( A ) WB analysis of ORMDL3 ( HA ) , SPTLC1 and SPTLC2 in HUVEC lysates expressing HA - ORMDL3 and treated with cycloheximide ( CHX , 10µM ) for the indicated period of time .

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(A) WB analysis of ORMDL3 (HA), SPTLC1 and SPTLC2 in HUVEC lysates expressing HA-ORMDL3 and treated with cycloheximide (CHX, 10µM) for the indicated period of time.


WB analysis of ORMDLs , SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P ( 300nM , 30 ' ) and with or without ( C ) DMOG ( 1mM , 1h pre - treatment ) , ( D ) Eeyarestatin 1 ( EER1 , 10µM , 1h pre - treatment ) ,

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

WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') and with or without (C) DMOG (1mM, 1h pre-treatment), (D) Eeyarestatin 1 (EER1, 10µM, 1h pre-treatment),


WB analysis of ORMDLs , SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P ( 300nM , 30 ' ) ( E ) MG132 ( 10µM , 1h pre - treatment ) .

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WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') (E) MG132 (10µM, 1h pre-treatment).


( F ) WB analysis of HA - ORMDL3 and HA - ORMDL3 - P137A in HUVEC lysates in absence or presence of S1P ( 300nM , 30 ' ) , DMOG ( 1mM , 1h ) , EER1 ( 10µM , 1h ) , and MG132 ( 10µM , 1h ) .

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(F) WB analysis of HA-ORMDL3 and HA-ORMDL3-P137A in HUVEC lysates in absence or presence of S1P (300nM, 30'), DMOG (1mM, 1h), EER1 (10µM, 1h), and MG132 (10µM, 1h).


( G , H ) ( G ) WB analysis of HA - ORMDL3 and HA - ORMDL3 - P137A , SPTLC1 and SPTLC2 in HUVEC lysates treated with CHX ( 10µM ) for the indicated period of time , and ( H ) relative

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

(G,H) (G) WB analysis of HA-ORMDL3 and HA-ORMDL3-P137A, SPTLC1 and SPTLC2 in HUVEC lysates treated with CHX (10µM) for the indicated period of time, and (H) relative quantification


( I ) WB analysis for Ubiquitin , HA and GFP of HEK293T transfected with GFP - ubiquitin and with the indicated HA - ORMDL3 plasmid , and immunoprecipitated with HA antibody .

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(I) WB analysis for Ubiquitin, HA and GFP of HEK293T transfected with GFP-ubiquitin and with the indicated HA-ORMDL3 plasmid, and immunoprecipitated with HA antibody.


SPT activity in S1pr1 , 3f / f and S1pr1 , 2ECKO mEC , in absence or presence of S1P ( 300nM , 30 ' ) ( B ) relative WB analysis for S1PR1 , ORMDLs , P - AKT and AKT .

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SPT activity in S1pr1,3f/f and S1pr1,2ECKO mEC , in absence or presence of S1P (300nM, 30') (B) relative WB analysis for S1PR1, ORMDLs, P-AKT and AKT.


( G ) WB analysis of ORMDLs and S1PR1 in HUVEC lysates in absence or presence of P - FTY720 ( 100nM , 30 ' )

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

(G) WB analysis of ORMDLs and S1PR1 in HUVEC lysates in absence or presence of P-FTY720 (100nM, 30') and


( A - C ) ( A ) WB analysis of P - VEGFR2 ( Y1175 ) , VEGFR2 , P - IR ( Y1150 / 1151 ) , IR , P - AKT ( S473 ) , AKT , P - eNOS ( S1176 ) , and eNOS in Spns2f / f and Spns2ECKO mEC lysates in absence or presence of VEGF ( 100ng / mL , 2 ' ) or Insulin ( 1U / mL , 2 ' ) and ( B - C ) relative quantification of the indicated phospho / total protein ratios ( n ≥ 3 biological replicates ) .

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(A-C) (A) WB analysis of P-VEGFR2 (Y1175), VEGFR2, P-IR (Y1150/1151), IR, P-AKT (S473), AKT, P-eNOS (S1176), and eNOS in Spns2f/f and Spns2ECKO mEC lysates in absence or presence of VEGF (100ng/mL, 2') or Insulin (1U/mL, 2') and (B-C) relative quantification of the indicated phospho/total protein ratios (n≥3 biological replicates).


( D , E ) ( D ) WB analysis of P - VEGFR2 ( Y1175 ) , VEGFR2 , P - eNOS ( S1176 ) , and eNOS in Spns2f / f and Spns2ECKO mEC lysates in absence or presence of VEGF ( 100ng / mL , 2 ' ) , and in absence or presence of Myriocin ( 300nM ) for the indicated time and ( E ) relative quantification of the indicated phospho / total protein ratios ( n ≥ 3 biological replicates ) .

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

(D,E) (D) WB analysis of P-VEGFR2 (Y1175), VEGFR2, P-eNOS (S1176), and eNOS in Spns2f/f and Spns2ECKO mEC lysates in absence or presence of VEGF (100ng/mL, 2'), and in absence or presence of Myriocin (300nM) for the indicated time and (E) relative quantification of the indicated phospho/total protein ratios (n≥3 biological replicates).


( 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 .

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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


( B ) Growth kinetics of PiCoVacc ( CN2 ) P5 stock in Vero cells .

(B) Growth kinetics of PiCoVacc (CN2) P5 stock in Vero cells.


( D ) Protein composition and purity evaluation of PiCoVacc by NuPAGE 4 - 12 % Bis - Tris Gel .

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(D) Protein composition and purity evaluation of PiCoVacc by NuPAGE 4-12% Bis-Tris Gel.


( E ) Representative electron micrograph of PiCoVacc .

(E) Representative electron micrograph of PiCoVacc.


isolates . BALB / c mice and Wistar rats were immunized with various doses of PiCoVacc or control ( adjuvant only as the sham group ) ( n = 10 ) . Serums from recovered COVID19 patients ( RECOV ) and non - infected people ( NI ) act as positive and negative controls , respectively . The antibody responses were analyzed in mice ( A ) , humans ( B ) and rats ( C ) . Top : SARS - CoV - 2 - specific IgG response measured by

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isolates.BALB/c mice and Wistar rats were immunized with various doses of PiCoVacc or control (adjuvant only as the sham group) (n=10). Serums from recovered COVID19 patients (RECOV) and non-infected people (NI) act as positive and negative controls, respectively. The antibody responses were analyzed in mice (A), humans (B) and rats (C). Top: SARS-CoV-2-specific IgG response measured by ELISA


The spectrum of neutralizing activities elicited by PiCoVacc was investigated in mice ( D ) and rats ( E ) . Neutralization assays against the other 9 isolated SARS - CoV - 2 strains were performed using mouse and rat serums collected 3 weeks post vaccination . Points represent individual animals and humans ; dotted lines indicate the limit of detection ; horizontal lines indicate the geometric mean titer ( GMT ) of EC50 for each group .

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The spectrum of neutralizing activities elicited by PiCoVacc was investigated in mice (D) and rats (E). Neutralization assays against the other 9 isolated SARS-CoV-2 strains were performed using mouse and rat serums collected 3 weeks post vaccination. Points represent individual animals and humans; dotted lines indicate the limit of detection; horizontal lines indicate the geometric mean titer (GMT) of EC50 for each group.


primates . Macaques were immunized three times through the intramuscular route with various doses of PiCoVacc or adjuvant only ( sham ) or placebo ( n = 4 ) . SARS - CoV - 2 - specific IgG response ( A ) and neutralizing antibody titer ( B ) were measured . Points represent individual macaques ; dotted lines indicate the limit of detection ; horizontal lines indicate the geometric mean titer ( GMT ) of EC50 for each group . Protective efficacy of PiCoVacc against SARS - CoV - 2 challenge at week 3 after immunization was evaluated in

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primates.Macaques were immunized three times through the intramuscular route with various doses of PiCoVacc or adjuvant only (sham) or placebo (n=4). SARS-CoV-2-specific IgG response (A) and neutralizing antibody titer (B) were measured. Points represent individual macaques; dotted lines indicate the limit of detection; horizontal lines indicate the geometric mean titer (GMT) of EC50 for each group. Protective efficacy of PiCoVacc against SARS-CoV-2 challenge at week 3 after immunization was evaluated in macaques


( C - F ) . Viral loads of throat ( C ) and anal ( D ) swab specimens collected from the inoculated macaques at day 3 , 5 and 7 pi were monitored . Viral loads in various lobe of lung tissue from all the inoculated macaques at day 7 post infection were measured ( E ) . RNA was extracted and viral load was determined by qRT - PCR . All data are presented as mean ± SEM .

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(C-F). Viral loads of throat (C) and anal (D) swab specimens collected from the inoculated macaques at day 3, 5 and 7 pi were monitored. Viral loads in various lobe of lung tissue from all the inoculated macaques at day 7 post infection were measured (E). RNA was extracted and viral load was determined by qRT-PCR. All data are presented as mean ± SEM.


Histopathological examinations ( F ) in lungs from all the inoculated macaques at day 7 post infection . Lung tissue was collected and stained with hematoxylin and eosin .

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Histopathological examinations (F) in lungs from all the inoculated macaques at day 7 post infection. Lung tissue was collected and stained with hematoxylin and eosin.


primates . Macaques were immunized three times at day 0 , 7 and 14 through the intramuscular route with low dose ( 1 . 5 μg per dose ) or high dose ( 6 μg per dose ) of PiCoVacc or adjuvant only ( sham ) or placebo . ( A and B ) Hematological analysis in all four groups of macaques ( n = 4 ) . Lymphocyte subset percents ( A ) , including CD3 + , CD4 + and CD8 + were monitored at day − 1 ( 1 day before vaccination ) , 18 ( 3 day after the second vaccination ) and 29 ( 7 day after the third vaccination ) . Key cytokines ( B ) , containing TNF - α , IFN - γ and IL - 2 were examined at day − 1 , 1 ( the day for the first vaccination ) , 4 , 18 and 29 after vaccination . Values are mean ± s . d .

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primates.Macaques were immunized three times at day 0, 7 and 14 through the intramuscular route with low dose (1.5 μg per dose) or high dose (6 μg per dose) of PiCoVacc or adjuvant only (sham) or placebo. (A and B) Hematological analysis in all four groups of macaques (n=4). Lymphocyte subset percents (A), including CD3+, CD4+ and CD8+ were monitored at day −1 (1 day before vaccination), 18 (3 day after the second vaccination) and 29 (7 day after the third vaccination). Key cytokines (B), containing TNF-α, IFN-γ and IL-2 were examined at day −1, 1 (the day for the first vaccination), 4, 18 and 29 after vaccination. Values are mean ± s.d.


( C ) Histopathological evaluations in lungs from four groups of macaques at day 29 . Lung tissue was collected and stained with hematoxylin and eosin .

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(C) Histopathological evaluations in lungs from four groups of macaques at day 29. Lung tissue was collected and stained with hematoxylin and eosin.


Left : representative conventional image of interphase MT network in the 1st quadrant of a Human Retinal Pigment Epithelium ( HRPE ) cell . Grey : α - tubulin ; Yellow : TGN - 46 ; Yellow dashed line : Golgi boundary . Right : the classified MT subgroups . Red : GaMTs ; Green : non - GaMTs . Scale bar : 5 μm . Box - whisker plot presents the proportion of GaMT and non - GaMTs . ( data were pooled from 3 independent experiments and n = 36 cells ) . The ends of whiskers set as the 10 % and 90 % of the entire population . * * * p < 0 . 001 , two - tailed Mann - Whitney .

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Left: representative conventional image of interphase MT network in the 1st quadrant of a Human Retinal Pigment Epithelium (HRPE) cell. Grey: α-tubulin; Yellow: TGN-46; Yellow dashed line: Golgi boundary. Right: the classified MT subgroups. Red: GaMTs; Green: non-GaMTs. Scale bar: 5 μm. Box-whisker plot presents the proportion of GaMT and non-GaMTs. (data were pooled from 3 independent experiments and n= 36 cells). The ends of whiskers set as the 10% and 90% of the entire population. ***p < 0.001, two-tailed Mann-Whitney.


Six representative images of MT networks extracted from MT STORM images of HRPE cells under combinative KDs . Cells were rotated to orient their leading edges locating in the 1st quadrant . Upper : control cell ; CLASPs KD cell ; GCC185 KD cell . Lower : CLASPs + GCC185 KD cell ; CAMSAP2 KD cell ; AKAP450 KD cell . Red : GaMT ; Green : non - GaMT . Scale bar : 5 μm . Box - whisker plot presents the ratio of GaMT / non - GaMT under combinative KDs . ( 1 representative of 3 independent experiments and n = 12 cells ) . The ends of the whiskers are set at 10 % and 90 % of the entire population . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , no significant difference , unpaired t - test .

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Six representative images of MT networks extracted from MT STORM images of HRPE cells under combinative KDs. Cells were rotated to orient their leading edges locating in the 1st quadrant. Upper: control cell; CLASPs KD cell; GCC185 KD cell. Lower: CLASPs + GCC185 KD cell; CAMSAP2 KD cell; AKAP450 KD cell. Red: GaMT; Green: non-GaMT. Scale bar: 5 μm. Box-whisker plot presents the ratio of GaMT/non-GaMT under combinative KDs. (1 representative of 3 independent experiments and n= 12 cells). The ends of the whiskers are set at 10% and 90% of the entire population. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference, unpaired t-test.


Time series show EB1 movement on individual GaMTs . Red : GaMT ; Green : non - GaMT ; White : EB1 tracks . Scale bar : 1 μm . Time series show EB1 movement on individual non - GaMTs . Red line : GaMT ; Green line : non - GaMT ; White line : EB1 tracks . Scale bar : 0 . 5 μm . EB1 velocity distributions of GaMTs and non - GaMTs . Red curve and green curve represent the Gaussian fittings ( data were pooled from 3 independent experiments and n = 92 tracks ) . * * * p < 0 . 001 , unpaired t - test .

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Time series show EB1 movement on individual GaMTs. Red: GaMT; Green: non-GaMT; White: EB1 tracks. Scale bar: 1 μm. Time series show EB1 movement on individual non-GaMTs. Red line: GaMT; Green line: non-GaMT; White line: EB1 tracks. Scale bar: 0.5 μm. EB1 velocity distributions of GaMTs and non-GaMTs. Red curve and green curve represent the Gaussian fittings (data were pooled from 3 independent experiments and n= 92 tracks). ***p <0.001, unpaired t-test.


Representative trajectories of E - cad cargos tracked over 120 s in an HRPE cell . Colors represent different average velocity ranges . Red : > 400 nm / s ; Purple : 200 ~ 400 nm / s ; Green : 100 ~ 200 nm / s ; Yellow : 0 ~ 100 nm / s . Scale bar : 20 μm . Total number of cargos within different velocity ranges in the 4 quadrants , respectively ( Data were pooled from 3 independent experiments and n = 45 cells ) . Proportions of cargos with different velocity in the 4 quadrants . Red : the 1st quadrant ; Dark grey : the 2nd quadrant ; Light grey : the 3rd quadrant ; White : the 4th quadrant ( Data were pooled from 3 independent experiments and n = 45 cells ) .

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Representative trajectories of E-cad cargos tracked over 120 s in an HRPE cell. Colors represent different average velocity ranges. Red: > 400 nm/s; Purple: 200~400 nm/s; Green: 100~200 nm/s; Yellow: 0~100 nm/s. Scale bar: 20 μm. Total number of cargos within different velocity ranges in the 4 quadrants, respectively (Data were pooled from 3 independent experiments and n= 45 cells). Proportions of cargos with different velocity in the 4 quadrants. Red: the 1st quadrant; Dark grey: the 2nd quadrant; Light grey: the 3rd quadrant; White: the 4th quadrant (Data were pooled from 3 independent experiments and n= 45 cells).


Representative time series show one fast cargo moving on a GaMT and a slow cargo moving on a non - GaMT . Upper , conventional imaging ; lower , STORM imaging . Red line : GaMT ; Green line : non - GaMT ; White circle : cargo position . Scale bar : 2 μm . Marginal distributions show the velocity on GaMT ( red dots ) or non - GaMTs ( green dots ) . Red curve and green curve , Gaussian fitting curves of velocity distribution on GaMT or non - GaMT , respectively . ( Data were pooled from 3 independent experiments and n = 69 MTs ) . Peak of red curve ( 431 . 14 ± 9 . 7 nm / s ) and peak of purple curve ( 188 . 96 ± 22 . 03 nm / s ) . * * * p < 0 . 001 , unpaired t - test .

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Representative time series show one fast cargo moving on a GaMT and a slow cargo moving on a non-GaMT. Upper, conventional imaging; lower, STORM imaging. Red line: GaMT; Green line: non-GaMT; White circle: cargo position. Scale bar: 2 μm. Marginal distributions show the velocity on GaMT (red dots) or non-GaMTs (green dots). Red curve and green curve, Gaussian fitting curves of velocity distribution on GaMT or non-GaMT, respectively. (Data were pooled from 3 independent experiments and n= 69 MTs). Peak of red curve (431.14 ± 9.7 nm/s) and peak of purple curve (188.96 ± 22.03 nm/s). ***p < 0.001, unpaired t-test.


Upper panel : time series show an example of several fast cargos present on GaMT . Lower panel : time series show one slow cargo on non - GaMT switching onto a GaMT and becoming faster . Red line : GaMT ; Green line : non - GaMT ; White circle : cargo position . White line , cargo trajectory . Scale bar : 2 μm . Box - whisker plot presents the averaged acetylation level of fast or slow MT tracks . ( Data were pooled from 2 independent experiments and n = 63 MTs ) . The ends of the whiskers are set at 10 % and 90 % of the entire population , ns , no significant difference , unpaired t - test . Marginal distributions of the mean velocity of cargos on segments of Ac - MTs ( orange dots ) and non - Ac - MTs ( purple dots ) . Orange curve and purple curve represent the Gaussian fittings ( Data were pooled from 2 independent experiments and n = 63 MTs ) . ns , no significant difference , unpaired t - test .

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Upper panel: time series show an example of several fast cargos present on GaMT. Lower panel: time series show one slow cargo on non-GaMT switching onto a GaMT and becoming faster. Red line: GaMT; Green line: non-GaMT; White circle: cargo position. White line, cargo trajectory. Scale bar: 2 μm. Box-whisker plot presents the averaged acetylation level of fast or slow MT tracks. (Data were pooled from 2 independent experiments and n= 63 MTs). The ends of the whiskers are set at 10% and 90% of the entire population, ns, no significant difference, unpaired t-test. Marginal distributions of the mean velocity of cargos on segments of Ac-MTs (orange dots) and non-Ac-MTs (purple dots). Orange curve and purple curve represent the Gaussian fittings (Data were pooled from 2 independent experiments and n= 63 MTs). ns, no significant difference, unpaired t-test.


Five representative images of MT repair sites in HRPE cells under combinative KDs . Cells were rotated to orient their leading edges locating in the 1st quadrant . From left to right , control cell , CLASPs & GCC185 KD cell , CAMSAP2 KD cell , AKAP450 KD cell and Centrinone - B treated cell . Grey : α - tubulin ; red : GTP - tubulin . Scale bar : 20 μm . Box - whisker plot presents intensity analysis of MT repair sites in HRPE cells under combinative KDs . ( 1 representative of 3 independent experiments and n = 20 cells ) . * * * p < 0 . 001 , unpaired t - test .

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Five representative images of MT repair sites in HRPE cells under combinative KDs. Cells were rotated to orient their leading edges locating in the 1st quadrant. From left to right, control cell, CLASPs&GCC185 KD cell, CAMSAP2 KD cell, AKAP450 KD cell and Centrinone-B treated cell. Grey: α-tubulin; red: GTP-tubulin. Scale bar: 20 μm. Box-whisker plot presents intensity analysis of MT repair sites in HRPE cells under combinative KDs. (1 representative of 3 independent experiments and n= 20 cells). ***p < 0.001, unpaired t-test.


Conventional image ( left ) and STORM image ( right ) of MTs and MT repair sites . Grey : α - tubulin ; yellow : GTP - tubulin ; green : Golgi . Scale bar : 5 μm .

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Conventional image (left) and STORM image (right) of MTs and MT repair sites. Grey: α-tubulin; yellow: GTP-tubulin; green: Golgi. Scale bar: 5 μm.


MT repair sites on GaMTs and non - GaMTs extracted from C . Red : GaMTs ; Green : non - GaMTs ; yellow : GTP - tubulin . MT repair sites on GaMTs and non - GaMTs separately presented in detail . White arrows indicates GTP - tubulin labeling sites . Scale bar : 5 μm . Box - whisker plot presents the ratio of MT repair sites on GaMTs and non - GaMTs separately . ( 1 representative of 3 independent experiments and n = 8 cells ) . * * * p < 0 . 001 , unpaired t - test .

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MT repair sites on GaMTs and non-GaMTs extracted from C. Red: GaMTs; Green: non-GaMTs; yellow: GTP-tubulin. MT repair sites on GaMTs and non-GaMTs separately presented in detail. White arrows indicates GTP-tubulin labeling sites. Scale bar: 5 μm. Box-whisker plot presents the ratio of MT repair sites on GaMTs and non-GaMTs separately. (1 representative of 3 independent experiments and n= 8 cells). ***p < 0.001, unpaired t-test.


Representative time series show one fast cargo moving on a GaMT and slowing down at the MT repaire site . Red : GaMT ; Green : non - GaMT ; Yellow : GTP - tubulin ; White circle : cargo position . Scale bar : 2 μm . Representative time series show one cargo moving fast between two MT repair sites and slowing down at the MTrepair sites . Red : GaMT ; Green : non - GaMT ; Yellow : GTP - tubulin ; White circle : cargo position . Scale bar : 2 μm . Pause ( I ) and reverse ( J ) events of cargos on MT repair segment and non - MTs repair segment , respectively . ( Data were pooled from 2 independent experiments and n = 22 cells ) . * * * p < 0 . 001 , ns , no significant difference , unpaired t - test .

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Representative time series show one fast cargo moving on a GaMT and slowing down at the MT repaire site. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm. Representative time series show one cargo moving fast between two MT repair sites and slowing down at the MTrepair sites. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm. Pause (I) and reverse (J) events of cargos on MT repair segment and non-MTs repair segment, respectively. (Data were pooled from 2 independent experiments and n= 22 cells). ***p <0.001, ns, no significant difference, unpaired t-test.


A representative image of MT networks extracted from MT STORM image of an HRPE cell . Red line : GaMT ; Green line : non - GaMT . Yellow circle : intersection . Blue circle : intersections formed by GaMTs and non GaMTs . Scale bar : 5 μm . Rose - plot presents the orientation of GaMTs and non - GaMTs . Red : GaMTs ; Green : non - GaMTs . Box - whisker plot presents the projections of GaMTs and non - GaMTs to the leading edge direction . ( Data were pooled from 3 independent experiments and n = 36 cells ) . The ends of the whiskers are set at 10 % and 90 % of the entire population , * * p < 0 . 01 , unpaired t - test . Box - whisker plot presents the intersection counts of GaMTs and non - GaMTs . ( data are pooled from 3 independent experiments and n = 36 cells ) . The ends of the whiskers are set at 10 % and 90 % of the entire population , * * * p < 0 . 001 , unpaired t - test .

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

A representative image of MT networks extracted from MT STORM image of an HRPE cell. Red line: GaMT; Green line: non-GaMT. Yellow circle: intersection. Blue circle: intersections formed by GaMTs and non GaMTs. Scale bar: 5 μm. Rose-plot presents the orientation of GaMTs and non-GaMTs. Red: GaMTs; Green: non-GaMTs. Box-whisker plot presents the projections of GaMTs and non-GaMTs to the leading edge direction. (Data were pooled from 3 independent experiments and n= 36 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, unpaired t-test. Box-whisker plot presents the intersection counts of GaMTs and non-GaMTs. (data are pooled from 3 independent experiments and n= 36 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ***p < 0.001, unpaired t-test.


Random migration of HRPE cells during 24 h . Upper : control cell ; Middle : CAMSAP2 KD cell ; Lower : CLASPs & GCC185 KD cell . Red arrow and white dashed lines indicate the migration direction . Different migration trajectories are distinguished with different colors . Scale bar : 50 μm . Rose - plot presents the representative directionality of an HRPE cell . ( B ) control cell ; ( C ) CAMSAP2 KD cell ; ( D ) CLASPs & GCC185 KD cell . Box - whisker plot presents the migration persistence of control cell , CAMSAP2 KD cell and CLASPs & GCC185 KD cell . ( 1 representative of 3 independent experiments and n = 34 cells ) . The ends of the whiskers are set at 10 % and 90 % of the entire population , * * * p < 0 . 001 , unpaired t - test .

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

Random migration of HRPE cells during 24 h. Upper: control cell; Middle: CAMSAP2 KD cell; Lower: CLASPs&GCC185 KD cell. Red arrow and white dashed lines indicate the migration direction. Different migration trajectories are distinguished with different colors. Scale bar: 50 μm. Rose-plot presents the representative directionality of an HRPE cell. (B) control cell; (C) CAMSAP2 KD cell; (D) CLASPs&GCC185 KD cell. Box-whisker plot presents the migration persistence of control cell, CAMSAP2 KD cell and CLASPs&GCC185 KD cell. (1 representative of 3 independent experiments and n= 34 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ***p < 0.001, unpaired t-test.


Controlled cell migration of 3 representative cells in PA - Rac1 assay . White spot : 440 nm laser spot ; Green area : the original position of the cell ; Red area : the current position of the cell ; Yellow area : the overlapped area . Scale bar : 10 μm .

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

Controlled cell migration of 3 representative cells in PA-Rac1 assay. White spot: 440 nm laser spot; Green area: the original position of the cell; Red area: the current position of the cell; Yellow area: the overlapped area. Scale bar: 10 μm.


Angle change curve of cell , nucleus and Golgi . Red - dashed line : laser angle ; Blue line : nucleus angle ; Orange line : cell angle ; Green line : Golgi angle . H : control cell ; I : CAMSAP2 KD cell ; J : CLASPs & GCC185 KD cell . Box - whisker plot shows the total time required for the nucleus , Golgi and cell body reaching the given laser angle . Blue : time required for nucleus ; Green : time required for Golgi ; Orange : time required for cell body . ( 1 representative of 3 independent experiments and n = 14 cells ) . The ends of the whiskers are set at 10 % and 90 % of the entire population , * * * p < 0 . 001 , ns , no significant difference , unpaired t - test .

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

Angle change curve of cell, nucleus and Golgi. Red-dashed line: laser angle; Blue line: nucleus angle; Orange line: cell angle; Green line: Golgi angle. H: control cell; I: CAMSAP2 KD cell; J: CLASPs&GCC185 KD cell. Box-whisker plot shows the total time required for the nucleus, Golgi and cell body reaching the given laser angle. Blue: time required for nucleus; Green: time required for Golgi; Orange: time required for cell body. (1 representative of 3 independent experiments and n= 14 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ***p < 0.001, ns, no significant difference, unpaired t-test.


Projections of 120 frame movies present leading edge delivery of two different types of cargos . Left : Ecad cargo ; Right : Rab5 cargo . Yellow line mark the cell boundary . Scale bar : 20 μm .

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

Projections of 120 frame movies present leading edge delivery of two different types of cargos. Left: Ecad cargo; Right: Rab5 cargo. Yellow line mark the cell boundary. Scale bar: 20 μm.


Examples of cargo delivery towards the leading edge , time series present at 40 s , 80 s and 120 s , respectively . B : control cell ; C : CAMSAP2 KD cell ; D : CLASPs & GCC185 KD cell . Red arrow heads indicate cargo current position . Size of cropped regions : 30 μm x 5 μm .

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

Examples of cargo delivery towards the leading edge, time series present at 40 s, 80 s and 120 s, respectively. B: control cell; C: CAMSAP2 KD cell; D: CLASPs&GCC185 KD cell. Red arrow heads indicate cargo current position. Size of cropped regions: 30 μm x 5 μm.


Box - whisker plot presents the Ecad cargo number on GaMTs and non - GaMTs . ( Data were pooled from 3 independent experiments and n = 36 cells ) . The ends of the whiskers are set at 10 % and 90 % of the entire population , * * p < 0 . 01 , unpaired t - test .

Box-whisker plot presents the Ecad cargo number on GaMTs and non-GaMTs. (Data were pooled from 3 independent experiments and n= 36 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, unpaired t-test.


Box - whisker plot presents the number of Ecad cargos finally reached the leading edge membrane during 120 s . ( 1 representative of 3 independent experiments and n = 24 cells ) . The ends of the whiskers are set at 10 % and 90 % of the entire population , * * p < 0 . 01 , two - tailed Mann - Whitney .

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

Box-whisker plot presents the number of Ecad cargos finally reached the leading edge membrane during 120 s. (1 representative of 3 independent experiments and n= 24 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, two-tailed Mann-Whitney.


Box - whisker plot shows the number of Rab5 cargos that finally reached the leading edge membrane during 120 s . ( 1 representative of 3 independent experiments and n = 23 cells ) . The ends of the whiskers are set at 10 % and 90 % of the entire population , ns , no significant difference , two - tailed Mann - Whitney .

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

Box-whisker plot shows the number of Rab5 cargos that finally reached the leading edge membrane during 120 s. (1 representative of 3 independent experiments and n= 23 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ns, no significant difference, two-tailed Mann-Whitney.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Body weight curve .

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

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Body weight curve.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Cumulative body weight .

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Cumulative body weight.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Fasting and fed blood glucose .

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

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Fasting and fed blood glucose.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Rectal temperature .

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

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Rectal temperature.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Locomotor tolerance .

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Locomotor tolerance.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . 24 - h oxygen consumption . Respiratory exchange ratio ( RER : VCO2 / VO2 ) . Energy expenditure .

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

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. 24-h oxygen consumption. Respiratory exchange ratio (RER: VCO2/VO2). Energy expenditure.


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or HFD for 3 months . Blood glucose levels and area under curve ( AUC ) during the glucose tolerance test . Blood glucose levels and AUC during the insulin tolerance test .

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

Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Blood glucose levels and area under curve (AUC) during the glucose tolerance test. Blood glucose levels and AUC during the insulin tolerance test.


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or HFD for 3 months . Glucose infusion rates ( GIR ) . Glucose disposal rate ( GDR ) . Hepatic glucose production ( HGP ) .

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

Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Glucose infusion rates (GIR). Glucose disposal rate (GDR). Hepatic glucose production (HGP).


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or HFD for 3 months . Percentage of suppression of hepatic glucose production ( HGP ) .

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

Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Percentage of suppression of hepatic glucose production (HGP).


Eight - week - old male WT and DOCK5 - / - mice Glucose infusion rates ( GIR ) during lipid infusion .

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

Eight-week-old male WT and DOCK5-/- mice Glucose infusion rates (GIR) during lipid infusion.


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or a HFD for 3 months . Mice were treated with insulin ( 1 U / kg ) or control ( PBS ) by intraperitoneal injection for 10 minutes ; animals were killed and livers were collected . The expression of expression of phosphoenolpyruvate carboxykinase ( PEPCK ) and glucose - 6 - phosphatase ( G6Pase ) mRNA and protein .

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

Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. The expression of expression of phosphoenolpyruvate carboxykinase(PEPCK) and glucose-6- phosphatase(G6Pase) mRNA and protein.


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or a HFD for 3 months . Mice were treated with insulin ( 1 U / kg ) or control ( PBS ) by intraperitoneal injection for 10 minutes ; animals were killed and livers were collected . Total and phosphorylated insulin receptor ( InsR ) , IRS - 1ser1101 and Akt .

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

Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. Total and phosphorylated insulin receptor (InsR), IRS-1ser1101 and Akt.


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or a HFD for 3 months . Mice were treated with insulin ( 1 U / kg ) or control ( PBS ) by intraperitoneal injection for 10 minutes ; animals were killed and livers were collected . Total and phosphorylated mTOR , S6K1 , and the levels of Raptor and Rictor protein .

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

Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. Total and phosphorylated mTOR, S6K1, and the levels of Raptor and Rictor protein.


Hep1 - 6 cells were infected with pCDNA3 . 1 - DOCK5 ( DOCK5 + ) or pCDNA3 . 1 ( DOCK5 - ) for 48 h . mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . The mRNA and protein expression of PEPCK and G6Pase in Hep1 - 6 cells .

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

Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. The mRNA and protein expression of PEPCK and G6Pase in Hep1-6 cells.


Hep1 - 6 cells were infected with pCDNA3 . 1 - DOCK5 ( DOCK5 + ) or pCDNA3 . 1 ( DOCK5 - ) for 48 h . - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt in Hep1 - 6 cells .

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

Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. - mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in Hep1-6 cells.


Hep1 - 6 cells were infected with pCDNA3 . 1 - DOCK5 ( DOCK5 + ) or pCDNA3 . 1 ( DOCK5 - ) for 48 h . mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated mTOR and S6K1 , and the levels of Raptor and Rictor protein in Hep1 - 6 cells .

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

Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated mTOR and S6K1, and the levels of Raptor and Rictor protein in Hep1-6 cells.


MPHs isolated from WT and DOCK5 - / - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . The mRNA and protein expression of PEPCK and G6Pase in MPHs .

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

MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. The mRNA and protein expression of PEPCK and G6Pase in MPHs.


MPHs isolated from WT and DOCK5 - / - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt in MPHs .

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

MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in MPHs.


MPHs isolated from WT and DOCK5 - / - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated mTOR and S6K1 , and the levels of Raptor and Rictor protein in MPHs .

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

MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated mTOR and S6K1, and the levels of Raptor and Rictor protein in MPHs.


Eight - week - old male Raptor flox / flox mice ( Raptorflox / flox Cre + ) were fed a HFD for 12 weeks and injected with AAV8 - shDOCK5 ± AAV8 - Cre or AAV8 - GFP ( at a dose of 3 × 1011 vg / 200 μL / mouse ) via the tail - vein 14 days prior to the in vivo study . Fasting and fed blood glucose 14 days post - infection .

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

Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Fasting and fed blood glucose 14 days post-infection.


Eight - week - old male Raptor flox / flox mice ( Raptorflox / flox Cre + ) were fed a HFD for 12 weeks and injected with AAV8 - shDOCK5 ± AAV8 - Cre or AAV8 - GFP ( at a dose of 3 × 1011 vg / 200 μL / mouse ) via the tail - vein 14 days prior to the in vivo study . Blood glucose levels and AUC during glucose tolerance tests . Blood glucose levels and AUC during insulin tolerance tests . Glucose infusion rates ( GIR ) . Glucose disposal rate ( GDR ) . Hepatic glucose production ( HGP ) . Percentage of suppression of hepatic glucose production ( HGP ) .

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

Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Blood glucose levels and AUC during glucose tolerance tests. Blood glucose levels and AUC during insulin tolerance tests. Glucose infusion rates (GIR). Glucose disposal rate (GDR). Hepatic glucose production (HGP). Percentage of suppression of hepatic glucose production (HGP).


Male Raptor flox / flox mice ( 8 weeks ) were fed a HFD for 12 weeks and injected with AAV8 - GFP or AAV8 - shDOCK5 or AAV8 - shDOCK5 + AAV8 - Cre via the tail vein . The mRNA and protein expression of PEPCK and G6Pase in the liver .

Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. The mRNA and protein expression of PEPCK and G6Pase in the liver.


Male Raptor flox / flox mice ( 8 weeks ) were fed a HFD for 12 weeks and injected with AAV8 - GFP or AAV8 - shDOCK5 or AAV8 - shDOCK5 + AAV8 - Cre via the tail vein . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt in the liver .

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

Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in the liver.


Male Raptor flox / flox mice ( 8 weeks ) were fed a HFD for 12 weeks and injected with AAV8 - GFP or AAV8 - shDOCK5 or AAV8 - shDOCK5 + AAV8 - Cre via the tail vein . The levels of DOCK5 and Raptor protein , and total and phosphorylated mTOR and S6K1 in the liver .

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

Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. The levels of DOCK5 and Raptor protein, and total and phosphorylated mTOR and S6K1 in the liver.


MPHs from Raptor flox / flox mice were infected with Ad - GFP or Ad - shDOCK5 or Ad - shDOCK5 + Ad - Cre . The lysates were immunoblotted with indicated antibodies or β - actin . The expression of PEPCK and G6Pase mRNA and protein .

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

MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. The expression of PEPCK and G6Pase mRNA and protein.


MPHs from Raptor flox / flox mice were infected with Ad - GFP or Ad - shDOCK5 or Ad - shDOCK5 + Ad - Cre . The lysates were immunoblotted with indicated antibodies or β - actin . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt .

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

MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. Total and phosphorylated InsR, IRS-1Ser1101 and Akt.


MPHs from Raptor flox / flox mice were infected with Ad - GFP or Ad - shDOCK5 or Ad - shDOCK5 + Ad - Cre . The lysates were immunoblotted with indicated antibodies or β - actin . The levels of DOCK5 and Raptor protein , and total and phosphorylated mTOR and S6K1 .

MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. The levels of DOCK5 and Raptor protein, and total and phosphorylated mTOR and S6K1.


Rac1 GEF activation in MPHs from WT and DOCK5 - / - mice .

Rac1 GEF activation in MPHs from WT and DOCK5-/- mice.


SEW2871 treatment promotes Rac1 GEF activation and eliminates the influence of a DOCK5 deficiency on Raptor / S6K1 signaling in the mouse primary hepatocytes ( MPHs ) of DOCK5 - / - mice .

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SEW2871 treatment promotes Rac1 GEF activation and eliminates the influence of a DOCK5 deficiency on Raptor / S6K1 signaling in the mouse primary hepatocytes (MPHs) of DOCK5-/- mice.


Total and phosphorylated mTOR and S6K1 and the level of Raptor protein expression in plasmids expressing a WT DOCK5 or mutant DOCK5 treated - Hep1 - 6 cells .

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Total and phosphorylated mTOR and S6K1 and the level of Raptor protein expression in plasmids expressing a WT DOCK5 or mutant DOCK5 treated-Hep1-6 cells.


Coimmunoprecipitation ( Co - IP ) analysis of DOCK5 and Raptor expression in Hepa1 - 6 cells . After transfected with indicated plasmids , lysates from cells were immunoprecipitated ( IP ) with anti - DOCK5 antibody or normal IgG , and then immunoblotted ( IB ) with the indicated antibody .

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Coimmunoprecipitation (Co-IP) analysis of DOCK5 and Raptor expression in Hepa1-6 cells. After transfected with indicated plasmids, lysates from cells were immunoprecipitated (IP) with anti-DOCK5 antibody or normal IgG, and then immunoblotted (IB) with the indicated antibody.


DOCK5 binding to Raptor fragments ( 445 - 887 aa ) during transient expression in Hepa1 - 6 cells .

DOCK5 binding to Raptor fragments (445 - 887 aa) during transient expression in Hepa1-6 cells.


The DOCK5 and Raptor binding sites were identified using mutant DOCK5 ( DHR - 2V1559A ) and Raptor fragments ( 445 - 887 aa ) based on a Co - IP assay . VA , mutant DOCK5 DHR - 2 ; His , His - DHR - 2 .

The DOCK5 and Raptor binding sites were identified using mutant DOCK5 (DHR-2V1559A) and Raptor fragments (445 - 887 aa) based on a Co-IP assay. VA, mutant DOCK5 DHR-2; His, His-DHR-2.


A ) Maximum intensity projections of pediatric ( 0 - 12 months ) small intestine showing the distribution of CD56 , CD90 and CD24 within the ENS . The first image of each row shows a low magnification overview and an inset depicting the DAPI channel in high magnification . The remaining panels correspond to the high magnification inset . Scale bar in overview images = 200 µm . Scale bar in high magnification panels = 40 µm .

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A) Maximum intensity projections of pediatric (0-12 months) small intestine showing the distribution of CD56, CD90 and CD24 within the ENS. The first image of each row shows a low magnification overview and an inset depicting the DAPI channel in high magnification. The remaining panels correspond to the high magnification inset. Scale bar in overview images = 200 µm. Scale bar in high magnification panels = 40 µm.


( F ) RT - qPCR from CD56 + CD90 + sorted cells vs control non - ENS cells for representative neuronal and glial markers , confirms high enrichment of both neurons and glial cells ( mean ± SD , N = 4 biological replicates , P * < 0 . 05 , P * * * < 0 . 001 , multiple Student ' s t test , FDR correction for multiple comparisons ) .

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(F) RT-qPCR from CD56+CD90+ sorted cells vs control non- ENS cells for representative neuronal and glial markers, confirms high enrichment of both neurons and glial cells (mean±SD, N=4 biological replicates, P * < 0.05, P *** < 0.001, multiple Student's t test, FDR correction for multiple comparisons).


B ) Representative plot showing full co - expression of TUBB3 ( neuronal marker ) and SOX10 ( glial marker ) , on ENS cells ( bottom ) and only minimal and low - intensity background on non - ENS cells ( Top ) ( N = 1 representative sample , for stats see Figure 2C ) . Intracellular staining was performed on formalin fixed cells from ileum , after pre - staining for the extracellular markers . Selection of the ENS cluster in fixed cells is shown in Appendix Figure S2F .

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B) Representative plot showing full co-expression of TUBB3 (neuronal marker) and SOX10 (glial marker), on ENS cells (bottom) and only minimal and low-intensity background on non-ENS cells (Top) (N=1 representative sample, for stats see Figure 2C). Intracellular staining was performed on formalin fixed cells from ileum, after pre-staining for the extracellular markers. Selection of the ENS cluster in fixed cells is shown in Appendix Figure S2F.


C ) Percentage of TUBB3 + and SOX10 + cells in ENS and non - ENS cells ( mean ± SD , TUBB3 : N = 4 biological replicates , SOX10 : N = 3 biological replicates ) . The single markers TUBB3 and SOX10 were gated vs SSC - A as shown in Appendix Figure S2G .

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C) Percentage of TUBB3+ and SOX10+ cells in ENS and non-ENS cells (mean±SD, TUBB3: N=4 biological replicates, SOX10: N=3 biological replicates). The single markers TUBB3 and SOX10 were gated vs SSC-A as shown in Appendix Figure S2G.


B ) The distribution of ENS cells and non - nucleated particles is represented in a CD56 / CD90 plot . Left : initial selection of ENS cells . Right : zoom on the ENS cluster , where ENS events are shown as non - nucleated , nucleated , merge . For additional details and stats see Appendix Figure S3E .

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B) The distribution of ENS cells and non-nucleated particles is represented in a CD56/CD90 plot. Left: initial selection of ENS cells. Right: zoom on the ENS cluster, where ENS events are shown as non-nucleated, nucleated, merge. For additional details and stats see Appendix Figure S3E.


C ) ENS cells and non - nucleated particles are shown in a CD56 / CD24 plot , as in Figure 3B . Nucleated and non - nucleated events largely overlap and define the same subclusters in CD56 / CD24 and CD56 / CD90 plots , although the average staining intensity of the three markers is lower for non - nucleated events . For additional details and stats see Appendix Figure S3E .

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C) ENS cells and non-nucleated particles are shown in a CD56/CD24 plot, as in Figure 3B. Nucleated and non-nucleated events largely overlap and define the same subclusters in CD56/CD24 and CD56/CD90 plots, although the average staining intensity of the three markers is lower for non-nucleated events. For additional details and stats see Appendix Figure S3E.


Confocal images of ENS events sorted as nucleated and non - nucleated , as shown in Figure . 3 . ( A ) Most of the staining belongs to what appears to be non - nucleated ( DAPI - ) cellular debris ( putative neuronal terminations ) attached to a nucleated cell ( putative glial cell ) . This is particularly true for TUBB3 and CD24 , which show ( as expected for neuronal selective markers ) a starker distinction than CD56 ( also expressed by glial cells ) . At a very low frequency among sorted ENS cells , we could observe putative neurons as represented in ( B ) , with a more homogenous distribution of the three markers . ( C ) Examples of sorted Hoechst - negative ENS debris , which are confirmed to be non - nucleated ( thus validating our gates on Hoechst 33342 - A ) , but positive for all neuronal markers .

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Confocal images of ENS events sorted as nucleated and non-nucleated, as shown in Figure. 3. (A) Most of the staining belongs to what appears to be non-nucleated (DAPI-) cellular debris (putative neuronal terminations) attached to a nucleated cell (putative glial cell). This is particularly true for TUBB3 and CD24, which show (as expected for neuronal selective markers) a starker distinction than CD56 (also expressed by glial cells). At a very low frequency among sorted ENS cells, we could observe putative neurons as represented in (B), with a more homogenous distribution of the three markers. (C) Examples of sorted Hoechst-negative ENS debris, which are confirmed to be non-nucleated (thus validating our gates on Hoechst 33342-A), but positive for all neuronal markers.


C ) RT - qPCR for neuronal markers on cells sorted for CD24TIP vs all the other ENS cells , confirms strong neuronal enrichment ( mean ± SD , N = 5 biological replicates , P * * * < 0 . 001 , multiple Student ' s t test , FDR correction for multiple comparisons ) .

C) RT-qPCR for neuronal markers on cells sorted for CD24TIP vs all the other ENS cells, confirms strong neuronal enrichment (mean±SD, N=5 biological replicates, P *** < 0.001, multiple Student's t test, FDR correction for multiple comparisons).


E ) Graph showing the proportion of the CD24 - based subclusters in the nucleated vs non - nucleated fraction of ganglionic and aganglionic colon ( mean ± SD , N = 4 biological replicates , P * * * < 0 . 001 , multiple Student ' s t test , FDR correction for multiple comparisons ) . Additional details and stats are shown in Appendix Figure S4E - J .

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E) Graph showing the proportion of the CD24-based subclusters in the nucleated vs non-nucleated fraction of ganglionic and aganglionic colon (mean±SD, N=4 biological replicates, P *** < 0.001, multiple Student's t test, FDR correction for multiple comparisons). Additional details and stats are shown in Appendix Figure S4E-J.


G ) Stacked violin plots showing enriched genes in specific neuronal subtypes .

G) Stacked violin plots showing enriched genes in specific neuronal subtypes.


A . Western blot analysis of Tace using lysates from WT and TaceFl / Fl ; P0 - Cre mousesciatic nerves at P10 ( 65ug of total protein lysate loaded ) . The arrowhead indicates the pro - protein and the arrow shows the cleaved active form of Tace . Representative of two independent experiments .

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A. Western blot analysis of Tace using lysates from WT and TaceFl/Fl;P0-Cre mousesciatic nerves at P10 (65ug of total protein lysate loaded). The arrowhead indicates the pro-protein and the arrow shows the cleaved active form of Tace. Representative of two independent experiments.


B . Western blot analysis ( 50ug of total protein lysate loaded ) shows that Tace expression in axons is maximal at P5 - P10 and then progressively declines in the adult . Representative of two independent experiments .

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B. Western blot analysis (50ug of total protein lysate loaded) shows that Tace expression in axons is maximal at P5-P10 and then progressively declines in the adult. Representative of two independent experiments.


C . Western blot analysis of Tace in Mtmr2 - / - sciatic nerves at P5 , P10 , and P20 . Representative of two independent experiments .

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C. Western blot analysis of Tace in Mtmr2-/- sciatic nerves at P5, P10, and P20. Representative of two independent experiments.


D . Western blot analysis of Tace using lysates from Mtmr2 - / - mouse sciatic nerves at 5 months , with quantification , p = 0 . 10 , two - tailed Mann Whitney U test . Representative of two independent experiments .

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D. Western blot analysis of Tace using lysates from Mtmr2-/- mouse sciatic nerves at 5 months, with quantification, p=0.10, two-tailed Mann Whitney U test. Representative of two independent experiments.


E . Phosphorylation of ErbB2 in Mtmr2 - / - sciatic nerves at P2 , P10 and P15 . At P2 , each lane is a pool of n = 7 animals per genotype , representative of six independent experiments using n = 6 different nerve pools per genotype , and quantification using WT values arbitrary set to1 , p = 0 . 0313 , Wilcoxon Rank Sum test . At P10 and P15 , mean values of two samples are shown . At P10 , each lane is a pool of n = 3 / 4 animals per genotype , representative of three independent experiments using n = 6 different pools per genotype . Nf - l is the neurofilament light chain and Clx is calnexin .

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E. Phosphorylation of ErbB2 in Mtmr2-/- sciatic nerves at P2, P10 and P15. At P2, each lane is a pool of n=7 animals per genotype, representative of six independent experiments using n=6 different nerve pools per genotype, and quantification using WT values arbitrary set to1, p=0.0313, Wilcoxon Rank Sum test. At P10 and P15, mean values of two samples are shown. At P10, each lane is a pool of n=3/4 animals per genotype, representative of three independent experiments using n=6 different pools per genotype. Nf-l is the neurofilament light chain and Clx is calnexin.


F . Phosphorylation of ErbB2 in Mtmr2 - / - Schwann cell / DRG neuron co - cultures after 4 days of ascorbic acid treatment . Each lane is a pool from at least 10 coverslips / DRG per genotype . Representative of two independent experiments using n = 4 different pools of coverslips / DRG per genotype , p = 0 . 20 , two - tailed Mann Whitney U test .

F. Phosphorylation of ErbB2 in Mtmr2-/- Schwann cell/DRG neuron co-cultures after 4 days of ascorbic acid treatment. Each lane is a pool from at least 10 coverslips/DRG per genotype. Representative of two independent experiments using n=4 different pools of coverslips/DRG per genotype, p=0.20, two-tailed Mann Whitney U test.


A . ShRNA targeting Nrg1 type III were validated in rat purified neurons . Western blot analysis for Nrg1 shows reduction of its expression ( pro - protein at 150 KDa ) using two different harpins , representative of two independent experiments . Clx is calnexin .

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A. ShRNA targeting Nrg1 type III were validated in rat purified neurons. Western blot analysis for Nrg1 shows reduction of its expression (pro-protein at 150 KDa) using two different harpins, representative of two independent experiments. Clx is calnexin.


B . ShRNA # 1 and 2 downregulate Nrg1 type III expression also in myelin - forming Schwann cell / DRG neuron co - cultures analyzed after 7 days of ascorbic acid treatment , as shown by western blot analysis , representative of two independent experiments . Clx is calnexin .

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B. ShRNA#1 and 2 downregulate Nrg1 type III expression also in myelin-forming Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, as shown by western blot analysis, representative of two independent experiments. Clx is calnexin.


C , D . Titration of shRNA # 1 ( C ) and of shRNA # 2 ( D ) LVs ( lentiviral vectors ) on WT Schwann cell / DRG neuron co - cultures analyzed after 7 days of ascorbic acid treatment , with quantification of the mean number of Mbp positive segments and Schwann cell number . In ( C ) n = 20 ( SCR , Scramble ) , n = 11 ( LV # sh1 , 12 . 5 % ) , n = 16 ( LV # sh1 , 25 % ) , n = 9 ( LV # sh1 , 50 % ) DRG / coverslips from two independent experiments . Results are mean ± SEM , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test . In ( D ) n = 14 ( SCR ) , n = 10 ( LV # sh2 , 12 . 5 % ) , n = 12 ( LV # sh2 , 25 % ) , n = 79 ( LV # sh2 , 50 % ) DRG / coverslips , representative of two independent experiments . Results are mean ± SEM , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test .

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C, D. Titration of shRNA #1 (C) and of shRNA #2 (D) LVs (lentiviral vectors) on WT Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, with quantification of the mean number of Mbp positive segments and Schwann cell number. In (C) n= 20 (SCR, Scramble), n= 11 (LV #sh1, 12.5%), n= 16 (LV #sh1, 25%), n= 9 (LV #sh1, 50%) DRG/coverslips from two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test. In (D) n= 14 (SCR), n= 10 (LV #sh2, 12.5%), n= 12 (LV #sh2, 25%), n= 79 (LV #sh2, 50%) DRG/ coverslips, representative of two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


E . Representative confocal images of Mtmr2 - / - co - cultures transduced using LV expressing Nrg1 type III shRNA # 1 , with quantification of the percentage of myelin outfoldings , n = number of DRG / coverslips , n = 9 , 382 fibers ( SCR ) , n = 9 , 368 fibers ( LV # sh1 , 12 . 5 % ) , n = 8 , 327 fibers ( LV # sh1 , 25 % ) . Asterisks indicate fibers with myelin outfoldings . Results are mean ± SEM , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test , representative of two independent experiments .

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E. Representative confocal images of Mtmr2-/- co-cultures transduced using LV expressing Nrg1 type III shRNA #1, with quantification of the percentage of myelin outfoldings, n= number of DRG/coverslips, n= 9, 382 fibers (SCR), n=9, 368 fibers (LV #sh1, 12.5%), n= 8, 327 fibers (LV #sh1, 25%). Asterisks indicate fibers with myelin outfoldings. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test, representative of two independent experiments.


E . Western blot analysis of Nrg1 and Akt phosphorylation ( S473 ) shows that activation of Akt decreases following Nrg1 type III downregulation ( p - Akt / Akt / Actin , ratio between Mtmr2 - / - Scramble - treated and Mtmr2 - / - sh # 1 - treated using 12 . 5 % LV is 1 : 0 . 85 ) . Each lane is a pool of at least 10 different DRG / coverslips , representative of two independent experiments . Bar in ( C , D ) is 100 μm ; in ( E ) is 20 μm .

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E. Western blot analysis of Nrg1 and Akt phosphorylation (S473) shows that activation of Akt decreases following Nrg1 type III downregulation (p-Akt/Akt/Actin, ratio between Mtmr2-/- Scramble-treated and Mtmr2-/- sh#1-treated using 12.5% LV is 1 : 0.85). Each lane is a pool of at least 10 different DRG/coverslips, representative of two independent experiments. Bar in (C, D) is 100 μm; in (E) is 20 μm.


A . Titration of rhTACE on wild - type ( WT ) co - cultures stained for Mbp with quantification of Mbp positive fibers and Schwann cell nuclei : n = 18 ( NT , not treated ) , n = 6 ( 5ng / ml ) , n = 20 ( 10ng / ml ) , n = 7 ( 50ng / ml ) DRGs / covers from two independent experiments , p = 0 . 0053 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test .

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A. Titration of rhTACE on wild-type (WT) co-cultures stained for Mbp with quantification of Mbp positive fibers and Schwann cell nuclei: n=18 (NT, not treated), n=6 (5ng/ml), n=20 (10ng/ml), n=7 (50ng/ml) DRGs/covers from two independent experiments, p=0.0053, nonparametric one-way ANOVA, followed by Dunn's post hoc test.


B . Immunohistochemistry and confocal microscopy of Mtmr2 - / - cultures treated with 5ng / ml and 10ng / ml rhTACE , with quantification of the percentage of myelin outfoldings ; n = 10 ( NT ) , n = 10 ( 5ng / ml ) , n = 11 ( 10ng / ml ) , DRGs / coverslips . A total of 547 , 557 , and 571 Mbp fibers were quantified , respectively , p < 0 . 0001 , nonparametric one - way ANOVA , followed by Dunn ' s post hoc test .

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B. Immunohistochemistry and confocal microscopy of Mtmr2-/- cultures treated with 5ng/ml and 10ng/ml rhTACE, with quantification of the percentage of myelin outfoldings; n=10 (NT), n=10 (5ng/ml), n=11 (10ng/ml), DRGs/coverslips. A total of 547, 557, and 571 Mbp fibers were quantified, respectively, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.