Figure 1 ( 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 ) ( 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 ) ( 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 ) ( 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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Figure 1(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;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)(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.(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.)


Figure 2 ( 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 . ( 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 . ( 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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Figure 2(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.(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.(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).


Figure 3Lyzs - 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 ) . 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 . 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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Figure 3Lyzs-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.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.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).


Figure 4Lyzs - 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 . 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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Figure 4Lyzs-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.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).


Figure 5Lyzs - 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 ) 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 . 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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Figure 5Lyzs-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).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).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).


Figure 6Lethally 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 . 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 . 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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Figure 6Lethally 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;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.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).


Figure 7 ( 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 . ( 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 ) . ( 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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Figure 7(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).(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).


Figure 8Osmotic 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 . 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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Figure 8Osmotic 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;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.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).


Figure 9 ( 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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Figure 9(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).


Figure 3Assays 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 .

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Figure 3Assays 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.


Figure 5A ) 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 .

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Figure 5A) 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.


Figure 6B ) 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 . 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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Figure 6B) 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.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


Figure 1 ( 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 . ( 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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Figure 1(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.(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).


Figure 2 ( 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 . ( 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 . ( 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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Figure 2(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.(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.(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.


Figure 3 ( 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 . ( 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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Figure 3(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.(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.


Figure 4 ( 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 . ( 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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Figure 4(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.(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.


Figure 5 ( 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 ) 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 . ( F ) The SARS - CoV - 2 titer in the pretreatment and full - time experiments was analyzed by real - time PCR .

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Figure 5(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)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.(F) The SARS-CoV-2 titer in the pretreatment and full-time experiments was analyzed by real-time PCR.


Figure 6 ( 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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Figure 6(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.


Figure 7 ( 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 ) . ( 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 .

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Figure 7(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).(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.


Figure 1 ( 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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Figure 1(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.


Figure 2 ( 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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Figure 2(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.


Figure 3 ( 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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Figure 3(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.


Figure 4D ) 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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Figure 4D) 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.


Figure 5 ( 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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Figure 5(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.


Figure 1C 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 * * * * . 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 . 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 * * *

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Figure 1C 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 * * * *.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.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 * * *


Figure 2A 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 ) . 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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Figure 2A 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).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 *.


Figure 3B 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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Figure 3B 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.


Figure 4F 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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Figure 4F 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* *


Figure 5C 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 * * * * 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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Figure 5C 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* * * *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* * * *


Figure 6A 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 * * * . 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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Figure 6A 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* * *.D Western blot analysis of steady state AMPK and pAMPK protein levels of starved and/or A769662 (AMPK activator) treated cells as indicated.


Figure 7A 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 ) 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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Figure 7A 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)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 * *


Figure 1F 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1F 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).


Figure 2C 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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Figure 2C 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)


Figure 3C 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3C 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.


Figure 4B 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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Figure 4B 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.


Figure 5C 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 inI Altered localization of the GFP - labeled mitochondria in the AIS of injured motor neurons of AnkG shRNA AAV - infected Rpt3 CKO mice at 5 days after injury . J Fluorescent intensity scans of GFP and AnkG corresponding to ( I ) . K Graph represents the percentage ( % ) of GFP intensity in the AIS , compared with that in the AIS of Atf3 : BAC2 Tg mouse treated with AnkG shRNA ( n = 5 mice per group ) . Data information : For data are shown as the mean ± s . e . m . , * * P = 0 . 0064 in ( K ) determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . In I ) , closed arrowheads denote the presence of the AIS , while open arrowheads show disappeared AIS . Scale bars , 5 μm in I ) L Thionine - stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury . Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs . Dashed lines outline hypoglossal nucleus . XII , hypoglossal nucleus ; cc , central canal . M The percentage of surviving motor neurons on the injured side compared with that on the control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs ( n = 4 mice per group ) . Data information : For M ) , data are shown as the mean ± s . e . m . , * P = 0 . 0405 , * * P = 0 . 003 in ( M ) , determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 100 μm in ( L ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5C 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 inI Altered localization of the GFP-labeled mitochondria in the AIS of injured motor neurons of AnkG shRNA AAV-infected Rpt3 CKO mice at 5 days after injury. J Fluorescent intensity scans of GFP and AnkG corresponding to (I). K Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Atf3:BAC2 Tg mouse treated with AnkG shRNA (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0064 in (K) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In I), closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in I)L Thionine-stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury. Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs. Dashed lines outline hypoglossal nucleus. XII, hypoglossal nucleus; cc, central canal. M The percentage of surviving motor neurons on the injured side compared with that on the control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs (n= 4 mice per group). Data information: For M), data are shown as the mean ± s.e.m. , * P = 0.0405, ** P = 0.003 in (M), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 100 μm in (L).


Figure 6B Whole - mount visualization of GFP - or CFP - labeled mitochondria ( mtGFP or mtCFP ) in sciatic nerve . Dashed lines indicate the boundary of axon . Scale bar , 5 μm . C Representative kymographs showing the behavior of mtCFP in uninjured sciatic nerves of Thy1 - Mito Tg and mtGFP in injured sciatic nerves of Atf3 ; BAC2 and Rpt3 CKO mice . D - F The number of mitochondria ( D ) , percentage of motile mitochondria ( E ) and stationary mitochondrial site ( F ) in axons . Data are shown as the mean ± s . e . m . * * * P = 0 . 0001 , * * * * P < 0 . 0001 in ( D ) , * * P = 0 . 0006 , * * * P = 0 . 0001 , * * * * P < 0 . 0001 in ( E ) , determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected , n = 5 mice for each group .

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Figure 6B Whole-mount visualization of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in sciatic nerve. Dashed lines indicate the boundary of axon. Scale bar, 5 μm. C Representative kymographs showing the behavior of mtCFP in uninjured sciatic nerves of Thy1-Mito Tg and mtGFP in injured sciatic nerves of Atf3;BAC2 and Rpt3 CKO mice.D-F The number of mitochondria (D), percentage of motile mitochondria (E) and stationary mitochondrial site (F) in axons. Data are shown as the mean ± s.e.m. *** P = 0.0001, **** P < 0.0001 in (D), ** P = 0.0006, *** P = 0.0001, **** P < 0.0001 in (E), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected, n = 5 mice for each group.


Figure 7C Immunostaining of spinal cord in Atf3 : BAC Tg and Atf3 ; SOD1 mice at P70 . The boxed regions are magnified in lower panels . Arrows indicate GFP - and ATF3 - positive motor neurons ( MNs ) . Dashed lines outline the edge of the gray matter in spinal cord . D The percentage ( % ) of GFP - positive MNs ( black bar ) in ChAT - positive MNs of Atf3 : BAC and Atf3 ; SOD1 mice at P70 ( n = 5 mice per each group ) . E The percentage ( % ) of ATF3 , MMP - 9 or osteopontin ( OPN ) - positive MNs ( black bar ) in GFP - positive MNs of Atf3 ; SOD1 mice at P65 - 70 ( n = 5 - 7 mice per group ) . Data information : For ( D , E , data are shown as the mean ± s . e . m . , Scale bars , 50 μm in ( C ; upper panel ) , 20 μm in ( C ; lower ) F Immunostaining of AnkG in ChAT - positive spinal MN of Atf3 : BAC and SOD1 and in GFP - positive spinal MN of Atf3 ; SOD1 mouse at P70 . G Quantification of the AIS length ( n = 5 mice per group ) . Data information : For G , data are shown as the mean ± s . e . m . , no significant difference in ( G ) determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 10 μm in ( FH The localization of AnkG and GFP - labeled mitochondria in uninjured spinal MN of Thy1 - Mito ( without ATF3 induction ) , traumatically sciatic nerve - injured MN of Atf3 : BAC ( with ATF3 induction ) and pathologically damaged MN of Atf3 ; SOD1 mice ( with ATF3 induction ) . Closed arrowheads denote the presence of the AIS , while open arrowheads show disappeared AIS . I Fluorescent intensity scan of GFP and AnkG in corresponding to ( H ) . J The percentage ( % ) of GFP intensity in the AIS , compared with that in the AIS of uninjured MNs in Thy1 - Mito mice ( n = 5 mice per group ) . Data information : For data are shown as the mean ± s . e . m . , * * P = 0 . 0008 , * * * P = 0 . 0007 in ( J ) , * * determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 5 μm in ( H ) . K Whole - mount observation of mtGFP or mtCFP in sciatic nerve . Dashed lines outline the axon . L The number of mtGFP or mtCFP in sciatic nerve ( n = 5 mice ) Data information : For L ) , data are shown as the mean ± s . e . m . , , * * P = 0 . 0036 , * * * * P < 0 . 0001 in ( L ) , determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 10 μm in K )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7C Immunostaining of spinal cord in Atf3:BAC Tg and Atf3;SOD1 mice at P70. The boxed regions are magnified in lower panels. Arrows indicate GFP- and ATF3- positive motor neurons (MNs). Dashed lines outline the edge of the gray matter in spinal cord. D The percentage (%) of GFP-positive MNs (black bar) in ChAT-positive MNs of Atf3:BAC and Atf3;SOD1 mice at P70 (n = 5 mice per each group). E The percentage (%) of ATF3, MMP-9 or osteopontin (OPN)-positive MNs (black bar) in GFP-positive MNs of Atf3;SOD1 mice at P65-70 (n = 5-7 mice per group). Data information: For (D, E, data are shown as the mean ± s.e.m., Scale bars , 50 μm in (C; upper panel), 20 μm in (C; lower)F Immunostaining of AnkG in ChAT-positive spinal MN of Atf3:BAC and SOD1 and in GFP-positive spinal MN of Atf3;SOD1 mouse at P70. G Quantification of the AIS length (n = 5 mice per group). Data information: For G, data are shown as the mean ± s.e.m., no significant difference in (G) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 10 μm in (FH The localization of AnkG and GFP-labeled mitochondria in uninjured spinal MN of Thy1-Mito (without ATF3 induction), traumatically sciatic nerve-injured MN of Atf3:BAC (with ATF3 induction) and pathologically damaged MN of Atf3;SOD1 mice (with ATF3 induction). Closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. I Fluorescent intensity scan of GFP and AnkG in corresponding to (H). J The percentage (%) of GFP intensity in the AIS, compared with that in the AIS of uninjured MNs in Thy1-Mito mice (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0008, *** P = 0.0007 in (J), ** determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 5 μm in (H).K Whole-mount observation of mtGFP or mtCFP in sciatic nerve. Dashed lines outline the axon. L The number of mtGFP or mtCFP in sciatic nerve (n = 5 mice) Data information: For L), data are shown as the mean ± s.e.m., , ** P = 0.0036, **** P <0.0001 in (L), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 10 μm in K)


Figure 1 ( A ) Inducible GFP ‐ S ‐ Ubqln1 293 cell line was treated with 1 μg / ml of doxycycline for 24 h . GFP ‐ S ‐ Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis . Red sequences indicate Ubqln4 peptides identified by mass spectrometry . ( B ) 293 cells were co ‐ transfected with GFP ‐ S or GFP ‐ S ‐ Ubqln1 and HA ‐ Ubqln4 . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ HA antibodies . Lower molecular weight GFP ‐ tag ‐ containing bands that appear in the GFP ‐ S ‐ Ubqln1 lane might represent degradation products . ( C ) Endogenous Ubqln1 in 293 cells was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG . The immunoprecipitates were analysed by western blotting using anti ‐ Ubqln1 and anti ‐ Ubqln4 antibodies . ( D ) Confocal images of Hela cells transfected with GFP ‐ S ‐ Ubqln1 and RFP ‐ Ubqln4 . Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross ‐ section . GFP , green fluorescent protein ; HA , haemagglutinin ; IP , immunoprecipitation ; Ubqln , Ubiquilin ; WCL , whole ‐ cell lysate .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1(A) Inducible GFP‐S‐Ubqln1 293 cell line was treated with 1 μg/ml of doxycycline for 24 h. GFP‐S‐Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis. Red sequences indicate Ubqln4 peptides identified by mass spectrometry.(B) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. Lower molecular weight GFP‐tag‐containing bands that appear in the GFP‐S‐Ubqln1 lane might represent degradation products.(C) Endogenous Ubqln1 in 293 cells was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG. The immunoprecipitates were analysed by western blotting using anti‐Ubqln1 and anti‐Ubqln4 antibodies.(D) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and RFP‐Ubqln4. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross‐section. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; Ubqln, Ubiquilin; WCL, whole‐cell lysate.