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 ) .

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

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

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 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

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

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

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 the indicated molar excess of TF2 ( analyzed as in A ) . Representative experiment shown ; n = 4 biological replicates

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

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

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

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

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

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

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

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 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 ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 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 .

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

(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 , 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 .

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

(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 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 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O 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 ) 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 .

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

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

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O 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 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 ) .

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

(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 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

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 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 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 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 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 ) .

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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 and K438R with ATP and ADP / ATP = 0 . 11 , respectively ( n = 3 , * t - test p - value < 0 . 05 , + t - test p - value < 0 . 1 ) .

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(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 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 .

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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 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 .

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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 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

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

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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 , 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 ) .

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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 , 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

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

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 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 ; amp ; E - stained liver sections . Scale Bar : 50μm . ( n = 11 - 74 )

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(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 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 ; amp ; E - stained liver sections . Scale Bar : 50μm . ( n = 11 - 9 )

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(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 ) 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 )

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(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 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 )

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(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 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 .

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(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 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 . )

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(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 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 .

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

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


( D ) TBARS and hydrogen peroxide detected in liver samples from mice fasted overnight after the 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.


( E ) qRT - PCR analysis of Col1a1 , Acta2 and Timp1 mRNA expression . mRNA expression was normalized to the amount of Gapdh mRNA .

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(E) qRT-PCR analysis of Col1a1, Acta2 and Timp1 mRNA expression. mRNA expression was normalized to the amount of Gapdh mRNA.


( F ) Representative Masson ' s trichrome - stained liver sections prepared from WT and p38γ / δ - / - mice fed a ND or the MCD diet for 3 weeks . Scale Bar : 50μm . Data are means ± SEM ( n = 5 - 10 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post tests ) .

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(F) Representative Masson's trichrome-stained liver sections prepared from WT and p38γ/δ-/- mice fed a ND or the MCD diet for 3 weeks. Scale Bar: 50μm. Data are means ± SEM (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a MCD diet for 3 weeks . ( A ) Representative H & amp ; amp ; E and oil red stained liver sections . Scale Bar : 50μm . ( n = 5 - 10 ) .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a MCD diet for 3 weeks. (A) Representative H&amp;E and oil red stained liver sections. Scale Bar: 50μm. (n=5-10).


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a MCD diet for 3 weeks . ( B ) Liver triglycerides ( C ) and plasma ALT at the end of the diet period .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a MCD diet for 3 weeks. (B) Liver triglycerides (C) and plasma ALT at the end of the diet period.


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a MCD diet for 3 weeks . ( D ) Measurement of plasmaTNF - α and IL - 6 .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a MCD diet for 3 weeks. (D) Measurement of plasmaTNF-α and IL-6.


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a MCD diet for 3 weeks . ( E ) qRT - PCR analysis of myeloid cell markers and cytokine mRNA expression from liver tissue ; mRNA expression was normalized to the amount of Gapdh mRNA .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a MCD diet for 3 weeks. (E) qRT-PCR analysis of myeloid cell markers and cytokine mRNA expression from liver tissue; mRNA expression was normalized to the amount of Gapdh mRNA.


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a MCD diet for 3 weeks . ( F ) qRT - PCR analysis of M1 and M2 polarization cell markers from liver infiltrated macrophages . mRNA expression was normalized to the amount of Gapdh mRNA . Data are means ± SEM ( n = 5 - 10 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post tests ) .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a MCD diet for 3 weeks. (F) qRT-PCR analysis of M1 and M2 polarization cell markers from liver infiltrated macrophages. mRNA expression was normalized to the amount of Gapdh mRNA. Data are means ± SEM (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a high - fat diet ( HFD ) for 10 weeks . ( A ) Body weight measured at the indicated times during HFD treatment .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a high-fat diet (HFD) for 10 weeks. (A) Body weight measured at the indicated times during HFD treatment.


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a high - fat diet ( HFD ) for 10 weeks . ( B ) Fat mass and lean mass determined by MRI at the end of the diet period .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a high-fat diet (HFD) for 10 weeks. (B) Fat mass and lean mass determined by MRI at the end of the diet period.


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a high - fat diet ( HFD ) for 10 weeks . ( C and D ) Liver mass and white fat ( WF ) mass .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a high-fat diet (HFD) for 10 weeks. (C and D) Liver mass and white fat (WF) mass.


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a high - fat diet ( HFD ) for 10 weeks . ( E ) Respiratory exchange quotient , energy expenditure , and locomotor activity , detected in metabolic cages . Data are means ± SEM . ( n = 5 - 10 ) * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post tests ) .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a high-fat diet (HFD) for 10 weeks. (E) Respiratory exchange quotient, energy expenditure, and locomotor activity, detected in metabolic cages. Data are means ± SEM. (n=5-10) *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a HFD for 10 weeks . ( A ) Representative H & amp ; amp ; E and oil red stained liver sections ( Scale Bar : 50μm )

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a HFD for 10 weeks. (A) Representative H&amp;E and oil red stained liver sections (Scale Bar: 50μm)


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a HFD for 10 weeks . ( B ) liver triglycerides ( n = 5 - 10 ) .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a HFD for 10 weeks. (B) liver triglycerides (n=5-10).


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a HFD for 10 weeks . ( C ) Plasma ALT at the end of the diet period .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a HFD for 10 weeks. (C) Plasma ALT at the end of the diet period.


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a HFD for 10 weeks . ( D ) Glucose tolerance measured at the end of the diet period . Blood glucose concentration was measured in mice given an intraperitoneal glucose injection ( 1g / kg ) after overnight fasting ( n = 5 - 10 ) .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a HFD for 10 weeks. (D) Glucose tolerance measured at the end of the diet period. Blood glucose concentration was measured in mice given an intraperitoneal glucose injection (1g/kg) after overnight fasting (n=5-10).


Lyzs - Cre and p38γ / δLyzs - KO mice were fed a ND or a HFD for 10 weeks . ( E ) Basal blood glucose in overnight - fasted ND and HFD - fed Lyzs - Cre and p38γ / δLyzs - KO mice ( n = 5 - 10 ) . Data are means ± SEM . ( n = 5 - 10 ) * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 refers to p38γ / δLyzs - KO versus Lyzs - Cre ; # # P < 0 . 01 ; # # # P < 0 . 001 refers to ND versus HFD ( 2 - way ANOVA coupled to Bonferroni ' s post tests or Newman - Keuls post test for liver triglycerides ) .

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Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a HFD for 10 weeks. (E) Basal blood glucose in overnight-fasted ND and HFD-fed Lyzs-Cre and p38γ/δLyzs-KO mice (n=5-10). Data are means ± SEM. (n=5-10) *P<0.05; **P<0.01; ***P<0.001 refers to p38γ/δLyzs-KO versus Lyzs-Cre; ##P<0.01; ###P<0.001 refers to ND versus HFD (2-way ANOVA coupled to Bonferroni's post tests or Newman-Keuls post test for liver triglycerides).


Lethally irradiated WT mice were reconstituted with BM from Lyzs - Cre ( Cx BMLyzs - Cre ) or p38γ / δLyzs - KOmice ( Cx BMp38γ / δLyzs - KO ) . Two months after the transplant , mice were fed the HFD for 10 weeks . ( A ) Freshly prepared CD45 . 2 whole BM mononuclear cells ( 2 × 107 ) were transplanted into lethally irradiated B6 . SJL ( CD45 . 1 ) mice , and engraftment by CD45 . 2 cells ( % ) was analyzed by antibody staining and FACS of peripheral blood and liverCD45 + cells . Charts show CD45 . 1 and CD45 . 2 expression in blood cells ( left ) and liver cells ( right ) isolated from transplanted mice ( n = 3 ) . Representative FACS dot plots of CD45 . 1 and CD45 . 2 expression are shown beneath the charts .

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Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BMLyzs-Cre) or p38γ/δLyzs-KOmice (Cx BMp38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (A) Freshly prepared CD45.2 whole BM mononuclear cells (2 × 107) were transplanted into lethally irradiated B6.SJL (CD45.1) mice, and engraftment by CD45.2 cells (%) was analyzed by antibody staining and FACS of peripheral blood and liverCD45+ cells. Charts show CD45.1 and CD45.2 expression in blood cells (left) and liver cells (right) isolated from transplanted mice (n = 3). Representative FACS dot plots of CD45.1 and CD45.2 expression are shown beneath the charts.


Lethally irradiated WT mice were reconstituted with BM from Lyzs - Cre ( Cx BM Lyzs - Cre ) or p38γ / δLyzs - KO mice ( Cx BM p38γ / δLyzs - KO ) . Two months after the transplant , mice were fed the HFD for 10 weeks . ( B ) Representative H & amp ; amp ; E and oil red - stained liver sections . Scale bar : 50 μm .

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Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BM Lyzs-Cre) or p38γ/δLyzs-KO mice (Cx BM p38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (B) Representative H&amp;E and oil red-stained liver sections. Scale bar: 50 μm.


Lethally irradiated WT mice were reconstituted with BM from Lyzs - Cre ( Cx BMLyzs - Cre ) or p38γ / δLyzs - KOmice ( Cx BMp38γ / δLyzs - KO ) . Two months after the transplant , mice were fed the HFD for 10 weeks . ( C ) Livertriglyceride content .

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Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BMLyzs-Cre) or p38γ/δLyzs-KOmice (Cx BMp38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (C) Livertriglyceride content.


Lethally irradiated WT mice were reconstituted with BM from Lyzs - Cre ( Cx BM Lyzs - Cre ) or p38γ / δLyzs - KO mice ( Cx BM p38γ / δLyzs - KO ) . Two months after the transplant , mice were fed the HFD for 10 weeks . ( D ) Plasma transaminase ALT activity .

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Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BM Lyzs-Cre) or p38γ/δLyzs-KO mice (Cx BM p38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (D) Plasma transaminase ALT activity.


Lethally irradiated WT mice were reconstituted with BM from Lyzs - Cre ( Cx BMLyzs - Cre ) or p38γ / δLyzs - KOmice ( Cx BMp38γ / δLyzs - KO ) . Two months after the transplant , mice were fed the HFD for 10 weeks . ( E ) Fasted glucose , detected at the end of the diet period in overnight - fasted mice . Data are means ± SEM . ( n = 5 - 10 ) * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post tests ) .

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Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BMLyzs-Cre) or p38γ/δLyzs-KOmice (Cx BMp38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (E) Fasted glucose, detected at the end of the diet period in overnight-fasted mice. Data are means ± SEM. (n=5-10) *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).


( A ) Flow cytometry analysis of liver myeloid subsets ( CD11b + Gr - 1high , CD11b + Gr - 1intermediate , CD11b + Gr - 1 - ) isolated from Lyzs - Cre and p38γ / δLyzs - KO mice fed a MCD for 3 weeks or HFD for 10 weeks . Representative dot plots are shown , and bar charts show the diet - induced increase in each population as a percentage of the total intra - hepatic CD11b + leukocyte population . Myeloid infiltrating cells isolated from livers were sorted by FACS and stain with H & amp ; amp ; E . Representative cells were shown next to the appropriate myeloid subsets .

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(A) Flow cytometry analysis of liver myeloid subsets (CD11b+ Gr-1high, CD11b+ Gr-1intermediate, CD11b+ Gr-1-) isolated from Lyzs-Cre and p38γ/δLyzs-KO mice fed a MCD for 3 weeks or HFD for 10 weeks. Representative dot plots are shown, and bar charts show the diet-induced increase in each population as a percentage of the total intra-hepatic CD11b+ leukocyte population. Myeloid infiltrating cells isolated from livers were sorted by FACS and stain with H&amp;E. Representative cells were shown next to the appropriate myeloid subsets.


( B and C ) Neutrophils and monocytes as a percentage of total circulating leukocytes , measured in total blood in animals fed the MCD diet for 3 weeks ( B ) or the HFD for 10 weeks ( C ) .

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(B and C) Neutrophils and monocytes as a percentage of total circulating leukocytes, measured in total blood in animals fed the MCD diet for 3 weeks (B) or the HFD for 10 weeks (C).


( D and E ) WT mice fed the MCD diet were i . v . injected with a 1 : 1 mix of DiO - labeled Lyzs - Cre neutrophil and DiD - labeled p38γ / δLyzs - KO neutrophils ( 6x106 cells in total ; n = 10 ) . One hour after injection , liver - infiltrating neutrophils were assessed by flow cytometry ( D ) and fluorescence micrography on liver sections ( E ) .

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(D and E) WT mice fed the MCD diet were i.v. injected with a 1:1 mix of DiO-labeled Lyzs-Cre neutrophil and DiD-labeled p38γ/δLyzs-KO neutrophils (6x106 cells in total; n=10). One hour after injection, liver-infiltrating neutrophils were assessed by flow cytometry (D) and fluorescence micrography on liver sections (E).


( F ) Intravital microscopy quantification of the rolling and adhesion frequencies and rolling velocities of neutrophils recruited to venules irrigating inflamed ( TNFα - injected ) cremaster muscle . Data are means ± SEM . ( n = 5 - 10 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post tests ) .

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(F) Intravital microscopy quantification of the rolling and adhesion frequencies and rolling velocities of neutrophils recruited to venules irrigating inflamed (TNFα-injected) cremaster muscle. Data are means ± SEM. (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).


Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs - Cre and p38γ / δLyzs - KO mice . These animals were fed a ND or MCD for 3 weeks . ( A ) Neutrophils and monocytes as a percentage of circulating leukocytes , measured in total blood .

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Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre and p38γ/δLyzs-KO mice. These animals were fed a ND or MCD for 3 weeks. (A) Neutrophils and monocytes as a percentage of circulating leukocytes, measured in total blood.


Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs - Cre and p38γ / δLyzs - KOmice . These animals were fed a ND or MCD for 3 weeks . ( B ) Representative H & amp ; amp ; E and oil red - stained liver sections after 3 weeks of treatment . Scale Bar : 50μm .

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Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre and p38γ/δLyzs-KOmice. These animals were fed a ND or MCD for 3 weeks. (B) Representative H&amp;E and oil red-stained liver sections after 3 weeks of treatment. Scale Bar: 50μm.


Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs - Cre and p38γ / δLyzs - KO mice . These animals were fed a ND or MCD for 3 weeks . ( C ) Liver triglyceride and ( D ) plasma transaminase activity ( ALT ) at the end of the diet period .

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Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre and p38γ/δLyzs-KO mice. These animals were fed a ND or MCD for 3 weeks. (C) Liver triglyceride and (D) plasma transaminase activity (ALT) at the end of the diet period.


Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs - Cre and p38γ / δLyzs - KO mice . These animals were fed a ND or MCD for 3 weeks . ( E ) Total RNA was extracted from livers , and chemokine and cytokine mRNA levels were determined by qRT - PCR . mRNA expression was normalized to the amount of Gapdh mRNA . Data are means ± SEM . ( n = 5 - 10 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post tests ) .

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Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre and p38γ/δLyzs-KO mice. These animals were fed a ND or MCD for 3 weeks. (E) Total RNA was extracted from livers, and chemokine and cytokine mRNA levels were determined by qRT-PCR. mRNA expression was normalized to the amount of Gapdh mRNA. Data are means ± SEM. (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).


( A , B and C ) Plasma levels of 3 - hydroxybutyrate in WT and p38γ / δ - / - ( A ) , Lyzs - Cre and p38γ / δLyzs - KO mice ( B ) and Lyzs - Cre Ly6G Ab treated after MCD diet ( C ) .

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(A, B and C) Plasma levels of 3-hydroxybutyrate in WT and p38γ/δ-/- (A), Lyzs-Cre and p38γ/δLyzs-KO mice (B) and Lyzs-Cre Ly6G Ab treated after MCD diet (C).


B Antiproliferative activities of XMU - MP - 5 and crizotinib against EML4 - ALK Ba / F3 and parental Ba / F3 cells , respectively . Cells were treated with increasing concentration of each drug for 48 hours and then analyzed by MTS assay . Each concentration was performed in triplicate . Data were presented as Mean ± SEM from three independent experiments ( n = 3 ) .

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B Antiproliferative activities of XMU-MP-5 and crizotinib against EML4-ALK Ba/F3 and parental Ba/F3 cells, respectively. Cells were treated with increasing concentration of each drug for 48 hours and then analyzed by MTS assay. Each concentration was performed in triplicate. Data were presented as Mean ± SEM from three independent experiments (n = 3) .


C XMU - MP - 5 downregulates ALK signaling pathways in EML4 - ALK Ba / F3 cells . Cells were treated with XMU - MP - 5 or crizotinib for 4 h , and then analyzed by immunobloting .

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C XMU-MP-5 downregulates ALK signaling pathways in EML4-ALK Ba/F3 cells. Cells were treated with XMU-MP-5 or crizotinib for 4 h, and then analyzed by immunobloting.


C XMU - MP - 5 suppressed ALK signaling pathway in H3122 cells .

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C XMU-MP-5 suppressed ALK signaling pathway in H3122 cells.


E Comparison of tumor volume of different treatment groups in 11 days . BALB / c nude mice bearing H3122 xenograft tumors were treated with vehicle , XMU - MP - 5 and crizotinib by tail vein injection once daily . Data were shown as Mean ± SEM ( n = 6 ) . Statistical comparasions were performed using a two - tailed , unpaired Student ' s t test . N . S . , P = 0 . 1063 ; * * P = 0 . 0097 .

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E Comparison of tumor volume of different treatment groups in 11 days. BALB/c nude mice bearing H3122 xenograft tumors were treated with vehicle, XMU-MP-5 and crizotinib by tail vein injection once daily. Data were shown as Mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test. N.S., P = 0.1063; **P = 0.0097.


G Representative images of tumors analyzed by H & E and IHC staining . Scale bars , 100 μm .

G Representative images of tumors analyzed by H&E and IHC staining. Scale bars, 100 μm.


A Cell viability was measured by MTS assay . Each concentration was performed in triplicate . The exact IC50 value was presented as Mean ± SEM from three independent experiments ( n = 3 ) and shown in Appendix Table S6 . IC50 fold changes of wild type and mutant EML4 - ALK Ba / F3 cell lines to parental Ba / F3 cell line of XMU - MP - 5 and crizotinib were determined .

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A Cell viability was measured by MTS assay. Each concentration was performed in triplicate. The exact IC50 value was presented as Mean ± SEM from three independent experiments (n = 3) and shown in Appendix Table S6. IC50 fold changes of wild type and mutant EML4-ALK Ba/F3 cell lines to parental Ba/F3 cell line of XMU-MP-5 and crizotinib were determined.


B Heatmap of the anti - proliferative activity of ALK inhibitors across Ba / F3 cell lines with different ALK mutations .

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B Heatmap of the anti-proliferative activity of ALK inhibitors across Ba/F3 cell lines with different ALK mutations.


C , D In vivo activity of XMU - MP - 5 against L1196M xenograft mouse model . BALB / c nude mice bearing L1196M Ba / F3 xenograft tumors were treated with vehicle , 30 mg / kg or 60 mg / kg XMU - MP - 5 by tail vein injection once daily . Tumor volume ( C ) and tumor weight ( D ) were shown as mean ± SEM ( n = 6 ) . Statistical comparasions were performed using a two - tailed , unpaired Student ' s t test for ( C ) , N . S . , P = 0 . 1482 ; * P = 0 . 037 . Statistical comparasions were performed using a one - way ANOVA with Dunnett test for ( D ) , N . S . , P = 0 . 2162 ; * P = 0 . 0122 .

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C, D In vivo activity of XMU-MP-5 against L1196M xenograft mouse model. BALB/c nude mice bearing L1196M Ba/F3 xenograft tumors were treated with vehicle, 30 mg/kg or 60 mg/kg XMU-MP-5 by tail vein injection once daily. Tumor volume (C) and tumor weight (D) were shown as mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test for (C), N.S., P = 0.1482; *P = 0.037. Statistical comparasions were performed using a one-way ANOVA with Dunnett test for (D), N.S., P = 0.2162; *P = 0.0122.


E , F Efficacy of XMU - MP - 5 in G1202R Ba / F3 xenograft mouse model . BALB / c nude mice bearing G1202R Ba / F3 cells xenograft tumors were administrated with vehicle or XMU - MP - 5 by tail vein injection twice daily . Lorlatinib was used as a positive control by oral administration . Tumor volume ( E ) and tumor weight ( F ) were shown as mean ± SEM ( n = 6 ) . Statistical comparasions were performed using a two - tailed , unpaired Student ' s t test for ( E ) , Vehicle vs XMU - MP - 5 30 mg / kg , * P = 0 . 0433 ; Vehicle vs XMU - MP - 5 30 mg / kg BID , * * P = 0 . 0093 ; Vehicle vs Lorlatinib , * * P = 0 . 0029 . Statistical comparasions were performed using a one - way ANOVA with Dunnett test for ( F ) . Vehicle vs XMU - MP - 5 30 mg / kg , * * * P = 0 . 0008 ; Vehicle vs XMU - MP - 5 30 mg / kg BID , * * * P = 0 . 0003 ; Vehicle vs Lorlatinib , * * * P = 0 . 0002 .

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E, F Efficacy of XMU-MP-5 in G1202R Ba/F3 xenograft mouse model. BALB/c nude mice bearing G1202R Ba/F3 cells xenograft tumors were administrated with vehicle or XMU-MP-5 by tail vein injection twice daily. Lorlatinib was used as a positive control by oral administration. Tumor volume (E) and tumor weight (F) were shown as mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test for (E), Vehicle vs XMU-MP-5 30 mg/kg, *P = 0.0433; Vehicle vs XMU-MP-5 30 mg/kg BID, **P = 0.0093; Vehicle vs Lorlatinib, **P = 0.0029. Statistical comparasions were performed using a one-way ANOVA with Dunnett test for (F). Vehicle vs XMU-MP-5 30 mg/kg, ***P = 0.0008; Vehicle vs XMU-MP-5 30 mg/kg BID, ***P = 0.0003; Vehicle vs Lorlatinib, ***P = 0.0002.


A Representative CT images ( upper panel ) and H & E staining ( lower panel ) of lungs from EML4 - ALK tumor - bearing mice are shown . The complete set of CT images refers to Appendix Figure S10A . Mice were treated with 30 mg / kg XMU - MP - 5 twice daily for 3 weeks . H : heart ; arrow indicated tumor . Scale bars , 200 μm . B IHC analyses of lungs from EML4 - ALK transgenic mice . pALK and Cl - caspase 3 were probed . Scale bars , 100 μm .

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A Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10A. Mice were treated with 30 mg/kg XMU-MP-5 twice daily for 3 weeks. H: heart; arrow indicated tumor. Scale bars, 200 μm. B IHC analyses of lungs from EML4-ALK transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.


C Representative CT images ( upper panel ) and H & E staining ( lower panel ) of lungs from EML4 - ALK ( L1196M ) tumor - bearing mice are shown . The complete set of CT images refers to Appendix Figure S10B . Mice were treated with 40 mg / kg XMU - MP - 5 twice daily for 1 week . H : heart ; arrow indicated tumor . Scale bars , 200 μm . D IHC analyses of lungs from EML4 - ALK ( L1196M ) transgenic mice . pALK and Cl - caspase 3 were probed . Scale bars , 100 μm .

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C Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK(L1196M) tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10B. Mice were treated with 40 mg/kg XMU-MP-5 twice daily for 1 week. H: heart; arrow indicated tumor. Scale bars, 200 μm. D IHC analyses of lungs from EML4-ALK(L1196M) transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.