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

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


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 .

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 O O O O O O 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 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 .

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

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 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 .

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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 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 .

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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 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 .

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

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 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 .

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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 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 .

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

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 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 ) .

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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 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 .

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

O B-MEASURED_VAR 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

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 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 .

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

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


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 .

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 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

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 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 .

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

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 , 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 .

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

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 ) , 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 .

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

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

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 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 .

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 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

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 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 .

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

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 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 .

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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 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 .

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

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 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 .

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

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 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 .

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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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 the presence of different AHT concentrations . The amount of HTL produced in each reaction was determined by thin layer chromatography .

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

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 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

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 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 .

O O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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 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

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 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 .

O O O O O O O O O B-CONTROLLED_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 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

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 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 .

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 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 O O O O O O O O

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 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 .

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

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 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 .

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

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 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

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

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 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 .

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 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

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 , 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 .

O O O O O O O O O O O O O B-CONTROLLED_VAR 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 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 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 ) , 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 ) .

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

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

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

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

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

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

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

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 proteins . Pulldown fractions were subjected to western blotting using Flag and 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 cells were treated with APN or P5 . The cell 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 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 )

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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 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 .

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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 and IF for versican of skin tissue ; scale bars : 100 µm .

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


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

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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 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

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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 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 ) .

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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 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 ) .

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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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 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

(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 . 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 .

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

(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 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 .

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

(D The 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 ) 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 .

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

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 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

(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 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 .

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 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

(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 . 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 .

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 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

(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 . 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 .

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

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 . 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 .

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

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 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 .

(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 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 .

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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 . 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 .

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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 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 .

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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 plants . Data information : The 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 , 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 .

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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 , 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 .

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

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 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

(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 : The experiments were performed three times as biological replicates , with similar results .

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

(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 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 .

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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 the flg22 - induced interaction of GmFLS2 with GmBAK1a ( D ) Data information : The 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.


E ) Suppression effect of GmLMM1 on the flg22 - induced interaction of GmFLS2 with GmBAK1b ( E ) . Data information : The experiments were performed two times E ) , as biological replicates , with similar results .

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E) Suppression effect of GmLMM1 on the flg22-induced interaction of GmFLS2 with GmBAK1b (E). Data information: The experiments were performed two times E), as biological replicates, with similar results.


Single - marker association results for the combined non - Jewish and Jewish samples using the Cochran - Mantel - Haenszel ( CMH ) test . Each chromosome is depicted as a different color . The red line indicates the threshold ( P 5 × 10 − 5 ) selected to define regions for evaluation in the replication studies . The P value thresholds for suggestive and significant associations are 3 . 28 × 10 − 6 and 1 . 64 × 10 − 7 , respectively , based on a Bonferroni correction for multiple testing . A modest correction factor ( λ = 1 . 056 ) is necessary to correct for stratification between cases and controls , but it does not modify the current results .

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Single-marker association results for the combined non-Jewish and Jewish samples using the Cochran-Mantel-Haenszel (CMH) test. Each chromosome is depicted as a different color. The red line indicates the threshold (P 5 × 10−5) selected to define regions for evaluation in the replication studies. The P value thresholds for suggestive and significant associations are 3.28 × 10−6 and 1.64 × 10−7, respectively, based on a Bonferroni correction for multiple testing. A modest correction factor (λ = 1.056) is necessary to correct for stratification between cases and controls, but it does not modify the current results.


( a - c ) We used quantitative real - time PCR to determine the expression patterns of ATG5 ( a ) , ATG7 ( b ) and ATG16L1 ( c ) in various human immune and epithelial cell lines . ATG5 was broadly expressed in the cell lines studied ( a ) , with between 1 . 5 - and fourfold variations in expression level across the cell lines . ATG7 expression ( b ) was broadly comparable to that of ATG5 , with the exception of THP1 cells , which showed 35 - fold higher expression than SW480 cells . ATG16L1 expression ( c ) was also broadly similar to that of ATG5 and ATG7 ( except for ATG7 expression in THP1 cells ) , with moderate variations across the cell lines tested ( 1 . 5 - to 3 . 5 - fold ) .

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(a-c) We used quantitative real-time PCR to determine the expression patterns of ATG5 (a), ATG7 (b) and ATG16L1 (c) in various human immune and epithelial cell lines. ATG5 was broadly expressed in the cell lines studied (a), with between 1.5- and fourfold variations in expression level across the cell lines. ATG7 expression (b) was broadly comparable to that of ATG5, with the exception of THP1 cells, which showed 35-fold higher expression than SW480 cells. ATG16L1 expression (c) was also broadly similar to that of ATG5 and ATG7 (except for ATG7 expression in THP1 cells), with moderate variations across the cell lines tested (1.5- to 3.5-fold).


( d ) Expression of ATG16L1 in human primary immune cells . In a resting human immune cell RNA panel ( Clontech ) , ATG16L1 showed a clear peak of expression in the T cell compartment , with CD4 + and CD8 + cells expressing high levels of transcript ( 15 - to 20 - fold more than control samples ) . We detected modest levels of ATG16L1 in CD19 + cells and low levels in CD14 + monocytes and unfractionated mononuclear cells . In all cases , we performed real - time quantitative RT - PCR reactions in duplicate and plotted the means ; error bars represent 1 s . d . We normalized expression levels by comparison with GAPDH controls and plotted arbitrary relative expression units , where the SW480 or placental RNA expression equals 1 ( for cell lines or primary cells , respectively ) . We isolated all RNAs from resting cells in the absence of stimulation or activation .

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(d) Expression of ATG16L1 in human primary immune cells. In a resting human immune cell RNA panel (Clontech), ATG16L1 showed a clear peak of expression in the T cell compartment, with CD4+ and CD8+ cells expressing high levels of transcript (15- to 20-fold more than control samples). We detected modest levels of ATG16L1 in CD19+ cells and low levels in CD14+ monocytes and unfractionated mononuclear cells. In all cases, we performed real-time quantitative RT-PCR reactions in duplicate and plotted the means; error bars represent 1 s.d. We normalized expression levels by comparison with GAPDH controls and plotted arbitrary relative expression units, where the SW480 or placental RNA expression equals 1 (for cell lines or primary cells, respectively). We isolated all RNAs from resting cells in the absence of stimulation or activation.


( a ) We achieved specific siRNA knockdown of overexpressed , Flag - tagged ATG16L1 in HEK293 cells within 48 h of transfection with oligonucleotide duplexes . Flag - ATG16L1 was undetectable by protein blotting after treatment with specific siRNA constructs , but expression was maintained with control duplexes .

(a) We achieved specific siRNA knockdown of overexpressed, Flag-tagged ATG16L1 in HEK293 cells within 48 h of transfection with oligonucleotide duplexes. Flag-ATG16L1 was undetectable by protein blotting after treatment with specific siRNA constructs, but expression was maintained with control duplexes.


( b ) Endogenous ATG16L1 mRNA knockdown ( mean relative abundance ± s . d . ) by siRNA 2 in HeLa cells within 48 h of transfection , as assessed by real - time quantitative RT - PCR normalized to GAPDH . Compared with control duplex , siRNA 2 yielded an 89 % reduction in transcripts . RT - PCR was performed in triplicate ; results represent two independent experiments .

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(b) Endogenous ATG16L1 mRNA knockdown (mean relative abundance ± s.d.) by siRNA 2 in HeLa cells within 48 h of transfection, as assessed by real-time quantitative RT-PCR normalized to GAPDH. Compared with control duplex, siRNA 2 yielded an 89% reduction in transcripts. RT-PCR was performed in triplicate; results represent two independent experiments.


c ) Knockdown of ATG16L1 prevented effective autophagy of S . typhimurium in HeLa cells . Forty - eight hours after cotransfection with control siRNA or duplex 2 and LC3 - GFP plasmid , HeLa cells were infected for 1 h with Salmonella typhimurium SL1344 , fixed and examined microscopically . Mean percentages ( ± s . e . m . ) of bacteria per cell encapsulated by LC3 + membranes ( autophagosomes ) are shown . Bacterial counts were pooled from two separate experiments ; each counted a minimum of 100 infected cells . Significance was assessed by two - tailed Students t - test , assuming unequal variances .

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c) Knockdown of ATG16L1 prevented effective autophagy of S. typhimurium in HeLa cells. Forty-eight hours after cotransfection with control siRNA or duplex 2 and LC3-GFP plasmid, HeLa cells were infected for 1 h with Salmonella typhimurium SL1344, fixed and examined microscopically. Mean percentages (± s.e.m.) of bacteria per cell encapsulated by LC3+ membranes (autophagosomes) are shown. Bacterial counts were pooled from two separate experiments; each counted a minimum of 100 infected cells. Significance was assessed by two-tailed Students t-test, assuming unequal variances.


( d ) Representative images of intracellular S . typhimurium show normal autophagic encapsulation by LC3 + membranes in control siRNA - treated cells but loss of autophagic targeting after ATG16L1 knockdown . Channels are separated to show both the LC3 - GFP fusion protein ( autophagosomal marker ) and S . typhimurium SL1344 dsRed2 ( SL1344 ) , with a merged image in the final column . Rows 2 and 4 show magnified views of boxed areas in rows 1 and 3 , respectively . Images were obtained using confocal microscopy and are a flat projection of z stacks taken throughout the plane of the infected cell . Scale bars = 5 μm .

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(d) Representative images of intracellular S. typhimurium show normal autophagic encapsulation by LC3+ membranes in control siRNA-treated cells but loss of autophagic targeting after ATG16L1 knockdown. Channels are separated to show both the LC3-GFP fusion protein (autophagosomal marker) and S. typhimurium SL1344 dsRed2 (SL1344), with a merged image in the final column. Rows 2 and 4 show magnified views of boxed areas in rows 1 and 3, respectively. Images were obtained using confocal microscopy and are a flat projection of z stacks taken throughout the plane of the infected cell. Scale bars = 5 μm.


( B ) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB - treated mMuSCs , human myoblasts and hiMPs . Red dots represent genes which display a positive fold - change in expression upon treatment with DLL4 & PDGF - BB whilst violet dots represent genes which are significantly downregulated . Differentially expressed genes required a P value of ≤ 0 . 05 to be considered significant .

(B) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB-treated mMuSCs, human myoblasts and hiMPs. Red dots represent genes which display a positive fold-change in expression upon treatment with DLL4 & PDGF-BB whilst violet dots represent genes which are significantly downregulated. Differentially expressed genes required a P value of ≤ 0.05 to be considered significant.


( C ) Heatmaps showing changes in expression of key myogenic ( MYOD , MYOGENIN ) , perivascular ( PDGFRB , NG2 , CD146 , ALPL ) and NOTCH target ( HEY1 , HES1 ) genes upon treatment with DLL4 & PDGF - BB in mMuSC - derived myoblasts ( left ) , human myoblasts ( middle ) and hiMPs ( right ) . Clustering was performed by genes / probes with Pearson correlation . Colour scale based on z - scores : red regions indicate high expression whilst blue regions indicate low expression . Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled .

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(C) Heatmaps showing changes in expression of key myogenic (MYOD, MYOGENIN), perivascular (PDGFRB, NG2, CD146, ALPL) and NOTCH target (HEY1, HES1) genes upon treatment with DLL4 & PDGF-BB in mMuSC-derived myoblasts (left), human myoblasts (middle) and hiMPs (right). Clustering was performed by genes/probes with Pearson correlation. Colour scale based on z-scores: red regions indicate high expression whilst blue regions indicate low expression. Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled.


( B ) Phase contrast images displaying morphology of untreated and treated hiMPs . Scale bar : 25 μm . ( C ) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs . Morphology was quantified as circularity ratio , where 1 = perfect circle and 0 = line ( experimental replicates = 3 ) . Statistical analysis ( paired t test ) was performed on the first quartile to enhance detection of morphological changes ( error bars : SD ) .

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(B) Phase contrast images displaying morphology of untreated and treated hiMPs. Scale bar: 25 μm. (C) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs. Morphology was quantified as circularity ratio, where 1 = perfect circle and 0 = line (experimental replicates = 3). Statistical analysis (paired t test) was performed on the first quartile to enhance detection of morphological changes (error bars: SD).


( E ) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours . Scale bar : 75 μm . ( F ) Bar graphs quantifying EdU experiment shown in ( E ) ( experimental replicates = 3 ; error bars : SEM ) . Statistical analysis based on an unpaired t - test .

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(E) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours. Scale bar: 75 μm. (F) Bar graphs quantifying EdU experiment shown in (E) (experimental replicates = 3; error bars: SEM). Statistical analysis based on an unpaired t-test.


( H ) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week , induced to differentiate for 4 days in the presence or absence of γ - secretase inhibitor L685458 and immunostained for myosin heavy chain ( MyHC ) . ( I ) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes ( experimental replicates = 3 ; error bars : SEM ) . Statistical significance based on one - way ANOVA with Tukey ' s multiple comparison .

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(H) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week, induced to differentiate for 4 days in the presence or absence of γ-secretase inhibitor L685458 and immunostained for myosin heavy chain (MyHC). (I) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes (experimental replicates = 3; error bars: SEM). Statistical significance based on one-way ANOVA with Tukey's multiple comparison.


( J ) Immunofluorescence panels showing human specific LAMIN A / C + ( nuclei ) and SPECTRIN ( sarcolemma ) staining in tibialis anterior muscles of immunodeficient mice ( N = 3 ) transplanted with treated ( n = 3 muscles ) and untreated ( n = 3 muscles ) N5 hiMPs . ( K ) Quantification of LAMIN A / C + grafted human cells across each muscle . Data information : full gene list for heatmaps in ( A ) and ( D ) available in Appendix Table S4 .

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(J) Immunofluorescence panels showing human specific LAMIN A/C+ (nuclei) and SPECTRIN (sarcolemma) staining in tibialis anterior muscles of immunodeficient mice (N = 3) transplanted with treated (n = 3 muscles) and untreated (n = 3 muscles) N5 hiMPs. (K) Quantification of LAMIN A/C+ grafted human cells across each muscle. Data information: full gene list for heatmaps in (A) and (D) available in Appendix Table S4.


( F ) Representative fluorescence images of CMFDA - stained untreated and DLL4 & PDGF - BB - treated hiMPs within the top perfusion channel , 15 minutes after delivery and kept on the OrganoFlow ® . ( G ) Bar graph quantifying adhesion images in ( E ) . Statistical significance was calculated based on a paired t - test ( experimental replicates = 3 ) . Each point on the plot represents the number of adhered cells after 15 minutes within a single chip .

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(F) Representative fluorescence images of CMFDA-stained untreated and DLL4 & PDGF-BB-treated hiMPs within the top perfusion channel, 15 minutes after delivery and kept on the OrganoFlow®. (G) Bar graph quantifying adhesion images in (E). Statistical significance was calculated based on a paired t-test (experimental replicates = 3). Each point on the plot represents the number of adhered cells after 15 minutes within a single chip.


( H ) Assessment of DLL4 & PDGF - BB - treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells . Representative images showing the lower side of the trans - well membrane on which treated and untreated hiMPs ( stained with the transient dye CFDA , in green ) are simultaneously seeded on HUVECs for 8 hours . Bar graphs quantifying the average number of CFDA - positive cells / mm2 , that have migrated through the endothelial layer in each considered condition . ( experimental replicates = 3 ) . A minimum of 10 ( 1 . 5 mm2 ) fields per condition was quantified ( error bars : SEM ) . ( I ) Bar graph showing fold - change in trans - endothelial migration ( error bars : SEM ) . Statistical significance based on one - way ANOVA with Bonferroni ' s multiple comparison .

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(H) Assessment of DLL4 & PDGF-BB-treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans-well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 hours. Bar graphs quantifying the average number of CFDA-positive cells/ mm2, that have migrated through the endothelial layer in each considered condition. (experimental replicates = 3). A minimum of 10 (1.5 mm2) fields per condition was quantified (error bars: SEM). (I) Bar graph showing fold-change in trans-endothelial migration (error bars: SEM). Statistical significance based on one-way ANOVA with Bonferroni's multiple comparison.


Sanger - sequencing chromatograms showing the target region of sgAkt1E17K in wild - type ( WT ) and base edited ( BE ) cells . Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3 - expressing NIH3T3 cells with Lenti - sgAkt1E17K . EditR ( Kluesner et al . , 2018 ) was used to calculate the frequency ( % ) of C - to - T conversion at C7 of the protospacer targeted by sgAkt1E17K in BE3 - expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors .

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Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgAkt1E17K. EditR (Kluesner et al., 2018) was used to calculate the frequency (%) of C-to-T conversion at C7 of the protospacer targeted by sgAkt1E17K in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.