E - F . qRT - PCR ( E ) and immunostaining ( F ) confirm differential expression of genes found in MS profiling . qRT - PCR is repeated as triplicate and is presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . Student ' s t - test was used . Scale bar : 20 μm .

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E-F. qRT-PCR (E) and immunostaining (F) confirm differential expression of genes found in MS profiling. qRT-PCR is repeated as triplicate and is presented as mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Student's t-test was used. Scale bar: 20 μm.


F - J . Representative images of GFAP immunostaining ( F ) and quantification ( G - J ) in controls , Daam2 icKO , Slc4a4 icKO , and double icKO 6 weeks after tamoxifen injection . Scale bar : 20 μm . Data is presented as mean ± SEM . n = 10 - 24 images , N = 3 - 4 mice per genotype . Scale bar : 20 μm . Two - way ANOVA is used for statistics . [ * ] and [ # ] indicates comparison with control and double icKO , respectively . * * p < 0 . 01 , p * * * < 0 . 001 , # # p < 0 . 01 .

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F-J. Representative images of GFAP immunostaining (F) and quantification (G-J) in controls, Daam2 icKO, Slc4a4 icKO, and double icKO 6 weeks after tamoxifen injection. Scale bar: 20 μm. Data is presented as mean ± SEM. n=10-24 images, N=3-4 mice per genotype. Scale bar: 20 μm. Two-way ANOVA is used for statistics. [*] and [#] indicates comparison with control and double icKO, respectively. **p<0.01, p***<0.001, ##p<0.01.


Photograph of male patient with FMR1G - ins . allele . Typical physical and behavioral features of FXS were noted in the patient , who shows moderate to severe intellectual disability .

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Photograph of male patient with FMR1G-ins. allele. Typical physical and behavioral features of FXS were noted in the patient, who shows moderate to severe intellectual disability.


FMR1 mRNA levels were analyzed via RT - qPCR in EBV - transformed lymphocyte cells derived from the patient ' s blood samples . Patient cells showed a ˜ 60 % decrease in FMR1 mRNA compared to control cells ( * P = 0 . 010 , 0 . 383 ± 0 . 110 SD , n = 3 ) . Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells ( * P = 0 . 048 , 0 . 383 ± 0 . 110 SD versus 1 . 25 ± 0 . 166 SD , n = 3 ) , suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense - mediated decay . RT - qPCR reactions were run in triplicate in three independent experiments . Fold changes in FMR1 expression , normalized to HRPT expression , were calculated using the ΔΔCT method , and analyzed statistically with a two - tailed t - test ( GraphPad ) . Error bars represent mean values with SD .

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FMR1 mRNA levels were analyzed via RT-qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔCT method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD.


Western blot analysis shows a significant decrease ( > 90 % ) in FMRP protein levels in patient - derived cells ( * * * P = 0 . 008 , 0 . 13 ± 0 . 031 SD , n = 2 ) , and the patient FMRP migrates slightly lower than the wild - type protein . Band intensities for the different cell lines were quantified , normalized for actin and analyzed statistically with a twot - tailed t - test ( GraphPad ) . Error bars represent mean values with SD .

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Western blot analysis shows a significant decrease (> 90%) in FMRP protein levels in patient-derived cells (***P = 0.008, 0.13 ± 0.031 SD, n = 2), and the patient FMRP migrates slightly lower than the wild-type protein. Band intensities for the different cell lines were quantified, normalized for actin and analyzed statistically with a twot-tailed t-test (GraphPad). Error bars represent mean values with SD.


A - C HEK293 ( A ) and SH - Sy5y ( B ) cells , as well as primary neurons from FMR1 KO mice ( C ) , were transfected with vectors encoding the GFP - tagged wild - type FMRP ( GFP - FMR1WT ) or the GFP - tagged patient FMRP ( GFP - FMR1G - insertion ) under the control of a β - actin promoter . Wild - type FMRP localizes predominantly in the cytoplasm , whereas the patient FMRP forms nuclear and nucleolar aggregates that colocalize with nucleophosmin . Scale bars in ( A , B ) represent 10 μm . Scale bar in ( C ) represents 5 μm .

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A-C HEK293 (A) and SH-Sy5y (B) cells, as well as primary neurons from FMR1 KO mice (C), were transfected with vectors encoding the GFP-tagged wild-type FMRP (GFP-FMR1WT) or the GFP-tagged patient FMRP (GFP-FMR1G-insertion) under the control of a β-actin promoter. Wild-type FMRP localizes predominantly in the cytoplasm, whereas the patient FMRP forms nuclear and nucleolar aggregates that colocalize with nucleophosmin. Scale bars in (A, B) represent 10 μm. Scale bar in (C) represents 5 μm.


Transfected HEK293 cells expressing a GFP - tagged FMRP in which the C - terminus is truncated following the G - ins . mutation site ( GFP - FMR1ΔCt ) . GFP - FMR1ΔCt protein is exclusively cytoplasmic , suggesting that the truncation of the C - terminus alone is not sufficient to explain the localization change observed for the patientFMRP . Transfected HEK293 cells expressing a modified version of GFP - tagged patientFMRP ( GFP - FMR1G - ins . [ NLSmutated ] ) . The novel amino acid sequence in the C - terminus of patientFMRP [ RKRTRRKRTWRRLQRKRRSLPNR ] contains stretches of arginines and lysines predicted to function as a nuclear localization signal ( based on Expasy protein motif scanner , ScanProsite ) . Mutating adjacent R and K residues into alanines [ RARTRAARTWARLQRAARSLPNR ] abolishes the nucleolar localization of the patientFMRP , strongly suggesting that the novel amino acid sequence present in the patientFMRP C - terminus is a functional nuclear localization signal ( NLS ) .

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Transfected HEK293 cells expressing a GFP-tagged FMRP in which the C-terminus is truncated following the G-ins. mutation site (GFP-FMR1ΔCt). GFP-FMR1ΔCt protein is exclusively cytoplasmic, suggesting that the truncation of the C-terminus alone is not sufficient to explain the localization change observed for the patientFMRP.Transfected HEK293 cells expressing a modified version of GFP-tagged patientFMRP (GFP-FMR1G-ins. [NLSmutated]). The novel amino acid sequence in the C-terminus of patientFMRP [RKRTRRKRTWRRLQRKRRSLPNR] contains stretches of arginines and lysines predicted to function as a nuclear localization signal (based on Expasy protein motif scanner, ScanProsite). Mutating adjacent R and K residues into alanines [RARTRAARTWARLQRAARSLPNR] abolishes the nucleolar localization of the patientFMRP, strongly suggesting that the novel amino acid sequence present in the patientFMRP C-terminus is a functional nuclear localization signal (NLS).


Transfected HEK293 cells expressing GFP - tagged wild - type FMRP fused to the patient NLS motif ( GFP - FMR1wt + NLS ) . Unlike the patient FMRP , GFP - FMR1wt + NLS protein is predominantly cytoplasmic and does not aggregate in the nucleus . However , treatment of HEK293 cells expressing GFP - FMR1wt + NLS with leptomycin B - an inhibitor of nuclear export - resulted in the appearance of nucleolar inclusions . This suggests that the presence of a full - length C - terminus facilitates the nuclear export of FMRP in this context . Transfected HEK293 cells expressing a modified version of GFP - tagged patient FMRP , in which the truncation of the C - terminus is reverted by restoring the open reading frame ( GFP - FMR1G - ins . [ Ct restored ] ) . The patient protein is not detected in the nucleus when the C - terminus is intact ; however , nucleolar retention of the protein is observed upon leptomycin B treatment of the transfected cells . In line with results from ( A ) , this suggests that an intact C - terminus enables nuclear export of the FMR1 protein . Data information : Scale bar represents 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 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 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

Transfected HEK293 cells expressing GFP-tagged wild-type FMRP fused to the patient NLS motif (GFP-FMR1wt+NLS). Unlike the patient FMRP, GFP-FMR1wt+NLS protein is predominantly cytoplasmic and does not aggregate in the nucleus. However, treatment of HEK293 cells expressing GFP-FMR1wt+NLS with leptomycin B-an inhibitor of nuclear export-resulted in the appearance of nucleolar inclusions. This suggests that the presence of a full-length C-terminus facilitates the nuclear export of FMRP in this context.Transfected HEK293 cells expressing a modified version of GFP-tagged patient FMRP, in which the truncation of the C-terminus is reverted by restoring the open reading frame (GFP-FMR1G-ins.[Ct restored]). The patient protein is not detected in the nucleus when the C-terminus is intact; however, nucleolar retention of the protein is observed upon leptomycin B treatment of the transfected cells. In line with results from (A), this suggests that an intact C-terminus enables nuclear export of the FMR1 protein.Data information: Scale bar represents 10 μm.


The morphology of small lateral ventral neurons ( sLNvs ) is sensitive to dfmr1 activity . Overexpression of wild - type dfmr1 in the sLNv neurons results in a consistent " axonal collapse " phenotype , where the branching of axonal termini of sLNvs is reduced . Scale bars represent 50 and 10 μm ( magnified images ) .

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The morphology of small lateral ventral neurons (sLNvs) is sensitive to dfmr1 activity. Overexpression of wild-type dfmr1 in the sLNv neurons results in a consistent "axonal collapse" phenotype, where the branching of axonal termini of sLNvs is reduced. Scale bars represent 50 and 10 μm (magnified images).


Overexpression of wild - type dfmr1 ( UAS - dfmr1wt ) using a LNv neuron - specific driver line ( Pdf - Gal4 ) causes reduction of branching area - " axonal collapse " - in the sLNv axonal termini . dfmr1ΔCt and dfmr1wt + NLS overexpression phenocopies overexpression of dfmr1wt , suggesting that the truncation of the C - terminus or the presence of the patient NLS alone does not impair or alter the functional efficacy of dfmr1 in this context . However , overexpression of dfmr1ΔCt + NLS fails to cause collapse of sLNv termini , indicating a significant loss or change of Dfmrp protein function . Axonal morphology is visualized by expressing CD8 - GFP under the control of the same neuronal driver . Scale bar represents 10 μm . Quantification of axonal termini branching in LNv neurons overexpressing various dfmr1 alleles . Overexpression of dfmr1wt , dfmr1ΔCt , or dfmr1wt + NLS significantly reduced axonal branching area [ * * * * P < 0 . 0001 ; control 101 . 3 ± 29 . 76 SD , n = 30 ; UAS - dfmrwt 45 . 41 ± 21 . 69 SD , n = 29 ; UAS - dfmrΔCt 52 . 31 ± 14 . 67 SD , n = 32 ; UAS - dfmrwt + NLS 72 . 45 ± 21 . 83 SD , n = 29 ; UAS - dfmrΔCt + NLS 117 . 8 ± 36 . 96 SD , n = 28 ] . dfmr1ΔCt + NLS overexpression does not result in a significant change of axonal branching area compared to the background control [ Pdf - Gal4 , UAS - GFP ] ( P = 0 . 672 ) . For quantification purposes , each branching area ( n ) was manually outlined , starting from the first point of defasculation of the axonal bundle . These measurements were normalized for variability in brain size across samples using LNv commissure length measurements for each brain . Two - tailed t - tests using Welch ' s correction were then used to compare controls with mutant phenotypes ( GraphPad ) . Error bars represent mean values with SD . Dots visible for box plots of UAS - dfmrwt and UAS - dfmrΔCt represent samples that outlier the whiskers .

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Overexpression of wild-type dfmr1 (UAS-dfmr1wt) using a LNv neuron-specific driver line (Pdf-Gal4) causes reduction of branching area-"axonal collapse"-in the sLNv axonal termini. dfmr1ΔCt and dfmr1wt+NLS overexpression phenocopies overexpression of dfmr1wt, suggesting that the truncation of the C-terminus or the presence of the patient NLS alone does not impair or alter the functional efficacy of dfmr1 in this context. However, overexpression of dfmr1ΔCt+NLS fails to cause collapse of sLNv termini, indicating a significant loss or change of Dfmrp protein function. Axonal morphology is visualized by expressing CD8-GFP under the control of the same neuronal driver. Scale bar represents 10 μm.Quantification of axonal termini branching in LNv neurons overexpressing various dfmr1 alleles. Overexpression of dfmr1wt, dfmr1ΔCt, or dfmr1wt+NLS significantly reduced axonal branching area [****P < 0.0001; control 101.3 ± 29.76 SD, n = 30; UAS-dfmrwt 45.41 ± 21.69 SD, n = 29; UAS-dfmrΔCt 52.31 ± 14.67 SD, n = 32; UAS-dfmrwt+NLS 72.45 ± 21.83 SD, n = 29; UAS-dfmrΔCt+NLS 117.8 ± 36.96 SD, n = 28]. dfmr1ΔCt+NLS overexpression does not result in a significant change of axonal branching area compared to the background control [Pdf-Gal4, UAS-GFP] (P = 0.672). For quantification purposes, each branching area (n) was manually outlined, starting from the first point of defasculation of the axonal bundle. These measurements were normalized for variability in brain size across samples using LNv commissure length measurements for each brain. Two-tailed t-tests using Welch's correction were then used to compare controls with mutant phenotypes (GraphPad). Error bars represent mean values with SD. Dots visible for box plots of UAS-dfmrwt and UAS-dfmrΔCt represent samples that outlier the whiskers.


Overexpression of dfmr1ΔCt + NLS results in aberrant axonal termini morphology . Axonal guidance defects were frequently observed ( arrowheads ) and scored manually for bifurcations of the axon bundle ( 9 / 48 ) , tangling of axons in the termini ( 13 / 48 ) , and apparent looping of axons ( 7 / 48 ) .

Overexpression of dfmr1ΔCt+NLS results in aberrant axonal termini morphology. Axonal guidance defects were frequently observed (arrowheads) and scored manually for bifurcations of the axon bundle (9/48), tangling of axons in the termini (13/48), and apparent looping of axons (7/48).


A - D Subcellular localization of transgenically expressed dfmr1wt , dfmr1ΔCt , dfmr1wt + NLS , and dfmr1ΔCt + NLS proteins in LNv neurons . DfmrpΔCt + NLS shows nuclear localization ( A ) , colocalizing with the genetically encoded nuclear marker RedStinger , while Dfmrpwt , DfmrpΔCt , and Dfmrpwt + NLS are predominantly cytoplasmic ( B - D ) . These results suggest that an intact C - terminus can mediate the nuclear export of the Dfmrp protein . Scale bar represents 5 μm .

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A-D Subcellular localization of transgenically expressed dfmr1wt, dfmr1ΔCt, dfmr1wt+NLS, and dfmr1ΔCt+NLS proteins in LNv neurons. DfmrpΔCt+NLS shows nuclear localization (A), colocalizing with the genetically encoded nuclear marker RedStinger, while Dfmrpwt, DfmrpΔCt, and Dfmrpwt+NLS are predominantly cytoplasmic (B-D). These results suggest that an intact C-terminus can mediate the nuclear export of the Dfmrp protein. Scale bar represents 5 μm.


( 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 .

(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 .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

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 )

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

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 ) .

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

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 .

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

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 ) .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

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 .

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

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

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

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

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 .

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

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 .

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

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 ) .

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

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 ) .

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

(A, 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).


Assays were performed in HEK293 cells for A ) F508del - CFTR For the F508del - CFTR measurements since this mutant does not respond well to Fsk alone ( Fsk + VX - 770 potentiator is required to obtain a detectable signal ) we compared the CFTR inh172 ( inhibitor ) response ( F1 / ' F - CFTRinh172 ' ; where F1 represents peak RFU immediately before inhibitor addition and ' F - CFTRinh172 ' is the minimum RFU reached at the end of the CFTR inh172 measurement cycle ) . For each of F508del - CFTR data for three proteins not detected as interactors ( first three bars following CFTR only ) are shown as negative controls . Fluorescence signals were normalized to the CFTR - only control and the statistical significance of differences assed by unpaired t - test ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Samples run in n = 3 replicates , and all values are mean ± s . d .

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

Assays were performed in HEK293 cells for A) F508del-CFTR For the F508del-CFTR measurements since this mutant does not respond well to Fsk alone (Fsk + VX-770 potentiator is required to obtain a detectable signal) we compared the CFTR inh172 (inhibitor) response (F1/'F-CFTRinh172'; where F1 represents peak RFU immediately before inhibitor addition and 'F-CFTRinh172' is the minimum RFU reached at the end of the CFTR inh172 measurement cycle). For each of F508del-CFTR data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d.


Assays were performed in HEK293 cells for B ) wt - CFTR . For the wildtype measurements , we compared the maximum Fsk response ( Maximum F / F0 ; where F0 represents baseline RFU right before the stimulation ) . For each of wt - CFTR data for three proteins not detected as interactors ( first three bars following CFTR only ) are shown as negative controls . Fluorescence signals were normalized to the CFTR - only control and the statistical significance of differences assed by unpaired t - test ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Samples run in n = 3 replicates , and all values are mean ± s . d .

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

Assays were performed in HEK293 cells for B) wt-CFTR. For the wildtype measurements, we compared the maximum Fsk response (Maximum F /F0; where F0 represents baseline RFU right before the stimulation). For each of wt-CFTR data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d.


A ) Blot images showing bands B and C as well as the calnexin internal control .

A) Blot images showing bands B and C as well as the calnexin internal control.


B ) Blot quantification showing relative fold change of band B and band C versus treatment with a non - targeting siRNA ( Neg1 ) . One - way Anova followed by Dunnett ' s post hoc test was performed to determine significance versus baseline ( siNeg1 + DMSO treatment , * p < 0 . 05 ) and 5 µM VX - 661 ( # p < 0 . 05 ) . Measurements were obtained from biological triplicates ( n = 3 ) and all values are mean ± s . d .

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

B) Blot quantification showing relative fold change of band B and band C versus treatment with a non-targeting siRNA (Neg1). One-way Anova followed by Dunnett's post hoc test was performed to determine significance versus baseline (siNeg1 + DMSO treatment, *p<0.05) and 5 µM VX-661 (#p<0.05). Measurements were obtained from biological triplicates (n=3) and all values are mean ± s.d.


B ) qPCR Validation of lentivirus - mediated shRNA knockdown of FGL2 and CFTR in intestinal organoids . Significance of sample differences was calculated using the one - tailed student t - test ( n = 2 biological replicates ) . All values are mean ± s . d . EV = empty vector .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR Validation of lentivirus-mediated shRNA knockdown of FGL2 and CFTR in intestinal organoids. Significance of sample differences was calculated using the one-tailed student t-test (n=2 biological replicates). All values are mean ± s.d. EV = empty vector.


C ) Representative confocal microscopy images of calcein green labelled FIS organoids of a healthy individual and a CF patient , before and 60 min after stimulation with Fsk ( 5 uM ) . Scale bar , 200 μm .

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C) Representative confocal microscopy images of calcein green labelled FIS organoids of a healthy individual and a CF patient, before and 60 min after stimulation with Fsk (5 uM). Scale bar, 200 μm.


D ) Quantification of FIS response in wt - CFTR organoids ( ORG - 01 ( HC ) ) shown in ( C ) as area under the curve at 60 min . Significance of sample differences was calculated using two - way ANOVA ( n = 2 biological replicates ) . All values are mean ± s . d . E ) Quantification of FIS response in F508del - CFTR organoids ( ORG - BX - 002 ( dF / dF ) ) shown in ( C ) as area under the curve at 60 min . Significance of sample differences was calculated using two - way ANOVA ( n = 2 biological replicates ) . All values are mean ± s . d .

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D) Quantification of FIS response in wt-CFTR organoids (ORG-01(HC)) shown in (C) as area under the curve at 60 min. Significance of sample differences was calculated using two-way ANOVA (n=2 biological replicates). All values are mean ± s.d. E) Quantification of FIS response in F508del-CFTR organoids (ORG-BX-002 (dF/dF)) shown in (C) as area under the curve at 60 min. Significance of sample differences was calculated using two-way ANOVA (n=2 biological replicates). All values are mean ± s.d. F


( B ) Western blot analysis of SARS - CoV - 2 Spike 1 - 1273 , 1 - 674 , and 319 - 591 expression in HEK293T cells . The black arrows indicate the location of the induced target proteins .

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(B) Western blot analysis of SARS-CoV-2 Spike 1-1273, 1-674, and 319-591 expression in HEK293T cells. The black arrows indicate the location of the induced target proteins.


( C ) Detection of decoy antibody ACE2 - Fc and Spike S1 - Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue staining .

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(C) Detection of decoy antibody ACE2-Fc and Spike S1-Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue staining.


( D ) Recognition of the ACE2 - Fc and Spike S1 - Fc by anti - human IgG Fc antibody in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( SDS - PAGE ) .

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(D) Recognition of the ACE2-Fc and Spike S1-Fc by anti-human IgG Fc antibody in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).


( E , F ) Deglycosylation of ACE2 - Fc and Spike 1 - 674 - Fc by PNGase F . PNGase F digested ACE2 - Fc ( 500 ng ) and Spike 1 - 674 - Fc ( 500 ng ) were subjected to Coomassie Brilliant Blue staining ( E ) and Western blot analysis by anti - human IgG Fc antibody ( F ) .

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(E, F) Deglycosylation of ACE2-Fc and Spike 1-674-Fc by PNGase F. PNGase F digested ACE2-Fc (500 ng) and Spike 1-674-Fc (500 ng) were subjected to Coomassie Brilliant Blue staining (E) and Western blot analysis by anti-human IgG Fc antibody (F).


( A ) Competitive ELISA . ACE2 - Fc can compete with ACE2 - Fc - Biotin for Spike 1 - 674 binding in a dose - dependent manner . The competition effects of ACE2 - Fc on ACE2 - Spike protein binding was normalized to PBS control . Error bars represent the standard deviation ( SD ) , n = 2 . Statistical analysis was performed by unpaired two tail t - test . * * P < 0 . 01 , * * * P < 0 . 001 .

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(A) Competitive ELISA. ACE2-Fc can compete with ACE2-Fc-Biotin for Spike 1-674 binding in a dose-dependent manner. The competition effects of ACE2-Fc on ACE2-Spike protein binding was normalized to PBS control. Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. **P < 0.01, ***P < 0.001.


( B ) Recognition of full - length Spike on H1975 ( left panel as negative control ) or H1975 - Spike ( right panel ) cells by ACE2 - Fc using flow cytometry analysis . Isotype control : 40 μg / mL mouse IgG - FITC .

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(B) Recognition of full-length Spike on H1975 (left panel as negative control) or H1975-Spike (right panel) cells by ACE2-Fc using flow cytometry analysis. Isotype control: 40 μg/mL mouse IgG-FITC.


( C ) Confocal microscopy of H1975 - Spike - overexpressing cells . Spikes on H1975 cells were stained with anti - Spike antibody and Alexa Fluor ® 594 - conjugated secondary antibody on ice for 1 hr . After that , ACE2 - Fc - FITC was incubated for another 1 hr . Scale bars equal to 20 µm . DAPI was used as a nuclear counterstain .

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(C) Confocal microscopy of H1975-Spike-overexpressing cells. Spikes on H1975 cells were stained with anti-Spike antibody and Alexa Fluor® 594-conjugated secondary antibody on ice for 1 hr. After that, ACE2-Fc-FITC was incubated for another 1 hr. Scale bars equal to 20 µm. DAPI was used as a nuclear counterstain.


( D ) Preservation of ACE2 - Fc peptidase activity . The peptidase activity of ACE2 - Fc was measured by cleavage of the fluorescent peptide substrates .

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(D) Preservation of ACE2-Fc peptidase activity. The peptidase activity of ACE2-Fc was measured by cleavage of the fluorescent peptide substrates.


( E ) Inhibition of angiotensin II ( Ang II ) - induced TNF - α production by ACE2 - Fc . Ang II was preincubated with indicated amounts of ACE2 - Fc or IgG for 30 minutes . After that , the mixtures were added to RAW264 . 7 cells for 12 hr . The concentration of TNF - α in the culture medium was determined by ELISA . Error bars represent the standard deviation ( SD ) , n = 3 . Statistical analysis was performed by unpaired two tail t - test . * P < 0 . 05 . The dotted line represents the mean value of TNF - α in control group .

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(E) Inhibition of angiotensin II (Ang II)-induced TNF-α production by ACE2-Fc. Ang II was preincubated with indicated amounts of ACE2-Fc or IgG for 30 minutes. After that, the mixtures were added to RAW264.7 cells for 12 hr. The concentration of TNF-α in the culture medium was determined by ELISA. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. *P < 0.05. The dotted line represents the mean value of TNF-α in control group.


( F ) Inhibition of Ang II - induced ADAM17 ( a disintegrin and metalloprotease 17 ) phosphorylation by ACE2 - Fc . The protein extracts from ( E ) were immunoblotted with the indicated antibodies ( left panel ) . β - actin served as the loading control . The signal intensity was normalized to cells only ( right panel ) . Data are representative of three independent experiments , and the values are expressed as the mean ± SD ( right panel ) . Error bars represent the standard deviation ( SD ) , n = 2 . Statistical analysis was performed by unpaired two tail t - test . * P < 0 . 05 . IgG represents the human normal IgG control .

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(F) Inhibition of Ang II-induced ADAM17 (a disintegrin and metalloprotease 17) phosphorylation by ACE2-Fc. The protein extracts from (E) were immunoblotted with the indicated antibodies (left panel). β-actin served as the loading control. The signal intensity was normalized to cells only (right panel). Data are representative of three independent experiments, and the values are expressed as the mean ± SD (right panel). Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. *P < 0.05. IgG represents the human normal IgG control.


( B ) HEK293 and H1975 cells were transduced with full - length ACE2 by lentivirus . Protein extracts were immunoblotted with the indicated antibodies . O / E represents overexpression .

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(B) HEK293 and H1975 cells were transduced with full-length ACE2 by lentivirus. Protein extracts were immunoblotted with the indicated antibodies. O/E represents overexpression.


( C ) GFP and full - length Spike co - transfected HEK293T cells were used as effector cells ( 293 - S ) . HEK293 / ACE2 ( HEK293T - overexpressing ACE2 ) ( left panel ) or H1975 / ACE2 ( H1975 - overexpressing ACE2 ) ( right panel ) cells were used as target cells . The 293 - S cells were preincubated with normal human IgG or ACE2 - Fc at 37 ° C for 1 hr . After that , the mixtures were added to target cells for 4 hr ( cell - cell fusion ) or 24 hr ( syncytia formation ) . The fluorescent areas were measured by inverted fluorescence microscopy and Metamorph software ( Metamorphosis ) . Scale bars equal to 50 µm . The white arrows indicate the cell - cell fusion or syncytia formation .

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(C) GFP and full-length Spike co-transfected HEK293T cells were used as effector cells (293-S). HEK293/ACE2 (HEK293T-overexpressing ACE2) (left panel) or H1975/ACE2 (H1975-overexpressing ACE2) (right panel) cells were used as target cells. The 293-S cells were preincubated with normal human IgG or ACE2-Fc at 37°C for 1 hr. After that, the mixtures were added to target cells for 4 hr (cell-cell fusion) or 24 hr (syncytia formation). The fluorescent areas were measured by inverted fluorescence microscopy and Metamorph software (Metamorphosis). Scale bars equal to 50 µm. The white arrows indicate the cell-cell fusion or syncytia formation.


( D ) The inhibition of cell - cell fusion and syncytia formation by ACE2 - Fc in HEK293 / ACE2 and H1975 / ACE2 was determined with the formula described in the Methods section . Error bars represent the standard deviation ( SD ) , n = 6 . Statistical analysis was performed by unpaired two tail t - test . * * P < 0 . 01 , * * * P < 0 . 001 .

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(D) The inhibition of cell-cell fusion and syncytia formation by ACE2-Fc in HEK293/ACE2 and H1975/ACE2 was determined with the formula described in the Methods section. Error bars represent the standard deviation (SD), n=6. Statistical analysis was performed by unpaired two tail t-test. ** P < 0.01, *** P < 0.001.


( A ) ACE2 - Fc blocked the entry of Spike - expressing pseudotyped lentivirus into HEK293T - ACE2 and H1975 - ACE2 cells . The relative luciferase activities , normalized to the only virus group , represent the efficiency of virus entry . MOI : Multiplicity of infection .

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(A) ACE2-Fc blocked the entry of Spike-expressing pseudotyped lentivirus into HEK293T-ACE2 and H1975-ACE2 cells. The relative luciferase activities, normalized to the only virus group, represent the efficiency of virus entry. MOI: Multiplicity of infection.


( B ) Confocal microscopy images of the airway organoids . The green fluorescence represents the specific staining of indicated monoclonal antibodies and Alexa Fluor 488 - conjugated secondary antibodies . DAPI was used as a nuclear counterstain . Hematoxylin and eosin ( H & E ) stain : hematoxylin stained the nuclei in blue color ; eosin stained the cytoplasm in pink color . Scale bars equal to 20 µm .

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(B) Confocal microscopy images of the airway organoids. The green fluorescence represents the specific staining of indicated monoclonal antibodies and Alexa Fluor 488-conjugated secondary antibodies. DAPI was used as a nuclear counterstain. Hematoxylin and eosin (H&E) stain: hematoxylin stained the nuclei in blue color; eosin stained the cytoplasm in pink color. Scale bars equal to 20 µm.


( C ) Lung cancer A549 cells , human normal bronchial epithelial cells ( HBEpc ) , and HBEpc - differentiated cells ( airway organoids ) were harvested for immunoblotting with the indicated antibodies .

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(C) Lung cancer A549 cells, human normal bronchial epithelial cells (HBEpc), and HBEpc-differentiated cells (airway organoids) were harvested for immunoblotting with the indicated antibodies.


( D ) Blockage of pseudovirus entry into airway organoids by ACE2 - Fc . Mixtures of pseudotyped lentivirus with or without ACE2 - Fc were cocultured with airway organoids for 72 hr . The virus entry was determined by quantifying the luciferase activity in the cell lysates .

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(D) Blockage of pseudovirus entry into airway organoids by ACE2-Fc. Mixtures of pseudotyped lentivirus with or without ACE2-Fc were cocultured with airway organoids for 72 hr. The virus entry was determined by quantifying the luciferase activity in the cell lysates.


( A ) Inhibition of SARS - CoV - 2 infection by ACE2 - Fc in the plaque assay . Mixtures of ACE2 - Fc and SARS - CoV - 2 were incubated for one hour before adding to Vero E6 cells for another 1 hr at 37 ° C . The ACE2 - Fc and SARS - CoV - 2 premixtures were removed , and the cells were washed once with PBS and overlaid with methylcellulose with 2 % FBS for 5 - 7 days before being stained with crystal violet . Those results showed increase of plaque formation was regarded as no inhibition of plaque formation . TPCK - treated trypsin : N - tosyl - L - phenylalanine chloromethyl ketone - treated trypsin .

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(A) Inhibition of SARS-CoV-2 infection by ACE2-Fc in the plaque assay. Mixtures of ACE2-Fc and SARS-CoV-2 were incubated for one hour before adding to Vero E6 cells for another 1 hr at 37°C. The ACE2-Fc and SARS-CoV-2 premixtures were removed, and the cells were washed once with PBS and overlaid with methylcellulose with 2% FBS for 5-7 days before being stained with crystal violet. Those results showed increase of plaque formation was regarded as no inhibition of plaque formation. TPCK-treated trypsin: N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin.


Yield reduction assay was performed to determine the inhibitory effects of ACE2 - Fc on virus titers The culture medium and cell extracts were harvested 24 hours post - infection for real - time PCR ( B )

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Yield reduction assay was performed to determine the inhibitory effects of ACE2-Fc on virus titers The culture medium and cell extracts were harvested 24 hours post-infection for real-time PCR (B)


Yield reduction assay was performed to determine the inhibitory effects of ACE2 - Fc on protein expression . The culture medium and cell extracts were harvested 24 hours post - infection for western blot ( C ) . The NP / PCNA represents the relative NP expression as compared to that of PCNA , which served as a loading control . The NP / PCNA numbers below the panel are the ratios of NP / PCNA normalized to that of the human IgG control group . PCNA : Proliferating cell nuclear antigen .

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Yield reduction assay was performed to determine the inhibitory effects of ACE2-Fc on protein expression. The culture medium and cell extracts were harvested 24 hours post-infection for western blot (C). The NP/PCNA represents the relative NP expression as compared to that of PCNA, which served as a loading control. The NP/PCNA numbers below the panel are the ratios of NP/PCNA normalized to that of the human IgG control group. PCNA: Proliferating cell nuclear antigen.


( E ) The cell lysates from the pretreatment and full - time experiments were harvested and immunoblotted with the indicated antibodies .

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(E) The cell lysates from the pretreatment and full-time experiments were harvested and immunoblotted with the indicated antibodies.


( F ) The SARS - CoV - 2 titer in the pretreatment and full - time experiments was analyzed by real - time PCR .

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(F) The SARS-CoV-2 titer in the pretreatment and full-time experiments was analyzed by real-time PCR.


( A ) The ( A ) NP proteins in the cell lysates were determined by Western blot analysis .

(A) The (A) NP proteins in the cell lysates were determined by Western blot analysis.


( B , C ) The virus RNA in the ( B ) culture medium and ( C ) cell lysates was quantified by real - time RT - PCR . The genetic information and sequence reference numbers for the 5 SARS - CoV - 2 strains were summarized in Table EV1 . Error bars represent the standard deviation ( SD ) , n = 3 . Statistical analysis was performed by unpaired two tail t - test . * * P < 0 . 01 , * * * P < 0 . 001 . Experiments were performed at least three times with similar results .

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(B, C) The virus RNA in the (B) culture medium and (C) cell lysates was quantified by real-time RT-PCR. The genetic information and sequence reference numbers for the 5 SARS-CoV-2 strains were summarized in Table EV1. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. ** P < 0.01, *** P < 0.001. Experiments were performed at least three times with similar results.


( A ) H1975 cells were transduced with full - length Spike by Lentiviral vector . Protein extracts were immunoblotted with the indicated antibodies . O / E represents overexpress .

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(A) H1975 cells were transduced with full-length Spike by Lentiviral vector. Protein extracts were immunoblotted with the indicated antibodies. O/E represents overexpress.


( B ) The effect of ACE2 - Fc activation on degranulative capacity of NK cells . The experiments were performed with the primary human NK cells derived from three independent donors . Error bars represent the standard deviation ( SD ) , n = 3 . Statistical analysis was performed by unpaired two tail t - test . * P < 0 . 05 ( each concentration of ACE2 - Fc vs control ) .

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(B) The effect of ACE2-Fc activation on degranulative capacity of NK cells. The experiments were performed with the primary human NK cells derived from three independent donors. Error bars represent the standard deviation (SD), n= 3. Statistical analysis was performed by unpaired two tail t-test. * P < 0.05 (each concentration of ACE2-Fc vs control).


( C ) The CD107a , IFN - γ , and TNF - α expression levels were determined by flow cytometry after the NK cells and H1975 - Spike cells coincubation in the presence or absence of ACE2 - Fc or ACE2 control . Error bars represent the standard deviation ( SD ) , n = 3 . Statistical analysis was performed by unpaired two tail t - test . * P < 0 . 05 . The percentage of positive cells in both groups were normalized to the NK / H1975 - Spike only group .

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(C) The CD107a, IFN-γ, and TNF-α expression levels were determined by flow cytometry after the NK cells and H1975-Spike cells coincubation in the presence or absence of ACE2-Fc or ACE2 control. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. * P < 0.05. The percentage of positive cells in both groups were normalized to the NK/H1975-Spike only group.


( D ) The differential expression of degranulation markers after the treatment of ACE2 - Fc or ACE2 by the flow cytometry . The numbers in each plot indicate the percentages of positive cells .

(D) The differential expression of degranulation markers after the treatment of ACE2-Fc or ACE2 by the flow cytometry. The numbers in each plot indicate the percentages of positive cells.


( G ) Fold change in total RNA modification levels upon loss of m5C . Fold changes were calculated by dividing the peak area ratio of noNSUN samples by the one of wild type samples . n = 3 independent biological replicates . Multiple t - tests . Data information : In G ) , data are presented as mean ± SEM .

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(G) Fold change in total RNA modification levels upon loss of m5C. Fold changes were calculated by dividing the peak area ratio of noNSUN samples by the one of wild type samples. n = 3 independent biological replicates. Multiple t-tests. Data information: In G), data are presented as mean ± SEM.


( A , B ) Site - specific methylation analysis by whole - transcriptome bisulfite sequencing . Scatter plots show individual cytosines and their respective non - conversion rates in rRNAs ( A ) and tRNAs ( B ) of wild type and noNSUN strains ; pie chart showing most frequently methylated tRNA isoacceptors . Data information : In ( A , B n = 3 independent biological replicates .

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(A, B) Site-specific methylation analysis by whole-transcriptome bisulfite sequencing. Scatter plots show individual cytosines and their respective non-conversion rates in rRNAs (A) and tRNAs (B) of wild type and noNSUN strains; pie chart showing most frequently methylated tRNA isoacceptors. Data information: In (A, B n = 3 independent biological replicates.


( A ) RNA bisulfite sequencing map for mitochondrial tRNA Met - CAU in wild type ( top ) and noNSUN ( bottom ) strains . Each row represents one sequence read and each column one cytosine . Data information : In ( A ) , a representative map of the replicates is shown , n = 3 independent biological replicates . Similar effects were observed in all replicates analysed .

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(A) RNA bisulfite sequencing map for mitochondrial tRNA Met-CAU in wild type (top) and noNSUN (bottom) strains. Each row represents one sequence read and each column one cytosine. Data information: In (A), a representative map of the replicates is shown, n = 3 independent biological replicates. Similar effects were observed in all replicates analysed.


D ) Viable progeny counts of wild - type and noNSUN strains at 20 ° C and 25 ° C ( D ) . Automatic counting was done using a Matlab script which processed plate images in real - time . Data information : In ( D ) , data are presented as mean ± SEM , unpaired two - tailed Student ' s t - test .

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D) Viable progeny counts of wild-type and noNSUN strains at 20°C and 25°C (D). Automatic counting was done using a Matlab script which processed plate images in real-time. Data information: In (D), data are presented as mean ± SEM, unpaired two-tailed Student's t-test.


( D ) Translation efficiency of UUG - enriched , leucine - enriched , proline - enriched and random genes in each sample . A gene was considered enriched in a certain codon when the proportion of this codon in the gene was at least 3 - fold higher than the proportion of the same codon across the transcriptome . Data information : In n = 3 biological replicates . In ( D ) , boxplots show the median ( central band ) and IQR ( boxes ) ± 1 . 5 x IQR ( whiskers ) , Welch ' s t - test , p - value < 0 . 05 .

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(D) Translation efficiency of UUG-enriched, leucine-enriched, proline-enriched and random genes in each sample. A gene was considered enriched in a certain codon when the proportion of this codon in the gene was at least 3-fold higher than the proportion of the same codon across the transcriptome. Data information: In n = 3 biological replicates. In (D), boxplots show the median (central band) and IQR (boxes) ± 1.5 x IQR (whiskers), Welch's t-test, p-value < 0.05.


C Western blot analysis of isolated mouse heart mitochondria from 12 - week - old animals .

C Western blot analysis of isolated mouse heart mitochondria from 12-week-old animals.


D Survival of indicated mice presented as percent of total number of mice analyzed . n = 60 ( per genotype ) , median survival of TAZG197V mice is 24 . 5 weeks , Mantel - Cox test : p < 0 . 0001 * * * * .

D Survival of indicated mice presented as percent of total number of mice analyzed. n=60 (per genotype), median survival of TAZG197V mice is 24.5 weeks, Mantel-Cox test: p < 0.0001 * * * *.


E Quantification of neutrophils , percent of white blood cells ( left ) , white blood cells ( middle ) , and red blood cells ( right ) of 12 - week - old mice . Mean ± SEM , n = 6 , unpaired t test : p < 0 . 0001 * * * * , ns ( non - significant ) .

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E Quantification of neutrophils, percent of white blood cells (left), white blood cells (middle), and red blood cells (right) of 12-week-old mice. Mean ± SEM, n=6, unpaired t test: p < 0.0001 * * * *, ns (non-significant).


I Picrosirius red staining of histology sections of hearts from WT and TAZ mutant mice .

I Picrosirius red staining of histology sections of hearts from WT and TAZ mutant mice.


J qPCR analysis of ANP ( left ) and BNP ( right ) from total mRNA isolated from heart lysates of 20 - week - old mice . Mean ± SEM , n = 5 for ANP , n = 4 for BNP , unpaired t test : p < 0 . 05 * , p < 0 . 001 * * *

J qPCR analysis of ANP (left) and BNP (right) from total mRNA isolated from heart lysates of 20-week-old mice. Mean ± SEM, n=5 for ANP, n=4 for BNP, unpaired t test: p < 0.05 *, p < 0.001 * * *


A Mass spectrometric analysis of Cardiolipin and Monolysocardiolipin in WT and TAZG197V mouse heart . Mean ± SEM , n = 3 , unpaired t test : p < 0 . 05 * , p < 0 . 01 * * .

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A Mass spectrometric analysis of Cardiolipin and Monolysocardiolipin in WT and TAZG197V mouse heart. Mean ± SEM, n=3, unpaired t test: p < 0.05 *, p < 0.01 * *.


B Western blot analysis of BN - PAGE separted protein complexes from digitonin solubilized heart mitochondria ( 12 - week - old mice ) .

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B Western blot analysis of BN-PAGE separted protein complexes from digitonin solubilized heart mitochondria (12-week-old mice).


D Steady state protein levels of isolated mitochondria from 12 - week - old mouse hearts .

D Steady state protein levels of isolated mitochondria from 12-week-old mouse hearts.


E and F Oxygen consumption rate of isolated heart mitochondria from 12 - week - old mice using succinate E and pyruvate F as substrates . Mean ± SEM , n = 3 , unpaired t test : p < 0 . 001 * * * , p < 0 . 0001 * * * G Flow cytometric analysis of isolated mitochondria using MitoSOX Red staining . Mean ± SEM , n = 3 , unpaired t test : p < 0 . 05 * .

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E and F Oxygen consumption rate of isolated heart mitochondria from 12-week-old mice using succinate E and pyruvate F as substrates. Mean ± SEM, n=3, unpaired t test: p < 0.001 * * *, p < 0.0001 * * * G Flow cytometric analysis of isolated mitochondria using MitoSOX Red staining. Mean ± SEM, n=3, unpaired t test: p < 0.05 *.


B Volcano plot of differentially expressed genes between 14 - week - old WT and TAZG197V mice . X - axis denotes fold change in expression ( log2 scale ) , y - axis denotes adjusted p - value ( negative log10 scale ) for the analyzed genes in the data ( each dot represents one single gene ) . Blue and red dots represent genes significantly downregulated ( DR ) and upregulated ( UR ) , respectively , in the TAZG197V mice compared to WT mice . The cut - offs used were : adjusted p - value < 0 . 05 and fold change > 1 . 15 or < - 1 . 15 . Non - significant ( NS ) genes are depicted in black . Selected genes involved in pathways relevant for the study are labelled in the plot .

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B Volcano plot of differentially expressed genes between 14-week-old WT and TAZG197V mice. X-axis denotes fold change in expression (log2 scale), y-axis denotes adjusted p-value (negative log10 scale) for the analyzed genes in the data (each dot represents one single gene). Blue and red dots represent genes significantly downregulated (DR) and upregulated (UR), respectively, in the TAZG197V mice compared to WT mice. The cut-offs used were: adjusted p-value < 0.05 and fold change > 1.15 or < -1.15. Non-significant (NS) genes are depicted in black. Selected genes involved in pathways relevant for the study are labelled in the plot.


F Western Blot analysis of BN - PAGE separated complexes from digitonin solubilized heart mitochondria ( 12 - week - old mice ) .

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F Western Blot analysis of BN-PAGE separated complexes from digitonin solubilized heart mitochondria (12-week-old mice).


G Steady state protein levels of isolated heart mitochondria ( 12 - week - old mice ) . CPT1B and CPT2 protein levels were normalized to VDAC . Mean ± SEM , n = 5 , unpaired t test : p < 0 . 01 * * , p < 0 . 001 * *

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G Steady state protein levels of isolated heart mitochondria (12-week-old mice). CPT1B and CPT2 protein levels were normalized to VDAC. Mean ± SEM, n=5, unpaired t test: p < 0.01* *, p < 0.001* *


C Western blot steady state protein levels of CPT1B and CPT2 . Values were normalized to β - tubulin in control and iTAZ iPSC - cardiomyocyte . Mean of WT values was set to 1 . Mean ± SEM , n = 5 , unpaired t test : p < 0 . 05 * , p < 0 . 0001 * * * * .

C Western blot steady state protein levels of CPT1B and CPT2. Values were normalized to β-tubulin in control and iTAZ iPSC-cardiomyocyte. Mean of WT values was set to 1. Mean ± SEM, n=5, unpaired t test: p < 0.05 *, p < 0.0001* * * *.


D Real time respirometry . Left , Oxygen consumption measured as basal , and upon addition of oligomycin , CCCP , and antimycin / rotenone using palmitate - BSA as substrate , ETO , etomoxir . Right , quantification of basal and maximal respiration . Mean ± SEM , n = 4 , unpaired t test : p < 0 . 01 * * , p < 0 . 0001 * * * *

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D Real time respirometry. Left, Oxygen consumption measured as basal, and upon addition of oligomycin, CCCP, and antimycin/rotenone using palmitate-BSA as substrate, ETO, etomoxir. Right, quantification of basal and maximal respiration. Mean ± SEM, n=4, unpaired t test: p < 0.01* *, p < 0.0001* * * *


E Extracellular acidification rate of control and iTAZ iPSC - cardiomyocytes . Basal state and upon sequential addition of glucose , oligomycin and 2 deoxyglucose ( 2 - DG ) . Right , quantification of glycolytic capacity . Mean ± SEM , n = 6 , unpaired t test : p < 0 . 001 * * * *

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E Extracellular acidification rate of control and iTAZ iPSC-cardiomyocytes. Basal state and upon sequential addition of glucose, oligomycin and 2 deoxyglucose (2-DG). Right, quantification of glycolytic capacity. Mean ± SEM, n=6, unpaired t test: p < 0.001* * * *


A Western blot analysis of steady state protein levels of untreated ( US ) and cells starved ( S ) for indicated times .

A Western blot analysis of steady state protein levels of untreated (US) and cells starved (S) for indicated times.


C Glycolytic and mitochondrial ATP production measured using a real time ATP rate assay . Mean ± SEM , n = 3 , unpaired t test : p < 0 . 001 * * * .

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C Glycolytic and mitochondrial ATP production measured using a real time ATP rate assay. Mean ± SEM, n=3, unpaired t test: p < 0.001* * *.


D Western blot analysis of steady state AMPK and pAMPK protein levels of starved and / or A769662 ( AMPK activator ) treated cells as indicated .

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D Western blot analysis of steady state AMPK and pAMPK protein levels of starved and/or A769662 (AMPK activator) treated cells as indicated.


A Ejection fraction from short axis expressed in % , B Fractional shortening in % , Mean ± SEM , n = 15 ( WT control ) ; 4 ( TAZG197V control ) ; 9 ( WT treated ) ; 7 ( TAZG197V treated ) , 2Way ANOVA : p < 0 . 05 * , p < 0 . 01 * *

A Ejection fraction from short axis expressed in %, B Fractional shortening in %, Mean ± SEM, n=15 (WT control); 4 (TAZG197V control); 9 (WT treated); 7 (TAZG197V treated), 2Way ANOVA: p < 0.05 *, p < 0.01 * *


F Gene expression addressed by qPCR of ANP ( left ) and BNP ( right ) in 24 - week - old WT and TAZG197V mice heart upon 6 - week treatment with A769662 . Mean ± SEM , n = 4 , unpaired t test : ns ( non - significant )

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F Gene expression addressed by qPCR of ANP (left) and BNP (right) in 24-week-old WT and TAZG197V mice heart upon 6-week treatment with A769662. Mean ± SEM, n=4, unpaired t test: ns (non-significant)


G Quantification of steady state levels of AMPK normalized to beta tubulin in tissue lysates from 24 - week - old WT and TAZG197V mice heart from untreated control or following a 6 - week treatment with A769662 . The mean of the WT values was set to 1 . Mean ± SEM , n = 7 ( control ) ; 5 ( treated ) , unpaired t test : p < 0 . 01 * *

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G Quantification of steady state levels of AMPK normalized to beta tubulin in tissue lysates from 24-week-old WT and TAZG197V mice heart from untreated control or following a 6-week treatment with A769662. The mean of the WT values was set to 1. Mean ± SEM, n=7 (control); 5 (treated), unpaired t test: p < 0.01 * *


I Real time respirometry , oxygen consumption rate , of isolated heart mitochondria from 24 - week - old TAZG197V mice , from untreated control or following a 6 - week treatment with A769662 , driven by palmitoyl / carnitine / malate / ADP . The OCR of the untreated WT samples was taken as 100 % . Mean ± SEM , n = 3 , unpaired t test : p < 0 . 01 * *

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I Real time respirometry, oxygen consumption rate, of isolated heart mitochondria from 24-week-old TAZG197V mice, from untreated control or following a 6-week treatment with A769662, driven by palmitoyl/carnitine/malate/ADP. The OCR of the untreated WT samples was taken as 100%. Mean ± SEM, n=3, unpaired t test: p < 0.01 * *


F Whole - mount visualization of mitochondria in injured hypoglossal motor axon at 5 days after injury . G Immunostaining of injured hypoglossal nerves for GFP and cytochrome c ( Cytc ) at 5 days after injury . H Number of mitochondria in injured axons of Atf3 : BAC2 Tg and Rpt3 CKO mice at 5 days after injury ( n = 5 mice per group ) . I Total number of GFP - positive injured motor neurons in Atf3 : BAC2 Tg and Rpt3 CKO mice at 5 days after injury ( n = 4 mice per group ) . J Number of GFP - positive mitochondria in soma at 5 days after injury . Data are shown as the mean ± s . e . m . ( n = 3 mice ) . Data information : Data are shown as the mean ± s . e . m . , * * P = 0 . 0005 in ( H ) , no significant difference ( P = 0 . 8915 ) in ( I ) and * P = 0 . 0068 in ( J ) , determined by unpaired t - test . Dashed lines reveal hypoglossal nucleus in axon in ( F and G ) . Scale bars , 5 μm in ( F ) and 2 . 5 μm in ( G ) .

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F Whole-mount visualization of mitochondria in injured hypoglossal motor axon at 5 days after injury. G Immunostaining of injured hypoglossal nerves for GFP and cytochrome c (Cytc) at 5 days after injury. H Number of mitochondria in injured axons of Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 5 mice per group). I Total number of GFP-positive injured motor neurons in Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 4 mice per group). J Number of GFP-positive mitochondria in soma at 5 days after injury. Data are shown as the mean ± s.e.m. (n= 3 mice). Data information: Data are shown as the mean ± s.e.m., ** P = 0.0005 in (H), no significant difference (P = 0.8915) in (I) and * P = 0.0068 in (J), determined by unpaired t-test. Dashed lines reveal hypoglossal nucleus in axon in (F and G). Scale bars, 5 μm in (F) and 2.5 μm in (G).


C Thionine - stained motor neurons of Atf3 : BAC2 Tg and Rpt3 CKO mice at 7 , 14 and 17 days after hypoglossal nerve axotomy . Arrows indicate the loss of motor neurons . D The graph showing the percentage of surviving motor neurons on the injured side compared with that on the control side . Data are shown as the mean ± s . e . m . ( * * P = 0 . 0002 , * * * P < 0 . 0001 , determined by unpaired t - test , n = 5 mice per group at each time point ) . Data information : Dashed lines outline hypoglossal nucleus in XII , hypoglossal nucleus ; cc , central canal . Scale bars , 150 μm in C )

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C Thionine-stained motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice at 7, 14 and 17 days after hypoglossal nerve axotomy. Arrows indicate the loss of motor neurons. D The graph showing the percentage of surviving motor neurons on the injured side compared with that on the control side. Data are shown as the mean ± s.e.m. (** P = 0.0002, *** P < 0.0001, determined by unpaired t-test, n = 5 mice per group at each time point). Data information: Dashed lines outline hypoglossal nucleus in XII, hypoglossal nucleus; cc, central canal. Scale bars, 150 μm in C)


C Hypoglossal motor neurons of Atf3 : BAC2 Tg and Rpt3 CKO mice before and at 5 days ( 5d ) and 28 days ( 28d ) after injury , immunostained by AnkG and GFP . Arrows indicate the AIS stained by AnkG . Scale bars , 20 μm . D Quantification of the number of the AIS in control ( Cont ) and injured ( Inj ) hypoglossal motor neurons of Atf3 : BAC2 and Rpt3 CKO mice . Data are expressed as mean ± s . e . m . * P = 0 . 043 , * * P = 0 . 0162 , * * * P = 0 . 008 , * * * * P < 0 . 0001 , determined by one - way ANOVA followed by Tukey post hoc analysis , n . d . , not detected , n = 5 mice for each group . E Quantification of the AIS length in control ( Cont ) and injured ( Inj ) hypoglossal motor neurons of Atf3 : BAC2 and Rpt3 CKO mice . Data are expressed as mean ± s . e . m . * P = 0 . 0087 , * * P = 0 . 0024 , * * * P = 0 . 0007 , determined by one - way ANOVA followed by Tukey post hoc analysis , n . d . , not detected , n = 5 mice for each group .

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C Hypoglossal motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice before and at 5 days (5d) and 28 days (28d) after injury, immunostained by AnkG and GFP. Arrows indicate the AIS stained by AnkG. Scale bars, 20 μm. D Quantification of the number of the AIS in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.043, ** P = 0.0162, *** P = 0.008, **** P < 0.0001, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group. E Quantification of the AIS length in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.0087, ** P = 0.0024, *** P = 0.0007, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group.


B Ubiquitination assay of AnkG protein . The lysates of COS - 7 cells expressing the indicated truncated AnkG tagged with GST and HA - Ubiquitin were pulled down with glutathione Sepharose beads , followed by western blotting with anti - HA and anti - GST antibodies .

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B Ubiquitination assay of AnkG protein. The lysates of COS-7 cells expressing the indicated truncated AnkG tagged with GST and HA-Ubiquitin were pulled down with glutathione Sepharose beads, followed by western blotting with anti-HA and anti-GST antibodies.


C Western blot of hypoglossal nuclei ( left ) and nerves ( right ) at 5 days after injury with anti - AnkG , RPT3 and GAPDH antibodies . AnkG products of 190 / 270 / 480 kDa are indicated by arrowheads . The positions of the degraded products by calpain are shown by asterisks at ~ 95 kDa and 72 kDa .

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C Western blot of hypoglossal nuclei (left) and nerves (right) at 5 days after injury with anti-AnkG, RPT3 and GAPDH antibodies. AnkG products of 190/ 270/ 480 kDa are indicated by arrowheads. The positions of the degraded products by calpain are shown by asterisks at ~95 kDa and 72 kDa.


C The localization of AnkG and GFP - labeled mitochondria in the AIS of uninjured MN of Thy1 - Mito Tg mice and injured motor neurons of Atf3 : BAC2 Tg and Rpt3 CKO mice . D Graphs show the GFP and AnkG fluorescent intensity scans corresponding to ( C ) over a 20 μm line running from the soma to the axon . E Graph represents the percentage ( % ) of GFP intensity in the AIS , compared with that in the AIS of Thy1 - Mito mouse ( n = 5 mice per group ) . Data information : For ( E , data are shown as the mean ± s . e . m . , * * P = 0 . 0003 , * * * P < 0 . 0001 in ( E determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . In closed arrowheads denote the presence of the AIS , while open arrowheads show disappeared AIS . Scale bars , 5 μm in C ,

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C The localization of AnkG and GFP-labeled mitochondria in the AIS of uninjured MN of Thy1-Mito Tg mice and injured motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice. D Graphs show the GFP and AnkG fluorescent intensity scans corresponding to (C) over a 20 μm line running from the soma to the axon. E Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Thy1-Mito mouse (n = 5 mice per group). Data information: For (E, data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in (E determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in C,


F Whole - mount observation of GFP - or CFP - labeled mitochondria ( mtGFP or mtCFP ) in the proximal region of peripheral hypoglossal ( XII ) nerve as shown in ( A ) . Dashed lines outline the axon . G Graph shows mitochondrial number in hypoglossal ( XII ) axon ( n = 5 mice ) . Data information : For G , data are shown as the mean ± s . e . m . , * * P = 0 . 0003 , * * * P < 0 . 0001 in G ) determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 5 μm

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F Whole-mount observation of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in the proximal region of peripheral hypoglossal (XII) nerve as shown in (A). Dashed lines outline the axon. G Graph shows mitochondrial number in hypoglossal (XII) axon (n = 5 mice). Data information: For G, data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in G) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 5 μm in