( B ) This group was enriched for sites that were compensated in an ATR - dependent manner in A - T . Box plots depict 9 phosphopeptides measured in two independent biological replicates . box indicates range from first to third quartiles , central band represents median . Upper lower whiskers extend from box to maximum minimum values which are not farther than 1 . 5 times interquartile range ( IQR ) .

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(B) This group was enriched for sites that were compensated in an ATR-dependent manner in A-T cells. Box plots depict 9 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).


( C ) Western blotting of pS343 / NBS1 , which is compensated upon ATM inhibition , pS824 / KAP - 1 pS395 / NUMA , which are not compensated under these conditions .

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(C) Western blotting analysis of pS343/NBS1, which is compensated upon ATM inhibition, and pS824/ KAP-1 and pS395/NUMA, which are not compensated under these conditions.


( A - F ) were either untreated ( UT ) or treated with neocarzinostatin ( NCS ) in presence or absence of PIKK inhibitors . Intensities from 3 independent biological replicates were normalized against reference synthetic standards are depicted as mean ± .

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(A-F) Cells were either untreated (UT) or treated with neocarzinostatin (NCS) in the presence or absence of PIKK inhibitors. Intensities from 3 independent biological replicates were normalized against reference synthetic standards and are depicted as mean ± SEM.


( C ) Time - lapse microscopy of containing a PsigV - YFP promoter reporter reveals heterogeneous activation of σV in response to lysozyme stress . stress ( red ) was added between 200 mins 300 min time points ( at 240 min ) . red arrow highlights a with a delayed activation of σV . Scale bar : 5 μm .

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(C) Time-lapse microscopy of cells containing a PsigV-YFP promoter reporter reveals heterogeneous activation of σV in response to lysozyme stress. The stress (red line) was added between the 200 mins and 300 min time points (at 240 min). The red arrow highlights a cell with a delayed activation of σV. Scale bar: 5 μm.


( B ) A priming ( 30 min ) stress of 1 µg / ml lysozyme followed by high lysozyme stress ( 20 µg / ml ) improves survival . solid blue lines are biological repeats for no priming experiment ( n = 2 ) whereas solid red lines are biological repeats for priming experiment ( n = 4 ) . ( Total number of shown for No Priming , N = 2013 Priming , N = 4937 ) .

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(B) A priming (30 min) stress of 1 µg/ml lysozyme followed by the high lysozyme stress (20 µg/ml) improves survival. The solid blue lines are the biological repeats for the no priming experiment (n=2) whereas the solid red lines are the biological repeats for the priming experiment (n=4). (Total number of cells shown for No Priming, N = 2013 and Priming, N = 4937).


( B , C ) - seq experiment on WT ( JLB130 ) ΔsigV ( JLB154 ) strains , showing of absolute expression of genes in presence absence of lysozyme stress . shaded grey box represents a ± 5 fold change . DEseq was used to identify genes which were differentially expressed with a 5 % p - value cutoff between WT ΔsigV mutant in response to lysozyme treatment ( default DEseq was used ) . ( B ) Only sigV operon is strongly activated ( > 5 fold change ) in response to lysozyme stress in WT ( JLB130 ) , as previously reported ( Guariglia - Oropeza Helmann 2011 ) . ( C ) No genes were strongly upregulated in ΔsigV ( JLB154 ) by lysozyme stress .

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(B, C) RNA-seq experiment on WT (JLB130) and ΔsigV (JLB154) strains, showing quantification of the absolute expression of individual genes in the presence and absence of lysozyme stress. The shaded grey box represents a ± 5 fold change. DEseq was used to identify genes which were differentially expressed with a 5% p-value cutoff between the WT and ΔsigV mutant in response to lysozyme treatment (default DEseq test was used). (B) Only the sigV operon is strongly activated (>5 fold change) in response to lysozyme stress in WT (JLB130), as previously reported (Guariglia-Oropeza and Helmann 2011). (C) No genes were strongly upregulated in ΔsigV (JLB154) by lysozyme stress.


( D ) Effect of overexpression of components of σV pathway ( Biological repeats : WT : n = 8 , sigV + : n = 4 , rsiV + : n = 3 oatA + : n = 4 , yrhK + : n = 3 , sipS + : n = 3 rasP + : n = 3 , pbpX + : n = 6 ) on fraction of activated . Only overexpression of sigV , rsiV or oatA changed observed dynamics compared to WT . histograms of shown data are shown in Appendix Figure S16 . ( E ) Deleting oatA did not alter σV activation dynamics . However , deleting sigV or rsiV resulted in no further activation of σV in response to 1 ug / ml lysozyme . n > = 3 biological repeats for data shown . histograms of shown data are shown in Appendix Figure S17 . (

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(D) Effect of the overexpression of individual components of the σV pathway (Biological repeats: WT: n=8, sigV+: n=4, rsiV+: n=3 oatA+: n=4, yrhK+: n=3, sipS+: n=3 and rasP+: n=3, pbpX+: n=6) on the fraction of activated cells. Only overexpression of sigV, rsiV or oatA changed the observed dynamics compared to WT. The histograms of the shown data are shown in Appendix Figure S16. (E) Deleting oatA did not alter the σV activation dynamics. However, deleting sigV or rsiV resulted in no further activation of σV in response to 1 ug/ml lysozyme. n>=3 biological repeats for all data shown. The histograms of the shown data are shown in Appendix Figure S17. (


( C ) As predicted , 2xsigV or 2xsigV - rsiV stains have a homogenous σV response to 1 µg / ml lysozyme , while 2xrsiV has increased heterogeneity . Each bar plot is average of three biological repeats . bars correspond to mean ± s . d . .

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(C) As predicted, the 2xsigV or 2xsigV-rsiV stains have a homogenous σV response to 1 µg/ml lysozyme, while the 2xrsiV strain has increased heterogeneity. Each bar plot is the average of three biological repeats. The bars correspond to the mean ± s.d..


( E , F ) For very long intervals between stress ( 12h ) , heterogeneity is same as if had not experienced a prior lysozyme stress ( control ) . N > 50 single - traces .

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(E, F) For very long intervals between stress (12h), the heterogeneity is the same as if the cells had not experienced a prior lysozyme stress (control). N>50 single-cell traces.


( A ) Yeast clones harbouring empty bait vector ( pYTH9 , mock ) or those encoding ScAtg8 , LC3B , Atg12 SUMO1 were transformed with empty prey vector ( pACT2 , mock ) or those encoding fragments of ATG4A , ATG4B , NBR1 , p62 full - Nix . Interaction was assessed by yeast growth on SD ‐ W / ‐ L / ‐ H medium with a β ‐ gal .

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(A) Yeast clones harbouring empty bait vector (pYTH9, mock) or those encoding ScAtg8, LC3B, Atg12 and SUMO1 were transformed with empty prey vector (pACT2, mock) or those encoding fragments of ATG4A, ATG4B, NBR1, p62 and full-length Nix. Interaction was assessed by yeast growth on the SD‐W/‐L/‐H medium with a β‐gal assay.


( B ) GST pulldown assays using extracts of Flag ‐ Nix ‐ expressing COS7 immobilized GST or indicated GST fusions . Coprecipitated Nix was detected with Flag antibodies .

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(B) GST pulldown assays using cell extracts of Flag‐Nix‐expressing COS7 cells and immobilized GST or the indicated GST fusions. Coprecipitated Nix was detected with Flag antibodies.


( C ) Purified GST ‐ tagged NixΔTM was from GST moiety by thrombin , purified used for precipitation by GST fusion . Precipitated were analysed by western blotting ( WB ) with Nix antibodies . Ponceau S was used to visualize GST ‐ fusion .

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(C) Purified GST‐tagged NixΔTM was cleaved from the GST moiety by thrombin, purified and used for precipitation by GST fusion proteins. Precipitated proteins were analysed by western blotting (WB) with Nix antibodies. Ponceau S staining was used to visualize GST‐fusion proteins.


( D ) HeLa were treated as indicated endogenous were immunoprecipitated ( IP ) from extracts using Nix antibody analysed by using Nix LC3 antibodies . β ‐ gal , β ‐ galactosidase ; BafA1 , bafilomycin ‐ A1 ; CCCP , carbonyl cyanide m ‐ chlorophenyl hidrazone ; GST , glutathione ‐ S ‐ transferase ; IgG , immunoglobulin G ; NixΔTM , recombinant Nix lacking a TM ; SUMO1 , small ubiquitin ‐ like modifier 1 ; TCL , total lysate ; TM , transmembrane domain .

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(D) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated (IP) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies. β‐gal, β‐galactosidase; BafA1, bafilomycin‐A1; CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; GST, glutathione‐S‐transferase; IgG, immunoglobulin G; NixΔTM, recombinant Nix lacking a TM; SUMO1, small ubiquitin‐like modifier 1; TCL, total cell lysate; TM, transmembrane domain.


( C ) Lysates of COS7 transfected with Flag ‐ wt ‐ Nix or indicated mutants were incubated with immobilized GST ‐ LC3A . coprecipitated were detected by western blotting ( WB ) with Flag antibodies . BNIP3 , Bcl2 / E1B 19kDa ‐ interacting 3 ‐ like ; Dr , Danio reio ; GST , glutathione ‐ S ‐ transferase ; Hs , Homo sapiens ; LIR , LC3 ‐ interacting region ; Mm , Mus musculus ; wt , wild type ; Xl , Xenopus laevis .

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(C) Lysates of COS7 cells transfected with Flag‐wt‐Nix or the indicated mutants were incubated with immobilized GST‐LC3A. The coprecipitated proteins were detected by western blotting (WB) with Flag antibodies. BNIP3, Bcl2/E1B 19kDa‐interacting protein 3‐like protein; Dr, Danio reio; GST, glutathione‐S‐transferase; Hs, Homo sapiens; LIR, LC3‐interacting region; Mm, Mus musculus; wt, wild type; Xl, Xenopus laevis.


Nix recruits EGFP ‐ GABARAP ‐ L1 to stressed mitochondria in reconstituted Nix − / − MEFs . Nix − / − MEFs cotransfected with EGFP ‐ GABARAP ‐ L1 wt ‐ Nix ( A ) , or Nix ‐ W35A ( B ) , were incubated with without mitochondrial poison CCCP for 3 h . were then fixed analysed for colocalization between EGFP ‐ GABARAP ‐ L1 Nix ( stained with Flag antibodies ) . final panel in each row shows colocalization between Flag ‐ Nix ‐ Cy3 GABARAP ‐ L1 using ' colocalization ' highlighter plug ‐ in for ImageJ software ( NIH , Bethesda , MA , USA ) .

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Nix recruits EGFP‐GABARAP‐L1 to stressed mitochondria in reconstituted Nix−/− MEFs. Nix−/− MEFs cotransfected with EGFP‐GABARAP‐L1 and wt‐Nix (A), or Nix‐W35A (B), were incubated with and without mitochondrial poison CCCP for 3 h. Cells were then fixed and analysed for colocalization between EGFP‐GABARAP‐L1 and Nix (stained with Flag antibodies). The final panel in each row shows colocalization between Flag‐Nix‐Cy3 and GABARAP‐L1 using the 'colocalization' highlighter plug‐in for ImageJ software (NIH, Bethesda, MA, USA).


( C ) was achieved by counting number of complete colocalizations , EGFP / Cy3 per for approximately 30 per condition . Results are expressed as a of colocalization ( EGFP : Cy3 ) in control CCCP ‐ treated . Error bars in ( C ) show standard deviation ( s . d . ) of three independent experiments . Statistical was performed using Mann - Whitney t ‐ . CCCP , carbonyl cyanide m ‐ chlorophenyl hidrazone ; DMSO , dimethyl sulfoxide ; EGFP , enhanced green ; MEF , mouse embryonic fibroblast ; wt , wild type .

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(C) Quantification was achieved by counting the number of complete colocalizations, EGFP/Cy3 per cell for approximately 30 cells per condition. Results are expressed as a percentage of colocalization (EGFP:Cy3) in control and CCCP‐treated cells. Error bars in (C) show the standard deviation (s.d.) of three independent experiments. Statistical analysis was performed using the Mann-Whitney t‐test. CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; wt, wild type.


( A ) Flow cytometry of reticulocyte ‐ enriched blood , cultured in vitro for 3 days , stained with MTR . blood was taken from mice transplanted with Nix − / − bone marrow reconstituted with wt ‐ Nix Nix ‐ W35A . Reticulocytes were induced by phenylhydrazine treatment . also express GFP .

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(A) Flow cytometry of reticulocyte‐enriched blood, cultured in vitro for 3 days, stained with MTR. The blood was taken from mice transplanted with Nix−/− bone marrow and reconstituted with wt‐Nix and Nix‐W35A viruses. Reticulocytes were induced by phenylhydrazine treatment. The viruses also express GFP.


( B ) Mitochondrial clearance in GFP ‐ positive fraction of reticulocyte ‐ enriched blood , as described in panel ( A ) . Mice were reconstituted with wt ‐ Nix , LIR ‐ W35A or empty vector . Data from Nix − / − Nix + / + mice are shown for comparison .

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(B) Mitochondrial clearance in the GFP‐positive fraction of the reticulocyte‐enriched blood, as described in panel (A). Mice were reconstituted with wt‐Nix, LIR‐W35A or empty vector viruses. Data from Nix−/− and Nix+/+ mice are shown for comparison.


( C ) Anti ‐ Flag , anti ‐ GFP anti ‐ β ‐ actin western blots of liver transduced with Flag ‐ Nix Flag ‐ W35A ‐ Nix , normalized to total . GFP , green ; LIR , LC3 ‐ interacting region ; MTR , MitoTracker Red ; wt , wild type .

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(C) Anti‐Flag, anti‐GFP and anti‐β‐actin western blots of fetal liver cells transduced with Flag‐Nix and Flag‐W35A‐Nix viruses, normalized to total protein. GFP, green fluorescent protein; LIR, LC3‐interacting region; MTR, MitoTracker Red; wt, wild type.


A , B MARS ( A ) MARS2 ( B ) mRNA expression were measured by real - time PCR in blood samples with 2 3 - 4 copies . Relative expression were normalized to GAPDH , data were quantified relative to mRNA expression of 2 copy samples . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Unpaired Student ' s t

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A, B MARS (A) and MARS2 (B) mRNA expression levels were measured by real-time PCR in blood samples with 2 and 3-4 copies. Relative expression levels were normalized to GAPDH levels, and data were quantified relative to the mRNA expression levels of 2 copy samples. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test


C , D Immunoblotting was performed to detect MARS in samples with 2 3 - 4 copies . - level shown in ( D ) was quantified using ImageJ divided by - level of actin . results were then graphed relative to samples with 2 copies . E , F MARS2 expression were detected ( E ) quantified ( F ) . Actin was used as a loading control . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Unpaired Student ' s t was

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C, D Immunoblotting analysis was performed to detect MARS protein levels in samples with 2 and 3-4 copies. Protein-level intensity shown in (D) was quantified using ImageJ and divided by the protein-level intensity of actin. The results were then graphed relative to samples with 2 copies. E, F MARS2 protein expression levels were detected (E) and quantified (F). Actin was used as a loading control. Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Unpaired Student's t test was used


G proliferation was assessed by Ki67 ( red ) immunofluorescent in mice neural tube sections at E10 . MARS ( green ) was co - stained . Scale bar : 150 μm .

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G Cell proliferation was assessed by Ki67 (red) immunofluorescent staining in mice neural tube sections at E10. MARS (green) was co-stained. Scale bar: 150 μm.


H Double immunofluorescent for MARS ( green ) Ki67 ( red ) in developing heart sections at E10 . Scale bar : 150 μm .

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H Double immunofluorescent staining for MARS (green) and Ki67 (red) in developing heart sections at E10. Scale bar: 150 μm.


I MARS expression was detected in E80 rhesus monkey brain sections ( n = 3 ) by . Local large magnification is shown as framed . Red arrow indicates germinar layer , blue indicates immature neurons . Scale bar : 1mm .

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I MARS expression was detected in E80 rhesus monkey brain sections (n=3) by IHC. Local large magnification is shown as framed. Red arrow indicates germinar layer, blue indicates immature neurons. Scale bar: 1mm.


MARS in embryonic chorion ( C ) their corresponding maternal deciduae ( D ) were determined by western blot . Each D / C lane pair was quantified relative to D as shown at bottom .

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MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. Each D/C lane pair was quantified relative to D as shown at the bottom.


MARS in embryonic chorion ( C ) their corresponding maternal deciduae ( D ) were determined by western blot . mean MARS in D C groups were quantified by ImageJ shown in ( K ) ( n = 6 ) . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs was

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MARS protein levels in embryonic chorion (C) cells and their corresponding maternal deciduae (D) cells were determined by western blot. The mean MARS levels in all D and C groups were quantified by ImageJ and shown in (K) (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


L MARS2 mRNA expression in embryonic chorion ( C ) maternal deciduae ( D ) were detected by qPCR ( n = 6 ) . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs was

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L MARS2 mRNA expression in embryonic chorion (C) and maternal deciduae (D) were detected by qPCR (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


M MARS in brains of foetal rats ( F ) their corresponding mothers ( M ) from experiment ( O ) ( n = 6 ) . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs was

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M MARS protein quantification in the brains of foetal rats (F) and their corresponding mothers (M) from experiment (O) (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


N MARS mRNA ( n = 6 ) in brains of foetal rats ( F ) their corresponding mothers ( M ) were determined by qPCR ( n = 6 ) . Data are presented as mean ± . * P ≤ 0 . 05 , * * P ≤ 0 . 01 . Wilcoxon matched pairs was

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N MARS mRNA levels (n=6) in the brains of foetal rats (F) and their corresponding mothers (M) were determined by qPCR (n=6). Data are presented as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01. Wilcoxon matched pairs test was used


O MARS in foetal rats ( F ) their corresponding mothers ( M ) were determined by western blot . Each M / F lane pair was quantified relative to M as shown at bottom .

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O MARS protein levels in foetal rats (F) and their corresponding mothers (M) were determined by western blot. Each M/F lane pair was quantified relative to M as shown at the bottom.


P Representative result of MARS expression was detected by in tissues ( T ) adjacent noncancer tissues ( N ) of UCC samples . Scale bar : 50 μm .

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P Representative result of MARS expression was detected by IHC in tumor tissues (T) and adjacent noncancer tissues (N) of UCC samples. Scale bar: 50 μm.


Q Ki67 MARS expression was analysed by in HCC samples . Scale bar : 20 μm .

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Q Ki67 and MARS expression was analysed by IHC in HCC samples. Scale bar: 20 μm.


B growth was determined in absence presence of 0 . 5 mM homocysteine or 0 . 5 mM methionine in culture media of NE4C transfected with pcDNA3 . 1 vector or pcDNA3 . 1 - Flag - Bcl - 2 plasmids ( n = 3 ) . 5μg plasmids were transfected in each 1 × 106 . index responds to changes in number adhesion . caspase 3 ( c - caspase3 ) were detected to determine apoptosis ( right ) . Data are presented as mean ± were compared using an unpaired Student ' s t .

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

B Cell growth was determined in the absence and presence of 0.5 mM homocysteine or 0.5 mM methionine in the culture media of NE4C cells transfected with pcDNA3.1 vector or pcDNA3.1-Flag-Bcl-2 plasmids (n=3). 5μg plasmids were transfected in each 1×106 cells. The cell index responds to changes in cell number and cell adhesion. Cleaved caspase 3 (c-caspase3) levels were detected to determine apoptosis levels (right). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test.


C , D Apoptotic were detected by flow cytometry . apoptotic rates of untreated , MARS ( C ) - , MARS2 ( D ) - over - expressing NE4C when cultured in absence presence of 20 μM homocysteine were normalized to untreated NE4C ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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

C, D Apoptotic cells were detected by flow cytometry. The apoptotic rates of untreated, MARS (C)-, and MARS2 (D)-over-expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


E were treated with different methionine concentrations as indicated . Apoptotic rates were detected by flow cytometry normalized to those of untreated ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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

E Cells were treated with different methionine concentrations as indicated. Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


F Apoptotic rates were determined in absence presence of 20 μM homocysteine in culture media of control MARS / MARS2 knockdown NE4C ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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

F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS/MARS2 knockdown NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


HTL ROS ( superoxide ) were compared ( n = 4 ) between NE4C transfected with empty vector , MARS ( A ) plasmids . Data were normalized against that of vector - transfected . HTL were determined by LC - MS , ROS were determined by DHE .

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

HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector, MARS (A) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.


HTL ROS ( superoxide ) were compared ( n = 4 ) between NE4C transfected with empty vector or MARS2 ( B ) plasmids . Data were normalized against that of vector - transfected . HTL were determined by LC - MS , ROS were determined by DHE .

HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector or MARS2 (B) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.


C MARS was knocked down by small hairpin RNAs in NE4C . MARS knockdown efficiencies were confirmed by western blot . HTL superoxide were quantified ( n = 4 ) relative to vector - transfected NE4C . were cultured in 20 μM Hcy - containing media .

O B-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 O O O O O O O O O O O O O O O O O O O

C MARS was knocked down by small hairpin RNAs in NE4C cells. MARS knockdown efficiencies were confirmed by western blot. HTL and superoxide levels were quantified (n=4) relative to vector-transfected NE4C cells. Cells were cultured in 20 μM Hcy-containing media.


D Relative superoxide were determined by DHE compared ( n = 4 ) between NE4C with without MARS knockdown by shRNA . were cultured in 20 μM Hcy - containing media . MARS2 knockdown efficiencies were confirmed by western blot .

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

D Relative superoxide levels were determined by DHE staining assay and compared (n=4) between NE4C cells with and without MARS knockdown by shRNA. Cells were cultured in 20 μM Hcy-containing media. MARS2 knockdown efficiencies were confirmed by western blot.


E NE4C with without MARS knockdown were treated with either Hcy ( 20 μM ) or Met ( 75 μM ) , superoxide were detected 6 h after start of respective treatment quantified relative to untreated NE4C ( n = 4 ) .

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

E NE4C cells with and without MARS knockdown were treated with either Hcy (20 μM) or Met (75 μM), and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells (n=4).


F ROS ( superoxide ) were determined ( n = 4 ) by DHE of NE4C treated with various HTL concentrations for 4 h . ROS were normalized to those of untreated NE4C .

O O O O O O O O O O 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 The cellular ROS (superoxide) levels were determined (n=4) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h. The ROS levels were normalized to those of untreated NE4C cells.


G Superoxide in response to Hcy treatment ( 20 μM , 4 h ) in MARS - expressing control were determined relative to untreated NE4C .

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

G Superoxide levels in response to Hcy treatment (20 μM, 4 h) in MARS-expressing and control cells were determined relative to the untreated NE4C cells.


H NE4C were cultured in DMEM supplemented with different HTL as indicated . N - Hcy in HTL - treated control were detected by western blot .

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

H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated. N-Hcy levels in HTL-treated and control cells were detected by western blot.


I N - Hcy in response to MARS knockdown ( by shRNA ) over - expression were detected by western blot .

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

I Protein N-Hcy levels in response to MARS knockdown (by shRNA) and over-expression were detected by western blot.


J , K Untreated MARS / MARS2 knockdown NE4C were treated with Hcy ( 20 μM ) or HTL ( 10 μM ) , N - Hcy ( J ) ( n = 4 ) in were detected 6 h after start of respective treatment quantified relative to untreated NE4C . Error bars indicate . Data were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and the N-Hcy (J) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


J , K Untreated MARS / MARS2 knockdown NE4C were treated with Hcy ( 20 μM ) or HTL ( 10 μM ) , superoxide ( K ) ( n = 4 ) in were detected 6 h after start of respective treatment quantified relative to untreated NE4C . Error bars indicate . Data were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and superoxide (K) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


C SOD activities were down - regulated by in vitro N - homocysteinylation . Flag - tagged SOD1 , SOD1K23W , K122W , K128W ( 3KW ) mutant , SOD2 , SOD2K44W , K51W , K98W , K106W , K178W ( 5KW ) mutant were expressed in HEK293T , affinity purified , incubated with or without 10 μM HTL in solution ( in vitro ) . After 12 h , reaction mixture was desalted concentrated with filter tubes . N - Hcy SOD activities were determined normalized to untreated WT SOD1 SOD2 , respectively ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

C SOD activities were down-regulated by in vitro N-homocysteinylation. Flag-tagged SOD1, SOD1K23W,K122W,K128W (3KW) mutant, SOD2, and SOD2K44W, K51W,K98W,K106W,K178W (5KW) mutant were expressed in HEK293T cells, affinity purified, and incubated with or without 10 μM HTL in solution (in vitro). After 12 h, the reaction mixture was desalted and concentrated with filter tubes. N-Hcy levels and SOD activities were determined and normalized to untreated WT SOD1 and SOD2, respectively (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


D Upon switching N - Hcy substrate lysine ( K ) sites to nonmodifiable tryptophan ( W ) , N - Hcy unmodifiable mutants displayed lower specific activity than wide - type ( WT ) SOD . SOD1 ( WT , K23W , K122W , K128W , 3KW ) SOD2 ( WT , K44W , K51W , K98W , K106W , K178W , 5KW ) were expressed in HEK293T . catalytic activity of affinity - purified SOD was determined normalized to . Wild - type SOD1 SOD2 activities were set at 100 % ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

D Upon switching N-Hcy substrate lysine (K) sites to nonmodifiable tryptophan (W), the N-Hcy unmodifiable mutants displayed lower specific activity than the wide-type (WT) SOD. SOD1 (WT, K23W, K122W, K128W, and 3KW) and SOD2 (WT, K44W, K51W, K98W, K106W, K178W, and 5KW) were expressed in HEK293T cells. The catalytic activity of affinity-purified SOD proteins was determined and normalized to protein levels. Wild-type SOD1 and SOD2 activities were set at 100% (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


E , F Hcy HTL treatment in NE4C increased N - homocysteinylation of SODs decreased their activities . Flag - tagged SOD1 SOD2 were each ectopically expressed in NE4C . were transferred to serum - free DMEM with or without 20 μM Hcy ( E ) or 10 μM HTL ( F ) 6 h before harvesting . After affinity purification , specific activities of SOD1 SOD2 were determined normalized to SOD1 SOD2 activity in untreated . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

E, F Hcy and HTL treatment in NE4C cells increased N-homocysteinylation of SODs and decreased their activities. Flag-tagged SOD1 and SOD2 were each ectopically expressed in NE4C cells. The cells were transferred to serum-free DMEM with or without 20 μM Hcy (E) or 10 μM HTL (F) 6 h before harvesting. After affinity purification, the specific activities of SOD1 and SOD2 were determined and normalized to SOD1 and SOD2 activity in untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


G N - Hcy SOD were down - regulated in MARS / MARS2 knockdown . Flag - tagged SOD1 SOD2 were each over - expressed in untreated MARS / MARS2 knockdown NE4C . After affinity purification , N - Hcy relative specific activities of purified SOD1 SOD2 were determined quantified relative to NE4C ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

G N-Hcy SOD levels were down-regulated in MARS/MARS2 knockdown cells. Flag-tagged SOD1 and SOD2 were each over-expressed in untreated and MARS/MARS2 knockdown NE4C cells. After affinity purification, the N-Hcy levels and relative specific activities of purified SOD1 and SOD2 were determined and quantified relative to the NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


A of β - catenin in response to Hcy treatment in NE4C were detected by western blot ( n = 3 ) . Representative western blots are shown , band intensities were quantified relative to untreated group . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Levels of β-catenin in response to Hcy treatment in NE4C cells were detected by western blot (n=3). Representative western blots are shown, and band intensities were quantified relative to the untreated group. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


B of β - catenin in response to HTL treatment in NE4C were detected . Band intensities were normalized to that of untreated . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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 O O O O O O O O O O O O O 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 Levels of β-catenin in response to HTL treatment in NE4C cells were detected. Band intensities were normalized to that of untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


C TCF activities were measured with a Topflash / Fopflash luciferase reporter system ( n = 4 ) . TCF activities responding to either 20 μM Hcy - or 10 μM HTL - treated were measured by quantifying Topflash / Fopflash relative to untreated groups . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

C TCF activities were measured with a Topflash/Fopflash luciferase reporter system (n=4). TCF activities responding to either 20 μM Hcy- or 10 μM HTL-treated were measured by quantifying Topflash/Fopflash relative to the untreated groups. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


D of β - catenin in untreated MARS knockdown NE4C were detected ( n = 3 ) with presence or absence of 20 μM Hcy in culture media . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

D Levels of β-catenin in untreated and MARS knockdown NE4C cells were detected (n=3) with the presence or absence of 20 μM Hcy in the culture media. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


NE4C were co - transfected with NRX - Flag DVL1 - Myc plasmids treated with different concentrations of Hcy ( E ) relative amount of DVL1 that coimmunoprecipitated ( co - IPed ) with NRX - Flag was determined ( n = 3 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O 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 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

NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of Hcy (E) The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


NE4C were co - transfected with NRX - Flag DVL1 - Myc plasmids treated with different concentrations of HTL ( F ) . relative amount of DVL1 that coimmunoprecipitated ( co - IPed ) with NRX - Flag was determined ( n = 3 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O O 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 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

NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of HTL (F). The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


G NRX - Flag DVL1 - Myc were co - transfected in untreated or MARS knockdown NE4C . relative DVL1 that co - IPed with NRX - Flag were determined quantified ( n = 3 ) for treated control cultured with or without 20 μM Hcy . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

G NRX-Flag and DVL1-Myc were co-transfected in untreated or MARS knockdown NE4C cells. The relative DVL1 levels that co-IPed with NRX-Flag were determined and quantified (n=3) for treated and control cells cultured with or without 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Flag - tagged SOD1 SOD2 were co - over - expressed in NE4C cultured in DMEM supplemented with Hcy or HTL . ( H ) of β - catenin in with without SOD1 / 2 expression were determined ( n = 3 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with Hcy or HTL. (H) The levels of β-catenin in cells with and without SOD1/2 expression were determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Flag - tagged SOD1 SOD2 were co - over - expressed in NE4C cultured in DMEM supplemented with HTL . ( I ) Relative of DVL1 that co - IPed with NRX - Flag were quantified in SOD - expressing control cultured with or without 10 μM HTL ( n = 3 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with HTL. (I) Relative levels of DVL1 that co-IPed with NRX-Flag were quantified in SOD-expressing and control cells cultured with or without 10 μM HTL (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


B MARS - or MARS2 - catalysed HTL production from Hcy ( 20 μM ) was carried out in vitro in presence of different AHT concentrations . amount of HTL produced in each reaction was determined by thin layer chromatography .

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

B MARS- or MARS2-catalysed HTL production from Hcy (20 μM) was carried out in vitro in the presence of different AHT concentrations. The amount of HTL produced in each reaction was determined by thin layer chromatography.


C MARS MARS2 activities were determined ( n = 4 ) in presence of indicated AHT concentrations in reactions . AHT IC50 values were determined for MARS MARS2 with 20 μM Hcy . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-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 B-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 O O O O O O O O O O O O O O O O O O O O O O O

C MARS and MARS2 activities were determined (n=4) in the presence of the indicated AHT concentrations in the reactions. AHT IC50 values were determined for MARS and MARS2 with 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


D Relative HTL were determined ( n = 4 ) for NE4C treated with various AHT concentrations for 4 h . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative HTL levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


E AHT ( 71 or 110 μg / kg per day ) was injected into C57BL / 6 mice for 6 days . Plasma HTL were detected by LC - MS . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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E AHT (71 or 110 μg/kg per day) was injected into C57BL/6 mice for 6 days. Plasma HTL levels were detected by LC-MS. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


F Hcy were compared in NE4C or H9C2 without with 100 μM AHT treatments . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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F Hcy levels were compared in NE4C or H9C2 cells without and with 100 μM AHT treatments. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


NE4C were cultured in DMEM with or without 20 μM Hcy supplemented with indicated AHT ; N - homocysteinylation were detected by western blot .

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NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; N-homocysteinylation levels were detected by western blot.


NE4C were cultured in DMEM with or without 20 μM Hcy supplemented with indicated AHT ; Band intensities were quantified relative to untreated group using Image J ( H ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; Band intensities were quantified relative to the untreated group using Image J (H). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


I superoxide were determined ( n = 4 ) for NE4C treated with various AHT concentrations for 4 h . Superoxide were normalized to those of untreated NE4C . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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I Cellular superoxide levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Superoxide levels were normalized to those of untreated NE4C cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


J Apoptotic rates were determined in absence or presence of 20 μM Hcy in culture media in control 70 μM AHT - treated NE4C ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

J Apoptotic rates were determined in the absence or presence of 20 μM Hcy in the culture media in control and 70 μM AHT-treated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


K of β - catenin in untreated , Hcy - , or HTL - treated NE4C were detected ( n = 3 ) with presence or absence of 70 μM AHT in culture media . Mean β - catenin in untreated NE4C were set at 100 % . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

K Levels of β-catenin in untreated, Hcy-, or HTL-treated NE4C cells were detected (n=3) with the presence or absence of 70 μM AHT in the culture media. Mean β-catenin levels in untreated NE4C cells were set at 100%. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


A , B Microscope of E10 neural tube sections with indicated treatments were stained with antibodies against MARS ( green ) N - Hcy ( red ) . Scale bar : 150 μm . relative ( to untreated ) intensities were quantified ( n = 3 ) are presented in ( B ) . C , D Double immunofluorescent ( C ) for MARS ( green ) N - Hcy ( red ) in E10 heart sections . Scale bar : 150 μm . relative ( to untreated ) intensities were quantified ( n = 3 ) are presented in ( D ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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A, B Microscope images of E10 neural tube sections with the indicated treatments were stained with antibodies against MARS (green) and N-Hcy (red). Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n=3) and are presented in (B). C, D Double immunofluorescent staining (C) for MARS (green) and N-Hcy (red) in E10 heart sections. Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n=3) and are presented in (D).Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


E Goss of E10 . 5 ATRA - treated untreated mice . ATRA - treated exhibits an open neural tube ( indicated by arrows ) compared to littermate controls . Scale bar : 500 μm .

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E Goss morphology of E10.5 ATRA-treated and untreated mice embryos. The ATRA-treated embryo exhibits an open neural tube (indicated by white arrows) compared to littermate controls. Scale bar: 500 μm.


F Examples of normally developed maldeveloped mice hearts : in normal heart ( i ) , ink travelled from right ventricle ( RV ) into pulmonary trunk ( PT ) ; however , in ATRA - treated group hearts , ink travelled into aorta ( Ao ) ( ii , transposition of great arteries ( TGA ) ) or both PT Ao ( iii , double outlet right ventricle ( DORV ) ) . Scale bar : 1000 μm .

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F Examples of normally developed and maldeveloped mice hearts: in the normal heart (i), the ink travelled from the right ventricle (RV) into the pulmonary trunk (PT); however, in ATRA-treated group hearts, ink travelled into the aorta (Ao) (ii, transposition of great arteries (TGA)) or both the PT and Ao (iii, double outlet right ventricle (DORV)). Scale bar: 1000 μm.


G Cardiac malformations were analysed by hematoxylin eosin ( H & E ) of heart sections from E14 . 5 mice . cardiac defects observed in ATRA treated mice are as follows : Ventricular septal defect ( VSD ) single ventricle defects ( SVD ) . Heart compartments are marked : RA : right atria , RV : right ventricle , LA : left atria , LV : left ventricle . Scale bar : 1000

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G Cardiac malformations were analysed by hematoxylin and eosin (H&E) staining of heart sections from E14.5 mice. The cardiac defects observed in ATRA treated mice are as follows: Ventricular septal defect (VSD) and single ventricle defects (SVD). Heart compartments are marked: RA: right atria, RV: right ventricle, LA: left atria, LV: left ventricle. Scale bar: 1000 μm


H Total N - Hcy in maternal mice liver homogenates were determined quantified in correlation to corresponding prevalence of NTDs in affected foetuses ( i ) . Western blot was used to detect N - Hcy ( ii ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

H Total protein N-Hcy levels in maternal mice liver homogenates were determined and quantified in correlation to the corresponding prevalence of NTDs in affected foetuses (i). Western blot was used to detect N-Hcy levels (ii). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


I Neural tube tissues at E10 were homogenized from litters of untreated , ATRA - treated , ATRA + AHT co - treated pregnant mice . Neural tube homogenates of each group were mixed from 6 . Endogenous SOD1 SOD2 were affinity purified from homogenates . N - Hcy specific activities of each purified SOD1 SOD2 were determined . SOD activities of untreated group were set at 100 % ; error bar indicates replicates ( n = 4 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

I Neural tube tissues at E10 were homogenized from litters of untreated, ATRA-treated, and ATRA+AHT co-treated pregnant mice. Neural tube homogenates of each group were mixed from 6 embryos. Endogenous SOD1 and SOD2 were affinity purified from the homogenates. N-Hcy levels and the specific activities of each purified SOD1 and SOD2 were determined. The SOD activities of the untreated group were set at 100%; error bar indicates assay replicates (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


J Superoxide ( a ) , apoptosis ( b ) , Wnt signalling ( c , d ) were over - activated in ATRA - treated mice neural tubes restored by AHT or NAC treatment . ( a ) Whole mount DHE of E10 . 5 mice . Scale bar : 500 μm . Neural tube defect ( arrows ) heart ( green arrow ) regions show especially higher intensities . ( b ) TUNEL ( green ) detected apoptotic in E8 . 5 neural tube ( E ) sections . MARS ( red ) . Scale bar : 300 μm . ( c , d ) for β - catenin ( green ) in E8 . 5 ( c ) E10 ( d ) mice neural tube sections . Scale bar : 300 μm ( c ) , 150 μm ( d ) . DAPI ( blue ) .

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J Superoxide (a), apoptosis (b), and Wnt signalling (c, d) were over-activated in ATRA-treated mice neural tubes and restored by AHT or NAC treatment. (a) Whole mount DHE staining of E10.5 mice embryos. Scale bar: 500 μm. Neural tube defect (white arrows) and heart (green arrow) regions show especially higher fluorescence intensities. (b) TUNEL (green) staining detected apoptotic cell death in E8.5 neural tube (E) sections. MARS (red). Scale bar: 300 μm. (c, d) IHC staining for β-catenin (green) in E8.5 (c) and E10 (d) mice neural tube sections. Scale bar: 300 μm (c), 150 μm (d). DAPI (blue).


K Average ( n = 3 ) DHE intensities were quantified relative to untreated group . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

K Average (n=3) DHE fluorescence intensities were quantified relative to the untreated group. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


L of TUNEL - positive Three sections from three were pooled for . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

L Quantification of TUNEL-positive cells Three sections from three embryos were pooled for analysis. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


M of β - catenin intensities in experiment J ( b ) J ( c ) . average ( n = 3 ) β - catenin level in untreated group was set at 100 % . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

M Quantification of β-catenin fluorescence intensities in experiment J (b) and J (c). The average (n=3) β-catenin level in the untreated group was set at 100%. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


N Hcy - treated eggs exhibited NTDs at embryonic stage 8 ( E8 ) . A normal ( a ) neural tube defect ( b , c ) are shown . Exencephaly ( arrows ) spina bifida ( blue arrows ) are indicated . Scale bar : 500 μm .

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N Hcy-treated eggs exhibited NTDs phenotypes at embryonic stage 8 (E8). A normal embryo (a) and neural tube defect phenotypes (b, c) are shown. Exencephaly (white arrows) and spina bifida (blue arrows) are indicated. Scale bar: 500 μm.


O Total N - Hcy of 72 h chicken homogenates from Hcy Hcy + AHT - treated groups were detected by western blot .

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O Total protein N-Hcy levels of 72 h chicken embryo homogenates from Hcy and Hcy+AHT-treated groups were detected by western blot.


P Whole mount DHE of 72 h chicken from untreated , Hcy - , Hcy + AHT - , Hcy + NAC - treated groups . Scale bar : 2000 μm . Q of DHE intensities in ( P ) ( n = 3 ) . Data are presented as mean ± were compared using an unpaired Student ' s t . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

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

P Whole mount DHE staining of 72 h chicken embryos from untreated, Hcy-, Hcy+AHT-, and Hcy+NAC-treated groups. Scale bar:2000 μm. Q Quantification of DHE fluorescence intensities in (P) (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


C GST pulldown of transiently expressed AdipoR1 ( residues 1 - 375 ) with immobilized GST - P5 or GST - P10 fusion . Pulldown fractions were subjected to western blotting using Flag GST antibodies .

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C GST pulldown of transiently expressed AdipoR1 (residues 1-375) with immobilized GST-P5 or GST-P10 fusion proteins. Pulldown fractions were subjected to western blotting using Flag and GST antibodies.


C AdipoR1 siRNA - transfected ORS or DP were treated with APN or P5 . lysates were analyzed for p - AMPK .

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C AdipoR1 siRNA-transfected ORS or DP cells were treated with APN or P5. The cell lysates were analyzed for p-AMPK.


D - H relative gene expression of growth factors in P5 - treated DP ( n = 4 ( VEGF , PDGFA ) , 5 ( IGF1 ) , 6 ( HGF , FGF7 ) biological replicates / group )

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D-H The relative gene expression levels of growth factors in P5-treated DP cells (n = 4 (VEGF, PDGFA), 5 (IGF1), 6 (HGF, FGF7) biological replicates / group)


C IF ( p - AMPK ) of HF bulb . Yellow arrow indicates p - AMPK signal ; scale bar : 100 µm . D IF ( Ki - 67 ) of HF bulb ; scale bar : 100 µm .

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C IF staining (p-AMPK) of the HF bulb area. Yellow arrow indicates p-AMPK signal; scale bar: 100 µm. D IF staining (Ki-67) of the HF bulb area; scale bar: 100 µm.


C H & E IF for versican of skin tissue ; scale bars : 100 µm .

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C H&E and IF for versican of skin tissue; scale bars: 100 µm.


G H & E IF for versican of skin tissue ; scale bars : 100 µm . H Anagen induction scores of vehicle - , or P5 - treated Adipoq - / - mice ( n = 6 tissues / mice , 4 mice / group ) ; Unpaired t

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G H&E and IF for versican of skin tissue; scale bars: 100 µm. H Anagen induction scores of vehicle-, or P5- treated Adipoq-/- mice (n = 6 tissues / mice, 4 mice / group); Unpaired t test


K . H & E IF for versican of skin tissue ; scale bars : 100 µm . L Anagen induction scores of vehicle - , or P5 - treated Adipor1 - / - mice ( n = 6 tissues / mice , 5 mice / group ) ; Unpaired t

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K. H&E and IF for versican of skin tissue; scale bars: 100 µm. L Anagen induction scores of vehicle-, or P5- treated Adipor1-/-mice (n = 6 tissues / mice, 5 mice / group); Unpaired t test


Pulldown of Flag - fused AdipoR1 input ( residues 89 - 375 ) with acid mutation ( s ) , as indicated , with GST - P5 . relative pulldown efficiency is expressed as of band intensities of bound AdipoR1 to those of AdipoR1 ( residue 89 - 375 ) inputs , as indicated ( n = 3 technical replicates / group ) .

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Pulldown of Flag-fused AdipoR1 input (residues 89-375) with amino acid mutation(s), as indicated, with GST-P5. The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).


F Pulldown of AdipoR1 ( residues 89 - 375 ) with GST - P5 or GST fused to two mutant peptides ( GLYDF GLYAF ) . relative pulldown efficiency is expressed as of band intensities of bound AdipoR1 to those of AdipoR1 ( residue 89 - 375 ) inputs , as indicated ( n = 3 technical replicates / group ) .

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F Pulldown of AdipoR1 (residues 89-375) with GST-P5 or GST fused to two mutant peptides (GLYDF and GLYAF). The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).


( A ) Phenotype appearance of Gmlmm1 - 1 mutant . Typical leaves ( left panel ; scale bar , 1 cm ) whole ( right panel ; scale bar , 10 cm . ) were photographed 60 118 days after seeding , respectively . Data information : experiments were performed three times as biological replicates , with similar results .

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(A) Phenotype appearance of the Gmlmm1-1 mutant. Typical leaves (left panel; scale bar, 1 cm) and whole plants (right panel; scale bar, 10 cm.) were photographed 60 and 118 days after seeding, respectively. Data information: The experiments were performed three times as biological replicates, with similar results.


( B ) Enhanced resistance of Gmlmm1 - 1 Gmlmm1 - 2 to Psg infection . Leaves from 2 - week - old soybean were infected with Psg number was determined at 0 4 dpi ( Mean ± SD , n = 8 , n represents sample number , * * * , p < 0 . 001 , Student ' s t - ) . Data information : experiments were performed three times , as biological replicates , with similar results .

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(B) Enhanced resistance of Gmlmm1-1 and Gmlmm1-2 to Psg infection. Leaves from 2-week-old soybean plants were infected with Psg and the bacteria number was determined at 0 and 4 dpi (Mean ± SD, n = 8, n represents sample number, ***, p < 0.001, Student's t-test). Data information: The experiments were performed three times , as biological replicates, with similar results.


( E ) Subcellular localization of GmLMM1 - GFP GmLMM1L407H - GFP in N . benthamiana . PI4P indicates localization . Scale bar , 10 μm . GFP , green . Data information : experiments were performed three times as biological replicates , with similar results .

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(E) Subcellular localization of GmLMM1-GFP and GmLMM1L407H-GFP in N. benthamiana. PI4P indicates the membrane localization. Scale bar, 10 μm. GFP, green fluorescent protein. Data information: The experiments were performed three times as biological replicates, with similar results.


( F ) Autophosphorylation activity of GmLMM1 intracellular domain in vitro . purified from E . coli were subjected to pMIAGO phosphorylation detection ( upper panel ) were detected using western blot ( lower panel ) with BIK1 - HIS as a control . GmLMM1K564E contains a mutation at predicted ATP binding site . Data information : experiments were performed two times ( F ) , as biological replicates , with similar results .

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(F) Autophosphorylation activity of the GmLMM1 intracellular domain in vitro. The proteins purified from E. coli were subjected to pMIAGO phosphorylation detection assay (upper panel) and were detected using western blot (lower panel) with BIK1-HIS as a control. GmLMM1K564E contains a mutation at the predicted ATP binding site. Data information: The experiments were performed two times (F), as biological replicates, with similar results.


( A ) ROS accumulation in Gmlmm1 - 1 Gmlmm1 - 2 mutant . Soybean leaves ( aged 3 ) were subjected to ROS accumulation examination by DAB . Scale bar , 1 cm . Data information : experiments were performed three times as biological replicates , with similar results . exact number ( n ) p values are shown in source data .

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(A) ROS accumulation in Gmlmm1-1 and Gmlmm1-2 mutant plants. Soybean leaves (aged 3 weeks) were subjected to ROS accumulation examination by DAB staining. Scale bar, 1 cm. Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.


( B ) ROS accumulation upon P . sojae treatment . leaves ( aged 3 ) were inoculated with P . sojae isolate P7076 , DAB was performed at 16 hpi . left panel was photographed under bright field ( scale bar , 1 cm ) . middle right panels were photographed under a microscope ( scale bar , 50 μm ) . Data information : experiments were performed three times as biological replicates , with similar results . exact number ( n ) p values are shown in source data .

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(B) ROS accumulation upon P. sojae treatment. The leaves (aged 3 weeks) were inoculated with P. sojae isolate P7076, and DAB staining was performed at 16 hpi. The left panel was photographed under bright field (scale bar, 1 cm). The middle and right panels were photographed under a microscope (scale bar, 50 μm). Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.


( D increased resistance of mutants to Psp ( D ) infection . indicated ( aged 2 ) were infected with indicated pathogens ( Mean ± SD , n ≥ 8 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - ) . lesions are shown by trypan blue . Scale bar , 1 cm . Data information : experiments were performed three times as biological replicates , with similar results . exact number ( n ) p values are shown in source data .

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(D The increased resistance of the mutants to Psp (D) infection. The indicated plants (aged 2 weeks) were infected with the indicated pathogens (Mean ± SD, n ≥ 8, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The lesions are shown by trypan blue staining. Scale bar, 1 cm. Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.


E ) increased resistance of mutants P . sojae ( E ) infection . indicated ( aged 2 ) were infected with indicated pathogens ( Mean ± SD , n ≥ 8 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - ) . lesions are shown by trypan blue . Scale bar , 1 cm . Data information : experiments were performed three times , as biological replicates , with similar results . exact number ( n ) p values are shown in source data .

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E) The increased resistance of the mutants P. sojae (E) infection. The indicated plants (aged 2 weeks) were infected with the indicated pathogens (Mean ± SD, n ≥ 8, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The lesions are shown by trypan blue staining. Scale bar, 1 cm. Data information: The experiments were performed three times , as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.


( A ) Flg22 - induced ROS burst in Gmlmm1 - 1 , Gmlmm1 - 2 Williams 82 . indicated leaves ( aged 2 ) were subjected to flg22 - induced ROS examination , peak relative luminescence unit ( RLU ) value was recorded ( Mean ± , n ≥ 12 , n represents sample number ) . Data information : experiments were performed three times ( biological replicates ) with similar results . exact n , values p values are shown in source data .

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(A) Flg22-induced ROS burst in Gmlmm1-1, Gmlmm1-2 and Williams 82. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the peak relative luminescence unit (RLU) value was recorded (Mean ± SEM, n ≥ 12, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.


( B ) Flg22 - induced ROS burst in CRISPR lines C9 - 24 - 13 DN50 . indicated leaves ( aged 2 ) were subjected to flg22 - induced ROS examination , relative RLU value was recorded ( Mean ± , n ≥ 9 , n represents sample number ) . Data information : experiments were performed three times ( biological replicates ) with similar results . exact n , values p values are shown in source data .

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(B) Flg22-induced ROS burst in the CRISPR lines C9-24-13 and DN50. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the relative RLU value was recorded (Mean ± SEM, n ≥ 9, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.


( C ) Suppression of flg22 - induced ROS production in Arabidopsis by GmLMM1 . GmLMM1 gene was introduced into Arabidopsis WT ( Col - 0 ) by Agrobacterium - mediated transformation . Two independent T2 lines were subjected to flg22 - induced ROS examination ( Mean ± SD , n ≥ 8 , n represents sample number , * * * , p < 0 . 001 , Student ' s t - ) . expression is shown . Data information : experiments were performed three times ( biological replicates ) with similar results . exact n , values p values are shown in source data .

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(C) Suppression of flg22-induced ROS production in Arabidopsis by GmLMM1. The GmLMM1 gene was introduced into Arabidopsis WT (Col-0) plants by Agrobacterium-mediated transformation. Two independent T2 lines were subjected to flg22-induced ROS examination (Mean ± SD, n ≥8, n represents sample number, ***, p < 0.001, Student's t-test). The protein expression is shown. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.


Suppression of flg22 - induced ( D ) ROS production in N . benthamiana by GmLMM1 . indicated constructs were transiently expressed by Agrobacterium - mediated transient expression for 2 days subjected to flg22 - induced ROS examination . GmLMM1K564E contains a mutation at predicted ATP binding site without kinase activity . GFP was used as a control ( Mean ± SD , n = 6 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - ) . expression is shown in lower panel . Data information : experiments were performed three times ( biological replicates ) with similar results . exact n , values p values are shown in source data .

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Suppression of flg22-induced (D) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to flg22-induced ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.


E ) Suppression of chitin - induced ( E ) ROS production in N . benthamiana by GmLMM1 . indicated constructs were transiently expressed by Agrobacterium - mediated transient expression for 2 days subjected to ROS examination . GmLMM1K564E contains a mutation at predicted ATP binding site without kinase activity . GFP was used as a control ( Mean ± SD , n = 6 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - ) . expression is shown in lower panel . Data information : experiments were performed three times ( biological replicates ) with similar results . exact n , values p values are shown in source data .

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E) Suppression of chitin-induced (E) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.


( F ) Inhibition of XEG1 - induced in N . benthamiana by GmLMM1 . indicated constructs were transiently expressed in N . benthamiana 1 day before infiltration of Agrobacterium containing XEG1 or GFP vector . were visualized 5 days later . expression of GmLMM1 - HA GmLMM1K564E - HA is shown in lower panel .

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(F) Inhibition of XEG1-induced cell death in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed in N. benthamiana 1 day before infiltration of Agrobacterium containing the XEG1 or GFP vector. The cell death phenotypes were visualized 5 days later. Protein expression of GmLMM1-HA and GmLMM1K564E-HA is shown in the lower panel.


( A ) Identification of GmLMM1 - interacting by IP - MS . GmLMM1 - FLAG was transiently expressed in Arabidopsis protoplasts , purified by anti - FLAG M2 agarose , subjected to mass spectrometry . complete list is shown in source data .

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(A) Identification of GmLMM1-interacting proteins by IP-MS analysis. GmLMM1-FLAG was transiently expressed in Arabidopsis protoplasts, purified by anti-FLAG M2 agarose, and subjected to mass spectrometry analysis. The complete list is shown in the source data.


( B ) Interactions of GmLMM1 with PRR receptors . indicated constructs were transiently expressed in N . benthamiana leaves subjected to luciferase complementation . GmFLS2 ( Gm08G083300 ) is closest homolog of FLS2 in soybean . GmILPA1 was used as a negative control . interaction is shown by relative luminescence unit ( RLU ) ( Mean ± SD , n ≥ 6 , n represents sample number , p < 0 . 05 , Student ' s t - , different letters indicate signiﬁcant difference ) . Immunoblots show of expression . Data information : experiments were performed three times as biological replicates , with similar results .

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(B) Interactions of GmLMM1 with PRR receptors. The indicated constructs were transiently expressed in N. benthamiana leaves and subjected to luciferase complementation assay. GmFLS2 (Gm08G083300) is the closest homolog of FLS2 in soybean. GmILPA1 was used as a negative control. The protein interaction intensity is shown by the relative luminescence unit (RLU) (Mean±SD, n ≥ 6, n represents sample number, p<0.05, Student's t-test, different letters indicate signiﬁcant difference). Immunoblots show the levels of protein expression. Data information: The experiments were performed three times as biological replicates, with similar results.


( C ) Co - IP of GmLMM1 with NbFLS2 GmFLS2 . indicated constructs were transiently expressed in N . benthamiana by Agrobacterium - mediated transient expression for 2 days interactions were examined by Co - IP . Data information : experiments were performed three times as biological replicates , with similar results .

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(C) Co-IP of GmLMM1 with NbFLS2 and GmFLS2. The indicated constructs were transiently expressed in N. benthamiana by Agrobacterium-mediated transient expression for 2 days and the protein interactions were examined by Co-IP assay. Data information: The experiments were performed three times as biological replicates, with similar results.


( D ) GmLMM1 interacts with NbRXEG1 in N . benthamiana . Data information : experiments were performed three times as biological replicates , with similar results .

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(D) GmLMM1 interacts with NbRXEG1 in N. benthamiana plants. Data information: The experiments were performed three times as biological replicates, with similar results.


( E ) Interaction of GmLMM1 with NbBAK1 , GmBAK1a , GmBAK1b . experiment statistical testing were performed as in ( B ) . Immunoblots show of expression . Data information : experiments were performed three times as biological replicates , with similar results .

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(E) Interaction of GmLMM1 with NbBAK1, GmBAK1a, and GmBAK1b. The experiment and statistical testing were performed as in (B). Immunoblots show the levels of protein expression. Data information: The experiments were performed three times as biological replicates, with similar results.


( F ) GmLMM1 interacts with NbBAK1 , GmBAK1a , GmBAK1b . indicated constructs were transiently expressed in N . benthamiana interactions were examined by Co - IP assays . Data information : experiments were performed two times ( F ) , as biological replicates , with similar results .

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(F) GmLMM1 interacts with NbBAK1, GmBAK1a, and GmBAK1b. The indicated constructs were transiently expressed in N. benthamiana and the interactions were examined by Co-IP assays. Data information: The experiments were performed two times (F), as biological replicates, with similar results.


( A ) Effect of flg22 on NbFLS2 - GmLMM1 interaction . indicated constructs were transiently expressed in N . benthamiana by Agrobacterium - mediated transient expression for 2 days , leaves were treated with flg22 ( 1 μM ) or water for 10 min before being subjected to Co - IP . Data information : experiments were performed three times , as biological replicates , with similar results .

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(A) Effect of flg22 on the NbFLS2-GmLMM1 interaction. The indicated constructs were transiently expressed in N. benthamiana by Agrobacterium-mediated transient expression for 2 days, and the leaves were treated with flg22 (1 μM) or water for 10 min before being subjected to Co-IP assay. Data information: The experiments were performed three times , as biological replicates, with similar results.


( B ) Regulation of NbBAK1 - GmLMM1 interaction by flg22 . Data information : experiments were performed three times as biological replicates , with similar results .

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(B) Regulation of NbBAK1-GmLMM1 interaction by flg22. Data information: The experiments were performed three times as biological replicates, with similar results.


( C ) Suppression of flg22 - induced NbFLS2 - NbBAK1 interaction by GmLMM1 . NbFLS2 NbBAK1 were co - expressed in N . benthamiana along with GmLMM1 or GmLMM1L407H , treated with H2O or flg22 subjected to co - IP assays . Data information : experiments were performed three times as biological replicates , with similar results .

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(C) Suppression of flg22-induced NbFLS2-NbBAK1 interaction by GmLMM1. NbFLS2 and NbBAK1 were co-expressed in N. benthamiana along with GmLMM1 or GmLMM1L407H, treated with H2O or flg22 and subjected to co-IP assays. Data information: The experiments were performed three times as biological replicates, with similar results.


( D Suppression effect of GmLMM1 on flg22 - induced interaction of GmFLS2 with GmBAK1a ( D ) Data information : experiments were performed two times ( D as biological replicates , with similar results .

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(D Suppression effect of GmLMM1 on the flg22-induced interaction of GmFLS2 with GmBAK1a (D) Data information: The experiments were performed two times (D as biological replicates, with similar results.