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

O O 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

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


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

O O 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

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


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

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

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


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

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

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


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

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

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


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

O O O 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

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


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

O O O 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 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

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


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

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

O 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

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


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

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

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


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

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

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


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

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

O 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

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


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

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

O B-MEASURED_VAR 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

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


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

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

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


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

O O 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 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

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


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

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 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

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


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

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

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


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

O O O O O O O O O O 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

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


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

O O 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

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


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

O O 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 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

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


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

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

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


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

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 B-CONTROLLED_VAR 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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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 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

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


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

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

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


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

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

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

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 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

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


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

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 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

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


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

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


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

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


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

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 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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


E ) Suppression effect of GmLMM1 on flg22 - induced interaction of GmFLS2 with GmBAK1b ( E ) . Data information : 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 combined non - Jewish Jewish samples using Cochran - Mantel - Haenszel ( CMH ) . Each chromosome is depicted as a different color . red indicates threshold ( P 5 × 10 − 5 ) selected to define regions for evaluation in replication studies . P value thresholds for suggestive significant associations are 3 . 28 × 10 − 6 1 . 64 × 10 − 7 , respectively , on a Bonferroni correction for multiple testing . A modest correction factor ( λ = 1 . 056 ) is necessary to correct for stratification between cases controls , but it does not modify 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 expression patterns of ATG5 ( a ) , ATG7 ( b ) ATG16L1 ( c ) in various human immune epithelial lines . ATG5 was broadly expressed in lines studied ( a ) , with between 1 . 5 - fourfold variations in expression level across lines . ATG7 expression ( b ) was broadly comparable to that of ATG5 , with exception of THP1 , which showed 35 - fold higher expression than SW480 . ATG16L1 expression ( c ) was also broadly similar to that of ATG5 ATG7 ( except for ATG7 expression in THP1 ) , with moderate variations across 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 immune . In a resting human immune panel ( Clontech ) , ATG16L1 showed a clear peak of expression in T compartment , with CD4 + CD8 + expressing high of transcript ( 15 - to 20 - fold more than control samples ) . We detected modest of ATG16L1 in CD19 + low in CD14 + monocytes unfractionated mononuclear . In cases , we performed real - time quantitative RT - PCR reactions in duplicate plotted means ; error bars represent 1 s . d . We normalized expression by comparison with GAPDH controls plotted arbitrary relative expression units , where SW480 or placental expression equals 1 ( for lines or , respectively ) . We isolated RNAs from resting in 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 within 48 h of transfection with oligonucleotide duplexes . Flag - ATG16L1 was undetectable by blotting after treatment with specific siRNA constructs , but expression was maintained with control duplexes .

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(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 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 . Forty - eight hours after cotransfection with control siRNA or duplex 2 LC3 - GFP plasmid , HeLa were infected for 1 h with Salmonella typhimurium SL1344 , fixed examined microscopically . Mean percentages ( ± s . e . m . ) of per encapsulated by LC3 + membranes ( autophagosomes ) are shown . were pooled from two separate experiments ; each counted a minimum of 100 infected . Significance was assessed by two - Students t - , 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 of intracellular S . typhimurium show normal autophagic encapsulation by LC3 + membranes in control siRNA - treated but loss of autophagic targeting after ATG16L1 knockdown . Channels are separated to show both LC3 - GFP fusion ( autophagosomal marker ) S . typhimurium SL1344 dsRed2 ( SL1344 ) , with a merged image in final column . Rows 2 4 show magnified views of boxed areas in rows 1 3 , respectively . were obtained using confocal microscopy are a flat projection of z stacks taken throughout plane of infected . 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 DLL4 & PDGFBB - treated mMuSCs , human myoblasts 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 .

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(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 ) NOTCH target ( HEY1 , HES1 ) genes upon treatment with DLL4 & PDGF - BB in mMuSC - derived myoblasts ( left ) , human myoblasts ( middle ) hiMPs ( right ) . Clustering was performed by genes / probes with Pearson correlation . Colour scale on z - scores : red regions indicate high expression whilst blue regions indicate low expression . Dendrograms indicate similarity of clusters as well as 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 displaying of untreated treated hiMPs . Scale bar : 25 μm . ( C ) Scatter dot plot showing morphometric of treated untreated hiMPs . was quantified as circularity , where 1 = perfect circle 0 = ( experimental replicates = 3 ) . Statistical ( paired t ) was performed on 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 of untreated 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 : ) . Statistical on an unpaired t - .

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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 of hiMPs expanded in control or treated conditions for 1 week , induced to differentiate for 4 days in presence or absence of γ - secretase inhibitor L685458 immunostained for myosin heavy chain ( MyHC ) . ( I ) Bar graph quantifying average of nuclei within MyHC positive myotubes ( experimental replicates = 3 ; error bars : ) . Statistical significance 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 ) SPECTRIN ( sarcolemma ) in tibialis anterior of immunodeficient mice ( N = 3 ) transplanted with treated ( n = 3 ) untreated ( n = 3 ) N5 hiMPs . ( K ) of LAMIN A / C + grafted human across each muscle . Data information : full gene list for heatmaps in ( A ) ( 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 of CMFDA - stained untreated DLL4 & PDGF - BB - treated hiMPs within top perfusion channel , 15 minutes after delivery kept on OrganoFlow ® . ( G ) Bar graph quantifying adhesion in ( E ) . Statistical significance was calculated on a paired t - ( experimental replicates = 3 ) . Each point on plot represents number of adhered 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 genetically corrected DMD hiMP migration through a layer of endothelial . Representative showing lower side of trans - well on which treated untreated hiMPs ( stained with transient dye CFDA , in green ) are simultaneously seeded on HUVECs for 8 hours . Bar graphs quantifying average number of CFDA - positive / mm2 , that have migrated through endothelial layer in each considered condition . ( experimental replicates = 3 ) . A minimum of 10 ( 1 . 5 mm2 ) fields per condition was quantified ( error bars : ) . ( I ) Bar graph showing fold - change in trans - endothelial migration ( error bars : ) . Statistical significance 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 target region of sgAkt1E17K in wild - type ( WT ) base edited ( BE ) . Arrowheads highlight cytosines of protospacer that show base editing 5 days after transduction of BE3 - expressing NIH3T3 with Lenti - sgAkt1E17K . EditR ( Kluesner et al . , 2018 ) was used to calculate frequency ( % ) of C - to - T conversion at C7 of protospacer targeted by sgAkt1E17K in BE3 - expressing NIH3T3 5 days after transduction with 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.


Kaplan - Meier curves showing mammary - specific survival for different models . WB1P - BE3 injected with Lenti - sgAkt1E17K - Myc ( n = 12 ) showed a reduced mammary - specific survival compared to WB1P - BE3 mice injected with Lenti - sgNT - Myc ( n = 11 ) vectors ( 58 days after injection vs 72 days after injection , P < 0 . 01 by Mantel - Cox ) .

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Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc (n=12) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (58 days after injection vs 72 days after injection, P < 0.01 by Mantel-Cox test).


Sanger - sequencing chromatograms showing target region of sgAkt1E17K in 3 independent tumors from WB1P - BE3 injected with Lenti - sgAkt1E17K - Myc . Arrowheads highlight cytosines of protospacer that show base editing . EditR was used to calculate average frequency ( % ) of C - to - T conversion at C7 of protospacer in tumors from WB1P - BE3 injected with Lenti - sgNT - Myc or Lenti - sgAkt1E17K - Myc .

Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in 3 independent tumors from WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc. Arrowheads highlight cytosines of the protospacer that show base editing. EditR was used to calculate the average frequency (%) of C-to-T conversion at C7 of the protospacer in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgAkt1E17K-Myc.


Sanger - sequencing chromatograms showing target regions of sgPik3caE542K , sgPik3caE545K sgPik3caE453K in wild - type ( WT ) base edited ( BE ) . Arrowheads highlight cytosines of protospacers that show base editing 5 days after transduction of BE3 - expressing NIH3T3 with Lenti - sgPik3caE542K , Lenti - sgPik3caE545K Lenti - sgPik3caE453K .

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Sanger-sequencing chromatograms showing the target regions of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacers that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPik3caE542K, Lenti-sgPik3caE545K and Lenti-sgPik3caE453K.


EditR was used to calculate frequency ( % ) of C - to - T conversion at indicated target cytosines of protospacers in BE3 - expressing NIH3T3 5 days after transduction with indicated sgRNA vectors .

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EditR was used to calculate the frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.


Kaplan - Meier curves showing mammary - specific survival for different models . WB1P - BE3 injected with Lenti - sgPik3caE542K - Myc ( n = 10 ) , Lenti - sgPik3caE545K - Myc ( n = 10 ) Lenti - sgPik3caE453K - Myc ( n = 10 ) showed a reduced mammary - specific survival compared to WB1P - BE3 mice injected with Lenti - sgNT - Myc ( n = 11 ) vectors ( respectively 47 , 44 49 days after injection vs 72 days after injection , P < 0 . 0001 by Mantel - Cox ) .

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Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgPik3caE542K-Myc (n=10), Lenti-sgPik3caE545K-Myc (n=10) and Lenti-sgPik3caE453K-Myc (n=10) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (respectively 47, 44 and 49 days after injection vs 72 days after injection, P < 0.0001 by Mantel-Cox test).


Sanger - sequencing chromatograms showing target region of sgPik3caE542K , sgPik3caE545K sgPik3caE453K in 3 independent tumors from WB1P - BE3 injected with corresponding Lenti - sgPik3ca - Myc vectors . Arrowheads highlight cytosines of protospacer that show base editing . EditR was used to calculate average frequency ( % ) of C - to - T conversion at indicated target cytosines of protospacers in tumors from WB1P - BE3 injected with Lenti - sgNT - Myc or Lenti - sgPik3caE542K - Myc , Lenti - sgPik3caE545K - Myc Lenti - sgPik3caE453K - Myc .

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Sanger-sequencing chromatograms showing the target region of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in 3 independent tumors from WB1P-BE3 females injected with the corresponding Lenti-sgPik3ca-Myc vectors. Arrowheads highlight cytosines of the protospacer that show base editing. EditR was used to calculate the average frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgPik3caE542K-Myc, Lenti-sgPik3caE545K-Myc and Lenti-sgPik3caE453K-Myc.


Sanger - sequencing chromatograms showing target region of sgPtenQ245 * in wild - type ( WT ) base edited ( BE ) . Arrowheads highlight cytosines of protospacer that show base editing 5 days after transduction of BE3 - expressing NIH3T3 with Lenti - sgPtenQ245 * . EditR was used to calculate frequency ( % ) of C - to - T conversion at C4 of protospacer targeted by sgPtenQ245 * in BE3 - expressing NIH3T3 5 days after transduction with indicated sgRNA vectors .

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

Sanger-sequencing chromatograms showing the target region of sgPtenQ245* 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-sgPtenQ245*. EditR was used to calculate the frequency (%) of C-to-T conversion at C4 of the protospacer targeted by sgPtenQ245* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.


Kaplan - Meier curves showing mammary - specific survival for different models . WB1P - BE3 injected with Lenti - sgPtenQ245 * - Myc ( n = 11 ) showed a reduced mammary - specific survival compared to WB1P - BE3 mice injected with Lenti - sgNT - Myc ( n = 11 ) vectors ( 37 days after injection vs 72 days after injection , P < 0 . 0001 by Mantel - Cox ) .

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

Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgPtenQ245*-Myc (n=11) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (37 days after injection vs 72 days after injection, P < 0.0001 by Mantel-Cox test).


BE Analyzer ( Hwang et al . , 2018 ) was used to assess from next - generation sequencing data fraction of wild - type Pten alleles , base edited alleles or alleles with insertions / deletions ( indels ) in tumors from WB1P - BE3 injected with Lenti - sgNT - Myc or Lenti - sgPtenQ245 * - Myc . TIDE showing spectrum of indels of targeted Pten alleles in two independent representative tumors from WB1P - BE3 mice injected with Lenti - sgPtenQ245 * - Myc . For two tumors shown in ( F ) , Sanger - sequencing chromatograms showing target region of sgPtenQ245 * ( PCR products were subcloned for clarity ) . Arrowheads highlight cytosines of protospacer that show base editing . In lower example gene was inactivated by indels at both alleles , while in upper one by Q245 * base editing in one allele a deletion at second copy of gene .

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

BE Analyzer (Hwang et al., 2018) was used to assess from next-generation sequencing data the fraction of wild-type Pten alleles, base edited alleles or alleles with insertions/deletions (indels) in tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc or Lenti-sgPtenQ245*-Myc. TIDE analysis showing the spectrum of indels of the targeted Pten alleles in two independent representative tumors from WB1P-BE3 mice injected with Lenti-sgPtenQ245*-Myc. For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgPtenQ245* (PCR products were subcloned for clarity). Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by indels at both alleles, while in the upper one by Q245* base editing in one allele and a deletion at the second copy of the gene.


Sanger - sequencing chromatograms showing target region of sgTrp53Q97 * in wild - type ( WT ) base edited ( BE ) . Arrowheads highlight cytosines of protospacer that show base editing 5 days after transduction of BE3 - expressing NIH3T3 with Lenti - sgTrp53Q97 EditR was used to calculate frequency ( % ) of C - to - T conversion at C8 of protospacer targeted by sgTrp53Q97 * in BE3 - expressing NIH3T3 5 days after transduction with indicated sgRNA vectors .

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

Sanger-sequencing chromatograms showing the target region of sgTrp53Q97* 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-sgTrp53Q97 EditR was used to calculate the frequency (%) of C-to-T conversion at C8 of the protospacer targeted by sgTrp53Q97* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.


Kaplan - Meier curves showing mammary - specific survival for different models . WapCre ; Brca1F / F ; Trp53F / + ; Col1a1invCAG - BE3 / + injected with Lenti - sgPik3caE545K / sgTrp53Q97 * - Myc ( n = 6 ) showed a reduced mammary - specific survival compared to injected with Lenti - sgTrp53Q97 * - Myc ( n = 5 ) vectors ( 76 days after injection vs 101 days after injection , P < 0 . 05 by Mantel - Cox ) . injected with Lenti - sgNT - Myc ( n = 11 ) did not develop any palpable tumors during 150 days observation period .

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

Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ females injected with Lenti-sgPik3caE545K/sgTrp53Q97*-Myc (n=6) showed a reduced mammary tumor-specific survival compared to animals injected with Lenti-sgTrp53Q97*-Myc (n=5) vectors (76 days after injection vs 101 days after injection, P < 0.05 by Mantel-Cox test). Females injected with Lenti-sgNT-Myc (n=11) did not develop any palpable tumors during the 150 days observation period.


BE Analyzer was used to assess from next - generation sequencing data fraction of wild - type Trp53 alleles , base edited alleles or alleles with indels in tumors from WapCre ; Brca1F / F ; Trp53F / + ; Col1a1invCAG - BE3 / + injected with Lenti - sgTrp53Q97 * - Myc . Tumors from WB1P - BE3 injected with Lenti - sgNT - Myc mice were used as control . TIDE showing spectrum of indels of targeted Trp53 alleles in two independent representative tumors from WapCre ; Brca1F / F ; Trp53F / + ; Col1a1invCAG - BE3 / + mice injected with Lenti - sgTrp53Q97 * - Myc . For two tumors shown in ( F ) , Sanger - sequencing chromatograms showing target region of sgTrp53Q97 * . Arrowheads highlight cytosines of protospacer that show base editing . In lower example gene was inactivated by a deletion , while in upper one by Q97 * base editing . Of note , allele with indel also displays base editing at C6 of protospacer .

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

BE Analyzer was used to assess from next-generation sequencing data the fraction of wild-type Trp53 alleles, base edited alleles or alleles with indels in tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ animals injected with Lenti-sgTrp53Q97*-Myc. Tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc mice were used as control. TIDE analysis showing the spectrum of indels of the targeted Trp53 alleles in two independent representative tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ mice injected with Lenti-sgTrp53Q97*-Myc. For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgTrp53Q97*. Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by a deletion, while in the upper one by Q97* base editing. Of note, the allele with the indel also displays base editing at C6 of the protospacer.


U2OS modified to expressed endogenous GFP - tagged ATG2A ( green ) were grown in complete media ( CM ) or starved ( EBSS ) for 2 h before fixation immunostaining with antibodies against ATG2B ( red ) LC3B ( magenta ) analysed by confocal microscopy . Arrows mark ATG2A / LC3B / ATG2B positive structures . Scale bar 10µm . ( B - D ) were treated as in ( A ) stained with anti - WIPI2 ( red ) or ( C ) anti - ATG16L1 ( red ) or ( D ) GABARAP - L1 ( red ) . Arrows mark structures of interest . Scale bar 10µm .

U2OS cells modified to expressed endogenous GFP-tagged ATG2A (green) were grown in complete media (CM) or starved (EBSS) for 2 h before fixation and immunostaining with antibodies against ATG2B (red) and LC3B (magenta) and analysed by confocal microscopy. Arrows mark ATG2A/LC3B/ATG2B positive structures. Scale bar 10µm. (B-D) Cells were treated as in (A) and stained with anti-WIPI2 (red) or (C) anti-ATG16L1 (red) or (D) GABARAP-L1 (red). Arrows mark structures of interest. Scale bar 10µm.


U2OS WT or U2OS GFP - ATG2A knock - in ( KI ) were grown in CM or starvation media for 2 h , lysed incubated with anti - GFP nanobody beads coupled to agarose to immunoprecipitate ( IP ) GFP - ATG2A . IP samples 2 % input lysates were run on 4 - 12 % gradient gel processed for western blotting . Anti - ATG2A , anti - WIPI4 anti - LC3B , anti - GABARAP ( pan ) were used to probe for presence / absence of autophagy in immunoprecipitated samples . p - p70S6K ( T389 ) was used as a marker for starved total p70S6K as loading control .

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

U2OS WT or U2OS GFP-ATG2A knock-in (KI) cells were grown in CM or starvation media for 2 h, lysed and incubated with anti-GFP nanobody beads coupled to agarose to immunoprecipitate (IP) GFP-ATG2A. IP samples and 2% input lysates were run on 4-12% gradient gel and processed for western blotting. Anti-ATG2A, anti-WIPI4 and anti-LC3B, anti-GABARAP (pan) were used to probe for the presence/absence of autophagy proteins in the immunoprecipitated samples. p-p70S6K (T389) was used as a marker for starved cells and total p70S6K as loading control.


Myc - tagged ATG2A - wild type ( WT ) ATG2A - mLIR ( orange ) were co - expressed with GFP - alone , GFP - LC3B or GFP - GABARAP in HEK293T , lysed anti - GFP nanobodies coupled to agarose were used to immunoprecipitate GFP - tagged . Samples were subjected to western blotting probed for presence of Myc - ATG2A in immunoprecipitated samples . Anti - p62 / SQSTM1 was used as an internal control for immunoprecipitated samples . As in ( B ) but using Myc - tagged ATG2B - WT or ATG2B - mLIR ( orange ) co - expressed with GFP - alone , GFP - LC3B or GFP - GABARAP . Samples were subjected to western blotting probed for presence of Myc - ATG2B in immunoprecipitated samples . Anti - p62 / SQSTM1 was used as an internal control for immunoprecipitated samples .

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

Myc-tagged ATG2A-wild type (WT) and ATG2A-mLIR (orange) were co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP in HEK293T cells, lysed and anti-GFP nanobodies coupled to agarose were used to immunoprecipitate GFP-tagged proteins. Samples were subjected to western blotting and probed for the presence of Myc-ATG2A in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.   As in (B) but using Myc-tagged ATG2B-WT or ATG2B-mLIR (orange) co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP. Samples were subjected to western blotting and probed for the presence of Myc-ATG2B in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.


HA - tagged ATG2A - WT , - mLIR ( orange ) - mYFS ( YFS / AAA ; purple ) were stably expressed in U2OS ATG2A / B double knock - out using retrovirus transduction . were transfected with GFP - GABARAP 24 h later grown in complete medium ( CM ) or starved for 2 h ( EBSS ) . were lysed anti - HA beads were used to immunoprecipitate HA - tagged ATG2A processed for western blot . Blots were then probed with antibodies against HA - tag ( ATG2A ) , anti - WIPI4 , anti - GFP for presence / absence in immunoprecipitated samples . blots are representative of at least n = 3 independent experiments . of WIPI4 GFP - GABARAP - ii co - precipitation with HA - ATG2A from ( D ) . bands were normalized against HA - ATG2A immunoprecipitate expressed as a fold change compared to HA - ATG2A - WT ( complete media ; CM ) . Each symbol represents an independent experiment ( n = 3 ) error bars mean ± SD .

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

HA-tagged ATG2A-WT, -mLIR (orange) and -mYFS (YFS/AAA; purple) were stably expressed in U2OS ATG2A/B double knock-out cells using retrovirus transduction. Cells were transfected with GFP-GABARAP and 24 h later grown in complete medium (CM) or starved for 2 h (EBSS). Cells were lysed and anti-HA beads were used to immunoprecipitate HA-tagged ATG2A and processed for western blot. Blots were then probed with antibodies against HA-tag (ATG2A), anti-WIPI4, and anti-GFP for the presence/absence in immunoprecipitated samples. All blots are representative of at least n=3 independent experiments.   Quantification of WIPI4 and GFP-GABARAP-ii co-precipitation with HA-ATG2A from (D). bands were normalized against HA-ATG2A immunoprecipitate and expressed as a fold change compared to HA-ATG2A-WT (complete media; CM). Each symbol represents an independent experiment (n=3) and error bars mean ± SD.


ATG2A / B do8uble knock - out ( DKO ) stably expressing HA - tagged ATG2A - WT , - mLIR ( orange ) or - mYFS ( YFS / AAA ; purple ) were transfected with GFP - GABARAP 24 h later grown in complete medium ( CM ) or starved for 2 h ( EBSS ) . were lysed GFP - GABARAP was immunoprecipitated using anti - GFP trap beads . Samples were processed for western blot probed with anti - HA - tag ( ATG2A ) , anti - WIPI4 , anti - GFP for their presence / absence in immunoprecipitated samples . blots are representative of at least n = 3 independent experiments . of HA - ATG2A WIPI4 co - precipitation with GFP - GABARAP from ( F ) . Results were expressed as a fold change compared to HA - ATG2A - WT ( complete media ; CM ) . Each symbol represents an independent experiment ( n = 3 ) with error bars mean ± SD .

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

ATG2A/B do8uble knock-out cells (DKO) stably expressing HA-tagged ATG2A-WT, -mLIR (orange) or -mYFS (YFS/AAA; purple) were transfected with GFP-GABARAP and 24 h later grown in complete medium (CM) or starved for 2 h (EBSS). Cells were lysed and GFP-GABARAP was immunoprecipitated using anti-GFP trap beads. Samples were processed for western blot and probed with anti-HA-tag (ATG2A), anti-WIPI4, and anti-GFP for their presence/absence in immunoprecipitated samples. All blots are representative of at least n=3 independent experiments.   Quantification of HA-ATG2A and WIPI4 co-precipitation with GFP-GABARAP from (F). Results were expressed as a fold change compared to HA-ATG2A-WT (complete media; CM). Each symbol represents an independent experiment (n=3) with error bars mean ± SD.


GFP alone , GFP - GABARAP or GFP - GABARAP with increasing concentrations of mCherry - WIPI4 were expressed in HEK293T for 24 h , lysed GFP - trap beads used to immunoprecipitate GFP alone or GFP - GABARAP . Samples were then probed with antibodies to detect endogenous ATG2A or ATG2B in immunoprecipitated samples . Blots are representative of n = 3 independent experiments . of ATG2A ( green , round symbols ) ATG2B ( grey squares ) co - precipitation with GFP - GABARAP in presence of increasing concentrations of mCherry - WIPI4 from ( H ) . Co - precipitation was normalised to GFP - GABARAP alone . error bars are mean ± SD of n = 3 independent experiments .

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

GFP alone, GFP-GABARAP or GFP-GABARAP with increasing concentrations of mCherry-WIPI4 were expressed in HEK293T cells for 24 h, lysed and GFP-trap beads used to immunoprecipitate GFP alone or GFP-GABARAP. Samples were then probed with antibodies to detect endogenous ATG2A or ATG2B in immunoprecipitated samples. Blots are representative of n=3 independent experiments.   Quantification of ATG2A (green line, round symbols) and ATG2B (grey line and squares) co-precipitation with GFP-GABARAP in the presence of increasing concentrations of mCherry-WIPI4 from (H). Co-precipitation was normalised to GFP-GABARAP alone. Line and error bars are mean ±SD of n=3 independent experiments.