-H2BΔ28 (I). yNap1 is shown in green circles and H2A-H2B as red and blue asterisks.


A Native PAGE gel showing result of an affinity shift scanned to detect AF488 - ( left ) or AF647 - labeled ( right ) . 31 nM - 4 µM of yNap1 - H2A - H2B ( lanes 6 - 13 ) were incubated with a mixture of 0 . 8 µM AF488 - labeled H3 - H4 - tetrasome complexes 0 . 8 µM AF647 - labeled ( lane 5 ) . Lanes 1 - 3 show migration of tetrasomes ( tetra ) , nucleosomes ( nucl ) or free H2A - H2B obtained by salt deposition onto AF647 - labeled .

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A Native PAGE gel showing the result of an affinity shift assay scanned to detect AF488- (left) or AF647-labeled DNA (right). 31 nM - 4 µM of yNap1-H2A-H2B (lanes 6-13) were incubated with a mixture of 0.8 µM AF488-labeled H3-H4-DNA tetrasome complexes and 0.8 µM AF647-labeled DNA (lane 5). Lanes 1-3 show migration of tetrasomes (tetra), nucleosomes (nucl) or free H2A-H2B obtained by salt deposition onto AF647-labeled DNA.


B of nucleosome band ( nucl ) is plotted . value at 4 µM of yNap1 - H2A - H2B ( lane 13 ) was set to 100 % . For non - specific binding , value in absence of H2A - H2B ( lane 5 ) was set to 100 % disappearance of free AF647 band was plotted . Each curve is representative of three independent experiments performed on different days . Standard deviations are shown .

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B Intensity of the nucleosome band (nucl) is plotted. The value at 4 µM of yNap1-H2A-H2B (lane 13) was set to 100%. For non-specific DNA binding, the value in the absence of H2A-H2B (lane 5) was set to 100% intensity and disappearance of the free DNA AF647 band was plotted. Each curve is representative of three independent experiments performed on different days. Standard deviations are shown.


C of MNase - digested fragments from wild - type ( BY4741 ) , nap1Δ , nap1Δ complemented with different yNap1 expression plasmids . Total MNase units per 50 ml of culture are indicated on bottom of lanes . Kb + : Marker .

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C Analysis of MNase-digested chromatin DNA fragments from wild-type (BY4741), nap1Δ, and nap1Δ cells complemented with different yNap1 expression plasmids. Total MNase units per 50 ml of culture are indicated on the bottom of the lanes. Kb+: DNA Marker.


a ) Kaplan - Meier to first fever ( ≥ 38 ° C ) episode within 24 hours of receiving vaccines administered at 4 months of ; control n = 88 4CMenB n = 89 .

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a) Kaplan-Meier to first fever (≥38°C) episode within 24 hours of receiving vaccines administered at 4 months of age; control n=88 and 4CMenB n=89.


b ) Principal component ( PC ) of - sequencing data ( 14837 genes , n = 253 ) from study time points . lines connect participant ' s pre - post - vaccination samples . ellipses are two - dimensional ( PC1 PC2 ) 95 % confidence intervals for each study time point . A contribution plot ( top right ) displays genes contributing most to PC1 / PC2 , i . e . 0 . 15 implies 0 . 15 % contribution of that variable to principal components displayed .

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b) Principal component (PC) analysis of RNA-sequencing data (14837 genes, n= 253) from all study time points. The lines connect participant's pre- and post-vaccination samples. The ellipses are the two-dimensional (PC1 and PC2) 95% confidence intervals for each study time point. A contribution plot (top right) displays the genes contributing most to PC1/PC2, i.e. 0.15 implies 0.15% contribution of that variable to the principal components displayed.


c ) Plotted are CIBERSORTx neutrophil fractions from whole blood - sequencing data , with median interquartile range . P - values were determined from a two sample Wilcoxon rank sum . number of individuals in each group is display in x - axis .

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c) Plotted are the CIBERSORTx neutrophil fractions from whole blood RNA-sequencing data, with median and interquartile range. P-values were determined from a two sample Wilcoxon rank sum test. The number of individuals in each group is display in the x-axis.


d ) Spearman ' s rank correlation between neutrophil measure by full blood ( n = 142 ) those estimated by CIBERSORTx , using LM22 signature matrix .

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d) Spearman's rank correlation between neutrophil counts measure by full blood counts (n= 142) and those estimated by CIBERSORTx, using the LM22 signature matrix.


a ) Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated blue downregulated ) at each study time point versus pre - vaccination ( 4 months of ) 4 hours post - vaccination ( 719 DEGs , n = 28 ) . P - values were obtained from moderated t - statistic , after adjustment for multiple testing ( Benjamini Hochberg ' s method ) . top 10 genes , ranked by FDR , are labelled .

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a) Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination (4 months of age) 4 hours post-vaccination (719 DEGs, n=28). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). The top 10 genes, ranked by FDR, are labelled.


Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated blue downregulated ) at each study time point versus pre - vaccination b ) Same as ( a ) but 24 hours post - vaccination ( 5553 DEGs , n = 31 )

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Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination b) Same as (a) but 24 hours post-vaccination (5553 DEGs, n=31)


Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated blue downregulated ) at each study time point versus pre - vaccination c ) Same as ( a ) but 3 days post - vaccination ( 159 DEGs , n = 30 )

Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination c) Same as (a) but 3 days post-vaccination (159 DEGs, n=30)


Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated blue downregulated ) at each study time point versus pre - vaccination d ) Same as ( a ) but 7 days post - vaccination ( 6 DEGs , n = 36 ) .

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Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination d) Same as (a) but 7 days post-vaccination (6 DEGs, n=36).


a ) Top genes differentially regulated between vaccine groups 4 hours post - vaccination . Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup . plotted lines are LOESS ( locally estimated scatterplot smoothing ) regression curves with 95 % confidence intervals in grey . FDR was derived by comparing fold - changes in gene expression in control group with group , from baseline to time point designated , is reported if statistically significant ( FDR < 0 . 05 ) . P - values were obtained from moderated t - statistic , after adjustment for multiple testing ( Benjamini Hochberg ' s method ) . Pre - vaccination samples n = 125 , 4 hour samples n = 28 , 24 hour samples n = 31 , 3 day sample n = 30 , 7 day samples n = 36 . expression E value , are gene expression value derived from voom - limma workflow ( Law et al . , 2014 ) .

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a) Top genes differentially regulated between vaccine groups 4 hours post-vaccination. Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). Pre-vaccination samples n= 125, 4 hour samples n= 28, 24 hour samples n = 31, 3 day sample n= 30, 7 day samples n= 36. The expression E value, are the gene expression value derived from the voom-limma workflow (Law et al., 2014).


Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup . plotted lines are LOESS ( locally estimated scatterplot smoothing ) regression curves with 95 % confidence intervals in grey . FDR was derived by comparing fold - changes in gene expression in control group with group , from baseline to time point designated , is reported if statistically significant ( FDR < 0 . 05 ) . b ) Same as ( a ) but top genes differentially regulated between vaccine groups 24 hours post - vaccination .

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Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). b) Same as (a) but top genes differentially regulated between vaccine groups 24 hours post-vaccination.


Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup . plotted lines are LOESS ( locally estimated scatterplot smoothing ) regression curves with 95 % confidence intervals in grey . FDR was derived by comparing fold - changes in gene expression in control group with group , from baseline to time point designated , is reported if statistically significant ( FDR < 0 . 05 ) . P - values were obtained from moderated t - statistic , after adjustment for multiple testing ( Benjamini Hochberg ' s method ) . c ) Same as ( a ) but top genes differentially regulated between vaccine groups 3 days post - vaccination .

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Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). c) Same as (a) but top genes differentially regulated between vaccine groups 3 days post-vaccination.


Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup . plotted lines are LOESS ( locally estimated scatterplot smoothing ) regression curves with 95 % confidence intervals in grey . FDR was derived by comparing fold - changes in gene expression in control group with group , from baseline to time point designated , is reported if statistically significant ( FDR < 0 . 05 ) . P - values were obtained from moderated t - statistic , after adjustment for multiple testing ( Benjamini Hochberg ' s method ) . d ) Same as ( a ) but top genes differentially regulated between vaccine groups 7 days post - vaccination .

Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). d) Same as (a) but top genes differentially regulated between vaccine groups 7 days post-vaccination.


False - discovery rate ( FDR ) are displayed above were derived from a two sample Wilcoxon rank sum ( Benjamini - Hochberg correction ) . Vertical lines represent interquartile range around median . red dashed horizontal is upper limit of normal CRP reference interval ( 0 - 5mg / l ) . number of individuals in each group is display in x - axis .

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False-discovery rate (FDR) are displayed above and were derived from a two sample Wilcoxon rank sum test (Benjamini-Hochberg correction). Vertical lines represent the interquartile range around the median. The red dashed horizontal line is upper limit of the normal CRP reference interval (0-5mg/l). The number of individuals in each group is display in the x-axis.


a , b ) Agreement plot . Red = differentially expressed ( DE ) in febrile infants , blue = DE in afebrile infants , purple = DE in febrile afebrile infants .

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a, b) Agreement plot. Red = differentially expressed (DE) in febrile infants, blue = DE in afebrile infants, purple =DE in febrile and afebrile infants.


c ) Exemplar genes differentially expressed at 4 hours 24 hours , respectively , between infants who experienced a fever within 24 hours of vaccination afebrile infants . plotted lines are LOESS ( locally estimated scatterplot smoothing ) regression curves with 95 % confidence intervals in grey . false - discovery rate ( FDR ) was derived by comparing fold - changes in gene expression between infants who experienced post - vaccination fever those who remained afebrile . P - values were obtained from moderated t - statistic , after adjustment for multiple testing ( Benjamini Hochberg ' s method ) . Pre - vaccination samples n = 125 , 4 hour samples n = 28 , 24 hour samples n = 31 , 3 day sample n = 30 , 7 day samples n = 36 .

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c) Exemplar genes differentially expressed at 4 hours and 24 hours, respectively, between infants who experienced a fever within 24 hours of vaccination and afebrile infants. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The false-discovery rate (FDR) was derived by comparing fold-changes in gene expression between infants who experienced post-vaccination fever and those who remained afebrile. P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). Pre-vaccination samples n= 125, 4 hour samples n= 28, 24 hour samples n = 31, 3 day sample n= 30, 7 day samples n= 36.


Boxplots represent median with interquartile ranges . False - discovery rate ( FDR ) are displayed above were derived from a two sample Wilcoxon rank sum ( Benjamini - Hochberg correction ) . N = 6 mice per vaccine group .

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Boxplots represent the median with interquartile ranges. False-discovery rate (FDR) are displayed above and were derived from a two sample Wilcoxon rank sum test (Benjamini-Hochberg correction). N = 6 mice per vaccine group.


Boxplots depicting change in from baseline to 24 hours after second 1μg dose of each of a variety of Escherichia coli lipopolysaccharide ( LPS ) peptidoglycan ( PGN ) preparations , with without alum , combinations compared with 1 / 5 of human dose of 4CMenB . Boxplots represent median with interquartile ranges . Vaccine group medians at each timepoint were compared with baseline for that vaccine using a Wilcoxon signed - rank . * < 0 . 05 , * * < 0 . 01 , * * * * < 0 . 0001 . N = 6 mice per vaccine group .

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Boxplots depicting the change in temperature from baseline to 24 hours after the second 1μg dose of each of a variety of Escherichia coli lipopolysaccharide (LPS) and peptidoglycan (PGN) preparations, with and without alum, and combinations compared with 1/5 of the human dose of 4CMenB. Boxplots represent the median with interquartile ranges. Vaccine group medians at each timepoint were compared with baseline for that vaccine using a Wilcoxon signed-rank test. * < 0.05, ** < 0.01, **** < 0.0001. N= 6 mice per vaccine group.


( A ) BECN1 expression of microglia from Becn1 + / - wild type mouse pups in full medium without Bafilomycin A1 ( Baf ) was quantified by Western blotting . Microglia from Becn1 + / - mice show a signifcant reduction of BECN1 ( 48 % ) compared to microglia from wild type mice ; mean ± , wild type n = 3 , Becn1 + / - n = 7 ; two - t - * * : p < 0 . 01 . Blot shows representative amounts of BECN1 after various treatments .

O O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O 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-GENEPROD O O O O

(A) BECN1 expression of microglia from Becn1+/- and wild type mouse pups in full medium without Bafilomycin A1 (Baf) was quantified by Western blotting. Microglia from Becn1+/- mice show a signifcant reduction of BECN1 (48%) compared to microglia from wild type mice; mean ± SEM, wild type n = 3, Becn1+/- n = 7; two-tailed t-test **: p < 0.01. Blot shows representative amounts of BECN1 after various treatments.


( B ) Microglia from newborn Becn1 + / - wild type mouse pups were kept for 2 h either in full medium or HBSS with or without Bafilomycin A1 ( Baf ) . LC3 ACTIN were determined by Western blot . LC3 - II was significantly reduced in microglia from Becn1 + / - mice after Baf treatment in full medium compared to wild type microglia ; mean ± , wild type n = 3 , Becn1 + / - n = 4 ; two - t - * : p < 0 . 05 .

O O O B-CELL_TYPE O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(B) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with or without Bafilomycin A1 (Baf). LC3 and ACTIN levels were determined by Western blot. LC3-II was significantly reduced in microglia from Becn1+/- mice after Baf treatment in full medium compared to wild type microglia; mean ± SEM, wild type n = 3, Becn1+/- n = 4; two-tailed t-test *: p < 0.05.


( C ) Microglia from newborn Becn1 + / - wild type mouse pups were kept for 2 h either in full medium or HBSS with Bafilomycin A1 ( Baf ) stained for endogenous LC3 . Exemplary of LC3 - positive vesicles / µm2 show reduced presence of LC3 - positive vesicles in microglia from Becn1 + / - induction of autophagy by HBSS ; mean ± , wild type n = 4 , Becn1 + / - n = 4 , 3 - 5 fields per view / n condition 73 - 271 / n condition ; two - t - * : p < 0 . 05 ; scale bar : 20

O O O B-CELL_TYPE O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with Bafilomycin A1 (Baf) and stained for endogenous LC3. Exemplary images and the quantification of LC3-positive vesicles/µm2 cell area show reduced presence of LC3-positive vesicles in microglia from Becn1+/- and induction of autophagy by HBSS; mean ± SEM, wild type n = 4, Becn1+/- n = 4, 3-5 fields per view/n and condition and 73-271 cells/n and condition; two-tailed t-test *: p < 0.05; scale bar: 20 µm


( A , B ) Microglia isolated from newborn Becn1 + / - wild type mouse pups were either non treated or treated with a pro - inflammatory stimulus ( LPS followed by ATP ) . As additional controls treated only with LPS or ATP were used in ( A ) . of pro - inflammatory cytokines IL - 1beta ( A ) IL - 18 ( B ) in supernatant was determined by ELISA . Reduction of BECN1 significantly enhanced release of both cytokines after stimulation with LPS / ATP ; mean ± , wild type n ( A / B ) = 9 / 2 , wild type LPS n = 3 / 0 , wild type ATP n = 3 / 0 , wild type LPS / ATP n = 9 / 9 , Becn1 + / - n = 23 / 5 , Becn1 + / - LPS / ATP n = 23 / 23 , two - t - * : p < 0 . 05 .

O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

(A, B) Microglia isolated from newborn Becn1+/- and wild type mouse pups were either non treated or treated with a pro-inflammatory stimulus (LPS followed by ATP). As additional controls cells treated only with LPS or ATP were used in (A). The concentration of the pro-inflammatory cytokines IL-1beta (A) and IL-18 (B) in the cell supernatant was determined by ELISA. Reduction of BECN1 significantly enhanced release of both cytokines after stimulation with LPS/ATP; mean ± SEM, wild type n (A/B) = 9/2, wild type LPS n = 3/0, wild type ATP n = 3/0, wild type LPS/ATP n = 9/9, Becn1+/- n = 23/5, Becn1+/- LPS/ATP n = 23/23, two-tailed t-test *: p < 0.05.


( C ) Microglia isolated from wild type mouse pups were pre - incubated in full medium or starvation medium HBSS before addition of a pro - inflammatory stimulus ( LPS followed by ATP ) . autophagy blocker 3 - MA was added for final 1 h 45 min of experiment . of pro - inflammatory cytokine IL - 1beta in supernatant was determined by ELISA . Incubation with HBSS reduced IL - 1beta release significantly while 3 - MA treatment increased it ; mean ± , full medium n = 6 , full medium LPS / ATP n = 6 , full medium LPS / ATP + 3MA n = 3 , HBSS n = 6 , HBSS LPS / ATP n = 6 , HBSS LPS / ATP + 3MA n = 6 ; two - t - * : p < 0 . 05 ; * * : p < 0 . 01 .

O O O B-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Microglia isolated from wild type mouse pups were pre-incubated in full medium or starvation medium HBSS before addition of a pro-inflammatory stimulus (LPS followed by ATP). The autophagy blocker 3-MA was added for the final 1 h 45 min of the experiment. The concentration of the pro-inflammatory cytokine IL-1beta in the cell supernatant was determined by ELISA. Incubation with HBSS reduced IL-1beta release significantly while 3-MA treatment increased it; mean ± SEM, full medium n = 6, full medium LPS/ATP n = 6, full medium LPS/ATP + 3MA n = 3, HBSS n = 6, HBSS LPS/ATP n = 6, HBSS LPS/ATP + 3MA n = 6; two-tailed t-test *: p < 0.05; **: p < 0.01.


( D ) Microglia isolated from wild type mouse pups were pre - incubated in starvation medium HBSS before addition of a pro - inflammatory stimulus ( LPS followed by ATP ) . autophagy blocker 3 - MA was added for final 1 h 45 min of experiment , microglia were stained for LC3 taken by confocal microscopy . Representative show a reduction of LC3 - positive vesicles in 3 - MA treated . Scale bar : 20 µm .

O O O B-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O

(D) Microglia isolated from wild type mouse pups were pre-incubated in starvation medium HBSS before addition of a pro-inflammatory stimulus (LPS followed by ATP). The autophagy blocker 3-MA was added for the final 1 h 45 min of the experiment, microglia were stained for LC3 and images taken by confocal microscopy. Representative images show a reduction of LC3-positive vesicles in 3-MA treated cells. Scale bar: 20 µm.


( E ) were extracted from brains of wild type , Becn1 + / - , APPPS1 APPPS1 Becn1 + / - mice at indicated ages . IL - 1beta was measured by electrochemiluminescence ( MesoScale ) compared within repective groups . IL - 1beta was significantly increased in TBS TX fraction ; mean ± , wild type : n ( per group ) = 2 / 6 / 3 , Becn1 + / - : n = 2 / 5 / 2 , APPPS1 : n = 5 / 5 / 6 , APPPS1 Becn1 + / - : n = 7 / 9 / 6 ; ANOVA with Dunnett ´ s post hoc with WT as control group ; * : p < 0 . 05 ; * * : p < 0 . 01 .

O O O O O O B-TISSUE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Proteins were extracted from brains of wild type, Becn1+/-, APPPS1 and APPPS1 Becn1+/- mice at the indicated ages. IL-1beta was measured by electrochemiluminescence (MesoScale) and compared within the repective age groups. IL-1beta was significantly increased in the TBS and TX fraction; mean ± SEM, wild type: n (per age group) = 2/6/3, Becn1+/-: n = 2/5/2, APPPS1: n = 5/5/6, APPPS1 Becn1+/-: n = 7/9/6; ANOVA with Dunnett´s post hoc test with WT as control group; *: p < 0.05; **: p < 0.01.


( F ) were extracted from brains of APPPS1 of APPPS1 Becn1 + / - mice at as indicated . Amyloid beta 1 - 40 1 - 42 were measured by electrochemiluminescence ( MesoScale ) compared within repective groups . No difference in amyloid beta load between genotypes is apparent ; mean ± , APPPS1 : n = 5 / 5 / 6 , APPPS1 Becn1 + / - : n = 7 / 9 / 6 ; ANOVA with Dunnett ´ s post hoc with APPPS1 as control group , ns .

O O O O O O B-TISSUE O O O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Proteins were extracted from brains of APPPS1 and of APPPS1 Becn1+/- mice at the age as indicated. Amyloid beta 1-40 and 1-42 were measured by electrochemiluminescence (MesoScale) and compared within the repective age groups. No difference in the amyloid beta load between the genotypes is apparent; mean ± SEM, APPPS1: n = 5/5/6, APPPS1 Becn1+/-: n = 7/9/6; ANOVA with Dunnett´s post hoc test with APPPS1 as control group, ns.


( A ) expression of NLRP3 in non treated LPS / ATP treated wild type Becn1 + / - microglia was determined by Western blot normalised to ACTIN . Reduction of BECN1 significantly enhanced expression of NLRP3 ; mean ± , wild type n = 4 , wild type LPS / ATP n = 9 , Becn1 + / - n = 4 , Becn1 + / - LPS / ATP n = 23 ; ANOVA with Tukey ´ s post hoc * : p < 0 . 05 .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(A) The expression of NLRP3 protein in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced expression of NLRP3; mean ± SEM, wild type n = 4, wild type LPS/ATP n = 9, Becn1+/- n = 4, Becn1+/- LPS/ATP n = 23; ANOVA with Tukey´s post hoc test *: p < 0.05.


( B ) expression of Nlrp3 mRNA in non treated LPS / ATP treated wild type Becn1 + / - microglia was determined by qPCR . No significant differences can be detected between wild type Becn1 + / - microglia ; mean ± , wild type : n = 7 , Becn1 + / - : n = 3 ; ANOVA with Tukey ´ s post hoc , ns for LPS / ATP treated wild type versus Becn1 + / - .

O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O

(B) The expression of Nlrp3 mRNA in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by qPCR. No significant differences can be detected between wild type and Becn1+/- microglia; mean ± SEM, wild type: n = 7, Becn1+/- : n = 3; ANOVA with Tukey ´s post hoc test, ns for LPS/ATP treated wild type versus Becn1+/-.


( C ) LPS / ATP treated non treated wild type Becn1 + / - microglia were immunolabeled for ASC ( red ) imaged by confocal microscopy to investigate assembly of inflammasomes . In non stimulated ASC is distributed diffusely in cytoplasm ( upper panels ) . However , after stimulation formation of inflammasomes ( arrowheads ) can be detected in a subpopulation of as well as release of inflammasomes after pyroptosis / ejection ( arrow ) . showed a significantly increased of inflammasomes in or around Becn1 + / - microglia ; mean ± , wild type LPS / ATP n = 4 , Becn1 + / - LPS / ATP n = 7 ; two - t - * : p < 0 . 05 ; scale bar : 50 µm , insert scale bar : 10 µm .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for ASC (red) and imaged by confocal microscopy to investigate the assembly of inflammasomes. In non stimulated cells ASC is distributed diffusely in the cytoplasm (upper panels). However, after stimulation formation of inflammasomes (arrowheads) can be detected in a subpopulation of cells as well as release of inflammasomes after pyroptosis/ejection (arrow). Quantification showed a significantly increased percentage of inflammasomes in or around Becn1+/- microglia; mean ± SEM, wild type LPS/ATP n = 4, Becn1+/- LPS/ATP n = 7; two-tailed t-test *: p < 0.05; scale bar: 50 µm, insert scale bar: 10 µm.


( D ) presence of CASP1 ( p10 ) in supernatant of LPS / ATP treated wild type Becn1 + / - microglia was determined by Western blot normalised to ACTIN . Reduction of BECN1 significantly enhanced release of CASP1 ; mean ± , wild type LPS / ATP n = 8 , Becn1 + / - LPS / ATP n = 18 ; two - t - * : p < 0 . 05 .

O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

(D) The presence of cleaved CASP1 (p10) protein in the supernatant of LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced release of CASP1; mean ± SEM, wild type LPS/ATP n = 8, Becn1+/- LPS/ATP n = 18; two-tailed t-test *: p < 0.05.


( A ) LPS / ATP treated non treated wild type Becn1 + / - microglia were immunolabeled for NLRP3 ( green ) LC3 ( red ) imaged by confocal microscopy . Stimulation resulted in appearance of many NLRP3 - containing of different sizes ( arrows ) . In non treated , only a few overlapping signals with LC3 - stained autophagosomes ( arrowheads ) are visible . Stimulated microglia however , showed multiple colocalizations of LC3 positive vesicles NLRP3 ; scale bar : 7 . 5 µm .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O

(A) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for NLRP3 (green) and LC3 (red) and imaged by confocal microscopy. Stimulation resulted in the appearance of many NLRP3-containing aggregates of different sizes (arrows). In non treated cells, only a few overlapping signals with LC3-stained autophagosomes (arrowheads) are visible. Stimulated microglia however, showed multiple colocalizations of LC3 positive vesicles and NLRP3 aggregates; scale bar: 7.5 µm.


( B ) LPS / ATP treated wild type microglia were immunolabeled for NLRP3 ( green ) LC3 ( red ) analysed by super resolution microscopy ( SIM ) . NLRP3 of different sizes coclustering with LC3 - positive autophagosomes are clearly visible . 3D volume rendering of large ( I ) small ( II ) NLRP3 from magnified ROIs showed engulfment of NLRP3 by autophagosomes ; scale bar : 10 µm ; magnified ROIs : 1 µm .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O

(B) LPS/ATP treated wild type microglia were immunolabeled for NLRP3 (green) and LC3 (red) and analysed by super resolution microscopy (SIM). NLRP3 aggregates of different sizes coclustering with LC3-positive autophagosomes are clearly visible. 3D volume rendering of large (I) and small (II) NLRP3 aggregates from the magnified ROIs showed engulfment of NLRP3 by autophagosomes; scale bar: 10 µm; magnified ROIs: 1 µm.


( C ) 3D Radial profiles of NLRP3 LC3 signals derived from SIM in wild type microglia , centered on maxima of NLRP3 clusters . radial profiles confirm that both colocalize to same organelle ; mean ± , n = 4 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(C) 3D Radial intensity profiles of NLRP3 and LC3 signals derived from SIM images in wild type microglia, centered on the maxima of NLRP3 clusters. The radial profiles confirm that both proteins colocalize to the same organelle; mean ± SEM, n = 4.


( A ) LPS / ATP treated wild type microglia were stained for NLRP3 ( green ) CALCOCO2 ( red ) analysed by confocal microscopy . Colocalizations of NLRP3 ( arrows ) CALCOCO2 ( arrowheads ) are apparent ; scale bar : 10 µm .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O

(A) LPS/ATP treated wild type microglia were stained for NLRP3 (green) and CALCOCO2 (red) and analysed by confocal microscopy. Colocalizations of NLRP3 aggregates (arrows) and CALCOCO2 (arrowheads) are apparent; scale bar: 10 µm.


( B ) LPS / ATP treated wild type microglia were stained for NLRP3 ( green ) CALCOCO2 ( red ) analysed by super resolution microscopy ( SIM ) . NLRP3 coclustering with CALCOCO2 - positive signals are visible . 3D volume rendering of NLRP3 from magnified ROIs showed close contact of NLRP3 with autophagic receptor CALCOCO2 ; scale bar : 10 µm ; magnified ROIs : 1 µm .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O

(B) LPS/ATP treated wild type microglia were stained for NLRP3 (green) and CALCOCO2 (red) and analysed by super resolution microscopy (SIM). NLRP3 aggregates coclustering with CALCOCO2-positive signals are visible. 3D volume rendering of NLRP3 aggregates from the magnified ROIs showed close contact of NLRP3 with the autophagic receptor CALCOCO2; scale bar: 10 µm; magnified ROIs: 1 µm.


( C ) 3D Radial profiles of NLRP3 CALCOCO2 signals derived from SIM in wild type microglia , centered on maxima of NLRP3 clusters . radial profiles confirm that both colocalize ; mean ± , n = 3 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

(C) 3D Radial intensity profiles of NLRP3 and CALCOCO2 signals derived from SIM images in wild type microglia, centered on the maxima of NLRP3 clusters. The radial profiles confirm that both proteins colocalize; mean ± SEM, n = 3.


( D ) In SIM from wild type microglia sub - autophagosomal localization of NLRP3 was analysed in relation to LC3 CALCOCO2 . LC3 CALCOCO2 intensities from center of mass of NLRP3 were normalised to their maximum within cluster . While center of NLRP3 shows near maximal CALCOCO2 , relative LC3 there was significantly lower ; mean ± , LC3 : n = 4 , CALCOCO2 : n = 3 ; two - t - * * : p < 0 . 01 .

O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(D) In SIM images from wild type microglia the sub-autophagosomal localization of NLRP3 was analysed in relation to LC3 and CALCOCO2. LC3 and CALCOCO2 intensities from the center of mass of NLRP3 aggregates were normalised to their maximum within the cluster. While the center of NLRP3 aggregates shows near maximal CALCOCO2 intensity, the relative LC3 intensity there was significantly lower; mean ± SEM, LC3: n = 4, CALCOCO2: n = 3; two-tailed t-test **: p < 0.01.


( E ) Microglia from Becn1 + / - mice were transfected with scrambled siRNA or with siRNA for CALCOCO2 . 6 d after transfection were stimulated with LPS / ATP . expression of CALCOCO2 was determined by Western blot . Application of CALCOCO2 siRNA resulted in significantly reduced expression of CALCOCO2 ; mean ± , n = 3 ; two - t - * : p < 0 . 05 .

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(E) Microglia from Becn1+/- mice were transfected with scrambled siRNA or with siRNA for CALCOCO2. 6 d after transfection cells were stimulated with LPS/ATP. The expression of CALCOCO2 protein was determined by Western blot. Application of CALCOCO2 siRNA resulted in significantly reduced expression of CALCOCO2; mean ± SEM, n = 3; two-tailed t-test *: p < 0.05.


( F ) amount of IL - 1beta in supernatant released from described in ( E ) was determined by ELISA . Microglia with a CALCOCO2 knockdown released significantly more IL - 1beta ; mean ± , n = 3 ; two - t - * : p < 0 . 05 .

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(F) The amount of IL-1beta in the supernatant released from the cells described in (E) was determined by ELISA. Microglia with a CALCOCO2 knockdown released significantly more IL-1beta; mean ± SEM, n = 3; two-tailed t-test *: p < 0.05.


okp1 - R164C suppressed - sensitive growth defect of cbf1 ∆ cse4 - R37A . Serial dilutions of strains with indicated genotypes were spotted on full medium grown for three days at 30ºC or 37ºC . original okp1 - R164C isolate from suppressor screen is indicated as " UV mutagenesis " .

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okp1-R164C suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC. The original okp1-R164C isolate from the suppressor screen is indicated as "UV mutagenesis".


Serial dilutions of strains with indicated genotypes were spotted on full medium grown for three days at 30ºC or 37ºC . original okp1 - R164C isolate from suppressor screen is indicated as " UV mutagenesis " . okp1 - R164C suppressed growth defect of cse4 - R37A with chl4 ∆ .

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Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC. The original okp1-R164C isolate from the suppressor screen is indicated as "UV mutagenesis". okp1-R164C suppressed the growth defect of cse4-R37A with chl4∆.


okp1 - R164C was unable to suppress lethality of cse4 - R37A with ame1 - 4 . Tetrad dissection of a genetic cross between a cse4 - R37A okp1 - R164C an ame1 - 4 is shown . four spores from asci are aligned in vertical rows .

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okp1-R164C was unable to suppress the lethality of cse4-R37A with ame1-4. Tetrad dissection of a genetic cross between a cse4-R37A okp1-R164C and an ame1-4 strain is shown. The four spores from individual asci are aligned in vertical rows.


okp1 - R164C suppressed maintenance defect of cse4 - R37A for plasmids lacking CDEI of CEN6 ( CEN ∆ CDEI , at 37ºC ) . Error bars give SD of at least three independent transformants . * p = 0 . 03 .

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okp1-R164C suppressed the maintenance defect of cse4-R37A for plasmids lacking the CDEI sequence of CEN6 (CEN ∆CDEI, at 37ºC). Error bars give SD of at least three independent transformants. * p = 0.03.


okp1 mutations I45T , S94T E208V suppressed - sensitive growth defect of cbf1 ∆ cse4 - R37A . Strains with indicated okp1 alleles on a plasmid ( derivatives of AEY5584 )

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The okp1 mutations I45T, S94T and E208V suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Strains with the indicated okp1 alleles on a plasmid (derivatives of AEY5584)


okp1 - R164C restored binding of Okp1 to centromeric sequences . ChIP of 9xmyc - tagged Okp1 was performed in strains with indicated genotypes that were grown at 37ºC for 4 hours before ChIP . Enrichment of CEN4 POL1 ( as a control ) relative to input is given . Below , Western blot of amounts of 9xmyc - Okp1 histone H2B ( loading control ) in whole extracts .

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okp1-R164C restored binding of Okp1 to centromeric sequences. ChIP of 9xmyc-tagged Okp1 was performed in strains with the indicated genotypes that were grown at 37ºC for 4 hours before ChIP. Enrichment of CEN4 and POL1 (as a control) relative to input is given. Below, Western blot analysis of the amounts of 9xmyc-Okp1 and histone H2B (loading control) in whole cell extracts.


okp1 - R164C restored binding of Ame1 to centromeric sequences . ChIP of 9xmyc - tagged Ame1 was performed as in ( A ) . Below , Western blot of amounts of 9xmyc - Ame1 H2B as in ( A ) .

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okp1-R164C restored binding of Ame1 to centromeric sequences. ChIP of 9xmyc-tagged Ame1 was performed as in (A). Below, Western blot analysis of the amounts of 9xmyc-Ame1 and H2B as in (A).


okp1 - R164C restored binding of Mtw1 to centromeric sequences . ChIP of 9xmyc - Mtw1 Western blots ( right )

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okp1-R164C restored binding of Mtw1 to centromeric sequences. ChIP of 9xmyc-Mtw1 and Western blots (right)


okp1 - R164C improved in vivo association of Okp1 with Cse4 - R37A . Co - immunoprecipitation of Cse4 - R37A Okp1 was carried out in carrying cse4 - R37A with ( + ) or without ( - ) 3xHA - tag OKP1 or okp1 - R164C with ( + ) or without ( - ) 9xmyc - tag ( AEY5040 , AEY5972 , AEY5973 , AEY6589 , AEY6592 ) . were grown at 23ºC or were shifted to 37ºC for 5 hours prior to harvesting . Cse4 - R37A was immunoprecipitated using an α - HA antibody , presence of Cse4 - R37A Okp1 or Okp1 - R164C in immunoprecipitate was tested by Western blotting with α - HA α - myc antibody , respectively . Left , inputs ( two bands indicate Okp1 a shorter degradation product ) ; right , α - HA immunoprecipitates .

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okp1-R164C improved in vivo association of Okp1 with Cse4-R37A. Co-immunoprecipitation of Cse4-R37A and Okp1 was carried out in cells carrying cse4-R37A with (+) or without (-) 3xHA-tag and OKP1 or okp1-R164C with (+) or without (-) 9xmyc-tag (AEY5040, AEY5972, AEY5973, AEY6589, AEY6592). Cells were grown at 23ºC or were shifted to 37ºC for 5 hours prior to harvesting. Cse4-R37A was immunoprecipitated using an α-HA antibody, and the presence of Cse4-R37A and Okp1 or Okp1-R164C in the immunoprecipitate was tested by Western blotting with α-HA and α-myc antibody, respectively. Left, inputs (the two bands indicate Okp1 and a shorter degradation product); right, α-HA immunoprecipitates.


Cse4 ( 21 - 129 , Cse4N ) Okp1 / Ame1 were expressed purified separately from co - incubated before separation by analytical gel filtration . Top , representative - exclusion chromatography ( SEC ) chromatogram with absorbance measurements at 280 nanometers ( nm ) is shown . Below , image of Coomassie Blue - stained SDS - PAGE gels with fractions from principal SEC peaks of runs with Okp1 / Ame1 alone ( top ) , Cse4N alone ( middle ) Okp1 / Ame1 pre - incubated with Cse4N before SEC ( bottom ) . Molecular standards are shown in kilodalton ( kDa ) .

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Cse4(21-129, Cse4N) and Okp1/ Ame1 were expressed and purified separately from bacteria and co-incubated before separation by analytical gel filtration. Top, representative size-exclusion chromatography (SEC) chromatogram with absorbance measurements at 280 nanometers (nm) is shown. Below, image of Coomassie Blue-stained SDS-PAGE gels with fractions from principal SEC peaks of runs with Okp1/ Ame1 alone (top), Cse4N alone (middle) and Okp1/ Ame1 pre-incubated with Cse4N before SEC (bottom). Molecular weight standards are shown in kilodalton (kDa).


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The ame1 mutations S59P, N159H, S275N and a C-terminal truncation after aa 273 (ame1-273*) suppressed the growth defect of cbf1∆ cse4-R37A. cbf1∆ cse4-R37A ame1∆ strains carrying the indicated plasmid-borne ame1 alleles


Cse4 - K49Ac - R37Me are increased in S - phase of cycle . carrying 3xHA - tagged Cse4 were used to immunoprecipitate Cse4 with an α - Cse4 - K49Ac ( left , top ) an α - HA antibody ( left , bottom ) . To detect Cse4 - R37 methylation , 3xHA - tagged Cse4 was immunoprecipitated with an α - Cse4 - R37Me2a ( right , top ) ( Samel et al . , 2012 ) an α - HA antibody ( right , bottom ) . Western blots were probed for presence of Cse4 with α - HA antibody . G1 , treatment with α - factor ; S , arrest with hydroxyurea ; G2 / M , arrest in nocodazole ; asynch , asynchronous culture .

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Cse4-K49Ac and -R37Me are increased in S-phase of the cell cycle. Cells carrying 3xHA-tagged Cse4 were used to immunoprecipitate Cse4 with an α-Cse4-K49Ac (left, top) and an α-HA antibody (left, bottom). To detect Cse4-R37 methylation, 3xHA-tagged Cse4 was immunoprecipitated with an α-Cse4-R37Me2a (right, top) (Samel et al., 2012) and an α-HA antibody (right, bottom). Western blots were probed for the presence of Cse4 with the α-HA antibody. G1, treatment with α-factor; S, arrest with hydroxyurea; G2/ M, arrest in nocodazole; asynch, asynchronous culture.


Mimicking unacetylated state of Cse4 - K49 ( cse4 - K49R ) partially suppressed - sensitivity of cbf1 ∆ cse4 - R37A . cbf1 ∆ cse4 ∆ strains carrying indicated cse4 alleles on a plasmid were spotted on full medium

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Mimicking the unacetylated state of Cse4-K49 (cse4-K49R) partially suppressed the temperature-sensitivity of cbf1∆ cse4-R37A. cbf1∆ cse4∆ strains carrying the indicated cse4 alleles on a plasmid were spotted on full medium and grown


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cse4-K49R suppressed the lethality of cse4-R37A with ctf19∆. Tetrad dissection of a ctf19∆ strain crossed with cse4-R37A (left) and cse4-R37A K49R (right). Representation


Acetylation of Cse4 - K49 was reduced in absence of Gcn5 SAGA , but not in absence of HAT Sas2 . Cse4 was precipitated as in ( A ) from strains wild - type ( wt ) or gcn5 ∆ ( top ) or sas2 ∆ strains ( bottom ) carrying HA - tagged Cse4 . w / o , control immunoprecipitation without antibody .

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Acetylation of Cse4-K49 was reduced in the absence of Gcn5 and the SAGA complex, but not in the absence of the HAT Sas2. Cse4 was precipitated as in (A) from strains wild-type (wt) or gcn5∆ (top) or sas2∆ strains (bottom) carrying HA-tagged Cse4. w/o, control immunoprecipitation without antibody.


Cse4 - K49Ac depended on SAGA components Ada2 Spt7 . Cse4 - K49Ac was determined as in ( A , left ) with carrying ada2 ∆ or spt7 ∆ .

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Cse4-K49Ac depended on the SAGA components Ada2 and Spt7. Cse4-K49Ac was determined as in (A, left) with cells carrying ada2∆ or spt7∆.


A synthetic 78 - aa Cse4 peptide ( aa 33 - 110 ) binds to Okp1 / Ame1 in vitro . Microscale thermophoresis ( MST ) measurement of binding of fluorescently labeled Cse4 ( 33 - 110 ) to Okp1 / Ame1 is shown . Normalized , from which of unbound Okp1 / Ame1 was subtracted ( ∆ F norm ) , is plotted against Cse4 ( 33 - 110 ) ( logarithmic scale ) . Mean values ± SD ( n = 3 ) are shown .

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A synthetic 78-aa Cse4 peptide (aa 33 - 110) binds to Okp1/ Ame1 in vitro. Microscale thermophoresis (MST) measurement of binding of fluorescently labeled Cse4 (33 - 110) to Okp1/ Ame1 is shown. Normalized fluorescence, from which the fluorescence of unbound Okp1/ Ame1 was subtracted (∆F norm), is plotted against Cse4(33-110) concentration (logarithmic scale). Mean values ± SD (n = 3) are shown.


Plot of MST measuring competition of fluorescently labelled Cse4 ( 33 - 110 ) bound to Okp1 / Ame1 ( black ) ( or Okp1 - R164C / Ame1 , red ) with unlabelled Cse4 ( 33 - 87 ) that is unmodified or monomethylated on R37 ( R37Me ) . Normalized from which of unbound Cse4 ( 33 - 110 ) was subtracted ( ∆ F norm ) is plotted against peptide . Mean values ± standard deviation ( n = 3 ) are given . Control , normalized of Cse4 ( 33 - 110 ) alone at increasing concentrations .

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Plot of MST measuring the competition of fluorescently labelled Cse4 (33 - 110) bound to Okp1/ Ame1 (black) (or Okp1-R164C/ Ame1, red) with unlabelled Cse4 (33 - 87) that is unmodified or monomethylated on R37 (R37Me). Normalized fluorescence from which the fluorescence of unbound Cse4 (33 - 110) was subtracted (∆F norm) is plotted against peptide concentration. Mean values ± standard deviation (n = 3) are given. Control, normalized fluorescence of Cse4 (33 - 110) alone at increasing concentrations.


E . Immunoblot ( IB ) of stable Ub replacement lines treated or not with DOX for 72 h . Data information : Data are representative of three ( E independent experiments with similar outcome .

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E. Immunoblot (IB) analysis of stable Ub replacement cell lines treated or not with DOX for 72 h. Data information: Data are representative of three (E independent experiments with similar outcome.


F , G . HA - Ub conjugates isolated as in ( D ) were immunoblotted with indicated antibodies . Data information : Data are representative of three independent experiments with similar outcome .

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F,G. HA-Ub conjugates isolated as in (D) were immunoblotted with indicated antibodies. Data information: Data are representative of three independent experiments with similar outcome.


B . Representative ( upper ) QIBC ( lower ) ( solid lines , median ; dashed lines , quartiles ; > 10000 analyzed per condition ) of Ub ( G76V ) - GFP expression in stable U2OS / Ub ( G76V ) - GFP treated or not with p97i for 4 h . Data information : Data are representative of three independent experiments with similar outcome .

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B. Representative images (upper) and QIBC analysis (lower) (solid lines, median; dashed lines, quartiles; >10000 cells analyzed per condition) of Ub(G76V)-GFP expression in stable U2OS/Ub(G76V)-GFP cells treated or not with p97i for 4 h. Data information: Data are representative of three independent experiments with similar outcome.


D . DOX - treated U2OS / shUb / HA - Ub ( WT ) U2OS / shUb / HA - Ub ( K27R ) lines were transfected with shUb - resistant Ub ( G76V ) - GFP construct treated or not with p97i for 4 h . extracts were subjected to GFP IP followed by immunoblotting with indicated antibodies . Data information : Data are representative of three independent experiments with similar outcome .

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D. DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines were transfected with shUb-resistant Ub(G76V)-GFP construct and treated or not with p97i for 4 h. Cell extracts were subjected to GFP IP followed by immunoblotting with indicated antibodies. Data information: Data are representative of three independent experiments with similar outcome.


E . Immunoblot of DOX - treated U2OS / shUb / HA - Ub lines treated or not with MG132 for 4 h . Data information : Data are representative of three independent experiments with similar outcome .

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E. Immunoblot analysis of DOX-treated U2OS/shUb/HA-Ub cell lines treated or not with MG132 for 4 h. Data information: Data are representative of three independent experiments with similar outcome.


G . DOX - treated U2OS / shUb / HA - Ub ( WT ) U2OS / shUb / HA - Ub ( K27R ) lines transfected with indicated Ub ( G76V ) - GFP expression constructs were subjected to GFP IP under denaturing conditions followed by immunoblotting with indicated antibodies . Data information : Data are representative of four ( G ) independent experiments with similar outcome .

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G. DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines transfected with indicated Ub(G76V)-GFP expression constructs were subjected to GFP IP under denaturing conditions followed by immunoblotting with indicated antibodies. Data information: Data are representative of four (G) independent experiments with similar outcome.


A . FLAG IPs from U2OS transfected with indicated FLAG - tagged expression constructs were incubated with purified K27 di - Ub analyzed by immunoblotting . Data information : Data are representative of three ( A independent experiments with similar outcome .

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A. FLAG IPs from U2OS cells transfected with indicated FLAG- tagged expression constructs were incubated with purified K27 di-Ub and analyzed by immunoblotting. Data information: Data are representative of three (A independent experiments with similar outcome.


C . transfected as in ( B ) treated or not with p97i for 4 h were subjected to GFP IP followed by immunoblotting . Data information : Data are representative of three independent experiments with similar outcome .

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C. Cells transfected as in (B) and treated or not with p97i for 4 h were subjected to GFP IP followed by immunoblotting. Data information: Data are representative of three independent experiments with similar outcome.


I . U2OS / Ub ( G76V ) - GFP U2OS / Ub ( G76V ) - 20AA - GFP lines were transfected or not with FLAG - UCHL3 C95S expression construct , treated with MG132 for 2 h subjected to GFP IP followed by immunoblotting . Data information : Data are representative of three independent experiments with similar outcome .

O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O

I. U2OS/Ub(G76V)-GFP and U2OS/Ub(G76V)-20AA-GFP cell lines were transfected or not with FLAG-UCHL3 C95S expression construct, treated with MG132 for 2 h and subjected to GFP IP followed by immunoblotting. Data information: Data are representative of three independent experiments with similar outcome.


B ) classification of total ( 3209 , left panel ) of glycoproteins ( 571 , right panel ) detected in 3 out of 3 experiments . More than 90 % of glycoproteins detected are classified as , as expected for surface labeling .

O O O O O O O O O O O O O O O O O O O O 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) Protein classification of the total proteins (3209, left panel) and of the glycoproteins (571, right panel) detected in 3 out of 3 experiments. More than 90% of the glycoproteins detected are classified as membrane proteins, as expected for the surface labeling.


C ) Changes of 3209 detected on surface of SEZ6KO neurons compared to WT . average log2 fold change of SEZ6KO neurons over WT ( log2 SEZ6KO / WT ) is plotted against negative log10 of each ' s p - value according to a one - sample t . Blue labeled dots are considered as hits ( including GluK2 GluK3 ) are with a p - value less than 0 . 05 ( neg . log10 ( 0 . 05 ) = 1 . 3 ; horizontal dotted ) a fold - change to less than - 0 . 5 or more than 0 . 5 ( vertical dotted lines ) . GluK2 GluK3 are labeled in pink , GluA1 GluA2 are indicated with green lines dots GluN1 GluN2B are marked with red lines dots .

O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O O

C) Changes of the 3209 proteins detected on the cell surface of SEZ6KO neurons compared to WT. The average log2 fold change of SEZ6KO neurons over WT (log2 SEZ6KO/WT) is plotted against the negative log10 of each protein's p-value according to a one-sample t test. Blue labeled dots are considered as hits (including GluK2 and GluK3) and are proteins with a p-value less than 0.05 (neg. log10(0.05)=1.3; horizontal dotted line) and a fold-change to less than -0.5 or more than 0.5 (vertical dotted lines). GluK2 and GluK3 are labeled in pink, GluA1 and GluA2 are indicated with green lines and dots and GluN1 and GluN2B are marked with red lines and dots.