Figure 7A , B Microscope images of E10 neural tube sections with the indicated treatments were stained with antibodies against MARS ( green ) and N - Hcy ( red ) . Scale bar : 150 μm . The relative ( to untreated ) fluorescence intensities were quantified ( n = 3 ) and are presented in ( B ) . C , D Double immunofluorescent staining ( C ) for MARS ( green ) and N - Hcy ( red ) in E10 heart sections . Scale bar : 150 μm . The relative ( to untreated ) fluorescence intensities were quantified ( n = 3 ) and are presented in ( D ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . E Goss morphology of E10 . 5 ATRA - treated and untreated mice embryos . The ATRA - treated embryo exhibits an open neural tube ( indicated by white arrows ) compared to littermate controls . Scale bar : 500 μm . F Examples of normally developed and maldeveloped mice hearts : in the normal heart ( i ) , the ink travelled from the right ventricle ( RV ) into the pulmonary trunk ( PT ) ; however , in ATRA - treated group hearts , ink travelled into the aorta ( Ao ) ( ii , transposition of great arteries ( TGA ) ) or both the PT and Ao ( iii , double outlet right ventricle ( DORV ) ) . Scale bar : 1000 μm . G Cardiac malformations were analysed by hematoxylin and eosin ( H & E ) staining of heart sections from E14 . 5 mice . The cardiac defects observed in ATRA treated mice are as follows : Ventricular septal defect ( VSD ) and single ventricle defects ( SVD ) . Heart compartments are marked : RA : right atria , RV : right ventricle , LA : left atria , LV : left ventricle . Scale bar : 1000 μmH Total protein N - Hcy levels in maternal mice liver homogenates were determined and quantified in correlation to the corresponding prevalence of NTDs in affected foetuses ( i ) . Western blot was used to detect N - Hcy levels ( ii ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . I Neural tube tissues at E10 were homogenized from litters of untreated , ATRA - treated , and ATRA + AHT co - treated pregnant mice . Neural tube homogenates of each group were mixed from 6 embryos . Endogenous SOD1 and SOD2 were affinity purified from the homogenates . N - Hcy levels and the specific activities of each purified SOD1 and SOD2 were determined . The SOD activities of the untreated group were set at 100 % ; error bar indicates assay replicates ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . J Superoxide ( a ) , apoptosis ( b ) , and Wnt signalling ( c , d ) were over - activated in ATRA - treated mice neural tubes and restored by AHT or NAC treatment . ( a ) Whole mount DHE staining of E10 . 5 mice embryos . Scale bar : 500 μm . Neural tube defect ( white arrows ) and heart ( green arrow ) regions show especially higher fluorescence intensities . ( b ) TUNEL ( green ) staining detected apoptotic cell death in E8 . 5 neural tube ( E ) sections . MARS ( red ) . Scale bar : 300 μm . ( c , d ) IHC staining for β - catenin ( green ) in E8 . 5 ( c ) and E10 ( d ) mice neural tube sections . Scale bar : 300 μm ( c ) , 150 μm ( d ) . DAPI ( blue ) . K Average ( n = 3 ) DHE fluorescence intensities were quantified relative to the untreated group . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . L Quantification of TUNEL - positive cells Three sections from three embryos were pooled for analysis . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . M Quantification of β - catenin fluorescence intensities in experiment J ( b ) and J ( c ) . The average ( n = 3 ) β - catenin level in the untreated group was set at 100 % . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . N Hcy - treated eggs exhibited NTDs phenotypes at embryonic stage 8 ( E8 ) . A normal embryo ( a ) and neural tube defect phenotypes ( b , c ) are shown . Exencephaly ( white arrows ) and spina bifida ( blue arrows ) are indicated . Scale bar : 500 μm . O Total protein N - Hcy levels of 72 h chicken embryo homogenates from Hcy and Hcy + AHT - treated groups were detected by western blot . P Whole mount DHE staining of 72 h chicken embryos from untreated , Hcy - , Hcy + AHT - , and Hcy + NAC - treated groups . Scale bar : 2000 μm . Q Quantification of DHE fluorescence intensities in ( P ) ( n = 3 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A, B Microscope images of E10 neural tube sections with the indicated treatments were stained with antibodies against MARS (green) and N-Hcy (red). Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n=3) and are presented in (B). C, D Double immunofluorescent staining (C) for MARS (green) and N-Hcy (red) in E10 heart sections. Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n=3) and are presented in (D).Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.E Goss morphology of E10.5 ATRA-treated and untreated mice embryos. The ATRA-treated embryo exhibits an open neural tube (indicated by white arrows) compared to littermate controls. Scale bar: 500 μm.F Examples of normally developed and maldeveloped mice hearts: in the normal heart (i), the ink travelled from the right ventricle (RV) into the pulmonary trunk (PT); however, in ATRA-treated group hearts, ink travelled into the aorta (Ao) (ii, transposition of great arteries (TGA)) or both the PT and Ao (iii, double outlet right ventricle (DORV)). Scale bar: 1000 μm.G Cardiac malformations were analysed by hematoxylin and eosin (H&E) staining of heart sections from E14.5 mice. The cardiac defects observed in ATRA treated mice are as follows: Ventricular septal defect (VSD) and single ventricle defects (SVD). Heart compartments are marked: RA: right atria, RV: right ventricle, LA: left atria, LV: left ventricle. Scale bar: 1000 μmH Total protein N-Hcy levels in maternal mice liver homogenates were determined and quantified in correlation to the corresponding prevalence of NTDs in affected foetuses (i). Western blot was used to detect N-Hcy levels (ii). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.I Neural tube tissues at E10 were homogenized from litters of untreated, ATRA-treated, and ATRA+AHT co-treated pregnant mice. Neural tube homogenates of each group were mixed from 6 embryos. Endogenous SOD1 and SOD2 were affinity purified from the homogenates. N-Hcy levels and the specific activities of each purified SOD1 and SOD2 were determined. The SOD activities of the untreated group were set at 100%; error bar indicates assay replicates (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.J Superoxide (a), apoptosis (b), and Wnt signalling (c, d) were over-activated in ATRA-treated mice neural tubes and restored by AHT or NAC treatment. (a) Whole mount DHE staining of E10.5 mice embryos. Scale bar: 500 μm. Neural tube defect (white arrows) and heart (green arrow) regions show especially higher fluorescence intensities. (b) TUNEL (green) staining detected apoptotic cell death in E8.5 neural tube (E) sections. MARS (red). Scale bar: 300 μm. (c, d) IHC staining for β-catenin (green) in E8.5 (c) and E10 (d) mice neural tube sections. Scale bar: 300 μm (c), 150 μm (d). DAPI (blue).K Average (n=3) DHE fluorescence intensities were quantified relative to the untreated group. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.L Quantification of TUNEL-positive cells Three sections from three embryos were pooled for analysis. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.M Quantification of β-catenin fluorescence intensities in experiment J (b) and J (c). The average (n=3) β-catenin level in the untreated group was set at 100%. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.N Hcy-treated eggs exhibited NTDs phenotypes at embryonic stage 8 (E8). A normal embryo (a) and neural tube defect phenotypes (b, c) are shown. Exencephaly (white arrows) and spina bifida (blue arrows) are indicated. Scale bar: 500 μm.O Total protein N-Hcy levels of 72 h chicken embryo homogenates from Hcy and Hcy+AHT-treated groups were detected by western blot.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.


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

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Figure 1C 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.


Figure 2C AdipoR1 siRNA - transfected ORS or DP cells were treated with APN or P5 . The cell lysates were analyzed for p - AMPK . D - H The relative gene expression levels of growth factors in P5 - treated DP cells ( n = 4 ( VEGF , PDGFA ) , 5 ( IGF1 ) , 6 ( HGF , FGF7 ) biological replicates / group )

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Figure 2C AdipoR1 siRNA-transfected ORS or DP cells were treated with APN or P5. The cell lysates were analyzed for p-AMPK.D-H The relative gene expression levels of growth factors in P5-treated DP cells (n = 4 (VEGF, PDGFA), 5 (IGF1), 6 (HGF, FGF7) biological replicates / group)


Figure 3C IF staining ( p - AMPK ) of the HF bulb area . Yellow arrow indicates p - AMPK signal ; scale bar : 100 µm . D IF staining ( Ki - 67 ) of the HF bulb area ; scale bar : 100 µm .

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Figure 3C 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.


Figure 4C H & E and IF for versican of skin tissue ; scale bars : 100 µm . G H & E and IF for versican of skin tissue ; scale bars : 100 µm . H Anagen induction scores of vehicle - , or P5 - treated Adipoq - / - mice ( n = 6 tissues / mice , 4 mice / group ) ; Unpaired t test K . H & E and IF for versican of skin tissue ; scale bars : 100 µm . L Anagen induction scores of vehicle - , or P5 - treated Adipor1 - / - mice ( n = 6 tissues / mice , 5 mice / group ) ; Unpaired t test

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


Figure 5Pulldown of Flag - fused AdipoR1 input ( residues 89 - 375 ) with amino acid mutation ( s ) , as indicated , with GST - P5 . The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 ( residue 89 - 375 ) inputs , as indicated ( n = 3 technical replicates / group ) . F Pulldown of AdipoR1 ( residues 89 - 375 ) with GST - P5 or GST fused to two mutant peptides ( GLYDF and GLYAF ) . The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 ( residue 89 - 375 ) inputs , as indicated ( n = 3 technical replicates / group ) .

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Figure 5Pulldown of Flag-fused AdipoR1 input (residues 89-375) with amino acid mutation(s), as indicated, with GST-P5. The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).F Pulldown of AdipoR1 (residues 89-375) with GST-P5 or GST fused to two mutant peptides (GLYDF and GLYAF). The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).


Figure 1 ( A ) Phenotype appearance of the Gmlmm1 - 1 mutant . Typical leaves ( left panel ; scale bar , 1 cm ) and whole plants ( right panel ; scale bar , 10 cm . ) were photographed 60 and 118 days after seeding , respectively . Data information : The experiments were performed three times as biological replicates , with similar results . ( B ) Enhanced resistance of Gmlmm1 - 1 and Gmlmm1 - 2 to Psg infection . Leaves from 2 - week - old soybean plants were infected with Psg and the bacteria number was determined at 0 and 4 dpi ( Mean ± SD , n = 8 , n represents sample number , * * * , p < 0 . 001 , Student ' s t - test ) . Data information : The experiments were performed three times , as biological replicates , with similar results . ( E ) Subcellular localization of GmLMM1 - GFP and GmLMM1L407H - GFP in N . benthamiana . PI4P indicates the membrane localization . Scale bar , 10 μm . GFP , green fluorescent protein . Data information : The experiments were performed three times as biological replicates , with similar results . ( F ) Autophosphorylation activity of the GmLMM1 intracellular domain in vitro . The proteins purified from E . coli were subjected to pMIAGO phosphorylation detection assay ( upper panel ) and were detected using western blot ( lower panel ) with BIK1 - HIS as a control . GmLMM1K564E contains a mutation at the predicted ATP binding site . Data information : The experiments were performed two times ( F ) , as biological replicates , with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Phenotype appearance of the Gmlmm1-1 mutant. Typical leaves (left panel; scale bar, 1 cm) and whole plants (right panel; scale bar, 10 cm.) were photographed 60 and 118 days after seeding, respectively. Data information: The experiments were performed three times as biological replicates, with similar results.(B) Enhanced resistance of Gmlmm1-1 and Gmlmm1-2 to Psg infection. Leaves from 2-week-old soybean plants were infected with Psg and the bacteria number was determined at 0 and 4 dpi (Mean ± SD, n = 8, n represents sample number, ***, p < 0.001, Student's t-test). Data information: The experiments were performed three times , as biological replicates, with similar results.(E) Subcellular localization of GmLMM1-GFP and GmLMM1L407H-GFP in N. benthamiana. PI4P indicates the membrane localization. Scale bar, 10 μm. GFP, green fluorescent protein. Data information: The experiments were performed three times as biological replicates, with similar results.(F) Autophosphorylation activity of the GmLMM1 intracellular domain in vitro. The proteins purified from E. coli were subjected to pMIAGO phosphorylation detection assay (upper panel) and were detected using western blot (lower panel) with BIK1-HIS as a control. GmLMM1K564E contains a mutation at the predicted ATP binding site. Data information: The experiments were performed two times (F), as biological replicates, with similar results.


Figure 3 ( A ) ROS accumulation in Gmlmm1 - 1 and Gmlmm1 - 2 mutant plants . Soybean leaves ( aged 3 weeks ) were subjected to ROS accumulation examination by DAB staining . Scale bar , 1 cm . Data information : The experiments were performed three times as biological replicates , with similar results . The exact number ( n ) and p values are shown in the source data . ( B ) ROS accumulation upon P . sojae treatment . The leaves ( aged 3 weeks ) were inoculated with P . sojae isolate P7076 , and DAB staining was performed at 16 hpi . The left panel was photographed under bright field ( scale bar , 1 cm ) . The middle and right panels were photographed under a microscope ( scale bar , 50 μm ) . Data information : The experiments were performed three times as biological replicates , with similar results . The exact number ( n ) and p values are shown in the source data . ( D The increased resistance of the mutants to Psp ( D ) infection . The indicated plants ( aged 2 weeks ) were infected with the indicated pathogens ( Mean ± SD , n ≥ 8 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - test ) . The lesions are shown by trypan blue staining . Scale bar , 1 cm . Data information : The experiments were performed three times as biological replicates , with similar results . The exact number ( n ) and p values are shown in the source data . E ) The increased resistance of the mutants P . sojae ( E ) infection . The indicated plants ( aged 2 weeks ) were infected with the indicated pathogens ( Mean ± SD , n ≥ 8 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - test ) . The lesions are shown by trypan blue staining . Scale bar , 1 cm . Data information : The experiments were performed three times , as biological replicates , with similar results . The exact number ( n ) and p values are shown in the source data .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) ROS accumulation in Gmlmm1-1 and Gmlmm1-2 mutant plants. Soybean leaves (aged 3 weeks) were subjected to ROS accumulation examination by DAB staining. Scale bar, 1 cm. Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.(B) ROS accumulation upon P. sojae treatment. The leaves (aged 3 weeks) were inoculated with P. sojae isolate P7076, and DAB staining was performed at 16 hpi. The left panel was photographed under bright field (scale bar, 1 cm). The middle and right panels were photographed under a microscope (scale bar, 50 μm). Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.(D The increased resistance of the mutants to Psp (D) infection. The indicated plants (aged 2 weeks) were infected with the indicated pathogens (Mean ± SD, n ≥ 8, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The lesions are shown by trypan blue staining. Scale bar, 1 cm. Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.E) The increased resistance of the mutants P. sojae (E) infection. The indicated plants (aged 2 weeks) were infected with the indicated pathogens (Mean ± SD, n ≥ 8, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The lesions are shown by trypan blue staining. Scale bar, 1 cm. Data information: The experiments were performed three times , as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.


Figure 4 ( A ) Flg22 - induced ROS burst in Gmlmm1 - 1 , Gmlmm1 - 2 and Williams 82 . The indicated leaves ( aged 2 weeks ) were subjected to flg22 - induced ROS examination , and the peak relative luminescence unit ( RLU ) value was recorded ( Mean ± SEM , n ≥ 12 , n represents sample number ) . Data information : All the experiments were performed three times ( biological replicates ) with similar results . The exact n , SEM values and p values are shown in the source data . ( B ) Flg22 - induced ROS burst in the CRISPR lines C9 - 24 - 13 and DN50 . The indicated leaves ( aged 2 weeks ) were subjected to flg22 - induced ROS examination , and the relative RLU value was recorded ( Mean ± SEM , n ≥ 9 , n represents sample number ) . Data information : All the experiments were performed three times ( biological replicates ) with similar results . The exact n , SEM values and p values are shown in the source data . ( C ) Suppression of flg22 - induced ROS production in Arabidopsis by GmLMM1 . The GmLMM1 gene was introduced into Arabidopsis WT ( Col - 0 ) plants by Agrobacterium - mediated transformation . Two independent T2 lines were subjected to flg22 - induced ROS examination ( Mean ± SD , n ≥ 8 , n represents sample number , * * * , p < 0 . 001 , Student ' s t - test ) . The protein expression is shown . Data information : All the experiments were performed three times ( biological replicates ) with similar results . The exact n , SEM values and p values are shown in the source data . Suppression of flg22 - induced ( D ) ROS production in N . benthamiana by GmLMM1 . The indicated constructs were transiently expressed by Agrobacterium - mediated transient expression for 2 days and subjected to flg22 - induced ROS examination . GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity . GFP was used as a control ( Mean ± SD , n = 6 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - test ) . The protein expression is shown in the lower panel . Data information : All the experiments were performed three times ( biological replicates ) with similar results . The exact n , SEM values and p values are shown in the source data . E ) Suppression of chitin - induced ( E ) ROS production in N . benthamiana by GmLMM1 . The indicated constructs were transiently expressed by Agrobacterium - mediated transient expression for 2 days and subjected to ROS examination . GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity . GFP was used as a control ( Mean ± SD , n = 6 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - test ) . The protein expression is shown in the lower panel . Data information : All the experiments were performed three times ( biological replicates ) with similar results . The exact n , SEM values and p values are shown in the source data . ( F ) Inhibition of XEG1 - induced cell death in N . benthamiana by GmLMM1 . The indicated constructs were transiently expressed in N . benthamiana 1 day before infiltration of Agrobacterium containing the XEG1 or GFP vector . The cell death phenotypes were visualized 5 days later . Protein expression of GmLMM1 - HA and GmLMM1K564E - HA is shown in the lower panel .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Flg22-induced ROS burst in Gmlmm1-1, Gmlmm1-2 and Williams 82. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the peak relative luminescence unit (RLU) value was recorded (Mean ± SEM, n ≥ 12, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.(B) Flg22-induced ROS burst in the CRISPR lines C9-24-13 and DN50. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the relative RLU value was recorded (Mean ± SEM, n ≥ 9, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.(C) Suppression of flg22-induced ROS production in Arabidopsis by GmLMM1. The GmLMM1 gene was introduced into Arabidopsis WT (Col-0) plants by Agrobacterium-mediated transformation. Two independent T2 lines were subjected to flg22-induced ROS examination (Mean ± SD, n ≥8, n represents sample number, ***, p < 0.001, Student's t-test). The protein expression is shown. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.Suppression of flg22-induced (D) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to flg22-induced ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.E) Suppression of chitin-induced (E) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.(F) Inhibition of XEG1-induced cell death in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed in N. benthamiana 1 day before infiltration of Agrobacterium containing the XEG1 or GFP vector. The cell death phenotypes were visualized 5 days later. Protein expression of GmLMM1-HA and GmLMM1K564E-HA is shown in the lower panel.


Figure 5 ( A ) Identification of GmLMM1 - interacting proteins by IP - MS analysis . GmLMM1 - FLAG was transiently expressed in Arabidopsis protoplasts , purified by anti - FLAG M2 agarose , and subjected to mass spectrometry analysis . The complete list is shown in the source data . ( B ) Interactions of GmLMM1 with PRR receptors . The indicated constructs were transiently expressed in N . benthamiana leaves and subjected to luciferase complementation assay . GmFLS2 ( Gm08G083300 ) is the closest homolog of FLS2 in soybean . GmILPA1 was used as a negative control . The protein interaction intensity is shown by the relative luminescence unit ( RLU ) ( Mean ± SD , n ≥ 6 , n represents sample number , p < 0 . 05 , Student ' s t - test , different letters indicate signiﬁcant difference ) . Immunoblots show the levels of protein expression . Data information : The experiments were performed three times as biological replicates , with similar results . ( C ) Co - IP of GmLMM1 with NbFLS2 and GmFLS2 . The indicated constructs were transiently expressed in N . benthamiana by Agrobacterium - mediated transient expression for 2 days and the protein interactions were examined by Co - IP assay . Data information : The experiments were performed three times as biological replicates , with similar results . ( D ) GmLMM1 interacts with NbRXEG1 in N . benthamiana plants . Data information : The experiments were performed three times as biological replicates , with similar results . ( E ) Interaction of GmLMM1 with NbBAK1 , GmBAK1a , and GmBAK1b . The experiment and statistical testing were performed as in ( B ) . Immunoblots show the levels of protein expression . Data information : The experiments were performed three times as biological replicates , with similar results . ( F ) GmLMM1 interacts with NbBAK1 , GmBAK1a , and GmBAK1b . The indicated constructs were transiently expressed in N . benthamiana and the interactions were examined by Co - IP assays . Data information : The experiments were performed two times ( F ) , as biological replicates , with similar results .

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Figure 5(A) Identification of GmLMM1-interacting proteins by IP-MS analysis. GmLMM1-FLAG was transiently expressed in Arabidopsis protoplasts, purified by anti-FLAG M2 agarose, and subjected to mass spectrometry analysis. The complete list is shown in the source data.(B) Interactions of GmLMM1 with PRR receptors. The indicated constructs were transiently expressed in N. benthamiana leaves and subjected to luciferase complementation assay. GmFLS2 (Gm08G083300) is the closest homolog of FLS2 in soybean. GmILPA1 was used as a negative control. The protein interaction intensity is shown by the relative luminescence unit (RLU) (Mean±SD, n ≥ 6, n represents sample number, p<0.05, Student's t-test, different letters indicate signiﬁcant difference). Immunoblots show the levels of protein expression. Data information: The experiments were performed three times as biological replicates, with similar results.(C) Co-IP of GmLMM1 with NbFLS2 and GmFLS2. The indicated constructs were transiently expressed in N. benthamiana by Agrobacterium-mediated transient expression for 2 days and the protein interactions were examined by Co-IP assay. Data information: The experiments were performed three times as biological replicates, with similar results.(D) GmLMM1 interacts with NbRXEG1 in N. benthamiana plants. Data information: The experiments were performed three times as biological replicates, with similar results.(E) Interaction of GmLMM1 with NbBAK1, GmBAK1a, and GmBAK1b. The experiment and statistical testing were performed as in (B). Immunoblots show the levels of protein expression. Data information: The experiments were performed three times as biological replicates, with similar results.(F) GmLMM1 interacts with NbBAK1, GmBAK1a, and GmBAK1b. The indicated constructs were transiently expressed in N. benthamiana and the interactions were examined by Co-IP assays. Data information: The experiments were performed two times (F), as biological replicates, with similar results.


Figure 6 ( A ) Effect of flg22 on the NbFLS2 - GmLMM1 interaction . The indicated constructs were transiently expressed in N . benthamiana by Agrobacterium - mediated transient expression for 2 days , and the leaves were treated with flg22 ( 1 μM ) or water for 10 min before being subjected to Co - IP assay . Data information : The experiments were performed three times , as biological replicates , with similar results . ( B ) Regulation of NbBAK1 - GmLMM1 interaction by flg22 . Data information : The experiments were performed three times as biological replicates , with similar results . ( C ) Suppression of flg22 - induced NbFLS2 - NbBAK1 interaction by GmLMM1 . NbFLS2 and NbBAK1 were co - expressed in N . benthamiana along with GmLMM1 or GmLMM1L407H , treated with H2O or flg22 and subjected to co - IP assays . Data information : The experiments were performed three times as biological replicates , with similar results . ( D Suppression effect of GmLMM1 on the flg22 - induced interaction of GmFLS2 with GmBAK1a ( D ) Data information : The experiments were performed two times ( D as biological replicates , with similar results . 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Effect of flg22 on the NbFLS2-GmLMM1 interaction. The indicated constructs were transiently expressed in N. benthamiana by Agrobacterium-mediated transient expression for 2 days, and the leaves were treated with flg22 (1 μM) or water for 10 min before being subjected to Co-IP assay. Data information: The experiments were performed three times , as biological replicates, with similar results.(B) Regulation of NbBAK1-GmLMM1 interaction by flg22. Data information: The experiments were performed three times as biological replicates, with similar results.(C) Suppression of flg22-induced NbFLS2-NbBAK1 interaction by GmLMM1. NbFLS2 and NbBAK1 were co-expressed in N. benthamiana along with GmLMM1 or GmLMM1L407H, treated with H2O or flg22 and subjected to co-IP assays. Data information: The experiments were performed three times as biological replicates, with similar results.(D Suppression effect of GmLMM1 on the flg22-induced interaction of GmFLS2 with GmBAK1a (D) Data information: The experiments were performed two times (D as biological replicates, with similar results.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.


figf1Single - marker association results for the combined non - Jewish and Jewish samples using the Cochran - Mantel - Haenszel ( CMH ) test . Each chromosome is depicted as a different color . The red line indicates the threshold ( P 5 × 10 − 5 ) selected to define regions for evaluation in the replication studies . The P value thresholds for suggestive and significant associations are 3 . 28 × 10 − 6 and 1 . 64 × 10 − 7 , respectively , based on a Bonferroni correction for multiple testing . A modest correction factor ( λ = 1 . 056 ) is necessary to correct for stratification between cases and controls , but it does not modify the current results .

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figf1Single-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.


figf2 ( a - c ) We used quantitative real - time PCR to determine the expression patterns of ATG5 ( a ) , ATG7 ( b ) and ATG16L1 ( c ) in various human immune and epithelial cell lines . ATG5 was broadly expressed in the cell lines studied ( a ) , with between 1 . 5 - and fourfold variations in expression level across the cell lines . ATG7 expression ( b ) was broadly comparable to that of ATG5 , with the exception of THP1 cells , which showed 35 - fold higher expression than SW480 cells . ATG16L1 expression ( c ) was also broadly similar to that of ATG5 and ATG7 ( except for ATG7 expression in THP1 cells ) , with moderate variations across the cell lines tested ( 1 . 5 - to 3 . 5 - fold ) . ( d ) Expression of ATG16L1 in human primary immune cells . In a resting human immune cell RNA panel ( Clontech ) , ATG16L1 showed a clear peak of expression in the T cell compartment , with CD4 + and CD8 + cells expressing high levels of transcript ( 15 - to 20 - fold more than control samples ) . We detected modest levels of ATG16L1 in CD19 + cells and low levels in CD14 + monocytes and unfractionated mononuclear cells . In all cases , we performed real - time quantitative RT - PCR reactions in duplicate and plotted the means ; error bars represent 1 s . d . We normalized expression levels by comparison with GAPDH controls and plotted arbitrary relative expression units , where the SW480 or placental RNA expression equals 1 ( for cell lines or primary cells , respectively ) . We isolated all RNAs from resting cells in the absence of stimulation or activation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a-c) We used quantitative real-time PCR to determine the expression patterns of ATG5 (a), ATG7 (b) and ATG16L1 (c) in various human immune and epithelial cell lines. ATG5 was broadly expressed in the cell lines studied (a), with between 1.5- and fourfold variations in expression level across the cell lines. ATG7 expression (b) was broadly comparable to that of ATG5, with the exception of THP1 cells, which showed 35-fold higher expression than SW480 cells. ATG16L1 expression (c) was also broadly similar to that of ATG5 and ATG7 (except for ATG7 expression in THP1 cells), with moderate variations across the cell lines tested (1.5- to 3.5-fold).(d) Expression of ATG16L1 in human primary immune cells. In a resting human immune cell RNA panel (Clontech), ATG16L1 showed a clear peak of expression in the T cell compartment, with CD4+ and CD8+ cells expressing high levels of transcript (15- to 20-fold more than control samples). We detected modest levels of ATG16L1 in CD19+ cells and low levels in CD14+ monocytes and unfractionated mononuclear cells. In all cases, we performed real-time quantitative RT-PCR reactions in duplicate and plotted the means; error bars represent 1 s.d. We normalized expression levels by comparison with GAPDH controls and plotted arbitrary relative expression units, where the SW480 or placental RNA expression equals 1 (for cell lines or primary cells, respectively). We isolated all RNAs from resting cells in the absence of stimulation or activation.


figf3 ( a ) We achieved specific siRNA knockdown of overexpressed , Flag - tagged ATG16L1 in HEK293 cells within 48 h of transfection with oligonucleotide duplexes . Flag - ATG16L1 was undetectable by protein blotting after treatment with specific siRNA constructs , but expression was maintained with control duplexes . ( b ) Endogenous ATG16L1 mRNA knockdown ( mean relative abundance ± s . d . ) by siRNA 2 in HeLa cells within 48 h of transfection , as assessed by real - time quantitative RT - PCR normalized to GAPDH . Compared with control duplex , siRNA 2 yielded an 89 % reduction in transcripts . RT - PCR was performed in triplicate ; results represent two independent experiments . c ) Knockdown of ATG16L1 prevented effective autophagy of S . typhimurium in HeLa cells . Forty - eight hours after cotransfection with control siRNA or duplex 2 and LC3 - GFP plasmid , HeLa cells were infected for 1 h with Salmonella typhimurium SL1344 , fixed and examined microscopically . Mean percentages ( ± s . e . m . ) of bacteria per cell encapsulated by LC3 + membranes ( autophagosomes ) are shown . Bacterial counts were pooled from two separate experiments ; each counted a minimum of 100 infected cells . Significance was assessed by two - tailed Students t - test , assuming unequal variances . ( d ) Representative images of intracellular S . typhimurium show normal autophagic encapsulation by LC3 + membranes in control siRNA - treated cells but loss of autophagic targeting after ATG16L1 knockdown . Channels are separated to show both the LC3 - GFP fusion protein ( autophagosomal marker ) and S . typhimurium SL1344 dsRed2 ( SL1344 ) , with a merged image in the final column . Rows 2 and 4 show magnified views of boxed areas in rows 1 and 3 , respectively . Images were obtained using confocal microscopy and are a flat projection of z stacks taken throughout the plane of the infected cell . Scale bars = 5 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) We achieved specific siRNA knockdown of overexpressed, Flag-tagged ATG16L1 in HEK293 cells within 48 h of transfection with oligonucleotide duplexes. Flag-ATG16L1 was undetectable by protein blotting after treatment with specific siRNA constructs, but expression was maintained with control duplexes.(b) Endogenous ATG16L1 mRNA knockdown (mean relative abundance ± s.d.) by siRNA 2 in HeLa cells within 48 h of transfection, as assessed by real-time quantitative RT-PCR normalized to GAPDH. Compared with control duplex, siRNA 2 yielded an 89% reduction in transcripts. RT-PCR was performed in triplicate; results represent two independent experiments.c) Knockdown of ATG16L1 prevented effective autophagy of S. typhimurium in HeLa cells. Forty-eight hours after cotransfection with control siRNA or duplex 2 and LC3-GFP plasmid, HeLa cells were infected for 1 h with Salmonella typhimurium SL1344, fixed and examined microscopically. Mean percentages (± s.e.m.) of bacteria per cell encapsulated by LC3+ membranes (autophagosomes) are shown. Bacterial counts were pooled from two separate experiments; each counted a minimum of 100 infected cells. Significance was assessed by two-tailed Students t-test, assuming unequal variances.(d) Representative images of intracellular S. typhimurium show normal autophagic encapsulation by LC3+ membranes in control siRNA-treated cells but loss of autophagic targeting after ATG16L1 knockdown. Channels are separated to show both the LC3-GFP fusion protein (autophagosomal marker) and S. typhimurium SL1344 dsRed2 (SL1344), with a merged image in the final column. Rows 2 and 4 show magnified views of boxed areas in rows 1 and 3, respectively. Images were obtained using confocal microscopy and are a flat projection of z stacks taken throughout the plane of the infected cell. Scale bars = 5 μm.


Figure 2 ( B ) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB - treated mMuSCs , human myoblasts and hiMPs . Red dots represent genes which display a positive fold - change in expression upon treatment with DLL4 & PDGF - BB whilst violet dots represent genes which are significantly downregulated . Differentially expressed genes required a P value of ≤ 0 . 05 to be considered significant . ( C ) Heatmaps showing changes in expression of key myogenic ( MYOD , MYOGENIN ) , perivascular ( PDGFRB , NG2 , CD146 , ALPL ) and NOTCH target ( HEY1 , HES1 ) genes upon treatment with DLL4 & PDGF - BB in mMuSC - derived myoblasts ( left ) , human myoblasts ( middle ) and hiMPs ( right ) . Clustering was performed by genes / probes with Pearson correlation . Colour scale based on z - scores : red regions indicate high expression whilst blue regions indicate low expression . Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB-treated mMuSCs, human myoblasts and hiMPs. Red dots represent genes which display a positive fold-change in expression upon treatment with DLL4 & PDGF-BB whilst violet dots represent genes which are significantly downregulated. Differentially expressed genes required a P value of ≤ 0.05 to be considered significant.(C) Heatmaps showing changes in expression of key myogenic (MYOD, MYOGENIN), perivascular (PDGFRB, NG2, CD146, ALPL) and NOTCH target (HEY1, HES1) genes upon treatment with DLL4 & PDGF-BB in mMuSC-derived myoblasts (left), human myoblasts (middle) and hiMPs (right). Clustering was performed by genes/probes with Pearson correlation. Colour scale based on z-scores: red regions indicate high expression whilst blue regions indicate low expression. Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled.


Figure 3 ( B ) Phase contrast images displaying morphology of untreated and treated hiMPs . Scale bar : 25 μm . ( C ) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs . Morphology was quantified as circularity ratio , where 1 = perfect circle and 0 = line ( experimental replicates = 3 ) . Statistical analysis ( paired t test ) was performed on the first quartile to enhance detection of morphological changes ( error bars : SD ) . ( E ) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours . Scale bar : 75 μm . ( F ) Bar graphs quantifying EdU experiment shown in ( E ) ( experimental replicates = 3 ; error bars : SEM ) . Statistical analysis based on an unpaired t - test . ( H ) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week , induced to differentiate for 4 days in the presence or absence of γ - secretase inhibitor L685458 and immunostained for myosin heavy chain ( MyHC ) . ( I ) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes ( experimental replicates = 3 ; error bars : SEM ) . Statistical significance based on one - way ANOVA with Tukey ' s multiple comparison . ( J ) Immunofluorescence panels showing human specific LAMIN A / C + ( nuclei ) and SPECTRIN ( sarcolemma ) staining in tibialis anterior muscles of immunodeficient mice ( N = 3 ) transplanted with treated ( n = 3 muscles ) and untreated ( n = 3 muscles ) N5 hiMPs . ( K ) Quantification of LAMIN A / C + grafted human cells across each muscle . Data information : full gene list for heatmaps in ( A ) and ( D ) available in Appendix Table S4 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Phase contrast images displaying morphology of untreated and treated hiMPs. Scale bar: 25 μm. (C) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs. Morphology was quantified as circularity ratio, where 1 = perfect circle and 0 = line (experimental replicates = 3). Statistical analysis (paired t test) was performed on the first quartile to enhance detection of morphological changes (error bars: SD).(E) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours. Scale bar: 75 μm. (F) Bar graphs quantifying EdU experiment shown in (E) (experimental replicates = 3; error bars: SEM). Statistical analysis based on an unpaired t-test.(H) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week, induced to differentiate for 4 days in the presence or absence of γ-secretase inhibitor L685458 and immunostained for myosin heavy chain (MyHC). (I) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes (experimental replicates = 3; error bars: SEM). Statistical significance based on one-way ANOVA with Tukey's multiple comparison.(J) Immunofluorescence panels showing human specific LAMIN A/C+ (nuclei) and SPECTRIN (sarcolemma) staining in tibialis anterior muscles of immunodeficient mice (N = 3) transplanted with treated (n = 3 muscles) and untreated (n = 3 muscles) N5 hiMPs. (K) Quantification of LAMIN A/C+ grafted human cells across each muscle. Data information: full gene list for heatmaps in (A) and (D) available in Appendix Table S4.


Figure 5 ( F ) Representative fluorescence images of CMFDA - stained untreated and DLL4 & PDGF - BB - treated hiMPs within the top perfusion channel , 15 minutes after delivery and kept on the OrganoFlow ® . ( G ) Bar graph quantifying adhesion images in ( E ) . Statistical significance was calculated based on a paired t - test ( experimental replicates = 3 ) . Each point on the plot represents the number of adhered cells after 15 minutes within a single chip . ( H ) Assessment of DLL4 & PDGF - BB - treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells . Representative images showing the lower side of the trans - well membrane on which treated and untreated hiMPs ( stained with the transient dye CFDA , in green ) are simultaneously seeded on HUVECs for 8 hours . Bar graphs quantifying the average number of CFDA - positive cells / mm2 , that have migrated through the endothelial layer in each considered condition . ( experimental replicates = 3 ) . A minimum of 10 ( 1 . 5 mm2 ) fields per condition was quantified ( error bars : SEM ) . ( I ) Bar graph showing fold - change in trans - endothelial migration ( error bars : SEM ) . Statistical significance based on one - way ANOVA with Bonferroni ' s multiple comparison .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(F) Representative fluorescence images of CMFDA-stained untreated and DLL4 & PDGF-BB-treated hiMPs within the top perfusion channel, 15 minutes after delivery and kept on the OrganoFlow®. (G) Bar graph quantifying adhesion images in (E). Statistical significance was calculated based on a paired t-test (experimental replicates = 3). Each point on the plot represents the number of adhered cells after 15 minutes within a single chip.(H) Assessment of DLL4 & PDGF-BB-treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans-well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 hours. Bar graphs quantifying the average number of CFDA-positive cells/ mm2, that have migrated through the endothelial layer in each considered condition. (experimental replicates = 3). A minimum of 10 (1.5 mm2) fields per condition was quantified (error bars: SEM). (I) Bar graph showing fold-change in trans-endothelial migration (error bars: SEM). Statistical significance based on one-way ANOVA with Bonferroni's multiple comparison.


Figure 1Sanger - 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Sanger-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 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 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.


Figure 2Sanger - 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Sanger-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 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 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 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.


Figure 3Sanger - 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Sanger-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 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 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.


Figure 4Sanger - 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Sanger-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 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 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.


Figure 1U2OS 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 ) 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1U2OS 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) 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.


Figure 2Myc - 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 ) 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Myc-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) 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 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 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.


Figure 3U2OS ATG2A / B double knock - out ( DKO ) CRISPR / Cas9 cells stably expressing Tandem tagged LC3B ( mCherry - GFP - LC3B ) were retrovirally transduced to express vector , or HA - tagged ATG2A - WT , - mLIR ( FCIL / AAAA ) or - mYFS ( YFS / AAA ) . Cells were grown in complete medium ( CM ) or starved for 2 h ( EBSS ) or treated with CM plus BafilomycinA1 ( 200nM , 4 h ) , fixed and analysed by confocal microscopy . Merged images of GFP ( green ) and mCherry ( red ) channels show the presence of autophagosomes / phagophores ( GFP and mCherry positive , yellow puncta ) or autolysosomes ( mCherry only , red puncta ) Scale bar 10µm . Images are representative of n = 3 independent experiments . Quantification using flow cytometry of measuring GFP and mCherry fluorescence . Cells were gated based on GFP and mCherry fluorescence and % mCherry positive cells gated used as an indication of autolysosome formation due to GFP quenching . Each symbol represents 1 independent experiment with 10 , 000 cells analysed per condition . A total of n = 3 independent experiments were performed , and horizontal bar indicates mean ± SD . U2OS ATG2A / B DKO cells reconstituted with vector only , HA - ATG2A - WT , - mLIR and - mYFS were stimulated with complete medium , ( CM ) , 2 h starvation ( EBSS ) or 4 h BafilomycinA1 ( BafA1 , 200nM ) , lysed in total cell lysis buffer and subjected to western blot analysis . Blots were probed for the presence of HA - tag ( ATG2A ) , p62 / SQSTM1 , LC3B , pan - GABARAP ( GABARAP , GABARAP - L1 and GABARAP - L2 ) and vinculin ( loading control ) . D , E p62 / SQSTM1 ( D ) and LC3B - II ( E ) levels were normalized to loading control and quantified as fold change of DKO proteins levels . Each symbol represents an independent experiment . Quantification of at least n = 3 independent experiments is shown . Horizontal bar represents mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3U2OS ATG2A/B double knock-out (DKO) CRISPR/Cas9 cells stably expressing Tandem tagged LC3B (mCherry-GFP-LC3B) were retrovirally transduced to express vector, or HA-tagged ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA). Cells were grown in complete medium (CM) or starved for 2 h (EBSS) or treated with CM plus BafilomycinA1 (200nM, 4 h), fixed and analysed by confocal microscopy. Merged images of GFP (green) and mCherry (red) channels show the presence of autophagosomes/phagophores (GFP and mCherry positive, yellow puncta) or autolysosomes (mCherry only, red puncta) Scale bar 10µm. Images are representative of n=3 independent experiments.Quantification using flow cytometry of measuring GFP and mCherry fluorescence. Cells were gated based on GFP and mCherry fluorescence and % mCherry positive cells gated used as an indication of autolysosome formation due to GFP quenching. Each symbol represents 1 independent experiment with 10,000 cells analysed per condition. A total of n=3 independent experiments were performed, and horizontal bar indicates mean ± SD.U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 2 h starvation (EBSS) or 4 h BafilomycinA1 (BafA1, 200nM), lysed in total cell lysis buffer and subjected to western blot analysis. Blots were probed for the presence of HA-tag (ATG2A), p62/SQSTM1, LC3B, pan-GABARAP (GABARAP, GABARAP-L1 and GABARAP-L2) and vinculin (loading control).D, E p62/SQSTM1 (D) and LC3B-II (E) levels were normalized to loading control and quantified as fold change of DKO proteins levels. Each symbol represents an independent experiment. Quantification of at least n=3 independent experiments is shown. Horizontal bar represents mean ± SD.


Figure 4U2OS ATG2A / B DKO cells reconstituted with vector only , HA - ATG2A - WT , - mLIR and - mYFS were stimulated with complete medium , ( CM ) , 4 h starvation ( EBSS ) plus BafilomycinA1 ( BafA1 , 200nM ) treatment . Cells were centrifuged and resuspended in PBS digitonin , spun , washed and the membrane fractions incubated with proteinase K with and without 0 . 1 % TritonX - 100 . Sampels were then subjected to western blotting using anti - p62 / SQSTM1 and anti - HA ( ATG2A ) antibodies . Percentage p62 / SQSTM1 remaining was calculated using densitometry analysis . Blots are representative of n = 3 independent experiments . U2OS ATG2A / B DKO cells reconstituted with vector only , HA - ATG2A - WT , - mLIR and - mYFS and stably expressing GFP - Syntaxin17 ( STX17 ) were stimulated starvation ( EBSS ) plus BafilomycinA1 ( BafA1 , 200nM ) for 4 hours to stimulate autophagosome generation and prevent their degradation in the lysosome . Cells were fixed and immune - stained for LC3B ( magenta ) . DAPI was included ( blue ) to mark the DNA / nucleus . Confocal analysis of LC3B and GFP - STX17 ( green ) localization was performed . Closed arrows ( ATG2A / B DKO and DKO + ATG2A mLIR ) highlight aggregate structures . Open arrows ( DKO + ATG2A - WT and ATG2A - mYFS ) highlight STX17 / LC3B positive vesicles . Scale bar 10µm . Quantification of ( C ) expressed as a percentage of cells with STX17 / LC3B positive vesicles . Each symbol represents a single field of cells with 5 - 10 cells per field . A total of 300 - 600 cells were analysed over n = 3 independent experiments . Data is shown as mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 4 h starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) treatment. Cells were centrifuged and resuspended in PBS digitonin, spun, washed and the membrane fractions incubated with proteinase K with and without 0.1% TritonX-100. Sampels were then subjected to western blotting using anti-p62/SQSTM1 and anti-HA (ATG2A) antibodies. Percentage p62/SQSTM1 remaining was calculated using densitometry analysis. Blots are representative of n=3 independent experiments.U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS and stably expressing GFP-Syntaxin17 (STX17) were stimulated starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) for 4 hours to stimulate autophagosome generation and prevent their degradation in the lysosome. Cells were fixed and immune-stained for LC3B (magenta). DAPI was included (blue) to mark the DNA/nucleus. Confocal analysis of LC3B and GFP-STX17 (green) localization was performed. Closed arrows (ATG2A/B DKO and DKO + ATG2A mLIR) highlight aggregate structures. Open arrows (DKO + ATG2A-WT and ATG2A-mYFS) highlight STX17/LC3B positive vesicles. Scale bar 10µm.   Quantification of (C) expressed as a percentage of cells with STX17/LC3B positive vesicles. Each symbol represents a single field of cells with 5-10 cells per field. A total of 300-600 cells were analysed over n=3 independent experiments. Data is shown as mean ± SD.


Figure 5 ( A - C ) U2OS ATG2A / B double knock out ( DKO ) cells or DKO reconstituted with ATG2A - WT , - mLIR ( FCIL / AAAA ) or - mYFS ( YFS / AAA ) were starved for 2 h , fixed and stained for LC3B ( green ) or WIPI2 ( magenta ) , ( B ) ATG9A ( magenta ) or ( C ) GABARAP ( Magenta ) and imaged using a Zeiss 880 Airyscan super - resolution confocal microscope . Images are representatives of n = 3 independent experiments . Scale bars 10µm . Transmission electron micrographs of ATG2A / B DKO cells ( upper left ) , DKO + ATG2A - WT ( upper right ) , DKO + ATG2A - mLIR ( lower left ) and DKO + ATG2A - mYFS ( lower right ) starved ( EBSS ) for 2 h . Clustered small vesicles ( open arrowheads ; DKO and DKO - mLIR ) and autophagosomes ( closed arrow heads , DKO + ATG2A - WT and - mYFS ) indicated . Images are representative of n = 3 independent experiments . Scale bars 500nm . ER = endoplasmic reticulum ; N = Nucleus ; Mt = Mitochondria ; G = Golgi ; AP = Autophagosome .

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Figure 5(A-C) U2OS ATG2A/B double knock out (DKO) cells or DKO reconstituted with ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA) were starved for 2 h, fixed and stained for LC3B (green) or WIPI2 (magenta), (B) ATG9A (magenta) or (C) GABARAP (Magenta) and imaged using a Zeiss 880 Airyscan super-resolution confocal microscope. Images are representatives of n=3 independent experiments. Scale bars 10µm.Transmission electron micrographs of ATG2A/B DKO cells (upper left), DKO + ATG2A-WT (upper right), DKO+ ATG2A-mLIR (lower left) and DKO+ATG2A-mYFS( lower right) starved (EBSS) for 2 h. Clustered small vesicles (open arrowheads; DKO and DKO-mLIR) and autophagosomes (closed arrow heads, DKO+ATG2A-WT and -mYFS) indicated. Images are representative of n=3 independent experiments. Scale bars 500nm. ER= endoplasmic reticulum; N = Nucleus; Mt = Mitochondria; G = Golgi; AP = Autophagosome.


Figure 1D Zygotic grk2i283 mutant at 4dpf compared to wild type ( N = 80 ) . Arrows indicate the position of swim bladder . Scale bar ,

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Figure 1D Zygotic grk2i283 mutant at 4dpf compared to wild type (N=80). Arrows indicate the position of swim bladder. Scale bar, 1mm