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

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

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

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


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

O O O O 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

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


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

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

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


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

O O O O 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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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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 ) of immunoblot of p38δ expression in liver extracts prepared from obese with NAFLD 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 of Mapk12 ( p38gamma ) Mapk13 ( p38delta ) mRNA expression in liver extracts prepared from wild - type mice ( WT ) fed a diet deficient in methionine choline ( MCD ) or control diet ( ND ) for 3 ; mRNA expression was normalized to amount of Gapdh mRNA . ( n = 5 - 10 )

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

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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 oil red - stained liver sections prepared from WT p38γ / δ - / - mice fed a ND or MCD diet for 3 . 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 ( C ) plasma transaminase activity ( ALT ) measured in WT p38γ / δ - / - mice after 3 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 hydrogen peroxide detected in liver samples from mice fasted overnight after 3 - week MCD diet .

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


( E ) qRT - PCR of Col1a1 , Acta2 Timp1 mRNA expression . mRNA expression was normalized to 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 p38γ / δ - / - mice fed a ND or MCD diet for 3 . Scale Bar : 50μm . Data are means ± ( n = 5 - 10 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post ) .

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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 p38γ / δLyzs - KO mice were fed a ND or a MCD diet for 3 . ( A ) Representative H & amp ; amp ; E 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 p38γ / δLyzs - KO mice were fed a ND or a MCD diet for 3 . ( B ) Liver triglycerides ( C ) plasma ALT at end of 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 p38γ / δLyzs - KO mice were fed a ND or a MCD diet for 3 . ( D ) Measurement of plasmaTNF - α IL - 6 .

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

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

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

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


Lyzs - Cre p38γ / δLyzs - KO mice were fed a ND or a HFD for 10 . ( 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 p38γ / δLyzs - KO mice were fed a ND or a HFD for 10 . ( C ) Plasma ALT at end of 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 p38γ / δLyzs - KO mice were fed a ND or a HFD for 10 . ( D ) Glucose tolerance measured at end of diet period . Blood glucose 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 p38γ / δLyzs - KO mice were fed a ND or a HFD for 10 . ( E ) Basal blood glucose in overnight - fasted ND HFD - fed Lyzs - Cre p38γ / δLyzs - KO mice ( n = 5 - 10 ) . Data are means ± . ( 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 or Newman - Keuls post 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 transplant , mice were fed HFD for 10 . ( A ) Freshly prepared CD45 . 2 whole BM mononuclear ( 2 × 107 ) were transplanted into lethally irradiated B6 . SJL ( CD45 . 1 ) mice , engraftment by CD45 . 2 ( % ) was analyzed by antibody FACS of peripheral blood liverCD45 + . Charts show CD45 . 1 CD45 . 2 expression in blood ( left ) liver ( right ) isolated from transplanted mice ( n = 3 ) . Representative FACS dot plots of CD45 . 1 CD45 . 2 expression are shown beneath 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 transplant , mice were fed HFD for 10 . ( B ) Representative H & amp ; amp ; E 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 transplant , mice were fed HFD for 10 . ( 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 transplant , mice were fed HFD for 10 . ( 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 transplant , mice were fed HFD for 10 . ( E ) Fasted glucose , detected at end of diet period in overnight - fasted mice . Data are means ± . ( n = 5 - 10 ) * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post ) .

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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 of liver myeloid subsets ( CD11b + Gr - 1high , CD11b + Gr - 1intermediate , CD11b + Gr - 1 - ) isolated from Lyzs - Cre p38γ / δLyzs - KO mice fed a MCD for 3 or HFD for 10 . Representative dot plots are shown , bar charts show diet - induced increase in each population as a of total intra - hepatic CD11b + leukocyte population . Myeloid infiltrating isolated from livers were sorted by FACS stain with H & amp ; amp ; E . Representative were shown next to 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 C ) Neutrophils monocytes as a of total circulating leukocytes , measured in total blood in fed MCD diet for 3 ( B ) or HFD for 10 ( 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 E ) WT mice fed MCD diet were i . v . injected with a 1 : 1 mix of DiO - labeled Lyzs - Cre neutrophil DiD - labeled p38γ / δLyzs - KO neutrophils ( 6x106 in total ; n = 10 ) . One hour after injection , liver - infiltrating neutrophils were assessed by flow cytometry ( D ) 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 of rolling adhesion frequencies rolling velocities of neutrophils recruited to venules irrigating inflamed ( TNFα - injected ) cremaster muscle . Data are means ± . ( n = 5 - 10 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post ) .

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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 p38γ / δLyzs - KO mice . These were fed a ND or MCD for 3 . ( A ) Neutrophils monocytes as a 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 p38γ / δLyzs - KOmice . These were fed a ND or MCD for 3 . ( B ) Representative H & amp ; amp ; E oil red - stained liver sections after 3 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 p38γ / δLyzs - KO mice . These were fed a ND or MCD for 3 . ( C ) Liver triglyceride ( D ) plasma transaminase activity ( ALT ) at end of 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 p38γ / δLyzs - KO mice . These were fed a ND or MCD for 3 . ( E ) Total was extracted from livers , chemokine cytokine mRNA were determined by qRT - PCR . mRNA expression was normalized to amount of Gapdh mRNA . Data are means ± . ( n = 5 - 10 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( 2 - way ANOVA coupled to Bonferroni ' s post ) .

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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 C ) Plasma of 3 - hydroxybutyrate in WT p38γ / δ - / - ( A ) , Lyzs - Cre p38γ / δLyzs - KO mice ( B ) 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 crizotinib against EML4 - ALK Ba / F3 parental Ba / F3 , respectively . were treated with increasing of each drug for 48 hours then analyzed by MTS . Each was performed in triplicate . Data were presented as Mean ± 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 . were treated with XMU - MP - 5 or crizotinib for 4 h , 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 .

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


E Comparison of volume of different treatment groups in 11 days . BALB / c nude mice bearing H3122 xenograft tumors were treated with vehicle , XMU - MP - 5 crizotinib by tail vein injection once daily . Data were shown as Mean ± ( n = 6 ) . Statistical comparasions were performed using a two - , unpaired Student ' s t . 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 of tumors analyzed by H & E . Scale bars , 100 μm .

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


A viability was measured by MTS . Each was performed in triplicate . exact IC50 value was presented as Mean ± from three independent experiments ( n = 3 ) shown in Appendix Table S6 . IC50 fold changes of wild type mutant EML4 - ALK Ba / F3 lines to parental Ba / F3 of XMU - MP - 5 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 of anti - proliferative activity of ALK inhibitors across Ba / F3 lines with different ALK mutations .

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 . volume ( C ) ( D ) were shown as mean ± ( n = 6 ) . Statistical comparasions were performed using a two - , unpaired Student ' s t for ( C ) , N . S . , P = 0 . 1482 ; * P = 0 . 037 . Statistical comparasions were performed using a one - way ANOVA with Dunnett 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 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 . volume ( E ) ( F ) were shown as mean ± ( n = 6 ) . Statistical comparasions were performed using a two - , unpaired Student ' s t 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 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 ( upper panel ) H & E ( lower panel ) of lungs from EML4 - ALK - bearing mice are shown . complete set of CT refers to Appendix Figure S10A . Mice were treated with 30 mg / kg XMU - MP - 5 twice daily for 3 . H : heart ; arrow indicated . Scale bars , 200 μm . B analyses of lungs from EML4 - ALK transgenic mice . pALK 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 ( upper panel ) H & E ( lower panel ) of lungs from EML4 - ALK ( L1196M ) - bearing mice are shown . complete set of CT refers to Appendix Figure S10B . Mice were treated with 40 mg / kg XMU - MP - 5 twice daily for 1 week . H : heart ; arrow indicated . Scale bars , 200 μm . D analyses of lungs from EML4 - ALK ( L1196M ) transgenic mice . pALK 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.


B Inhibition zone - for synergy between AMSIN HNP1 & 2 or lysozyme C ( abbreviated as hLZc ) to two oral Streptococcus ( SSAN SSAL ) . Inset ( upper panel , boxed ) , a representative example of inhibition - zone showing synergy . Peptide doses used here were 0 . 05 nmol / well for HNP1 & 2 hLZc at which no visible inhibition zones were observed for both of them . At same dose , AMSIN produced a small inhibition zone . For synergetic observation , two peptides were applied to same well according to doses indicated . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - ( * * * P < 0 . 001 Note : nanomolar of HNP1 & 2 was calculated from average molecular of HNP1 HNP2 .

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B Inhibition zone-based assay for synergy between AMSIN and HNP1&2 or lysozyme C (abbreviated as hLZc) to the two oral Streptococcus (SSAN and SSAL). Inset (upper panel, boxed), a representative example of the inhibition-zone test showing the synergy. Peptide doses used here were 0.05 nmol/well for HNP1&2 and hLZc at which no visible inhibition zones were observed for both of them. At the same dose, AMSIN produced a small inhibition zone. For synergetic observation, two peptides were applied to the same well according to the doses indicated. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test (***P<0.001 Note: the nanomolar concentration of HNP1&2 was calculated from the average molecular weight of HNP1 and HNP2.


C FIC index for synergy between AMSIN HNP1 & 2 or hLZc to SSAL SSAN . dashed represents cutoff of FIC index ( 0 . 5 ) for synergy .

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C The FIC index assay for synergy between AMSIN and HNP1&2 or hLZc to SSAL and SSAN. The dashed line represents the cutoff of the FIC index (0.5) for synergy.


A Growth kinetic measurements of SM exposed to AMSIN or various with known targets . peptide or compounds used were 4 × CL . VAN , vancomycin ; RIF , rifampicin ; KAN , kanamycin . OD600 were calculated as measured values subtracting background value from broth medium ( OD600 = 0 . 356 ) .

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A Growth kinetic measurements of SM exposed to AMSIN or various antibiotics with known cellular targets. The peptide or the compounds used were 4×CL. VAN, vancomycin; RIF, rifampicin; KAN, kanamycin. OD600 were calculated as measured values subtracting the background value from broth medium (OD600 = 0.356).


D Scanning electron microscopic observation of AMSIN - induced BM deformation . doses used were 10 × CL for AMSIN vancomycin .

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D Scanning electron microscopic observation of AMSIN-induced BM deformation. The doses used were 10×CL for AMSIN and vancomycin.


E accumulation of soluble - wall precursor UMP in vancomycin - treated ( blue ) , AMSIN - treated ( red dashed ) or not treated ( green ) culture of MRSA P1374 . Inset : UMP identified by UPLC - HRMS with singlely doubly protonated m / z observed .

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E Cytoplasmic accumulation of the soluble cell-wall precursor UMP in vancomycin-treated (blue line), AMSIN-treated (red dashed line) or not treated (green line) culture of MRSA P1374. Inset: UMP identified by UPLC-HRMS with singlely and doubly protonated m/z observed.


H ESI - MS detecting non - covalent between AMSIN UMP . quadruply protonated ( m / z 1304 . 8 ) is observed . Inset : comparison of intensities of between two ratios of UMP : AMSIN Here , AMSIN is abbreviated as AMS due to space limitation .

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H ESI-MS detecting the non-covalent complex between AMSIN and UMP. The quadruply protonated complex (m/z 1304.8) is observed. Inset: comparison of intensities of the complex between two ratios of UMP:AMSIN Here, AMSIN is abbreviated as AMS due to space limitation.


J dose - response unit curve of UMP binding to AMSIN , which is fitted by BIAevaluation v2 . 0 . 1 . software using 1 : 1 Langmuir binding model .

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J The dose-response unit curve of UMP binding to AMSIN, which is fitted by the BIAevaluation v2.0.1. software using 1:1 Langmuir binding model.


B Hemolysis by AMSIN . Meucin - 18 was used as a positive control . Mean ± SD of three technical replicates is displayed .

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B Hemolysis by AMSIN. Meucin-18 was used as a positive control. Mean ± SD of three technical replicates is displayed.


C Comparison of cytotoxic effects of AMSIN LL - 37 on HL - 60 . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - or Mann - Whitney U ( latter indicated by a red asterisk ; * P < 0 . 05 , * * * P < 0 . 001 , ns : no

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C Comparison of cytotoxic effects of AMSIN and LL-37 on HL-60 cells. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk; *P<0.05, ***P<0.001, ns: no significance


D An inhibition - zone showing inability of AMSIN to bind . bacterium used was MRSA P1374 peptide dose was 1 . 0 nmol / well . Mean ± SD of three biological replicates is displayed . P values were obtained by Mann - Whitney U ( ns : no

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D An inhibition-zone assay showing the inability of AMSIN to bind DNA. The bacterium used was MRSA P1374 and the peptide dose was 1.0 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann-Whitney U (ns: no significance


E effect of AMSIN on human ion channels expressed in CNS heart . asterisks mark steady state current traces after administering 100 μM AMSIN .

E The effect of AMSIN on human ion channels expressed in the CNS and heart. The asterisks mark steady state current traces after administering 100 μM AMSIN.


F Peptide - induced IL - 8 release . A549 were treated with AMSIN or LL - 37 for 24 h . IL - 8 in culture supernatants from FBS - containing medium ( left ) or serum - free medium ( right ) were measured with an ELISA , as described in Materials Methods . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - or Mann - Whitney U ( latter indicated by a red asterisk or ns ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : no significance G Peptide - induced MCP - 1 release . HL - 60 were treated with AMSIN or LL - 37 for 24 h . MCP - 1 in culture supernatants from FBS - containing medium ( left ) or serum - free medium ( right ) were measured with an ELISA . Mean ± SD of three biological replicates is displayed . P values were obtained by Student ' s t - or Mann - Whitney U ( latter indicated by a red asterisk or ns ; * P < 0 . 05 , * * P < 0 . 01 , ns : no

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F Peptide-induced IL-8 release. A549 cells were treated with AMSIN or LL-37 for 24 h. IL-8 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA, as described in Materials and Methods. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ***P<0.001, ns: no significance G Peptide-induced MCP-1 release. HL-60 cells were treated with AMSIN or LL-37 for 24 h. MCP-1 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ns: no significance


J Survival after peritoneal infection with MRSA P1374 . survival fraction of vehicle - treated control group was zero out of 11 mice treatment group contained six mice survived .

J Survival after peritoneal infection with MRSA P1374. The survival fraction of the vehicle-treated control group was zero out of 11 mice and the treatment group contained six mice and all survived.


A , B Functional freeMitos association with PMN . Overlay dot plot population , representative of 4 biological replicates of flow cytometry gating strategy used to quantify interaction between PMN ( anti - CD11b - PE ) freeMitos . Freshly isolated platelets were labeled with MitoTrackerTM Deep Red freeMitos isolation was subsequently performed . CD11b - labeled PMN were incubated in absence ( A ) or presence ( B ) of freeMitos at a 1 : 5 ( PMN : mitochondria ) .

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A, B Functional freeMitos association with PMN. Overlay dot plot population, representative of 4 biological replicates of the flow cytometry gating strategy used to quantify the interaction between PMN (anti-CD11b-PE) and freeMitos. Freshly isolated platelets were labeled with MitoTrackerTM Deep Red and freeMitos isolation was subsequently performed. CD11b-labeled PMN were incubated in absence (A) or presence (B) of freeMitos at a 1:5 ratio (PMN:mitochondria).


C Confocal microscopy imaging of PMN ( red ) labeled with CellMask Orange in absence ( upper panel ) or presence ( lower panel ) of MitoTrackerTM Deep Red - labeled freeMitos ( magenta ) . PMN nuclei were stained with DAPI ( cyan ) . scale bars shown in lower right of represent 10 μm in .

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C Confocal microscopy imaging of PMN cell membrane (red) labeled with CellMask Orange in absence (upper panel) or presence (lower panel) of MitoTrackerTM Deep Red-labeled freeMitos (magenta). PMN nuclei were stained with DAPI (cyan). The white scale bars shown in the lower right of the images represent 10 μm in size.


D , E FreeMitos associate with PMN modulate gene expression . Volcano plot , of 4 biological replicates , representing gene expression modulation induced by mitochondrial DAMPs preparation ( D ) freshly isolated functional freeMitos ( E ) in PMN .

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D, E FreeMitos associate with PMN and modulate gene expression. Volcano plot, of 4 biological replicates, representing the gene expression modulation induced by mitochondrial DAMPs preparation (D) and freshly isolated functional freeMitos (E) in PMN.


FreeMitos effect on respiratory state of PMN . O2 consumption rates of resting or activated ( incubated with TNF - a GM - CSF ) PMN were measured using high - resolution O2 respirometry ( Oroboros Oxygraph ) revealed no significant differences between basal state ( left panel ) , leak state ( middle panel ) maximum electron transport system capacity ( ETS , right panel ) of respiration ( F ) . Data information : For panels F data are shown as means ± , of 3 biological replicates . Two - way ANOVA followed by Tukey ' s multiple comparisons for each sample was performed for panels F Values without at least one common superscript letter are different ( P < 0 . 05 ) .

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FreeMitos effect on the respiratory state of PMN. O2 consumption rates of resting or activated (incubated with TNF-a and GM-CSF) PMN were measured using high-resolution O2 respirometry (Oroboros Oxygraph) and revealed no significant differences between the basal state (left panel), leak state (middle panel) and the maximum electron transport system capacity (ETS, right panel) of cellular respiration (F). Data information: For panels F data are shown as means ± SEM, of 3 biological replicates. Two-way ANOVA tests followed by Tukey's multiple comparisons tests for each sample was performed for panels F Values without at least one common superscript letter are different (P < 0.05).


Immunoblot of mitochondrial cytochrome c oxidase ( COX IV ) histone H3 as a loading control of PMN after co - incubations with freeMitos ( G ) with subsequent densitometry ( H ) . Data information : For panels H , data are shown as means ± , of 3 biological replicates . Two - way ANOVA followed by Tukey ' s multiple comparisons for each sample was performed for panels H . Values without at least one common superscript letter are different ( P < 0 . 05 ) .

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Immunoblot of mitochondrial cytochrome c oxidase (COX IV) and histone H3 as a loading control of PMN after co-incubations with freeMitos (G) with subsequent densitometry analysis (H). Data information: For panels H, data are shown as means ± SEM, of 3 biological replicates. Two-way ANOVA tests followed by Tukey's multiple comparisons tests for each sample was performed for panels H. Values without at least one common superscript letter are different (P < 0.05).


A , B FreeMitos induce PMN intracellular calcium release in PMN . Representative visualization of intracellular calcium monitoring by flow cytometry of 1 x 106 PMN co - incubated with 5 x 106 mitochondria ( A ) . Calcium mobilization was monitored by flow cytometry for 120 seconds using probe Fluo - 3 AM . gray represents PMN with vehicle ( HBSS ) while represents PMN incubated with mitochondria . PMN intracellular calcium mobilization induced by either vehicle ( HBSS ) , freeMitos or mitochondrial DAMPs quantified by flow cytometry ( B ) . Data information : Data are shown as means ± of 3 biological replicates ( panel B , One - way ANOVA , followed by Tukey ' s multiple comparisons were performed for panels B , Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

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A, B FreeMitos induce PMN intracellular calcium release in PMN. Representative visualization of intracellular calcium monitoring by flow cytometry of 1 x 106 PMN co-incubated with 5 x 106 mitochondria (A). Calcium mobilization was monitored by flow cytometry for 120 seconds using the fluorescent probe Fluo-3 AM. The gray line represents PMN with the vehicle (HBSS) while the dark line represents PMN incubated with mitochondria. PMN intracellular calcium mobilization induced by either the vehicle (HBSS), freeMitos or mitochondrial DAMPs and quantified by flow cytometry (B). Data information: Data are shown as means ± SEM of 3 biological replicates (panel B, One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels B, Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.


Functional freeMitos induce release of PMN - derived microvesicles . A dose - response of various mitochondria to PMN ratios was performed to measure release of PMN microvesicles . PMN microvesicles labeling was performed with either anti - CD66b - FITC labelling ( C , left panel ) or anti - CD11b - PE ( C , right panel ) . Data information : Data are shown as means ± of 4 biological replicates ( panel C two - way ( panel C ) ANOVA , followed by Dunnett ' s multiple comparisons were performed compared to vehicle ( HBSS ) ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

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Functional freeMitos induce the release of PMN-derived microvesicles. A dose-response analysis of various mitochondria to PMN ratios was performed to measure the release of PMN microvesicles. PMN microvesicles labeling was performed with either anti-CD66b-FITC labelling (C, left panel) or anti-CD11b-PE (C, right panel). Data information: Data are shown as means ± SEM of 4 biological replicates (panel C two-way (panel C) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.


PMN - derived microparticle gating strategy is shown in panel D . Flow cytometry panel established to distinguish PMNL - MVs from background using leucocyte specific integrin alpha - M beta - 2 ( CD11b ) , granulocyte specific carcinoembryonic antigen - related adhesion molecule 8 ( CD66b ) , reference beads . PMN were either incubated with vehicle ( Tyrode ' s Buffer , upper panels ) or freeMitos ( lower panels ) . Following co - incubation , PMN were washed twice resuspended in HBSS before processing on flow cytometer . Only single events , identified by singlets gate ( left upper lower panels ) using side scatter ( SSC - A ) vs side scatter heights ( SSC - H ) were analyzed . scatter ( side scatter ( SSC ) forward scatter ( FSC ) ) approach for determining background noise in HBSS . Histogram displaying of samples labelled with either CD11b - FITC or CD66b - PE . Overlay dot plot of PMNL - MVs population reference beads , representative of 4 biological replicates , each performed in 2 technical replicates .

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PMN-derived microparticle gating strategy is shown in panel D. Flow cytometry panel established to distinguish the PMNL-MVs from the background using the leucocyte specific integrin alpha-M beta-2 (CD11b), the granulocyte specific carcinoembryonic antigen-related cell adhesion molecule 8 (CD66b), and size reference beads. PMN were either incubated with the vehicle (Tyrode's Buffer, upper panels) or freeMitos (lower panels). Following co-incubation, PMN were washed twice and resuspended in HBSS before processing on flow cytometer. Only single events, identified by the singlets gate (left upper and lower panels) using side scatter area (SSC-A) vs side scatter heights (SSC-H) were analyzed. Light scatter (side scatter (SSC) and forward scatter (FSC)) approach for determining the background noise in HBSS. Histogram displaying the fluorescence intensity of samples labelled with either CD11b-FITC or CD66b-PE. Overlay dot plot of PMNL-MVs population and size reference beads, representative of 4 biological replicates, each performed in 2 technical replicates.


E Visual confirmation of PMN PMN - derived microvesicles . Transmission electron microscopy ( ) imaging of PMN - derived microvesicles ( black arrowhead ) , representative of 3 biological replicates , from resting or activated PMN ( 106 ) incubated with either mitochondria , mitochondrial DAMPs or fMLP as positive control . black scale bars are shown in lower right of for vehicles ( 400 nm ) , mitochondria ( 600 nm ) , DAMPs ( 600 nm ) fMLP ( 500 nm ) conditions .

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E Visual confirmation of PMN and PMN-derived microvesicles. Transmission electron microscopy (TEM) imaging of PMN-derived microvesicles (black arrowhead), representative of 3 biological replicates, from resting or activated PMN (106 cells) incubated with either mitochondria, mitochondrial DAMPs or fMLP as positive control. The black scale bars are shown in the lower right of the images for the vehicles (400 nm), mitochondria (600 nm), DAMPs (600 nm) and fMLP (500 nm) conditions.


F of resting PMN microvesicles produced by PMN ( 106 ) incubated with 107 freeMitos or 25 µg of mitochondrial DAMPs for 2 hr . Data information : Data are shown as means ± of 3 biological replicates ( panel One - way ANOVA , followed by Tukey ' s multiple comparisons were performed for panels Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

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F Quantification of resting PMN microvesicles produced by PMN (106 cells) incubated with 107 freeMitos or 25 µg of mitochondrial DAMPs for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.


G of calpain activity , by , in lysate of either resting ( left panel ) or activated ( right panel ) PMN ( 2 x 105 ) incubated in presence of freeMitos for 2 hr . Data information : Data are shown as means ± of 4 biological replicates ( panel G ) . One - way ANOVA , followed by Tukey ' s multiple comparisons were performed for panels G . Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

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G Quantification of calpain activity, by fluorescence assay, in cell lysate of either resting (left panel) or activated (right panel) PMN (2 x 105 cells) incubated in presence of freeMitos for 2 hr. Data information: Data are shown as means ± SEM of 4 biological replicates (panel G). One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels G. Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.


H ELISA of IL - 8 concentrations present in supernatant following incubation of PMN ( 106 ) with freeMitos ( 107 ) , DAMPs ( 25 µg ) or fMLP ( 1 µM ) for 2 hr . Data information : Data are shown as means ± of 3 biological replicates ( panel H ) One - way ( panel H ) ANOVA , followed by Dunnett ' s multiple comparisons were performed compared to vehicle ( HBSS ) ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

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H ELISA quantification of IL-8 concentrations present in the supernatant following the incubation of PMN (106 cells) with freeMitos (107), DAMPs (25 µg) or fMLP (1 µM) for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel H) One-way (panel H) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.


A Structure of autoinhibited ScV1 . catalytic A ( pink ) non catalytic B ( green ) subunits are arranged as a heterohexamer around central rotor subunits D ( red ) F ( cyan ) . three peripheral stalks ( EG heterodimers 1 - 3 , blue orange , respectively ) are bound at periphery of B subunits . inhibitory H subunit ( yellow ) is unique to eukaryotic V - ATPase . Unless otherwise stated , coloring scheme used here for V1 subunits will be used from here on .

A Structure of autoinhibited ScV1. The catalytic A (pink) and non catalytic B (green) subunits are arranged as a heterohexamer around the central rotor subunits D (red) and F (cyan). The three peripheral stalks (EG heterodimers 1-3, blue and orange, respectively) are bound at the periphery of the B subunits. The inhibitory H subunit (yellow) is unique to the eukaryotic V-ATPase. Unless otherwise stated, the coloring scheme used here for the V1 subunits will be used from here on.


B Interaction of peripheral stalks with catalytic hexamer . Each peripheral stalk crosses a non - catalytic A / B interface on its way from top of V1 to base of catalytic . Representative view of interaction is shown for EG3 .

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B Interaction of the peripheral stalks with the catalytic hexamer. Each peripheral stalk crosses a non-catalytic A/B interface on its way from the top of the V1 to the base of the catalytic core. Representative view of the interaction is shown for EG3.


C Interaction of peripheral stalks EG1 2 with catalytic hexamer . peripheral stalks are bound to top of B subunits via their C - termini ( boxed ) . electron density ( contoured at 1 . 2 σ ) for C - terminal domains of EG1 EG2 is shown in panels to left right . D structure of autoinhibited ScV1rotated 90 ° around vertical from view shown in panel A to highlight peripheral stalks EG2 EG3 . Note bent appearance of EG3 compared to EG2 along with presence of a partially unstructured region known as " bulge " in subunit G ( indicated with an arrow ) . box shows electron density ( contoured at 1 . 2 σ ) for EG3 N - termini contacting C - terminal domain of A subunit of a closed catalytic site ( see asterisk ) . horizontal lines on structure indicate sections shown in panels E ( upper two lines ) F ( lower two lines ) .

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C Interaction of peripheral stalks EG1 and 2 with the catalytic hexamer. The peripheral stalks are bound to the top of the B subunits via their C-termini (boxed). The electron density (contoured at 1.2 σ) for the C-terminal domains of EG1 and EG2 is shown in the panels to the left and right.D The structure of autoinhibited ScV1rotated 90° around the vertical from the view shown in panel A to highlight peripheral stalks EG2 and EG3. Note the bent appearance of EG3 compared to EG2 along with the presence of a partially unstructured region known as the "bulge" in subunit G (indicated with an arrow). The box shows electron density (contoured at 1.2 σ) for the EG3 N-termini contacting the C-terminal domain of the A subunit of a closed catalytic site (see asterisk). The horizontal lines on the structure indicate the sections shown in panels E (upper two lines) and F (lower two lines).


E Section through ScV1 electron density map showing interaction of A3B3 catalytic hexamer with peripheral stalks ( EG1 - 3 ) . View is rotated 90 ° from view in A , looking up central rotor . A / B pairs forming three catalytic sites are designated ( AB ) 1 - 3 according to which of three peripheral stalks is bound to each B subunit . For example , ( AB ) 1 has peripheral stalk EG1 bound to its B subunit . black arrowheads indicate non - catalytic A / B interfaces .

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E Section through the ScV1 electron density map showing interaction of the A3B3 catalytic hexamer with the peripheral stalks (EG1-3). View is rotated 90° from the view in A, looking up the central rotor. The A/B pairs forming the three catalytic sites are designated (AB)1-3 according to which of the three peripheral stalks is bound to each B subunit. For example, (AB)1 has peripheral stalk EG1 bound to its B subunit. The black arrowheads indicate the non-catalytic A/B interfaces.


F Section through ScV1 electron density map at level of N - termini of peripheral stalks . Note change in position of N - termini of peripheral stalks , which have each crossed a non - catalytic interface ( black arrowheads in E ) are now in proximity to C - termini of catalytic A subunits ( ACT ) of adjacent A / B pairs . While EG1 EG2 are now near ACT from ( AB ) 3 ( AB ) 1 , respectively ( see double headed arrows ) , only bent EG3 peripheral stalk is close enough to contact corresponding ACT from ( AB ) 2 ( see asterisk ) .

F Section through the ScV1 electron density map at the level of the N-termini of the peripheral stalks. Note the change in position of the N-termini of the peripheral stalks, which have each crossed a non-catalytic interface (black arrowheads in E) and are now in proximity to the C-termini of the catalytic A subunits (ACT) of the adjacent A/B pairs. While EG1 and EG2 are now near ACT from (AB)3 and (AB)1, respectively (see double headed arrows), only the bent EG3 peripheral stalk is close enough to contact the corresponding ACT from (AB)2 (see asterisk).


A A3B3DF shown with subunits unique to eukaryotic V1 removed , highlighting conserved catalytic labeled as " Catalytic " . three peripheral stalks inhibitory subunit H are found only in eukaryotic V1 , whereas A3B3DF represents full complement of subunits in prokaryotic V1 . Notably , prokaryotic V1 is an active ATP hydrolase ( Arai et al , 2009 ) . orientation of A B subunits with respect to is indicated by arrow on left .

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A The A3B3DF complex shown with the subunits unique to the eukaryotic V1 removed, highlighting the core conserved catalytic complex labeled as "Catalytic core". The three peripheral stalks and inhibitory subunit H are found only in the eukaryotic V1, whereas A3B3DF represents the full complement of subunits in the prokaryotic V1 complex. Notably, the prokaryotic V1 is an active ATP hydrolase (Arai et al, 2009). The orientation of the A and B subunits with respect to the membrane is indicated by the arrow on the left.


B Conformational differences in catalytic A subunits . catalytic A subunits are shown aligned by their N - terminal β barrels , highlighting change in conformation of C - terminal α helical bundles associated with closed ( ( AB ) 2 ( AB ) 3 ; gray , respectively ) open ( ( AB ) 1 ; magenta ) catalytic sites . Movement of this domain in response to nucleotide occupancy is thought to drive rotation of central rotor during catalysis .

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B Conformational differences in the catalytic A subunits. The catalytic A subunits are shown aligned by their N-terminal β barrels, highlighting the change in conformation of C-terminal α helical bundles associated with the closed ((AB)2 and (AB)3; dark and light gray, respectively) and open ((AB)1; magenta) catalytic sites. Movement of this domain in response to nucleotide occupancy is thought to drive rotation of the central rotor during catalysis.