( A - F ) Wing imaginal discs stained with : ( A , C , D , F ) antibody to DHR3 , ( B , E ) antibody to GFP . ( A , B ) Overexpression of DHR3 - RS USA - GFP in posterior compartment was induced using an engrailed - Gal4 driver . ( C ) ( D ) ap - Gal4 drives overexpression of DHR3 - RS DHR3 - EP , respectively . ( E , F ) Flip - out clone ( induced 3 days prior to dissection ) in a prepupal wing imaginal disc expressing UAS - DHR3 - RNAi UAS - GFP . ( E ) Decrease in DHR3 in a flip - out clone labeled by GFP ( F ) , indicating that DHR3 is expressed in this tissue .

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(A-F) Wing imaginal discs stained with: (A, C, D, F) antibody to DHR3, and (B, E) antibody to GFP. (A, B) Overexpression of DHR3-RS and USA-GFP in the posterior compartment was induced using an engrailed-Gal4 driver. (C) and (D) ap-Gal4 drives overexpression of DHR3-RS and DHR3-EP, respectively. (E, F) Flip-out clone (induced 3 days prior to dissection) in a prepupal wing imaginal disc expressing UAS-DHR3-RNAi and UAS-GFP. (E) Decrease in DHR3 staining in a flip-out clone labeled by GFP (F), indicating that DHR3 is expressed in this tissue.


( G ) Western blot of DHR3 in larval extracts from control ( Co ) heat shock - induced UAS lines expressing DHR3 - RA ( RA ) , DHR3 - RB ( RB ) , DHR3 - RS ( RS ) transcripts , or DHR3 - EPs ( E1 E2 ) .

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(G) Western blot analysis of DHR3 in larval protein extracts from control (Co) and heat shock-induced UAS lines expressing DHR3-RA (RA), DHR3-RB (RB), and DHR3-RS (RS) transcripts, or the DHR3-EPs (E1 and E2).


( H ) Western blot of endogenous DHR3 in late third - instar larvae ( L3 ) , prepupae ( pp ) , prepupae expressing an RNAi to DHR3 ( RNAipp ) . In ( G , H ) , arrows at left indicate position of DBD - containing ( high ) DBD - lacking ( low ) DHR3 .

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(H) Western blot of the endogenous DHR3 protein in late third-instar larvae (L3), prepupae (pp), and prepupae expressing an RNAi to DHR3 (RNAipp). In (G, H), the arrows at the left indicate the position of the DBD-containing (high) and DBD-lacking (low) DHR3 proteins.


( A , B ) ap - Gal4 driver induces dS6K in combination with either ( A ) DHR3 - EP transgene or ( B ) EP with EMS DHR3

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(A, B) The ap-Gal4 driver induces dS6K in combination with either (A) the DHR3-EP transgene or (B) the EP with the EMS DHR3 mutation


( D ) dS6K activity in larval extracts measured by H2B target phosphorylation ( E ) corresponding western blot of dS6K in trans - heterozygote LBD - mutant combination ( LBD ) , as compared to heterozygous larvae ( Co ) .

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(D) dS6K activity in larval protein extracts measured by H2B target phosphorylation and (E) corresponding western blot of dS6K in the trans-heterozygote LBD-mutant combination (LBD), as compared to heterozygous larvae (Co).


( F , G ) eyeless promoter directs flipase recombinase during eye development . Flipase allows recombination on right arm of second chromosome eventually leads to an adult eye ( F ) that is homozygous for wild - type allele of DHR3 . ( G ) Homozygosity for a DHR3K243X mutation results in a decrease in of adult eye . A dotted yellow surrounding control eye in ( F ) has been copied pasted on mutant eyes ( G ) ; insets are higher magnification of ommatidia showing misorientation of bristles .

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(F, G) The eyeless promoter directs the flipase recombinase during eye development. Flipase allows recombination on the right arm of the second chromosome and eventually leads to an adult eye (F) that is homozygous for the wild-type allele of DHR3. (G) Homozygosity for a DHR3K243X mutation results in a decrease in the size of the adult eye. A dotted yellow line surrounding the control eye in (F) has been copied and pasted on the mutant eyes (G); insets are higher magnification images of ommatidia showing misorientation of bristles.


( H ) At scutellum , a y - marked homozygous DHR3K243X mutant bristle ( arrow ) , recognizable by its color , is smaller than symmetrical neighboring bristles .

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(H) At the scutellum, a y-marked homozygous DHR3K243X mutant bristle (arrow), recognizable by its light color, is smaller than the symmetrical neighboring bristles.


( A ) Normalized eye ( mm2 ) of control ( black bar ; n = 20 ) , DHR3W284X ( grey bar ; n = 18 ) DHR3K243X ( bar ; n = 18 ) mutants . As compared to control recombined eyes ( Co ) , DHR3W284X DHR3K243X mutant eyes exhibit a highly significant reduction ( * * * P < 0 . 001 ) . ( B ) Ommatidia of control ( black bar ) , DHR3W284X ( grey bar ) , DHR3K243X ( bar ) mutants . As compared to control recombined eyes ( Co ) , DHR3W284X DHR3K243X mutant ommatidia exhibit a significant reduction ( * * P < 0 . 01 ) .

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(A) Normalized eye size (mm2) of control (black bar; n = 20), DHR3W284X (grey bar; n = 18) and DHR3K243X (white bar; n = 18) mutants. As compared to control recombined eyes (Co), DHR3W284X and DHR3K243X mutant eyes exhibit a highly significant size reduction (***P<0.001). (B) Ommatidia size of control (black bar), DHR3W284X (grey bar), and DHR3K243X (white bar) mutants. As compared to control recombined eyes (Co), DHR3W284X and DHR3K243X mutant ommatidia exhibit a significant size reduction (**P<0.01).


( C - E ) Comparison of y - marked homozygous clonal bristles ( black arrows ) to their neighboring heterozygous bristles ( arrows ) at wing margin . Note that control clonal bristles are of normal orientation ( C ) , whereas those of DHR3W284X ( D ) DHR3K243X ( E ) are reduced in are misoriented . ( F ) measurements ( µm ) of of homozygous clones ( y − ) their neighboring heterozygous ( y + ) bristles ( n = 51 for each bar ) . reduction of clonal versus neighbor bristles is highly significant for DHR3K243X ( * * * P < 0 . 001 ) DHR3W284X ( * * * P < 0 . 001 ) mutants , but not for control ( Co ) bristles ( P = 0 . 028 ) .

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(C-E) Comparison of y-marked homozygous clonal bristles (black arrows) to their neighboring heterozygous bristles (white arrows) at the wing margin. Note that control clonal bristles are of normal size and orientation (C), whereas those of DHR3W284X (D) and DHR3K243X (E) are reduced in size and are misoriented. (F) Size measurements (µm) of the length of homozygous clones (y−) and their neighboring heterozygous (y+) bristles (n = 51 for each bar). The size reduction of clonal versus neighbor bristles is highly significant for DHR3K243X (***P<0.001) and DHR3W284X (***P<0.001) mutants, but not for the control (Co) bristles (P = 0.028).


Exponential growth of E . coli K - 12 , measured using optical density ( OD600 ) , in lysogenic broth ( LB , circles ) or minimal medium supplemented with different carbon sources , listed in legend on bottom right in a greyscale . cultures are grown in batch mode , see Methods .

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Exponential growth of E. coli K-12, measured using optical density (OD600), in lysogenic broth (LB, white circles) or minimal medium supplemented with different carbon sources, listed in the legend on the bottom right in a greyscale. All the cultures are grown in batch mode, see Methods.


viability in colony forming units ( CFU ) per ml of cultures from panel A , grown until OD600 0 . 5 , washed re - suspended in carbon - free minimal medium . rates ( slopes of exponential fits ) are listed in Table EV1 . Initial density for fast growth ( lighter shades ) is lower than for slow growth ( darker shades ) , due larger sizes that lead to lower densities per OD600 .

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Bacterial viability in colony forming units (CFU) per ml of cultures from panel A, grown until OD600 0.5, washed and re-suspended in carbon-free minimal medium. Death rates (slopes of the exponential fits) are listed in Table EV1. Initial density for fast growth (lighter shades) is lower than for slow growth (darker shades), due larger cell sizes that lead to lower cell densities per OD600.


rates of panel B plotted against growth rates of panel A shown as circles in a grey - scale . Squares show data obtained using eight other carbon substrates , also listed in Table EV1 . Color circles show data obtained by varying growth rate in a chemostat . black shows a linear weighted least square fit to log - transformed data , that takes uncertainty in data points into account ( see Methods ) . Data shown as mean ± ( standard deviation ) SD . Two or three replicates per condition .

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Death rates of panel B plotted against growth rates of panel A shown as circles in a grey-scale. Squares show data obtained using eight other carbon substrates, also listed in Table EV1. Color circles show data obtained by varying growth rate in a chemostat. The black line shows a linear weighted least square fit to log-transformed data, that takes uncertainty in data points into account (see Methods). Data shown as mean ± (standard deviation) SD. Two or three replicates per condition.


rates of wild - type GlpK22 mutants plotted versus growth rates . Wild type is grown in glycerol minimal medium in batch cultures ( black circle ) in ' chemostat ' continuous cultures with growth rates coded in colors , see legend on center right . GlpK22 mutants ( circle ) are grown in glycerol minimal medium in batch culture . Data shown as mean ± ( standard deviation ) SD . Two replicates per condition .

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Death rates of wild-type and GlpK22 mutants plotted versus growth rates. Wild type is grown in glycerol minimal medium in batch cultures (black circle) and in 'chemostat' continuous cultures with growth rates coded in colors, see legend on the center right. GlpK22 mutants (white circle) are grown in glycerol minimal medium in batch culture. Data shown as mean ± (standard deviation) SD. Two replicates per condition.


Example experiment of sketched in panel C . previously grown at 0 . 3 h − 1 in continuous culture are starved ( green circles ) . After 2 days of starvation , a sample is ( 1 ) extracted ( 2 ) UV - sterilized , see dashed , followed by ( 3 ) inoculation of 1 % of starved ( 4 ) growth of culture . difference between maximum viability after growth , initial viability at point ( 3 ) is absolute yield .

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Example experiment of the assay sketched in panel C. Cells previously grown at 0.3 h − 1 in continuous culture are starved (green circles). After 2 days of starvation, a sample is (1) extracted and (2) UV-sterilized, see dashed line, followed by (3) inoculation of 1% of starved and (4) growth of the culture. The difference between the maximum viability after growth, and the initial viability at point (3) is the absolute yield.


absolute yield measured at three different time points for each of growth conditions shown in panel A is plotted against viability of starvation culture at extraction time . recycling yield is extracted as slope of linear fits . Recycling yield from panel E plotted versus growth rate . Data shown as mean ± SD . Two replicates per condition .

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The absolute yield measured at three different time points for each of the growth conditions shown in panel A is plotted against the viability of the starvation culture at the extraction time. The recycling yield is extracted as the slope of the linear fits. Recycling yield from panel E plotted versus growth rate. Data shown as mean ± SD. Two replicates per condition.


Example experiment from a starved culture previously grown at a rate of 0 . 3 h − 1 with a viability after 5 days of starvation . After ( 1 ) extraction ( 2 ) addition of 40 µM of glycerol decay of viability is delayed ( black circles ) compared to a control without glycerol ( green symbols ) . Per viable , glycerol addition in this experiment is 0 . 33 fmol CFU − 1 . After an initial period of survival , culture with added glycerol ( black ) dies at same rate as control ( green ) . ' lag time ' fitted to this data is 1 day .

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Example experiment from a starved culture previously grown at a rate of 0.3 h − 1 with a viability after 5 days of starvation. After (1) extraction and (2) addition of 40 µM of glycerol the decay of viability is delayed (black circles) compared to a control without glycerol (green symbols). Per viable cell, the glycerol addition in this experiment is 0.33 fmol CFU − 1. After an initial period of survival, the culture with added glycerol (black) dies at the same rate as the control (green). The 'lag time' fitted to this data is 1 day.


Lag time of experiments of panel B for different glycerol concentrations . Lag increases linearly with glycerol maintenance rate is extracted as inverse of slope of linear fits .

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Lag time of the experiments of panel B for different glycerol concentrations. Lag increases linearly with glycerol concentration and maintenance rate is extracted as the inverse of the slope of the linear fits.


Maintenance rate values extracted from panel I plotted versus growth rate . Data shown as mean ± SD . Two replicates per condition .

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Maintenance rate values extracted from panel I plotted versus growth rate. Data shown as mean ± SD. Two replicates per condition.


( K ) Consistency check of equation in panel A . Fold changes of maintenance rate , FC ( β ) , divided by fold changes of recycling yield , FC ( α ) , plotted versus fold changes of rate , FC ( γ ) . fold changes are relative to wild - type in batch , growing at 0 . 7 h − 1 . dashed shows unity FC ( γ ) = FC ( β ) / FC ( α ) .

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(K) Consistency check of the equation in panel A. Fold changes of maintenance rate, FC(β), divided by the fold changes of the recycling yield, FC(α), plotted versus the fold changes of death rate, FC(γ). All fold changes are relative to wild-type in batch, growing at 0.7 h − 1. The dashed line shows the unity line FC(γ)= FC(β)/FC(α).


during starvation . width of are measured with phase - contrast microscopy volume is computed considering shape as a cylinder with two semi - spheres , as described in graphical synopsis at top ( see also Table EV3 Methods ) . Each measurement is an average of 200 . ( A ) Measured width of wild type starved in batch cultures previously grown in chemostat at different steady - state growth rates of GlpK22 starved previously grown in batch culture at μ = 0 . 9 h − 1 ( see upper color legend ) . Note that , as shown in Schink et al . ( 2019 ) , widths do not change from steady state growth to starvation , while lengths decrease . Both width increase exponentially with growth rate . ( B ) Starvation volume of described in panel A , computed as explained in graphical synopsis ( see also Methods ) . In agreement with literature ( Schaechter et al . , 1958 ) , it increases exponentially with growth rate ( black ) .

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Cell size during starvation. Length and width of the cells are measured with phase-contrast microscopy and the volume is computed considering cell shape as a cylinder with two semi-spheres, as described in the graphical synopsis at the top (see also Table EV3 and Methods). Each measurement is an average of 200 cells. (A) Measured length and width of wild type cells starved in batch cultures and previously grown in the chemostat at different steady-state growth rates and of GlpK22 cells starved and previously grown in batch culture at μ = 0.9 h − 1 (see upper color legend). Note that, as shown in Schink et al. (2019), cell widths do not change from steady state growth to starvation, while lengths decrease. Both length and width increase exponentially with growth rate. (B) Starvation volume of the cells described in panel A, computed as explained in the graphical synopsis (see also Methods). In agreement with literature (Schaechter et al., 1958), it increases exponentially with growth rate (black line).


Recycling yield maintenance rate per volume . ( C ) Recycling yields ( D ) maintenance rate measured as described in Fig . 2 are normalized per volume plotted versus previous growth rate of cultures they refer to ( see color legend at top Table EV3 ) . normalized yield is constant within uncertainty . Maintenance rate increases significantly with a slope of ( 0 . 99 ± 0 . 55 ) h , matching increase of rate , ( 1 . 1 ± 0 . 4 ) h , shown in Fig 2B . Solid black lines show linear fits on log transformed data , using weighted least square fits .

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Recycling yield and maintenance rate per cell volume. (C) Recycling yields and (D) maintenance rate measured as described in Fig. 2 are normalized per cell volume and plotted versus the previous growth rate of the cultures they refer to (see color legend at the top and Table EV3). The normalized yield is constant within the uncertainty. Maintenance rate increases significantly with a slope of (0.99±0.55) h, matching the increase of the death rate, (1.1±0.4) h, shown in Fig 2B. Solid black lines show linear fits on log transformed data, using weighted least square fits.


rates of ∆ rpoS mutants versus growth rate ( grey symbols ) , compared to wild - type E . coli K - 12 ( symbols , data from Fig . 1 ) . rate increases with growth rate with a smaller slope ( ∆ rpoS : 0 . 57 ± 0 . 13 h ) compared to wild - type ( WT : 0 . 88 ± 0 . 13 h ) . Black grey lines show linear weighted least square fits to log - transformed data , which take uncertainty in data points into account ( see Methods ) . Data shown as mean ± SD . Two or three replicates per condition .

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Death rates of ∆rpoS mutants versus the growth rate (grey symbols), compared to wild-type E. coli K-12 (white symbols, data from Fig. 1). Death rate increases with growth rate with a smaller slope (∆rpoS: 0.57±0.13 h) compared to wild-type (WT: 0.88±0.13 h). Black and grey lines show linear weighted least square fits to log-transformed data, which take uncertainty in data points into account (see Methods). Data shown as mean ± SD. Two or three replicates per condition.


optimal growth rate , i . e . one that maximizes fitness over a cycle of famine feast , depends on of feast famine periods . For long periods of feast interrupted by short periods of famine , E . coli optimal strategy is to grow fast . For long periods of starvation intermitted by short periods of growth , optimal strategy is long survival slow growth . For scenario of panel A B , $ \ frac { T _ { - } } { T _ { + } } = 48 $ , optimum is μ = 0 . 86 h − 1 γ = 0 . 5 d − 1 , see dashed .

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The optimal growth rate, i.e. the one that maximizes fitness over a cycle of famine and feast, depends on the ratio of feast and famine periods. For long periods of feast interrupted by short periods of famine, E. coli the optimal strategy is to grow fast. For long periods of starvation intermitted by short periods of growth, the optimal strategy is long survival and slow growth. For the scenario of panel A and B, $\frac{T_{-}}{T_{+}} = 48$, the optimum is μ = 0.86 h − 1 and γ = 0.5 d − 1, see dashed line.


Adaptation at end of growth . When nutrient decreases , nutrient uptake rate slows down with a Michaelis - Menten type kinetics ( top ) . Depending on uptake affinity , will experience either a sharp or smooth decrease of growth rate ( bottom left , bright orange : 10 µM , orange : 1 mM ) . During this slow down , proteome adapts ( bottom center ) , with lower affinities ( orange ) showing a slight improvement of proteome adaptation , which results in a slightly lower rate ( bottom right ) .

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Adaptation at the end of growth. When the nutrient concentration decreases, the nutrient uptake rate slows down with a Michaelis-Menten type kinetics (top). Depending on the uptake affinity, bacteria will experience either a sharp or smooth decrease of growth rate (bottom left, bright orange: 10 µM, dark orange: 1 mM). During this slow down, the proteome adapts (bottom center), with lower affinities (dark orange) showing a slight improvement of proteome adaptation, which results in a slightly lower death rate (bottom right).


Adaptation in media . A medium supplemented with multiple nutrients ( one - red , two - green , ten - blue ) leads to a step - wise decrease of growth rate during nutrient depletion ( bottom left ) . Due to extended periods of slow growth after exhaustion of nutrients , can adapt their proteome substantially ( bottom center ) , decrease their rate ( bottom right ) .

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Adaptation in complex media. A medium supplemented with multiple nutrients (one - red, two - green, ten - blue) leads to a step-wise decrease of growth rate during nutrient depletion (bottom left). Due to the extended periods of slow growth after exhaustion of primary nutrients, bacteria can adapt their proteome substantially (bottom center), and decrease their death rate (bottom right).


A . In vivo genetic complementation of E . coli MC4100 wild - type ( WT ) secB or / tig knock - out derivatives by either an empty vector or one carrying secB or tig ( encoding TF ) genes or derivatives ( see " Plasmid " table in Appendix ) , as indicated . Serial dilutions of a culture ( OD600 = 0 . 5 ) were spotted ( 12μl ) on LB - Ampicillin plates containing or not anhydrotetracycline ( AHT ; 5 ng / ml ) grown at 16 ° C or 30 ° C ( as indicated ) . n = 3 - 7 biological replicates .

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

A. In vivo genetic complementation of the E. coli MC4100 wild-type (WT) and secB or/and tig knock-out derivatives by either an empty vector or one carrying the secB or tig (encoding TF) genes or derivatives (see "Plasmid" table in Appendix), as indicated. Serial dilutions of a culture (OD600= 0.5) were spotted (12μl) on LB-Ampicillin plates containing or not anhydrotetracycline (AHT; 5 ng/ml) and grown at 16°C or 30°C (as indicated). n= 3-7 biological replicates.


C . SecA ATPase activity determination in solution ( basal ; B ) , or plus SecYEG IMVs ; ( ; M ) or plus proOmpA ( pre - treated with 10 mM DTT ; 5 mM EDTA ; 20 min ; 4 ° C ; translocation ; T ) . n = 21 biological replicates . Mean values ( ± ) shown .

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

C. SecA ATPase activity determination in solution (basal; B), or plus SecYEG IMVs; (membrane; M) or plus proOmpA (pre-treated with 10 mM DTT; 5 mM EDTA; 20 min; 4°C; translocation; T). n= 21 biological replicates. Mean values (± SEM) shown.


D . proOmpA translocation ATPase activity stimulation of SecA ( as in C ) in absence or presence of TF2 or / SecB4 , added at indicated molar excess over proOmpA1 . T / M ratios were calculated ; one in absence of chaperones was considered as 100 % , other values were expressed as % of this . n = 2 - 4 biological replicates . Mean values ( ± ) shown . Unpaired parametric t - , 95 % confidence interval : ns : not significant ( p = 0 . 1032 ) ; * * * * : p < 0 . 0001 ; * * : p = 0 . 0037 .

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

D. proOmpA translocation ATPase activity stimulation of SecA (as in C) in the absence or presence of TF2 or/and SecB4, added at the indicated molar excess over proOmpA1. The T/M ratios were calculated; the one in the absence of chaperones was considered as 100%, all other values were expressed as % of this. n= 2-4 biological replicates. Mean values (± SEM) shown. Unpaired parametric t-test, 95% confidence interval: ns: not significant (p = 0.1032); ****: p < 0.0001; **: p = 0.0037.


E . Titrated TF - mediated inhibition ( 0 - 6 µM range ) of proOmpA - stimulated translocation ATPase activity ( as in C ) . Values were normalized ( effectmax = 100 % ; effectmin = 0 % ) plotted ( n = 1 - 4 biological replicates ; mean values ± ) versus log10 [ TF2 ] . Ki was determined using a variable slope fit ( log [ inhibitor ] versus normalized response ; GraphPad Prism ) .

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

E. Titrated TF-mediated inhibition (0-6 µM range) of proOmpA-stimulated translocation ATPase activity (as in C). Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 1-4 biological replicates; mean values ± SEM) versus the log10[TF2]. The Ki was determined using a variable slope fit (log[inhibitor] versus normalized response; GraphPad Prism).


F . SecB - mediated relief of TF - mediated inhibition of proOmpA translocation ATPase activity ( as in C ) . TF2 ( 1μM ) SecB4 ( 0 - 6 µM ) were used . T / M ratios were calculated , normalized ( as in D ) plotted ( n = 2 - 3 biological replicates ; mean values ± ) versus log10 [ SecB4 ] . An apparent relief constant ( Krel ) was determined ( as in E ) .

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

F. SecB-mediated relief of TF-mediated inhibition of proOmpA translocation ATPase activity(as in C). TF2 (1μM) and SecB4 (0-6 µM) were used. The T/M ratios were calculated, normalized (as in D) and plotted (n= 2-3 biological replicates; mean values ± SEM) versus the log10[SecB4]. An apparent relief constant (Krel) was determined (as in E).


A . TF - proOmpA physical interaction in solution . proOmpA1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 37 ° C ; 60 min ) with indicated molar excess of TF2 . Soluble were analyzed on 10 % native - PAGE ( 4 mA ; 16h ; 4 ° C ) Coomassie - blue stained . A representative experiment is shown ; n = 4 biological replicates

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A. TF-proOmpA physical interaction in solution. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) with the indicated molar excess of TF2. Soluble proteins were analyzed on 10% native-PAGE (4 mA; 16h; 4°C) and Coomassie-blue stained. A representative experiment is shown; n= 4 biological replicates


C . TF solubilizes proOmpA * . proOmpA * 1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 37 ° C ; 60 min ) alone or in presence of TF . in soluble fraction were analyzed on 15 % SDS - PAGE Coomassie - blue stained . Lane 2 : 2 . 84 μg proOmpA * . Representative experiment shown ; n = 5 biological replicates

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

C. TF solubilizes proOmpA*. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) alone or in the presence of TF. Proteins in the soluble fraction were analyzed on 15% SDS-PAGE and Coomassie-blue stained. Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 5 biological replicates


D . proOmpA preincubated at 37oC fails to stimulate SecA ATPase activity . proOmpA ( 0 . 5 μM ) was either diluted directly ( lane 1 ) or after preincubation alone ( 10 min ; buffer D ; < 0 . 2 M urea ; either at 4 ° C or 37 ° C ) into reactions containing SecYEG - SecA alone or together with TF2 or SecB4 ( 2 4 x molar excess over proOmpA , respectively ) . T / M ratios ( as in Fig . 1D ) are shown ( n = 8 biological replicates ; mean values ± ; Unpaired parametric t - , 95 % confidence interval : * * : p = 0 . 0017 ; * * * * : p < 0 . 0001 )

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

D. proOmpA preincubated at 37oC fails to stimulate SecA ATPase activity. proOmpA (0.5 μM) was either diluted directly (lane 1) or after preincubation alone (10 min; buffer D; < 0.2 M urea; either at 4°C or 37°C) into reactions containing SecYEG-SecA alone or together with TF2 or SecB4 (2 and 4 x molar excess over proOmpA, respectively). T/M ratios (as in Fig. 1D) are shown (n= 8 biological replicates; mean values ± SEM; Unpaired parametric t-test, 95% confidence interval: **: p = 0.0017; ****: p < 0.0001)


E . proOmpA pre - incubated at 37oC does not interact with TF . proOmpA1 ( 5 μM ) was preincubated ( Buffer D ; < 0 . 2 M urea ; 37 ° C ; 10 min ) or not ( lane 3 ) , prior to addition of indicated molar excess of TF2 ( analyzed as in A ) . Representative experiment shown ; n = 4 biological replicates

O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD 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 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

E. proOmpA pre-incubated at 37oC does not interact with TF. proOmpA1 (5 μM) was preincubated (Buffer D; < 0.2 M urea; 37°C; 10 min) or not (lane 3), prior to addition of the indicated molar excess of TF2 (analyzed as in A). Representative experiment shown; n= 4 biological replicates


B . SecB protects proOmpA * from aggregation . proOmpA * 1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; 37 ° C ; 60 min ) alone or in presence of indicated molar excess of SecB4 , proOmpA * in soluble fraction ( after centrifugation ) was analyzed by SDS - PAGE ( as in Fig . 2C ) . Lane 2 : 2 . 84 μg proOmpA * . Representative experiment shown ; n = 4 biological

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

B. SecB protects proOmpA* from aggregation. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; 37°C; 60 min) alone or in the presence of the indicated molar excess of SecB4, and proOmpA* in the soluble fraction (after centrifugation) was analyzed by SDS-PAGE (as in Fig. 2C). Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 4 biological replicates


C . SecB physically interacts with proOmpA . proOmpA1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 4 ° C ; 60 min ) , with indicated molar excess of SecB4 ( filled arrow ) , analyzed by native - PAGE as in Fig . 2A , stained as indicated . brace : higher order proOmpA species . Representative experiment shown ; n = 5 biological replicates

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

C. SecB physically interacts with proOmpA. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 4°C; 60 min), with the indicated molar excess of SecB4 (filled arrow), analyzed by native-PAGE as in Fig. 2A, and stained as indicated. brace: higher order proOmpA species. Representative experiment shown; n= 5 biological replicates


B . Blow up of ESI - mass spectrum of TF : proOmpA : SecB4 super - from A ( complete dataset in Fig . EV3 ) . Left : charge state distributions , at indicated m / z range . Right : masses deconvoluted from mass spectra ( as in Fig . 2B ) . Inset cartoon depicts stoichiometry . Representative spectrum shown ; n = 2 biological replicates .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O 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-EXP_ASSAY I-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

B. Blow up of the ESI-mass spectrum of the TF:proOmpA:SecB4 super-complex from A (complete dataset in Fig. EV3). Left: charge state distributions, at the indicated m/z range. Right: masses deconvoluted from the mass spectra (as in Fig. 2B). Inset cartoon depicts stoichiometry. Representative spectrum shown; n=2 biological replicates.


C . TF : proOmpA : SecB supercomplex using GPC - MALS in Buffer A . TF1 : proOmpA1 ( 10 µM each ; green ) , SecB4 ( 40 µM ; blue ) TF1 : proOmpA1 : SecB4 ( 10 , 10 40 µM , respectively ; black ) . n = 5 biological replicates ; mean mass : 137 kDa ( ± 6 kDa ) representative experiment shown ; red circles : determined mass ( kDa ) ; AU : arbitrary units .

O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD 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 O O O O

C. TF:proOmpA:SecB supercomplex analysis using GPC-MALS in Buffer A. TF1:proOmpA1 (10 µM each; green), SecB4 (40 µM; blue) and TF1:proOmpA1:SecB4 (10, 10 and 40 µM, respectively; black). n= 5 biological replicates; mean mass: 137 kDa (± 6 kDa) representative experiment shown; red circles: determined mass (kDa); AU: arbitrary units.


A . SecB fails to restore proOmpA translocation ATPase activity stimulation of SecA ( noC - tail ) in presence of TF . proOmpA translocation ATPase activity stimulation ( as in Fig . 1C ; SecB molar excess over proOmpA indicated ) . T / M of SecA ( noC - tail ) in absence of TF or SecB = 100 % ; lane 1 ; other values were expressed as a % of this value . n = 4 biological replicates ; mean values ± . Unpaired parametric t - , 95 % confidence interval : * * : p = 0 . 0048 ; * * * * : p < 0 . 0001 .

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

A. SecB fails to restore proOmpA translocation ATPase activity stimulation of SecA(noC-tail) in the presence of TF. proOmpA translocation ATPase activity stimulation (as in Fig. 1C; SecB molar excess over proOmpA indicated). T/M ratio of SecA(noC-tail) in the absence of TF or SecB= 100%; lane 1; all other values were expressed as a % of this value. n= 4 biological replicates; mean values ± SEM. Unpaired parametric t-test, 95% confidence interval: **: p= 0.0048; ****: p < 0.0001.


B . Excess of SecB inhibits proOmpA - stimulated translocation ATPase activity of SecA ( noCtail ) . ATPase measured as in Fig . 1C ; SecB range : 0 - 6 µM . Values were normalized ( effectmax = 100 % ; effectmin = 0 % ) plotted ( n = 3 biological replicates ; mean values ± ) versus log10 [ SecB4 ] . Ki was determined as in Fig . 1E .

O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR 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 B-GENEPROD O O O O O O O O O O O

B. Excess of SecB inhibits the proOmpA-stimulated translocation ATPase activity of SecA(noCtail). ATPase measured as in Fig. 1C; SecB concentration range: 0-6 µM. Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 3 biological replicates; mean values ± SEM) versus the log10[SecB4]. The Ki was determined as in Fig. 1E.


C . TF SecB - regulated , SecA - dependent in vitro translocation of proOmpA into SecYEG / A - IMVs , analyzed after proteolytic treatment . Translocated , protease - resistant proOmpA was visualized using immunostaining ( α - His ) in absence or presence of TF2 or / SecB4 ( as indicated ) . Translocation in absence of TF or SecB was considered 100 % ( lane 3 ) ; other values were expressed as a of this . A summary of repeats is presented ( mean ± ; n = 6 biological replicates ; ns : not significant ( p = 0 . 1568 for lane 3 vs 4 ; p = 0 . 4835 for lane 3 vs 6 ) ; Unpaired parametric t - , 95 % confidence interval : * * * * : p < 0 . 0001 ) with a representative western blot ( bottom ) .

O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O

C. TF and SecB-regulated, SecA-dependent in vitro translocation of proOmpA into SecYEG/A-IMVs, analyzed after proteolytic treatment. Translocated, protease-resistant proOmpA was visualized using immunostaining (α-His) in the absence or presence of TF2 or/and SecB4 (as indicated). Translocation in the absence of TF or SecB was considered 100% (lane 3); all other values were expressed as a percentage of this. A summary of all repeats is presented (mean ± SEM; n= 6 biological replicates; ns: not significant (p = 0.1568 for lane 3 vs 4; p = 0.4835 for lane 3 vs 6); Unpaired parametric t-test, 95% confidence interval: ****: p < 0.0001) with a representative western blot (bottom).


pH - dependent dissociation of E M . TBEV preparations treated at pH 8 ( left ) pH 6 ( right ) were solubilized with Triton X - 100 subjected to sucrose gradient centrifugation . fractions were analyzed by SDS - PAGE densitometry . Representative examples of at least two independent experiments are shown . Positions of E dimers ( D ) E trimers ( T ) in gradients are indicated .

O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-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

pH-dependent dissociation of E and M. TBEV preparations treated at pH 8 (left) and pH 6 (right) were solubilized with Triton X-100 and subjected to sucrose gradient centrifugation. The fractions were analyzed by SDS-PAGE and densitometry. Representative examples of at least two independent experiments are shown. Positions of E dimers (D) and E trimers (T) in the gradients are indicated.


Trimer stability expressed as of fraction detected in trimer peak normalized with respect to corresponding Ev fraction in sedimentation analyses after incubation at 70 ° C relative to 37 ° C ( 100 % ) . Data are from at least three independent experiments .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY I-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

Trimer stability expressed as percentage of the fraction detected in the trimer peak and normalized with respect to the corresponding Ev fraction in sedimentation analyses after incubation at 70°C relative to 37°C (100%). Data are from at least three independent experiments.


Difference in deuterium uptake by sE404r sE419r trimers ( D404 - D419 ) . Left panel , deuteration difference for each peptide reported against 1 - p value obtained via Student ' s t - between two different constructs . In right panel , peptides are reported as short horizontal lines , with position corresponding to peptide location along ( x - axis ) with their deuteration delta on y - axis . In both panels , red ( positive ) blue ( negative ) indicate changes above Student ' s t - threshold , in grey those not reaching this threshold . Four peptides , termed HDX - a , HDX - b , HDX - c HDX - d , were identified as showing a strong increase in deuteration in sE404r compared to sE419r . same pattern was observed in comparison between sE404r sE428r , whereas no difference deemed " significant " by same was seen when comparing sE419r with sE428r ( see Fig EV3 ) .

O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O 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 O O O O O O O O O O O O O O O B-EXP_ASSAY I-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 O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O 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 O O O O O

Difference in deuterium uptake by sE404r and sE419r trimers (D404-D419). Left panel, deuteration difference for each peptide reported against the 1-p value obtained via Student's t-test between the two different constructs. In the right panel, peptides are reported as short horizontal lines, with length and position corresponding to the peptide length and location along the protein sequence (x-axis) and with their deuteration delta on the y-axis. In both panels, red (positive) and blue (negative) indicate changes above the Student's t-test threshold, and in grey those not reaching this threshold. Four peptides, termed HDX-a, HDX-b, HDX-c and HDX-d, were identified as showing a strong increase in deuteration in sE404r compared to sE419r. The same pattern was observed in the comparison between sE404r and sE428r, whereas no difference deemed "significant" by the same test was seen when comparing sE419r with sE428r (see Fig EV3).


Blocking ELISA with mabs A1 , A2 , A3 B4 . Serial dilutions of trimers were pre - incubated with a predetermined of respective mabs . fraction of mab not blocked by antigen was detected in ELISA with TBEV - coated plates ( Materials Methods ) . bar charts show results expressed relative to reactivity with sE401v trimer . Data are from at least three independent experiments .

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Blocking ELISA with mabs A1, A2, A3 and B4. Serial dilutions of the trimers were pre-incubated with a predetermined concentration of the respective mabs. The fraction of mab not blocked by the antigen was detected in ELISA with TBEV-coated plates (Materials and Methods). The bar charts show the results expressed relative to the reactivity with the sE401v trimer. Data are from at least three independent experiments.


( A - B ) cilia are enriched at branch points greater curvature of aorta of wild type mice , schematized in ( A ) quantified in ( B ) . color in ( A ) indicates which side of aorta is quantified in ( B ) . Numbers indicate region of aorta ( A ) whose proportion of ciliated is quantified in ( B ) . Error bars are + / - 1 S . E . M n = 7 mice . ( C ) Aortic endothelial cilia are ablated in Tek - Cre Ift88C / - mice . Endothelial cilia are labeled by for Pecam1 Arl13b , respectively . Scale bars are 10μm .

O O O O O B-SUBCELLULAR O O O O O O O O B-TISSUE 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-TISSUE O O O O O O O O O O O B-TISSUE O O O O O 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-ORGANISM O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O B-CELL_TYPE B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD O O O O O O O O

(A-B) Primary cilia are enriched at branch points and the greater curvature of the aorta of wild type mice, schematized in (A) and quantified in (B). The color in (A) indicates which side of the aorta is quantified in (B). Numbers indicate the region of the aorta (A) whose proportion of ciliated cells is quantified in (B). Error bars are +/- 1 S.E.M and n=7 mice. (C) Aortic endothelial cilia are ablated in Tek-Cre Ift88C/- mice. Endothelial cells and cilia are labeled by staining for Pecam1 and Arl13b, respectively. Scale bars are 10μm.


( A ) vasculature in P5 retinas from ( A ) mTmG reporter mice with without EC cilia was visualized by EGFP . ( B ) fractional migrated to edge of retina was quantified . There was no statistically significant difference ( p = 0 . 305 , Student ' s 2 - t - , n = 6 mice for each group ) . average value of 8 measurements ( 4 retina leaflets measured per eye on both eyes ) was used for each mouse . ( C ) number of vascular branchpoints was counted . There was no statistically significant difference ( p = 0 . 25 , Student ' s 2 - t - , n = 6 for control n = 5 for mutant ) .

O O O B-TISSUE O O B-TISSUE O O O O O O B-ORGANISM O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O 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-ORGANISM O O O O O O O O O O O O B-TISSUE O O O B-TISSUE O O B-TISSUE O 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 O O O O O O O O O O O O O O O O O O O O O O O

(A) The vasculature in P5 retinas from (A) mTmG reporter mice with and without EC cilia was visualized by EGFP fluorescence.(B) The fractional distance migrated to the edge of the retina was quantified. There was no statistically significant difference (p=0.305, Student's 2-tailed t-test, n=6 mice for each group). The average value of 8 measurements (4 retina leaflets measured per eye on both eyes) was used for each mouse.(C) The number of vascular branchpoints was counted. There was no statistically significant difference (p=0.25, Student's 2-tailed t-test, n=6 for control and n=5 for mutant).


( D ) Control mutant adult mouse aortas were stained for GM130 ( green ) to mark Golgi , CD144 ( red ) to mark ECs DAPI ( blue ) to label . ( E ) angle between Golgi , nucleus tissue axis was measured binned as ' left ' , ' right ' , ' upstream ' or ' downstream ' . ( F ) Six areas in aorta were analyzed . ( G ) There was no difference in Golgi distribution at any single region or overall , nor was there a difference in variance of un - binned angles . Error bars are + 1 S . E . M . Scale bars are 500μm . n = 3 control mutant mice for each region except region 2 where n = 3 control n = 2 mutant mice .

O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O 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-TISSUE 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 O O O 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-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O

(D) Control and mutant adult mouse aortas were stained for GM130 (green) to mark the Golgi, CD144 (red) to mark ECs and DAPI (blue) to label DNA.(E) The angle between the Golgi, nucleus and tissue axis was measured and binned as 'left', 'right', 'upstream' or 'downstream'.(F) Six areas in the aorta were analyzed.(G) There was no difference in Golgi distribution at any single region or overall, nor was there a difference in the variance of the individual un-binned angles. Error bars are +1 S.E.M. Scale bars are 500μm. n=3 control and mutant mice for each region except region 2 where n=3 control and n=2 mutant mice.


( B ) Loss of EC cilia by Tek - Cre increases atherosclerosis in aortas of Apoe - / - mice on a high - fat high - cholesterol diet as visualized by Oil Red O ( ORO ) ( left panels ) whereas loss of cilia genes in blood by Mx1 - Cre has no effect ( right panels ) . ( C ) Cropped views of boxed areas in ( B ) showing increased atherosclerosis in mice with Tek - Cre but not Mx1 - Cre mediated loss of Ift88 in aortic arch . ( D - G ) Scatter plots of ORO - positive of thoracic aorta in ( D - E ) Tek - Cre ( F - G ) Mx1 - Cre mice , separated by ( D , F ) ( E , G ) . Tek - Cre Apoe - / - Ift88C / - mice had a 58 . 9 % ( p = 0 . 0135 , Student ' s 2 - t - , n = 13 control n = 10 mutant mice ) 67 % ( p = 0 . 0106 , Student ' s 2 - t - , n = 17 control n = 14 mutant ) increase in ORO - positive versus control in , respectively . Mx1 - Cre Apoe - / - Ift88C / - mice had no significant difference in ORO - positive in ( p = 0 . 6643 , Student ' s 2 - t - , n = 7 control mutant mice ) or ( p = 0 . 9381 , Student ' s 2 - t - , n = 9 control n = 7 mutant mice ) . Black bars represent mean error bars are + / - 1 S . E . M . Aortas depicted in ( B - C ) are red in scatter plots .

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

(B) Loss of EC cilia by Tek-Cre increases atherosclerosis in the aortas of Apoe-/- mice on a high-fat high-cholesterol diet as visualized by Oil Red O (ORO) staining (left panels) whereas loss of cilia genes in blood by Mx1-Cre has no effect (right panels).(C) Cropped views of the boxed areas in (B) showing increased atherosclerosis in mice with Tek-Cre but not Mx1-Cre mediated loss of Ift88 in the aortic arch.(D-G) Scatter plots of the percent ORO-positive area of the thoracic aorta in (D-E) Tek-Cre and (F-G) Mx1-Cre mice, separated by (D, F) females and (E, G) males. Tek-Cre Apoe-/- Ift88C/- mice had a 58.9% (p=0.0135, Student's 2-tailed t-test, n=13 control and n=10 mutant mice) and 67% (p=0.0106, Student's 2-tailed t-test, n=17 control and n=14 mutant) increase in ORO-positive area versus control littermates in females and males, respectively. Mx1-Cre Apoe-/- Ift88C/- mice had no significant difference in ORO-positive area in females (p=0.6643, Student's 2-tailed t-test, n=7 control and mutant mice) or males (p=0.9381, Student's 2-tailed t-test, n=9 control and n=7 mutant mice). Black bars represent the mean and error bars are +/- 1 S.E.M. Aortas depicted in (B-C) are red in the scatter plots.


( A ) Transcripts for inflammatory cytokines Il1b , Il6 , Tnf1 Tgfb1 , adhesion molecules Vcam1 Sele , lymphocyte marker Nos2 macrophage marker Cd68 , NFκB responsive genes Tnfaip3 Hmox1 were upregulated in aortas from Apoe - / - mice lacking EC cilia by RT - qPCR , while transcripts of Hh target genes Gli1 , Ptc1 , Ptc2 were not . Error bars are + 1 S . E . M . n = 8 control n = 10 mutant mice of mixed sex . P - values calculated by Student ' s 2 - t - .

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

(A) Transcripts for the inflammatory cytokines Il1b, Il6, Tnf1 and Tgfb1, the adhesion molecules Vcam1 and Sele, the lymphocyte marker Nos2 and macrophage marker Cd68, and the NFκB responsive genes Tnfaip3 and Hmox1 were upregulated in aortas from Apoe-/- mice lacking EC cilia by RT-qPCR, while transcripts of the Hh target genes Gli1, Ptc1, and Ptc2 were not. Error bars are +1 S.E.M. n=8 control and n=10 mutant mice of mixed sex. P-values calculated by Student's 2-tailed t-test.


( B ) Pkd2 localizes to EC cilia in vivo . Adult mouse aortas were stained for Pkd2 , acetylated α - Tubulin , Hoechst to label . Pkd2 localized strongly to cilia but also to other membranous structures in endothelial . Arrowheads point out cilia . Example cilium highlighted in inset .

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

(B) Pkd2 localizes to EC cilia in vivo. Adult mouse aortas were stained for Pkd2, acetylated α-Tubulin, and Hoechst to label DNA. Pkd2 localized strongly to primary cilia but also to other membranous structures in endothelial cells. Arrowheads point out cilia. Example cilium highlighted in inset.


( C ) Phosphorylation at Serine 1176 of eNOS was reduced in aortas of mice lacking EC cilia . Aortas from control mutant mice were stained for eNOS , p - eNOS - S1176 , acetylated α - Tubulin , Hoechst to label .

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

(C) Phosphorylation at Serine 1176 of eNOS was reduced in the aortas of mice lacking EC cilia. Aortas from control and mutant mice were stained for eNOS, p-eNOS-S1176, acetylated α-Tubulin, and Hoechst to label DNA.


( D ) of total p - eNOS - S1176 to total eNOS in upper thoracic aorta ( near first intercostal artery ) was calculated for control mutant mice . relative p - eNOS - S1176 to eNOS was reduced in mutant to 72 . 7 % level of control siblings ( p = 0 . 009 , paired 2 - t - , n = 7 sets of mice ) . Error bar is + 1 S . E . M . Four sets consisted of one control mouse one mutant mouse . Three sets consisted of two control or mutant mice average value of two was used . Scale bars are 10μm .

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

(D) The ratio of the total fluorescence p-eNOS-S1176 to total eNOS in the upper thoracic aorta (near the first intercostal artery) was calculated for control and mutant mice. The relative p-eNOS-S1176 to eNOS ratio was reduced in the mutant to 72.7% the level of control siblings (p=0.009, paired 2-tailed ratio t-test, n=7 sets of mice). Error bar is +1 S.E.M. Four sets consisted of one control mouse and one mutant mouse. Three sets consisted of two control or mutant mice and the average value of the two was used. Scale bars are 10μm.


( B , C ) Functional classification subcellular localization of identified lysine - acetylated . Lysine - acetylated identified over experiments were classified according to MapMan categoriesOver - or underrepresentation of categories was determined using a Fisher ' s exact with identified at 1 % FDR as background population . Blue red arrows mark categories significantly enriched at 5 % FDR ( Benjamini - Hochberg ) a 1 . 5 - fold - change cut - off .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) Functional classification and subcellular localization of identified lysine-acetylated proteins. Lysine-acetylated proteins identified over all experiments were classified according to MapMan categoriesOver- or underrepresentation of categories was determined using a Fisher's exact test with all proteins identified at 1% FDR as background population. Blue and red arrows mark categories significantly enriched at 5% FDR (Benjamini-Hochberg) and a 1.5-fold-change cut-off.


Figure 3 . Differential lysine acetylation expression in Arabidopsis leaves after inhibitor treatment . Vacuum - infiltration of leaf strips with solutions containing either of two deacetylase inhibitors apicidin ( A , C ) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites . Volcano plots depict lysine acetylation site ratios ( A , B ) for inhibitor treatment vs . control , with p - values determined using LIMMA package . Orange , with localization according to SUBA4 database . Blue , with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing indicates that significance threshold was not reached by any of data points .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors apicidin (A, C) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict lysine acetylation site ratios (A, B)for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points.


Figure 3 . Differential lysine acetylation expression in Arabidopsis leaves after inhibitor treatment . Vacuum - infiltration of leaf strips with solutions containing either of two deacetylase inhibitors trichostatin A ( B , D ) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites . Volcano plots depict lysine acetylation site ratios ( A , B ) for inhibitor treatment vs . control , with p - values determined using LIMMA package . Orange , with localization according to SUBA4 database . Blue , with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing indicates that significance threshold was not reached by any of data points .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors trichostatin A (B, D) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict lysine acetylation site ratios (A, B)for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points.


Figure 3 . Differential lysine acetylation expression in Arabidopsis leaves after inhibitor treatment . Vacuum - infiltration of leaf strips with solutions containing either of two deacetylase inhibitors apicidin ( A , C ) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites . Volcano plots depict ratios ( C , D ) for inhibitor treatment vs . control , with p - values determined using LIMMA package . Orange , with localization according to SUBA4 database . Blue , with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing indicates that significance threshold was not reached by any of data points . cancel

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors apicidin (A, C)versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict protein ratios (C, D) for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points. cancel


Figure 3 . Differential lysine acetylation expression in Arabidopsis leaves after inhibitor treatment . Vacuum - infiltration of leaf strips with solutions containing either of two deacetylase inhibitors trichostatin A ( B , D ) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites . Volcano plots depict ratios ( C , D ) for inhibitor treatment vs . control , with p - values determined using LIMMA package . Orange , with localization according to SUBA4 database . Blue , with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing indicates that significance threshold was not reached by any of data points .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors trichostatin A (B, D) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict protein ratios (C, D) for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points.


Figure 4 . HDA14 localizes to chloroplasts mitochondria in Arabidopsis , its activity is dependent on cofactors can be inhibited by deacetylase inhibitors . ( A ) GFP localization ( green ) of HDA14 - GFP fusion construct in Arabidopsis protoplasts ( 35S : HDA14 : GFP ) from stable transformants . mitochondrial marker TMRM is depicted in purple . GFP + TMRM shows overlay image of 35S : HDA14 : GFP TMRM , AF indicates chlorophyll autofluorescence BF

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

Figure 4. HDA14 protein localizes to the chloroplasts and mitochondria in Arabidopsis, its activity is dependent on cofactors and can be inhibited by deacetylase inhibitors. (A) GFP localization (green) of the HDA14-GFP fusion construct in Arabidopsis protoplasts (35S:HDA14:GFP) from stable transformants. The mitochondrial marker TMRM is depicted in purple. GFP+TMRM shows the overlay image of 35S:HDA14:GFP and TMRM, AF indicates the chlorophyll autofluorescence and BF the


( B ) Deacetylase activity of recombinant 6xHis - HDA14 using a colorimetric . Co2 + Zn2 + were used as cofactors , apicidin ( 100 µM ) trichostatin A ( 5 µM ) as deacetylase - inhibitors ( n = 5 , ± SD ) .

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

(B) Deacetylase activity of the recombinant 6xHis-HDA14 protein using a colorimetric assay. Co2+ and Zn2+ were used as cofactors, apicidin (100 µM) and trichostatin A (5 µM) as deacetylase-inhibitors (n = 5, ±SD).


Figure 5 . Differential lysine acetylation expression in hda14 vs . wild - type leaves under growth ( A , D ) Volcano plots depict lysine acetylation site ratios ( A - C , top row ) for mutant vs . control , with p - values determined using LIMMA package . Orange , with localization ; Green , with plastidial localization ; purple triangles , of Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , of reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR indicates that 5 % threshold was not reached by any of data points . cancel

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves under growth light (A, D)Volcano plots depict lysine acetylation site ratios (A-C, top row) for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points. cancel


Figure 5 . Differential lysine acetylation expression in hda14 vs . wild - type leaves in isolated thylakoids ( B , E ) Volcano plots depict lysine acetylation site ratios ( A - C , top row ) for mutant vs . control , with p - values determined using LIMMA package . Orange , with localization ; Green , with plastidial localization ; purple triangles , of Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , of reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR indicates that 5 % threshold was not reached by any of data points .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves in isolated thylakoids (B, E)Volcano plots depict lysine acetylation site ratios (A-C, top row)for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.


Figure 5 . Differential lysine acetylation expression in hda14 vs . wild - type leaves under low conditions ( C , F ) . Volcano plots depict lysine acetylation site ratios ( A - C , top row ) for mutant vs . control , with p - values determined using LIMMA package . Orange , with localization ; Green , with plastidial localization ; purple triangles , of Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , of reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR indicates that 5 % threshold was not reached by any of data points .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves under low light conditions (C,F). Volcano plots depict lysine acetylation site ratios (A-C, top row) for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.


Figure 5 . Differential lysine acetylation expression in hda14 vs . wild - type leaves under growth ( A , D ) Volcano plots depict ratios ( D - F , bottom row ) for mutant vs . control , with p - values determined using LIMMA package . Orange , with localization ; Green , with plastidial localization ; purple triangles , of Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , of reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR indicates that 5 % threshold was not reached by any of data points .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves under growth light (A, D) Volcano plots depict protein ratios (D-F, bottom row) for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.


Figure 5 . Differential lysine acetylation expression in hda14 vs . wild - type leaves in isolated thylakoids ( B , E ) Volcano plots depict ratios ( D - F , bottom row ) for mutant vs . control , with p - values determined using LIMMA package . Orange , with localization ; Green , with plastidial localization ; purple triangles , of Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , of reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR indicates that 5 % threshold was not reached by any of data points .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves in isolated thylakoids (B, E) Volcano plots depict protein ratios (D-F, bottom row) for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.


Figure 5 . Differential lysine acetylation expression in hda14 vs . wild - type leaves under low conditions ( C , F ) . Volcano plots depict ratios ( D - F , bottom row ) for mutant vs . control , with p - values determined using LIMMA package . Orange , with localization ; Green , with plastidial localization ; purple triangles , of Calvin - Benson ( CB ) cycle ; blue diamonds ( top row ) , of reaction ; localization information according to SUBA4 database . Blue circles ( bottom row ) , with lysine acetylation sites identified . Dashed lines indicate significance thresholds of either uncorrected p - values < 5 % or Benjamini - Hochberg corrected FDR < 5 % . A missing FDR indicates that 5 % threshold was not reached by any of data points .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5. Differential lysine acetylation and protein expression in hda14 vs. wild-type leaves under low light conditions (C,F). Volcano plots depict protein ratios (D-F, bottom row) for mutant vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization; Green, protein with plastidial localization; purple triangles, proteins of the Calvin-Benson (CB) cycle; blue diamonds (top row), proteins of the light reaction; localization information according to SUBA4 database. Blue circles (bottom row), proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5% or Benjamini-Hochberg corrected FDR < 5%. A missing FDR line indicates that the 5% threshold was not reached by any of the data points.


Figure 6 . RuBisCO activity RuBisCO activation state is increased in hda14 mutant under low conditions . ( A ) RuBisCO initial total activity in WT hda14 in low treated . Initial activity was measured directly upon extraction . For total activity samples were incubated with H2CO3 for 3 minutes to fully carbamylate active site of RuBisCO ( n = 10 , * t - p - value < 0 . 05 ) .

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

Figure 6. RuBisCO activity and RuBisCO activation state is increased in the hda14 mutant under low light conditions. (A) RuBisCO initial and total activity in WT and hda14 in low light treated plants. Initial activity was measured directly upon extraction. For the total activity samples were incubated with H2CO3 for 3 minutes to fully carbamylate the active site of RuBisCO (n = 10, *t-test p-value < 0.05).


( C ) ATPase activity of recombinant 6x - HisRCAß1 WT , K438Q K438R with ATP ADP / ATP = 0 . 11 , respectively ( n = 3 , * t - p - value < 0 . 05 , + t - p - value < 0 . 1 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-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 O O O O O

(C) ATPase activity of recombinant 6x-HisRCAß1 WT, K438Q and K438R with ATP and ADP/ATP= 0.11, respectively (n = 3, *t-test p-value < 0.05, +t-test p-value< 0.1).


B Induction of NF - κB targets in Jurkat T in response to 20 ng / ml TNF treatment for 1 , 2 , 4 hours as measured by RT - qPCR . Target values were normalized to GAPDH are reported as fold change relative to basal expression . Data are presented as mean ± standard deviation ( SD ) of 3 biological replicates .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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

B Induction of NF-κB targets in Jurkat T cells in response to 20 ng/ml TNF treatment for 1, 2, and 4 hours as measured by RT-qPCR. Target values were normalized to GAPDH and are reported as fold change relative to basal expression. Data are presented as mean ± standard deviation (SD) of 3 biological replicates.


D Maximum projections of smFISH microscopy z - stacks of basal Jurkat T stained for indicated genes . Nfkbia , Tnfaip3 , Tnf , Il6 were labeled with Quasar 670 , Il8 Csf2 were labeled with fluorescein . were filtered as described in Materials Methods . Brightness contrast were enhanced for visualization . Scale bars : 10 μm .

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

D Maximum intensity projections of smFISH fluorescence microscopy z-stacks of basal Jurkat T cells stained for the indicated genes. Nfkbia, Tnfaip3, Tnf, and Il6 were labeled with Quasar 670, and Il8 and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm.


A Maximum projections of smFISH microscopy z - stacks of Jurkat T stained for indicated genes after 1 - hour ( Nfkbia , Tnfaip3 , Tnf , Il8 ) or 4 - hour ( Il6 , Csf2 ) treatment with 20 ng / mL TNF . Nfkbia , Tnfaip3 , Tnf , Il6 were labeled with Quasar 670 , Il8 Csf2 were labeled with fluorescein . were filtered as described in Materials Methods . Brightness contrast were enhanced for visualization . Scale bars : 10 μm . B Bar graphs of mean of smFISH distributions before after TNF treatment for indicated genes . were combined from three replicates ( Nfkbia 1h ; Tnfaip3 1h ; Tnf 1h , 2h ; Il6 2h , 4h ) two replicates ( Nfkbia 2h ) or one replicate ( Tnfaip3 2h ; Il8 1h , 2h ; Csf2 2h , 4h ) . Basal data are same as in Fig . 1E . Error bars indicate bootstrapped 95 % CIs for samples in ( C ) . Significant differences indicated by non - overlapping CIs . C

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

A Maximum intensity projections of smFISH fluorescence microscopy z-stacks of Jurkat T cells stained for the indicated genes after 1-hour (Nfkbia, Tnfaip3, Tnf, Il8) or 4-hour (Il6, Csf2) treatment with 20 ng/mL TNF. Nfkbia, Tnfaip3, Tnf, and Il6 were labeled with Quasar 670, and Il8 and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm. B Bar graphs of mean of smFISH distributions before and after TNF treatment for the indicated genes. Cells were combined from three replicates (Nfkbia 1h; Tnfaip3 1h; Tnf 1h, 2h; and Il6 2h, 4h) two replicates (Nfkbia 2h) or one replicate (Tnfaip3 2h; Il8 1h, 2h; Csf2 2h, 4h). Basal data are same as in Fig. 1E. Error bars indicate bootstrapped 95% CIs for the samples in (C). Significant differences indicated by non-overlapping CIs. C


B - C Burst ( B ) burst frequency ( C ) parameter fits from two - state model in basal state after treatment with 20 ng / mL TNF for 1 , 2 or 4 hours . Error bars indicate bootstrapped 95 % CIs . Significant differences indicated by non - overlapping CIs . fit for Nfkbia ( 1h TNF ) was unstable thus is not

O O O O O 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 B-GENEPROD O O B-GENEPROD O O O O O O

B-C Burst size (B) and burst frequency (C) parameter fits from the two-state model in the basal state and after treatment with 20 ng/mL TNF for 1, 2 or 4 hours. Error bars indicate bootstrapped 95% CIs. Significant differences indicated by non-overlapping CIs. The fit for Nfkbia (1h TNF) was unstable and thus is not reported


A Enrichment of histone H3 , AcH3 , total RNPII , ser5 - p RNPII , ser2 - p RNPII , NELF - E in basal state ( 0 hours ) after treatment ( 2 4 hours ) with 20 ng / ml TNF quantified using ChIP shown as % input ( non - IP control ) . Data are presented as mean ± standard error of mean ( s . e . m . ) of three biological replicates . Significance calculated by Dunnett ' s multiple comparison ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) .

O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Enrichment of histone H3, AcH3, total RNPII, ser5-p RNPII, ser2-p RNPII, and NELF-E in the basal state (0 hours) and after treatment (2 and 4 hours) with 20 ng/ml TNF quantified using ChIP and shown as % input (non-IP control). Data are presented as mean ± standard error of the mean (s.e.m.) of three biological replicates. Significance calculated by Dunnett's multiple comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).


C Bar graphs of mean mRNA level for basal condition , 1h TNF , 1h TNF + 4h pretreatment with 300nM A - 485 , 1h TNF + 62 . 5nM JQ1 cotreatment measured by smFISH for Tnfaip3 ( left ) Tnf ( right ) . were combined from two replicates ( Tnfaip3 A - 485 , JQ1 ; Tnf A - 485 ) or one replicate ( Tnf JQ1 ) . Error bars indicated bootstrapped 95 % CIs . Samples with non - overlapping CIs are significant . D Change in enrichment of total Serine - 5 - phosphorylated RNAPII after 20ng / mL TNF treatment for 1 hour with 62 . 5nM JQ1 measured by ChIP - qPCR shown as % input ( non - IP control ) normalized to uninhibited control for each gene . Data are presented as mean for two biological replicates . E

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

C Bar graphs of mean mRNA level for basal condition, 1h TNF, 1h TNF + 4h pretreatment with 300nM A-485, and 1h TNF+62.5nM JQ1 cotreatment measured by smFISH for Tnfaip3 (left) and Tnf (right). Cells were combined from two replicates (Tnfaip3 A-485, JQ1; Tnf A-485) or one replicate (Tnf JQ1). Error bars indicated bootstrapped 95% CIs. Samples with non-overlapping CIs are significant. D Change in enrichment of total and Serine-5-phosphorylated RNAPII after 20ng/mL TNF treatment for 1 hour with 62.5nM JQ1 measured by ChIP-qPCR and shown as % input (non-IP control) normalized to the uninhibited control for each gene. Data are presented as mean for two biological replicates. E


( A ) Left : qRT - PCR of mRNA expression of MAPK12 ( p38gamma ) MAPK13 ( p38delta ) in liver extracts prepared from obese with alcoholic fatty liver disease ( NAFLD ) control individuals without NAFLD . mRNA expression was normalized to amount of Gapdh mRNA . Right : Representative H & amp ; amp ; E - stained liver sections . Scale Bar : 50μm . ( n = 11 - 74 )

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

(A) Left: qRT-PCR analysis of mRNA expression of MAPK12 (p38gamma) and MAPK13 (p38delta) in liver extracts prepared from obese patients with alcoholic fatty liver disease (NAFLD) and control individuals without NAFLD. mRNA expression was normalized to the amount of Gapdh mRNA. Right: Representative H&amp;E-stained liver sections. Scale Bar: 50μm. (n= 11-74)


( B ) Left : qRT - PCR of mRNA expression of MAPK12 ( p38gamma ) MAPK13 ( p38delta ) in liver extracts prepared from control with NAFLD / non - alcoholic steatohepatitis ( NASH ) control individuals without NAFLD / NASH . mRNA expression was normalized to amount of Gapdh mRNA . Right : Representative H & amp ; amp ; E - stained liver sections . Scale Bar : 50μm . ( n = 11 - 9 )

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

(B) Left: qRT-PCR analysis of mRNA expression of MAPK12 (p38gamma) and MAPK13 (p38delta) in liver extracts prepared from control patients with NAFLD/non-alcoholic steatohepatitis (NASH) and control individuals without NAFLD/NASH. mRNA expression was normalized to the amount of Gapdh mRNA. Right: Representative H&amp;E-stained liver sections. Scale Bar: 50μm. (n= 11-9)


( C ) of immunoblot of p38δ expression in liver extracts prepared from obese with NAFLD individuals without NAFLD . Representative blots are shown . ( n = 7 - 40 )

O O O O B-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of immunoblot analysis of p38δ expression in liver extracts prepared from obese patients with NAFLD and individuals without NAFLD. Representative blots are shown. (n= 7-40)


( D ) qRT - PCR of Mapk12 ( p38gamma ) Mapk13 ( p38delta ) mRNA expression in liver extracts prepared from wild - type mice ( WT ) fed a diet deficient in methionine choline ( MCD ) or control diet ( ND ) for 3 ; mRNA expression was normalized to amount of Gapdh mRNA . ( n = 5 - 10 )

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

(D) qRT-PCR analysis of Mapk12 (p38gamma) and Mapk13 (p38delta) mRNA expression in liver extracts prepared from wild-type mice (WT) fed a diet deficient in methionine and choline (MCD) or control diet (ND) for 3 weeks; mRNA expression was normalized to the amount of Gapdh mRNA. (n=5-10)


( E ) Immunoprecipitation of activation of p38γ δ isoforms in liver extracts prepared from WT fed a MCD or ND diet for times indicated .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O

(E) Immunoprecipitation analysis of activation and protein levels of p38γ and δ isoforms in liver extracts prepared from WT fed a MCD or ND diet for the times indicated.


( F ) Immunoprecipitation of activation of p38γ δ isoforms in liver extracts prepared from WT fed a MCD or ND for 3 ( n = 5 ) . Western blot against vinculin was used to amount in total lysate ( TL ) used for each IP . expression was normalized to vinculin . Data are means ± . * P < 0 . 05 ; * * P < 0 . 01 . ( Statistical significance by two - Student ' s t . Characteristics of controls were compared by means of χ2 or Mann - Whitney U . )

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

(F) Immunoprecipitation analysis of activation and protein levels of p38γ and δ isoforms in liver extracts prepared from WT fed a MCD or ND for 3 weeks (n=5). Western blot against vinculin was used to assay the protein amount in the total lysate (TL) used for each IP. Protein expression was normalized to vinculin. Data are means ± SEM. *P<0.05; **P<0.01. (Statistical significance by two-tailed Student's t test. Characteristics of patients and controls were compared by means of χ2 or Mann-Whitney U tests.)


( A ) Representative H & amp ; amp ; E oil red - stained liver sections prepared from WT p38γ / δ - / - mice fed a ND or MCD diet for 3 . Scale Bar : 50μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O 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

(A) Representative H&amp;E and oil red-stained liver sections prepared from WT and p38γ/δ-/- mice fed a ND or the MCD diet for 3 weeks. Scale Bar: 50μm.


( B ) Liver triglycerides ( C ) plasma transaminase activity ( ALT ) measured in WT p38γ / δ - / - mice after 3 of MCD diet .

O O O B-TISSUE B-SMALL_MOLECULE O O O B-TISSUE O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O

(B) Liver triglycerides and (C) plasma transaminase activity (ALT) measured in WT and p38γ/δ-/- mice after 3 weeks of MCD diet.


( D ) TBARS hydrogen peroxide detected in liver samples from mice fasted overnight after 3 - week MCD diet .

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(D) TBARS and hydrogen peroxide detected in liver samples from mice fasted overnight after the 3-week MCD diet.