( 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 .

O O O O O O O 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 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 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 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 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 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

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 .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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

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

(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 .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 ) .

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

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


C Bar graphs of mean mRNA level for basal condition , 1h TNF , 1h TNF + 4h pretreatment with 300nM A - 485 , 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

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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 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 ) .

(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 .

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.


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 .

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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 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 .

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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 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 ) .

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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 . The doses used were 10 × CL for AMSIN and 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 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 .

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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 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 .

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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 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 .

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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 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

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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 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

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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 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 .

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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 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

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

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 . 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 ) 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 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 ) .

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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).