B Co ‐ localization of HA ‐ Flag ‐ ATG9A with TGN46 .

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B Co‐localization of HA‐Flag‐ATG9A with TGN46.


C Lysates of U2OS transfected with siRNA targeting AP2A1 , or siRNA control AllStars analyzed by SDS - PAGE .

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C Lysates of U2OS cells transfected with siRNA targeting AP2A1, or siRNA control AllStars analyzed by SDS-PAGE.


D U2OS stably expressing mCherry − ATG9 were transiently transfected with GFP − Dynamin 2 or GFP − Dynamin 2 ( K44A ) , 20 h post − transfection were fixed with anti − TBC1D5 antibody . ATG9A Dynamin 2co − localization is indicated by arrows .

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D U2OS cells stably expressing mCherry−ATG9 were transiently transfected with GFP−Dynamin 2 or GFP−Dynamin 2 (K44A), 20 h post−transfection cells were fixed and with anti−TBC1D5 antibody. ATG9A and Dynamin 2co−localization is indicated by arrows.


A U2OS stably expressing HA − Flag − ATG9A treated with DMSO or Dynasore ( 100 μM ) 15 min , fixed immunostained with anti − HA anti − TBC1D5 antibodies .

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A U2OS cells stably expressing HA−Flag−ATG9A treated with DMSO or Dynasore (100 μM) 15 min, fixed and immunostained with anti−HA and anti−TBC1D5 antibodies.


B of ATG9Aand AP2A1co ‐ localization from experiment in ( A ) , pictures from 3 independent experiments were analyzed co ‐ localization quantified , statistics is calculated as described in Supplementary Materials Methods .

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B Quantification of ATG9Aand AP2A1co‐localization from experiment in (A), pictures from 3 independent experiments were analyzed and co‐localization quantified, statistics is calculated as described in Supplementary Materials and Methods.


C RGB ‐ profile across vesicle indicated with arrow in ( B ) ; red - AP2A1 ; green - ATG9A ; blue - TBC1D5 .

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C RGB‐line profile across the vesicle indicated with arrow in (B); red-AP2A1; green-ATG9A; blue-TBC1D5.


D of ATG9A AP2 co ‐ localization . were starved in EBSS media ( 45 min ) or pretreated with Dynasore ( 15 min ) subsequently starved for 45 min in combination with Dynasore . Control were treated with DMSO .

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D Quantification of ATG9A and AP2 co‐localization. Cells were starved in EBSS media (45 min) or pretreated with Dynasore (15 min) and subsequently starved for 45 min in combination with Dynasore. Control cells were treated with DMSO.


E of LC3 in stably expressing HA ‐ Flag ‐ ATG9A , treated presented as in ( D ) . 100 were quantified in 3 independent experiments .

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E Quantification of LC3 puncta in cells stably expressing HA‐Flag‐ATG9A, treated and presented as in (D). 100 cells were quantified in 3 independent experiments.


F T ‐ REx HeLa were transfected with siRNA Control oligo or siRNA oligo # 1 targeting ( 40 nM ) . 72 h post − transfection , expression of HA ‐ Flag − TBC1D5 was induced with doxycycline ( 50 ng / ml ) , 96 h post − transfection were treated with Dynasore for 15 min , or pretreated with Dynasore subsequently starved in combination with Dynasore for additional 45 min . were lysed in co ‐ immunoprecipitation buffer , lysates were incubated with Myc antibody or M2 antibody coupled with agarose overnight at 4 ° C . Beads were washed 3 times with incubation buffer subjected to SDS ‐ PAGE .

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F T‐REx HeLa cells were transfected with siRNA Control oligo or siRNA oligo#1 targeting (40 nM). 72 h post−transfection, expression of HA‐Flag−TBC1D5 was induced with doxycycline (50 ng/ml), and 96 h post−transfection cells were treated with Dynasore for 15 min, or pretreated with Dynasore and subsequently starved in combination with Dynasore for additional 45 min. Cells were lysed in co‐immunoprecipitation buffer, and lysates were incubated with Myc antibody or M2 antibody coupled with agarose overnight at 4°C. Beads were washed 3 times with incubation buffer and subjected to SDS‐PAGE.


A , Distribution classification of MAP4K3 / GLK - positive in 20 ml BALFs of 12 individuals ( A ) , Φ denotes macrophages ; N denotes neutrophils ; T denotes T ; E denotes epithelial .

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A, Distribution and classification of MAP4K3/GLK-positive cells in 20 ml BALFs of all 12 individuals (A), Φ denotes macrophages; N denotes neutrophils; T denotes T cells; E denotes epithelial cells.


3 healthy controls ( HC , # 1 - # 3 ) , 3 mild COVID - 19 ( # 1 - # 3 ) , 6 severe COVID - 19 ( # 1 - # 6 ) ( B ) from Cohort # 1 . Data were shown in UMAP . Single - gene expression of MAP4K3 ( GLK ) in was shown in color scale .

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3 healthy controls (HC, #1 - #3), 3 mild COVID-19 patients (#1 - #3), and 6 severe COVID-19 patients (#1 - #6) (B) from Cohort #1. Data were shown in UMAP. Single-cell gene expression of MAP4K3 (GLK) in individual cells was shown in color scale.


C , D percentages of MAP4K3 / GLK - positive KRT18 + epithelial ( C ) or KRT18 + TPPP3 + ciliated epithelial ( D ) in BALFs from Cohort # 1 . Data information : In ( C , D ) , * , P value < 0 . 05 ( ANOVA ) ; § , P value < 0 . 05 ( Dunnett ' s ) .

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C, D The percentages of MAP4K3/GLK-positive KRT18+ epithelial (C) or KRT18+ TPPP3+ ciliated epithelial cells (D) in BALFs from Cohort #1. Data information: In (C, D), *, P value < 0.05 (ANOVA test); §, P value < 0.05 (Dunnett's test).


E , F GLK mRNA in KRT18 + epithelial ( E ) or KRT18 + TPPP3 + ciliated epithelial ( F ) of Cohort # 1 . n ( number ) = 3 , 14 , 33 for HC ; n = 65 , 43 , 74 for mild COVID - 19 ; n = 164 , 62 , 10 , 4 , 74 , 66 for severe COVID - 19 ( E ) . n = 25 , 43 , 36 for HC ; n = 209 , 244 , 141 for mild COVID - 19 ; n = 189 , 223 , 330 , 792 , 80 , 243 for severe COVID - 19 ( F ) . Data information : In ( E , F ) , * , P value < 0 . 05 ; * * , P value < 0 . 01 ; * * * , P value < 0 . 001 ; * * * * , P value < 0 . 0001 ( Kruskal - Wallis ) .

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E, F GLK mRNA levels in KRT18+ epithelial (E) or KRT18+ TPPP3+ ciliated epithelial cells (F) of Cohort #1. n (cell number) = 3, 14, 33 for HC; n = 65, 43, 74 for mild COVID-19 patients; n = 164, 62, 10, 4, 74, 66 for severe COVID-19 patients (E). n = 25, 43, 36 for HC; n = 209, 244, 141 for mild COVID-19 patients; n = 189, 223, 330, 792, 80, 243 for severe COVID-19 patients (F). Data information: In (E, F), *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001 (Kruskal-Wallis test).


Immunoblotting of endogenous GLK vinculin from lysates of HCC827 lung epithelial infected with SARS - CoV - 2 pseudovirus ( A ) Data information : Data shown are representative results of three ( A , independent experiments .

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Immunoblotting analysis of the endogenous GLK and vinculin proteins from the lysates of HCC827 lung epithelial cells infected with SARS-CoV-2 pseudovirus (A) Data information: Data shown are representative results of three (A, independent experiments.


Real - time PCR of mouse GLK mRNA in HCC827 lung epithelial infected with SARS - CoV - 2 pseudovirus ( B ) mRNA of GLK were normalized to GAPDH mRNA . n = 2 ( technical replicates ) per group . Data information : In ( B data are presented as means . Data shown are representative results of three B , independent experiments .

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Real-time PCR analysis of mouse GLK mRNA levels in HCC827 lung epithelial cells infected with SARS-CoV-2 pseudovirus (B) The mRNA levels of GLK were normalized to GAPDH mRNA levels. n = 2 (technical replicates) per group. Data information: In (B data are presented as means. Data shown are representative results of three B, independent experiments.


Endogenous GLK were increased by SARS - CoV - 2 spike . Immunoblotting of endogenous GLK vinculin from lysates of HCC827 lung epithelial treated with SARS - CoV - 2 spike ( C ) . Data information : Data shown are representative results of three C , independent experiments .

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Endogenous GLK levels were increased by the SARS-CoV-2 spike protein. Immunoblotting analysis of the endogenous GLK and vinculin proteins from the lysates of HCC827 lung epithelial cells treated with the SARS-CoV-2 spike protein (C). Data information: Data shown are representative results of three C, independent experiments.


Real - time PCR of mouse GLK mRNA in HCC827 lung epithelial treated with SARS - CoV - 2 spike ( D ) . mRNA of GLK were normalized to GAPDH mRNA . n = 2 ( technical replicates ) per group . Data information : In D ) , data are presented as means . Data shown are representative results of three D ) independent experiments .

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Real-time PCR analysis of mouse GLK mRNA levels in HCC827 lung epithelial cells treated with the SARS-CoV-2 spike protein (D). The mRNA levels of GLK were normalized to GAPDH mRNA levels. n = 2 (technical replicates) per group. Data information: In D), data are presented as means. Data shown are representative results of three D) independent experiments.


A ZetaView nanoparticle tracking of particle numbers sizes of CD63 + extracellular vesicles ( EVs ) isolated from supernatants of HCC827 lung epithelial cancer , which were transfected with GLK wild - type or GLK kinase - dead ( K45E ) mutant . EVs were isolated sequentially using ExoQuick kits then ExoQuick ULTRA columns . Data information : Data shown are representative results of two ( A ) independent experiments .

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A ZetaView nanoparticle tracking analysis of particle numbers and sizes of CD63+ extracellular vesicles (EVs) isolated from the supernatants of HCC827 lung epithelial cancer cells, which were transfected with GLK wild-type or GLK kinase-dead (K45E) mutant. EVs were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Data information: Data shown are representative results of two (A) independent experiments.


B Immunoblotting of ACE2 CD63 in exosomes isolated from GLK wild - type - or GLK ( K45E ) kinase - dead - overexpressing HCC827 . Exosomes were isolated sequentially using ExoQuick kits then ExoQuick ULTRA columns . Arrowhead denotes glycosylated ACE2 ; asterisk denotes non - glycosylated ACE2 . Data information : Data shown are representative results of three ( B , independent experiments .

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B Immunoblotting of ACE2 and CD63 proteins in exosomes isolated from GLK wild-type- or GLK (K45E) kinase-dead-overexpressing HCC827 cells. Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins. Data information: Data shown are representative results of three (B, independent experiments.


C Immunoblotting of ACE2 CD63 in serum exosomes isolated from sera of 4 healthy controls ( HC ) 7 COVID - 19 from NHRI Biobank for COVID - 19 in Taiwan ( Cohort # 3 ) . Serum collection days from onset are shown ; treatment of oxygen therapy on is also indicated . Exosomes were isolated sequentially using ExoQuick kits then ExoQuick ULTRA columns . Arrowhead denotes glycosylated ACE2 ; asterisk denotes non - glycosylated ACE2 .

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C Immunoblotting of ACE2 and CD63 proteins in serum exosomes isolated from the sera of 4 healthy controls (HC) and 7 COVID-19 patients from NHRI Biobank for COVID-19 patients in Taiwan (Cohort #3). Serum collection days from the onset are shown; treatment of oxygen therapy on the patient is also indicated. Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins.


D Confocal microscopy of Flag - tagged ACE2 ( red ) in recipient after incubation with exosomes for 72 h . Exosomes were isolated sequentially from supernatants of Flag - ACE2 - overexpressing or GFP - GLK plus Flag - ACE2 - coexpressing HCC827 using ExoQuick kits then ExoQuick ULTRA columns . Arrows denote Flag - tagged ACE2 . Original magnification , × 630 ; scale bars , 10 μm . Data information : Data shown are representative results of three D , independent experiments .

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D Confocal microscopy analysis of Flag-tagged ACE2 proteins (red) in recipient cells after incubation with exosomes for 72 h. Exosomes were isolated sequentially from the supernatants of Flag-ACE2-overexpressing or GFP-GLK plus Flag-ACE2-coexpressing HCC827 cells using ExoQuick kits and then ExoQuick ULTRA columns. Arrows denote Flag-tagged ACE2 proteins. Original magnification, ×630; scale bars, 10 μm. Data information: Data shown are representative results of three D, independent experiments.


E Confocal microscopy of SARS - CoV - 2 spike ( S ) ( green ) RFP - labeled early endosomes ( red ) in exosome - recipient HCC827 epithelial . After incubation with exosomes for 72 h , recipient were treated with spike ( S ) for another 24 h . Yellow color suggests localization of S in early endosomes . Original magnification , × 630 ; scale bars , 25 μm . Data information : Data shown are representative results of three E , independent experiments .

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E Confocal microscopy analysis of SARS-CoV-2 spike (S) protein (green) and RFP-labeled early endosomes (red) in exosome-recipient HCC827 epithelial cells. After incubation with exosomes for 72 h, recipient cells were treated with the spike protein (S) for another 24 h. Yellow color suggests the localization of S protein in early endosomes. Original magnification, ×630; scale bars, 25 μm. Data information: Data shown are representative results of three E, independent experiments.


F entry eﬃciencies of SARS - CoV - 2 pseudovirus into exosome - recipient HCC827 epithelial were measured by luciferase activity presented as relative units ( RLU ) at 24 h post infection . n = 3 ( biological replicates ) . Data information : In ( F ) , data are presented as means ± . * * , P value < 0 . 01 ( ANOVA ) . Data shown are representative results of three F ) independent experiments .

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F Cell entry eﬃciencies of SARS-CoV-2 pseudovirus into exosome-recipient HCC827 epithelial cells were measured by luciferase activity and presented as relative light units (RLU) at 24 h post infection. n = 3 (biological replicates). Data information: In (F), data are presented as means ± SEM. **, P value < 0.01 (ANOVA test). Data shown are representative results of three F) independent experiments.


B Co - immunoprecipitation of Flag - tagged ACE2 with Myc - tagged GLK from lysates of HEK293T transfectants . NS , normal serum . Data information : Data shown are representative results of three ( B , independent experiments .

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B Co-immunoprecipitation of Flag-tagged ACE2 with Myc-tagged GLK proteins from lysates of HEK293T transfectants. NS, normal serum. Data information: Data shown are representative results of three (B, independent experiments.


C In situ PLA assays of interaction between ACE2 Flag - tagged GLK in HEK293T . Nuclei were stained with DAPI . Red dots represent direct interaction signals . Original magnification , x200 . Scale bars , 50 μm . Data information : Data shown are representative results of three C , independent experiments .

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C In situ PLA assays of the interaction between ACE2 and Flag-tagged GLK proteins in HEK293T cells. Nuclei were stained with DAPI. Red dots represent direct interaction signals. Original magnification, x200. Scale bars, 50 μm. Data information: Data shown are representative results of three C, independent experiments.


D Immunoblotting analyses of endogenous ACE2 , ACE1 , CD147 , tubulin , as well as transfected Flag - GLK in HCC827 lung epithelial , which were transfected with either GLK wild - type or kinase - dead ( K45E ) mutant . Arrowhead denotes glycosylated ACE2 ; asterisk denotes non - glycosylated ACE2 . Data information : Data shown are representative results of three D , independent experiments .

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D Immunoblotting analyses of the endogenous ACE2, ACE1, CD147, and tubulin proteins, as well as the transfected Flag-GLK proteins in HCC827 lung epithelial cells, which were transfected with either GLK wild-type or kinase-dead (K45E) mutant. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins. Data information: Data shown are representative results of three D, independent experiments.


E Immunoblotting of Flag - tagged ACE2 ( anti - Flag ) , Myc - tagged GLK ( anti - GLK ) , tubulin from HEK293T co - transfected with Flag - ACE2 plus increasing amounts of Myc - GLK plasmids . Data information : Data shown are representative results of three E , independent experiments .

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E Immunoblotting of Flag-tagged ACE2 (anti-Flag), Myc-tagged GLK (anti-GLK), and tubulin proteins from HEK293T cells co-transfected with Flag-ACE2 plus increasing amounts of Myc-GLK plasmids. Data information: Data shown are representative results of three E, independent experiments.


F Immunoblotting of ACE2 , GLK , tubulin in lung tissues of wild - type or GLK - deficient mice . Arrowhead denotes glycosylated ACE2 ; asterisk denotes non - glycosylated ACE2 . Data information : Data shown are representative results of three F , independent experiments .

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F Immunoblotting of ACE2, GLK, and tubulin proteins in the lung tissues of wild-type or GLK-deficient mice. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins. Data information: Data shown are representative results of three F, independent experiments.


G Cycloheximide - chase experiments in HEK293T . Immunoblotting of Flag - tagged ACE2 ( anti - Flag ) , Myc - tagged GLK ( anti - GLK ) , tubulin from HEK293T co - transfected with Flag - ACE2 Myc - GLK . Transfected were treated with 100 μg / ml cycloheximide ( CHX ) for up to 24 h . Data information : Data shown are representative results of three G , independent experiments .

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G Cycloheximide pulse-chase experiments in HEK293T cells. Immunoblotting of Flag-tagged ACE2 (anti-Flag), Myc-tagged GLK (anti-GLK), and tubulin proteins from HEK293T cells co-transfected with Flag-ACE2 and Myc-GLK. Transfected cells were treated with 100 μg/ml cycloheximide (CHX) for up to 24 h. Data information: Data shown are representative results of three G, independent experiments.


H Flag - tagged ACE2 were immunoprecipitated from lysates of HEK293T co - transfected with Flag - ACE2 Myc - GLK , followed by immunoblotting with anti - Lsy48 - linked ubiquitination or anti - Flag antibody . were treated with 25 μM MG132 for 2 h before being harvested . NS , normal serum . Data information : Data shown are representative results of three H , independent experiments .

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H Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-ACE2 and Myc-GLK, followed by immunoblotting with anti-Lsy48-linked ubiquitination or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested. NS, normal serum. Data information: Data shown are representative results of three H, independent experiments.


I , J entry eﬃciencies of SARS - CoV - 2 pseudovirus ( I ) or VSV - G pseudovirus ( J ) into GLK wild - type - or GLK ( K45E ) - overexpressing HCC827 epithelial were measured by luciferase activity presented as relative units ( RLU ) at 24 h post infection . n = 3 ( biological replicates ) . Data information : In ( I , J ) , data are presented as means ± . * * , P value < 0 . 01 ( ANOVA ) ; § § , P value < 0 . 01 ( Dunnett ' s ) . Data shown are representative results of three I , J ) independent experiments .

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I, J Cell entry eﬃciencies of SARS-CoV-2 pseudovirus (I) or VSV-G pseudovirus (J) into GLK wild-type- or GLK (K45E)-overexpressing HCC827 epithelial cells were measured by luciferase activity and presented as relative light units (RLU) at 24 h post infection. n = 3 (biological replicates). Data information: In (I, J), data are presented as means ± SEM. **, P value < 0.01 (ANOVA test); §§, P value < 0.01 (Dunnett's test). Data shown are representative results of three I, J) independent experiments.


A Immunoblotting analyses of ACE2 Flag - tagged GLK in HCC827 transfected with ether Flag - GLK or Flag - GLK ( K45E ) kinase - dead mutant . For immunoblotting of phosphorylated ACE2 , Phos - tagged SDS - PAGE gel ( Phos - tagTM ) was used , followed by immunoblotting with anti - ACE2 antibody . Asterisk denotes unglycosylated ACE2 . Data information : Data shown are representative results of three ( A independent experiments .

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A Immunoblotting analyses of ACE2 and Flag-tagged GLK in HCC827 cells transfected with ether Flag-GLK or Flag-GLK (K45E) kinase-dead mutant. For immunoblotting of phosphorylated ACE2, Phos-tagged SDS-PAGE gel (Phos-tagTM) was used, followed by immunoblotting with anti-ACE2 antibody. Asterisk denotes unglycosylated ACE2 proteins. Data information: Data shown are representative results of three (A independent experiments.


B In vitro kinase assays using purified . ACE2 serine phosphorylation , Flag - tagged GLK ( anti - GLK ) , Flag - tagged GLK ( K45E ) kinase - dead mutant ( anti - GLK ) , Flag - tagged ACE2 ( anti - ACE2 ) were detected by immunoblotting . Data information : Data shown are representative results of three B , independent experiments .

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B In vitro kinase assays using purified proteins. ACE2 serine phosphorylation, Flag-tagged GLK (anti-GLK), Flag-tagged GLK (K45E) kinase-dead mutant (anti-GLK), and Flag-tagged ACE2 proteins (anti-ACE2) were detected by immunoblotting. Data information: Data shown are representative results of three B, independent experiments.


C Mass spectrometry of GLK - phosphorylated ACE2 after in vitro kinase assays . ACE2 Ser776 Ser783 residues were phosphorylated by GLK wild - type but not GLK ( K45E ) kinase - dead mutant .

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C Mass spectrometry analysis of GLK-phosphorylated ACE2 proteins after in vitro kinase assays. ACE2 Ser776 and Ser783 residues were phosphorylated by GLK wild-type but not GLK (K45E) kinase-dead mutant.


D Mass spectrometry of ACE2 peptides from serum exosomes of COVID - 19 . ACE2 peptide sequences containing phospho - Ser776 or phospho - Ser783 residue of ACE2 detected in serum exosomes of COVID - 19 ( Cohort # 3 ) are shown . Exosomes were isolated using ExoQuick kits , soluble were removed by ExoQuick ULTRA columns . Exosomes were further purified by immunoprecipitation using a combination of anti - CD9 , anti - CD63 , CD81 magnetic beads .

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D Mass spectrometry analysis of the ACE2 peptides from the serum exosomes of COVID-19 patients. The ACE2 protein peptide sequences containing phospho-Ser776 or phospho-Ser783 residue of ACE2 proteins detected in the serum exosomes of COVID-19 patients (Cohort #3) are shown. Exosomes were isolated using ExoQuick kits, and soluble proteins were removed by ExoQuick ULTRA columns. Exosomes were further purified by immunoprecipitation using a combination of anti-CD9, anti-CD63, and CD81 magnetic beads.


E In vitro kinase assays using immunoprecipitated Flag - tagged ACE2 or Flag - tagged GLK immunocomplexes . ACE2 serine phosphorylation , ACE2 , GLK were detected by immunoblotting using anti - phospho - serine , anti - ACE2 , anti - GLK antibodies , respectively . Data information : Data shown are representative results of three E , independent experiments .

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

E In vitro kinase assays using immunoprecipitated Flag-tagged ACE2 or Flag-tagged GLK immunocomplexes. ACE2 serine phosphorylation, ACE2, and GLK were detected by immunoblotting using anti-phospho-serine, anti-ACE2, and anti-GLK antibodies, respectively. Data information: Data shown are representative results of three E, independent experiments.


F Immunoblotting analyses of Myc - tagged GLK , Flag - tagged ACE2 , tubulin in HEK293T co - transfected with Flag - ACE2 wild - type or ACE2 ( S776 / 783A , S776A , or S783A ) mutants plus either empty vector or Myc - GLK . Data information : Data shown are representative results of three F , independent experiments .

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F Immunoblotting analyses of Myc-tagged GLK, Flag-tagged ACE2, and tubulin proteins in HEK293T cells co-transfected with Flag-ACE2 wild-type or individual ACE2 (S776/783A, S776A, or S783A) mutants plus either empty vector or Myc-GLK. Data information: Data shown are representative results of three F, independent experiments.


G Immunoblotting analyses of Flag - tagged ACE2 tubulin in HEK293T transfected with Flag - ACE2 wild - type or phosphomimetic ACE2 ( S776 / 783E , S776E , or S783E ) mutants . Data information : Data shown are representative results of three G , independent experiments .

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G Immunoblotting analyses of Flag-tagged ACE2 and tubulin proteins in HEK293T cells transfected with Flag-ACE2 wild-type or individual phosphomimetic ACE2 (S776/783E, S776E, or S783E) mutants. Data information: Data shown are representative results of three G, independent experiments.


H Flag - tagged ACE2 were immunoprecipitated from lysates of HEK293T transfected with Flag - ACE2 wild - type or a phospho - mimetic ACE2 ( S776 / 783E ) mutant , followed by immunoblotting with anti - Lsy48 - linked ubiquitination or anti - Flag antibody . were treated with 25 μM MG132 for 2 h before being harvested . Data information : Data shown are representative results of three H ) independent experiments .

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

H Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells transfected with Flag-ACE2 wild-type or a phospho-mimetic ACE2 (S776/783E) mutant, followed by immunoblotting with anti-Lsy48-linked ubiquitination or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested. Data information: Data shown are representative results of three H) independent experiments.


B Immunoblotting of Myc - tagged ACE2 ( anti - ACE2 ) , Flag - tagged UBR4 ( anti - Flag ) , tubulin from HEK293T co - transfected with Myc - ACE2 Flag - UBR4 . Arrowhead denotes Myc - tagged ACE2 ; asterisk denotes endogenous ACE2 . Data information : Data shown are representative results of three B , independent experiments .

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B Immunoblotting of Myc-tagged ACE2 (anti-ACE2), Flag-tagged UBR4 (anti-Flag), and tubulin proteins from HEK293T cells co-transfected with Myc-ACE2 and Flag-UBR4. Arrowhead denotes Myc-tagged ACE2 proteins; asterisk denotes endogenous ACE2 proteins. Data information: Data shown are representative results of three B, independent experiments.


C Immunoblotting of Flag - tagged ACE2 ( anti - Flag ) , Myc - tagged UBR4 ( anti - Myc ) , tubulin from HEK293T co - transfected with Flag - ACE2 Myc - UBR4 . co - transfected were treated with 25 μM MG132 for 2 h before being harvested . Data information : Data shown are representative results of three C , independent experiments .

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C Immunoblotting of Flag-tagged ACE2 (anti-Flag), Myc-tagged UBR4 (anti-Myc), and tubulin proteins from HEK293T cells co-transfected with Flag-ACE2 and Myc-UBR4. The co-transfected cells were treated with 25 μM MG132 for 2 h before being harvested. Data information: Data shown are representative results of three C, independent experiments.


D Flag - tagged ACE2 were immunoprecipitated from lysates of HEK293T co - transfected with Flag - ACE2 Myc - UBR4 , followed by immunoblotting with anti - Lsy48 - linked ubiquitination , anti - Myc , or anti - Flag antibody . were treated with 25 μM MG132 for 2 h before being harvested . Data information : Data shown are representative results of three D , independent experiments .

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

D Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-ACE2 and Myc-UBR4, followed by immunoblotting with anti-Lsy48-linked ubiquitination, anti-Myc, or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested. Data information: Data shown are representative results of three D, independent experiments.


E In situ PLA assays of interaction between Flag - tagged ACE2 ( wild - type or mutant ) Myc - tagged UBR4 in HEK293T treated with 25 μM MG132 for 2 h . Nuclei were stained with DAPI . Red dots represent direct interaction signals . Original magnification , x200 . Scale bars , 20 μm . Data information : Data shown are representative results of three E , independent experiments .

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

E In situ PLA assays of the interaction between Flag-tagged ACE2 (wild-type or mutant) and Myc-tagged UBR4 proteins in HEK293T cells treated with 25 μM MG132 for 2 h. Nuclei were stained with DAPI. Red dots represent direct interaction signals. Original magnification, x200. Scale bars, 20 μm. Data information: Data shown are representative results of three E, independent experiments.


F Mass spectrometry of ACE2 peptides containing ubiquitination residues from Flag - ACE2 - transfected HEK293T treated with 25 μM MG132 for 2 h .

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F Mass spectrometry analysis of the ACE2 peptides containing ubiquitination residues from Flag-ACE2-transfected HEK293T cells treated with 25 μM MG132 for 2 h.


G Immunoblotting analyses of Flag - tagged ACE2 , Myc - tagged UBR4 , tubulin in HEK293T co - transfected with Flag - ACE2 wild - type or ( K26R , K94R , K112R , or K114R ) mutants plus either empty vector or Myc - UBR4 . Data information : Data shown are representative results of three G , independent experiments .

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

G Immunoblotting analyses of Flag-tagged ACE2, Myc-tagged UBR4, and tubulin proteins in HEK293T cells co-transfected with Flag-ACE2 wild-type or individual (K26R, K94R, K112R, or K114R) mutants plus either empty vector or Myc-UBR4. Data information: Data shown are representative results of three G, independent experiments.


H Flag - tagged ACE2 were immunoprecipitated from lysates of HEK293T co - transfected with Flag - ACE2 wild - type or Flag - ACE2 ( K26 / 112 / 114R ) mutant plus Myc - UBR4 plasmids , followed by immunoblotting with anti - Lsy48 - linked ubiquitination or anti - Flag antibody . were treated with 25 μM MG132 for 2 h before being harvested . Data information : Data shown are representative results of three H ) independent experiments .

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

H Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-ACE2 wild-type or Flag-ACE2 (K26/112/114R) mutant plus Myc-UBR4 plasmids, followed by immunoblotting with anti-Lsy48-linked ubiquitination or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested. Data information: Data shown are representative results of three H) independent experiments.


Wild - type hACE2 KI mice were intranasally infected with SARS - CoV - 2 pseudovirus . Infection eﬃciencies of SARS - CoV - 2 pseudovirus into mouse tissues were measured by IVIS presented as luminescence at 4 days post infection ( B ) . Data information : WT , wild - type mice ; KI , hACE2 knockin mice .

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Wild-type and hACE2 KI mice were intranasally infected with SARS-CoV-2 pseudovirus. Infection eﬃciencies of SARS-CoV-2 pseudovirus into mouse tissues were measured by IVIS and presented as luminescence counts at 4 days post infection (B). Data information: WT, wild-type mice; KI, hACE2 knockin mice.


Wild - type hACE2 KI mice were intranasally infected with SARS - CoV - 2 pseudovirus . Immunoblotting of ACE2 , GLK , tubulin in lung tissues of infected or non - infected mice ( C ) . Data information : Arrowhead denotes glycosylated ACE2 ; asterisk denotes non - glycosylated ACE2 . WT , wild - type mice ; KI , hACE2 knockin mice .

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Wild-type and hACE2 KI mice were intranasally infected with SARS-CoV-2 pseudovirus. Immunoblotting of ACE2, GLK, and tubulin proteins in the lung tissues of infected or non-infected mice (C). Data information: Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins. WT, wild-type mice; KI, hACE2 knockin mice.


Wild - type hACE2 KI mice were intranasally infected with SARS - CoV - 2 pseudovirus . Representative immunohistochemistry of SARS - CoV - 2 spike ( in red ) GLK ( in green ) in lung tissues of infected hACE2 KI wild - type mice ( D ) . Scale bars , 20 μm ( D ) . Data information : WT , wild - type mice ; KI , hACE2 knockin mice .

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Wild-type and hACE2 KI mice were intranasally infected with SARS-CoV-2 pseudovirus. Representative immunohistochemistry of the SARS-CoV-2 spike protein (in red) and GLK protein (in green) in the lung tissues of the infected hACE2 KI and wild-type mice (D). Scale bars, 20 μm (D). Data information: WT, wild-type mice; KI, hACE2 knockin mice.


Wild - type hACE2 KI mice were intranasally infected with SARS - CoV - 2 pseudovirus . Immunoblotting of CD9 mouse / human ACE2 in exosomes isolated from BALFs ( E ) of infected mice . Data information : Arrowhead denotes glycosylated ACE2 ; asterisk denotes non - glycosylated ACE2 . WT , wild - type mice ; KI , hACE2 knockin mice .

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Wild-type and hACE2 KI mice were intranasally infected with SARS-CoV-2 pseudovirus. Immunoblotting of CD9 and mouse/human ACE2 in the exosomes isolated from the BALFs (E) of the infected mice. Data information: Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins. WT, wild-type mice; KI, hACE2 knockin mice.


Wild - type hACE2 KI mice were intranasally infected with SARS - CoV - 2 pseudovirus . Exosomes were isolated sequentially using ExoQuick kits then ExoQuick ULTRA columns . hACE2 - containing exosomes in lung tissues of wild - type hACE2 KI mice were determined by in situ proximity ligation assays ( PLA ) of close proximity ( < 40 nm ) between hACE2 exosome marker CD9 using anti - human ACE2 antibody plus anti - CD9 antibody ( F ) . Scale bars , 20 μm ( F ) . Data information : WT , wild - type mice ; KI , hACE2 knockin mice .

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Wild-type and hACE2 KI mice were intranasally infected with SARS-CoV-2 pseudovirus. Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. The hACE2-containing exosomes in the lung tissues of wild-type and hACE2 KI mice were determined by in situ proximity ligation assays (PLA) of close proximity (<40 nm) between hACE2 and the exosome marker CD9 using anti-human ACE2 antibody plus anti-CD9 antibody (F). Scale bars, 20 μm (F). Data information: WT, wild-type mice; KI, hACE2 knockin mice.


Human ACE2 transgenic ( EF1α - hACE2 Tg ) mice were intranasally infected with 2x105 pfu of live SARS - CoV - 2 . survival rates of in EF1α - hACE2 Tg mice challenged with / without SARS - CoV - 2 were monitored ( H ) . Data information : Tg , transgenic mice .

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Human ACE2 transgenic (EF1α-hACE2 Tg) mice were intranasally infected with 2x105 pfu of live SARS-CoV-2. The survival rates of in EF1α-hACE2 Tg mice challenged with/without SARS-CoV-2 were monitored (H). Data information: Tg, transgenic mice.


Human ACE2 transgenic ( EF1α - hACE2 Tg ) mice were intranasally infected with 2x105 pfu of live SARS - CoV - 2 . lung tissues of infected mice at day 3 days post infection were collected then analyzed by immunohistochemistry ( ) PLA assays . Representative data of hACE2 ( in red ) GLK ( in green ) in lung tissues of infected wild - type EF1α - hACE2 Tg mice were shown ( I ) . nuclei were stained with DAPI . Original magnification , × 630 . Scale bars , 10 μm . Data information : WT , wild - type mice ; Tg , transgenic mice .

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Human ACE2 transgenic (EF1α-hACE2 Tg) mice were intranasally infected with 2x105 pfu of live SARS-CoV-2. The lung tissues of infected mice at day 3 days post infection were collected and then analyzed by immunohistochemistry (IHC) and PLA assays. Representative IHC data of hACE2 (in red) and GLK (in green) in the lung tissues of the infected wild-type and EF1α-hACE2 Tg mice were shown (I). Cell nuclei were stained with DAPI. Original magnification, ×630. Scale bars, 10 μm. Data information: WT, wild-type mice; Tg, transgenic mice.


Human ACE2 transgenic ( EF1α - hACE2 Tg ) mice were intranasally infected with 2x105 pfu of live SARS - CoV - 2 . hACE2 - containing exosomes in lung tissues of infected wild - type EF1α - hACE2 Tg mice were determined by PLA ( J ) . Original magnification , × 630 . Scale bars , 10 μm . Data information : WT , wild - type mice ; Tg , transgenic mice .

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Human ACE2 transgenic (EF1α-hACE2 Tg) mice were intranasally infected with 2x105 pfu of live SARS-CoV-2. The hACE2-containing exosomes in the lung tissues of infected wild-type and EF1α-hACE2 Tg mice were determined by PLA (J). Original magnification, ×630. Scale bars, 10 μm. Data information: WT, wild-type mice; Tg, transgenic mice.


K , Adoptive transfer of ACE2 - containing exosomes facilitates SARS - CoV - 2 pseudovirus infection . Serum exosomes ( exo ) isolated from wild - type ( WT ) mice hACE2 KI / activated GLK transgenic ( PolII - GLK E351K Tg ) mice were adoptively transferred into wild - type recipient mice every 3 days for 12 days , followed by SARS - CoV - 2 pseudovirus infection . Infection eﬃciencies into mouse tissues were measured by IVIS presented as luminescence at 3 days post infection ( K ) . Data information : WT , wild - type mice ; KI , hACE2 knockin mice . Tg , transgenic mice .

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K, Adoptive transfer of ACE2-containing exosomes facilitates SARS-CoV-2 pseudovirus infection. Serum exosomes (exo) isolated from wild-type (WT) mice and hACE2 KI/activated GLK transgenic (PolII-GLK E351K Tg) mice were adoptively transferred into wild-type recipient mice every 3 days for 12 days, followed by SARS-CoV-2 pseudovirus infection. Infection eﬃciencies into mouse tissues were measured by IVIS and presented as luminescence counts at 3 days post infection (K). Data information: WT, wild-type mice; KI, hACE2 knockin mice. Tg, transgenic mice.


L Adoptive transfer of ACE2 - containing exosomes facilitates SARS - CoV - 2 pseudovirus infection . Serum exosomes ( exo ) isolated from wild - type ( WT ) mice hACE2 KI / activated GLK transgenic ( PolII - GLK E351K Tg ) mice were adoptively transferred into wild - type recipient mice every 3 days for 12 days , followed by SARS - CoV - 2 pseudovirus infection . hACE2 - containing exosomes in lung tissues of exosome - recipient wild - type mice were determined by PLA using anti - human ACE2 antibody plus anti - CD9 antibody ( L ) . Scale bars , 10 μm ( L ) . Data information : WT , wild - type mice ; KI , hACE2 knockin mice . Tg , transgenic mice .

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L Adoptive transfer of ACE2-containing exosomes facilitates SARS-CoV-2 pseudovirus infection. Serum exosomes (exo) isolated from wild-type (WT) mice and hACE2 KI/activated GLK transgenic (PolII-GLK E351K Tg) mice were adoptively transferred into wild-type recipient mice every 3 days for 12 days, followed by SARS-CoV-2 pseudovirus infection. hACE2-containing exosomes in the lung tissues of exosome-recipient wild-type mice were determined by PLA using anti-human ACE2 antibody plus anti-CD9 antibody (L). Scale bars, 10 μm (L). Data information: WT, wild-type mice; KI, hACE2 knockin mice. Tg, transgenic mice.


( a ) Wild - type ( WT ) , atg17Δ , atg7Δ atg19Δ strains containing RPL25 - GFP were grown to mid log phase , starved in SD - N for 48 hours examined under a microscope ( left panel ) . arrows indicate vacuolar accumulation of GFP signal . CVT , cytoplasm - to - vacuole - targeting pathway . After times indicated , at least 100 per sample were scored for presence of Rpl25p - GFP in vacuole ( right panel ) . Scale bar , 5 μm .

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(a) Wild-type (WT), atg17Δ, atg7Δ and atg19Δ strains containing RPL25-GFP were grown to mid log phase, starved in SD-N for 48 hours and examined under a fluorescence microscope (left panel). The arrows indicate vacuolar accumulation of the GFP signal. CVT, cytoplasm-to-vacuole-targeting pathway. After the times indicated, at least 100 cells per sample were scored for the presence of Rpl25p-GFP in the vacuole (right panel). Scale bar, 5 μm.


( b ) Wild - type atg7Δ strains containing RPL25 - GFP or GFP - ATG8 were grown starved as in a . Samples were taken at indicated time points GFP cleavage was analysed by western blotting .

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(b) Wild-type and atg7Δ strains containing RPL25-GFP or GFP-ATG8 were grown and starved as in a. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting.


( c ) of endogenous Rps3p ( top ) Rpl3p ( middle ) were analysed by immunblotting in wild - type atg7Δ . Ponceau ( bottom ) controlled for equal loading . Uncropped of blots are shown in Supplementary Information , Fig . S6 . Starv , starvation .

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(c) The levels of endogenous Rps3p (top) and Rpl3p (middle) were analysed by immunblotting in wild-type and atg7Δ cells. Ponceau staining (bottom) controlled for equal loading. Uncropped images of blots are shown in Supplementary Information, Fig. S6. Starv, starvation.


( d ) Wild - type pep4Δ expressing RPL25 - GFP were analysed as in b ; 0 . 1 mM of PMSF was added to pep4Δ at time of starvation to block vacuolar proteases completely . LI - COR imaging system was used to quantify between GFP full - as described in Methods . last time point of wild - type containing RPL25 - GFP was set to 1 arbitrary unit ( a . u . ) . One of two independent experiments is shown .

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(d) Wild-type and pep4Δ cells expressing RPL25-GFP were analysed as in b; 0.1 mM of PMSF was added to pep4Δ cells at the time of starvation to block all vacuolar proteases completely. The LI-COR imaging system was used to quantify the ratio between cleaved GFP and full-length protein as described in Methods. The last time point of wild-type cells containing RPL25-GFP was set to 1 arbitrary unit (a.u.). One of two independent experiments is shown.


( e ) Wild - type containing either RPL25 - GFP or control GFP ( HOG1 - GFP ) were analysed as in d . average of four independent experiments is shown , standard deviation was less than 10 % ( Supplementary Information , Table S3 ) .

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(e) Wild-type cells containing either RPL25-GFP or control GFP (HOG1-GFP) were analysed as in d. The average of four independent experiments is shown, and the standard deviation was less than 10% (Supplementary Information, Table S3).


( a ) Wild - type , ubp3Δ or bre5Δ strains containing RPL25 - GFP either a control plasmid ( pRS315 ) , or plasmids expressing Ubp3p or catalytically inactive Ubp3p - C469A from endogenous promoter , were grown starved as described in Fig . 1a . were revealed by phase contrast ( right ) , localization of Rpl25p - GFP was analysed by GFP microscopy ( left ) . At least 100 per sample were analysed . Scale bar , 5 μm .

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(a) Wild-type, ubp3Δ or bre5Δ strains containing RPL25-GFP and either a control plasmid (pRS315), or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter, were grown and starved as described in Fig. 1a. Cells were revealed by phase contrast (right), and the localization of Rpl25p-GFP was analysed by GFP microscopy (left). At least 100 cells per sample were analysed. Scale bar, 5 μm.


( b ) Ribosomes from wild - type ubp3Δ grown as indicated for 16 h under rich or starvation ( Starv ) conditions were enriched through a high - salt cushion ( pellet ) of endogenous Rpl3p were analysed by immunoblotting . Ponceau of blot revealed total amount of ( bottom panel ) . Uncropped of blots are shown in Supplementary Information , Fig . S6 .

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(b) Ribosomes from wild-type and ubp3Δ cells grown as indicated for 16 h under rich or starvation (Starv) conditions were enriched through a high-salt cushion (pellet) and the levels of endogenous Rpl3p were analysed by immunoblotting. Ponceau staining of the blot revealed the total amount of proteins (bottom panel). Uncropped images of blots are shown in Supplementary Information, Fig. S6.


c ) Wild - type ubp3Δ strains containing RPL25 - GFP were grown starved as in Fig . 1a , GFP cleavage was analysed by western blotting at times indicated ( hours ) .

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c) Wild-type and ubp3Δ strains containing RPL25-GFP were grown and starved as in Fig. 1a, and GFP cleavage was analysed by western blotting at the times indicated (hours).


( d , e ) GFP cleavage of Rpl25p - GFP was analysed in wild - type , ubp3Δ , atg9Δ bre5Δ strains containing indicated plasmids , quantified at times indicated as described in Fig . 1d . One of three representative experiments is shown for d . average of three independent experiments is shown in e , statistical showed that standard deviation was less than 13 % ( Supplementary Information , Table S3 ) .

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(d, e) GFP cleavage of Rpl25p-GFP was analysed in wild-type, ubp3Δ, atg9Δ and bre5Δ strains containing the indicated plasmids, and quantified at the times indicated as described in Fig. 1d. One of three representative experiments is shown for d. The average of three independent experiments is shown in e, and the statistical analysis showed that the standard deviation was less than 13% (Supplementary Information, Table S3).


( a ) vacuolar localization of GFP - Atg8p was analysed in more than 100 wild - type , ubp3Δ , bre5Δ atg7Δ containing GFP - ATG8 after 10 - 15 h of nitrogen starvation ( right panel ) . Corresponding GFP phase contrast of representative are shown in left panel .

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(a) The vacuolar localization of GFP-Atg8p was analysed in more than 100 wild-type, ubp3Δ, bre5Δ and atg7Δ cells containing GFP-ATG8 after 10-15 h of nitrogen starvation (right panel). Corresponding GFP and phase contrast images of representative cells are shown in the left panel.


( b ) Vacuolar cleavage of GFP - Atg8p was examined at indicated time points ( hours ) by anti - GFP western blotting .

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(b) Vacuolar cleavage of GFP-Atg8p was examined at the indicated time points (hours) by anti-GFP western blotting.


( c ) Wild - type , ubp3Δ bre5Δ strains containing APE1 - RFP were analysed by RFP microscopy ( left ) phase contrast ( right ) 15 h after nitrogen starvation . Scale bars , 5 μm .

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(c) Wild-type, ubp3Δ and bre5Δ strains containing APE1-RFP were analysed by RFP microscopy (left) and phase contrast (right) 15 h after nitrogen starvation. Scale bars, 5 μm.


( a ) Wild - type , ubp3Δ or bre5Δ strains containing either a control plasmid ( pRS315 ) or plasmids expressing Ubp3p or catalytically inactive Ubp3p - C469A from endogenous promoter were spotted in serial dilutions onto YPD plates with ( left ) or without ( right ) 5 nM rapamycin . plates were photographed after 2 days at 30 ° C .

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(a) Wild-type, ubp3Δ or bre5Δ strains containing either a control plasmid (pRS315) or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter were spotted in serial dilutions onto YPD plates with (left) or without (right) 5 nM rapamycin. The plates were photographed after 2 days at 30 °C.


( b ) Induction of a GCN4 - LacZ reporter was measured 0 , 2 4 h after - acid starvation , is plotted as Miller units .

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(b) Induction of a GCN4-LacZ reporter was measured 0, 2 and 4 h after amino-acid starvation, and is plotted as Miller units.


( c ) Polysome profiles of exponentially growing wild - type ubp3Δ were determined in rich medium or 3 h after nitrogen starvation . arrows mark 60S peak .

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(c) Polysome profiles of exponentially growing wild-type and ubp3Δ cells were determined in rich medium or 3 h after nitrogen starvation. The arrows mark the 60S peak.


( d ) Wild - type , atg7Δ ubp3Δ containing either RPL25 - GFP , control GFP ( HOG1 - GFP ) or RPS2 - GFP were analysed as in Fig . 1c . One of three representative experiments is shown .

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(d) Wild-type, atg7Δ and ubp3Δ cells containing either RPL25-GFP, control GFP (HOG1-GFP) or RPS2-GFP were analysed as in Fig. 1c. One of three representative experiments is shown.


( e ) Ribosomes associated were immunopurified from starved wild - type ubp3Δ containing ( RPL25 - GFP ) ( 9 × MYC - UBI ) as indicated . GFP immunoprecipitates ( IP ; left ) total extracts ( TCA , right ) were analysed by immunoblotting with anti - Myc antibodies to reveal ubiquitinated ( top ) . Ub , ubiquitin . Bottom : immunoblotting with anti - GFP antibodies controls for presence of Rpl25p - GFP , anti - Rpl1p antibodies confirm equal loading of extract . asterisk marks G on beads recognized by secondary antibody .

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(e) Ribosomes and associated proteins were immunopurified from starved wild-type and ubp3Δ cells containing (RPL25-GFP) and (9 × MYC-UBI) as indicated. The GFP immunoprecipitates (IP; left) and total extracts (TCA, right) were analysed by immunoblotting with anti-Myc antibodies to reveal ubiquitinated proteins (top). Ub, ubiquitin. Bottom: immunoblotting with anti-GFP antibodies controls for the presence of Rpl25p-GFP, and anti-Rpl1p antibodies confirm equal loading of the extract. The asterisk marks protein G on the beads recognized by the secondary antibody.


( a ) Structure of GBA1 gene on chromosome 1 schematic overview of gene correction using ZFNs . Black boxes represent exons . ( b ) Mutated gene - corrected ( GC ) iPSCs after sequencing ( upper lanes : iPSCs with heterozygous mutations ; lower lanes : iPSCs with homozygous wt - GBA1 sequences as indicated by arrows ) .

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(a) Structure of the GBA1 gene on chromosome 1 and schematic overview of gene correction using ZFNs. Black boxes represent exons. (b) Mutated and gene-corrected (GC) iPSCs after sequencing (upper lanes: iPSCs with heterozygous mutations; lower lanes: iPSCs with homozygous wt-GBA1 sequences as indicated by arrows).


Immunostaining of indicated differentiated iPSC cultures at DIV65 . were stained for TH ( red ) β - TubIII , FOXA2 , NURR1 , GIRK2 VMAT2 ( green ) . nuclei were counterstained with Hoechst ( blue ) . ( Bars , 20 μm ) .

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Immunostaining of indicated differentiated iPSC cultures at DIV65. Cells were stained for TH (red) and β-TubIII, FOXA2, NURR1, GIRK2 and VMAT2 (all green). Cell nuclei were counterstained with Hoechst (blue). (Bars, 20 μm).


a ) Gating strategy used for neuronal sorting . Live as determined by FSC / SSC gating were selected for CD184 - APC - negative / low surface expression subsequently separated for CD24 - PE versus CD15 / CD29 / CD44 expression , detected in FITC channel as indicated in dot plots . Post - sort reanalysis shows representative samples of both fractions immediately after FACS enrichment , which were then plated for comparative in vitro ( phase contrast . Bars , 50 μm ) .

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a) Gating strategy used for neuronal cell sorting. Live cells as determined by FSC/SSC gating were selected for CD184-APC-negative/low surface expression and subsequently separated for CD24-PE versus CD15/CD29/CD44 expression, detected in the FITC channel as indicated in the dot plots. Post-sort reanalysis shows representative samples of both fractions immediately after FACS enrichment, which were then plated for comparative in vitro analysis (phase contrast images. Bars, 50 μm).


( b , c ) Immunocytochemistry at DIV2 post - sort for TH ( green ) neuronal marker MAP2 ( red ) performed on neuronal ( b ) mixed ( c ) populations of a representative control GBA - PD . Nuclei were counterstained with Hoechst ( blue ) . FACS enriches for both TH neuronal marker positivity in neuronal population compared with non - neuronal population . ( Bars , 50 μm . )

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(b,c) Immunocytochemistry at DIV2 post-sort for TH (green) and the neuronal marker MAP2 (red) performed on the neuronal (b) and mixed (c) populations of a representative control and GBA-PD line. Nuclei were counterstained with Hoechst (blue). FACS enriches for both TH and neuronal marker positivity in the neuronal population compared with the non-neuronal population. (Bars, 50 μm.)


( a ) Ganglioside composition of human fibroblasts , iPSCs , differentiated iPSCs enriched iPSC - derived neurons ( after FACS ) from control ( C1 - 1 ) , GBA - PD ( PD1 - 1 ) GD ( GD1 - 1 ) subjects . lipids were metabolically labelled with [ 1 - 3H ] sphingosine visualized by digital autoradiography .

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(a) Ganglioside composition of human fibroblasts, iPSCs, differentiated iPSCs and enriched iPSC-derived neurons (after FACS) from control (line C1-1), GBA-PD (line PD1-1) and GD (line GD1-1) subjects. Cell lipids were metabolically labelled with [1-3H]sphingosine and visualized by digital autoradiography.


( b ) Activities of glycohydrolases ( GCase , GBA2 , β - galactosidase , β - hexosaminidase ) in control iPSCs iPSC - derived enriched neurons at DIV65 . Enzyme activities are expressed as pmoles mg − 1 h − 1 . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; Student ' s t - .

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(b) Activities of glycohydrolases (GCase, GBA2, β-galactosidase, β-hexosaminidase) in control iPSCs and iPSC-derived enriched neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test.


c ) GBA1 , GBA2 , β - galactosidase HEXA HEXB gene expression in control iPSCs iPSC - derived enriched neurons at DIV65 . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 05 ; * * P0 . 01 , Student ' s t - .

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c) GBA1, GBA2, β-galactosidase and HEXA and HEXB gene expression levels in control iPSCs and iPSC-derived enriched neurons at DIV65. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01, Student's t-test.


( d ) Representative western blots showing GCase , GBA2 , β - galactosidase HEXA HEXB in control iPSCs corresponding iPSC - derived neurons ( DIV65 ) .

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(d) Representative western blots showing GCase, GBA2, β-galactosidase and HEXA and HEXB protein levels in control iPSCs and corresponding iPSC-derived neurons (DIV65).


( a ) LC - MS / MS showing level of Glc - Cer in control , GBA - PD , isogenic controls GD iPSC - derived neurons . Data are represented as mean + s . d . ; n = 3 , * P0 . 01 , One - way ANOVA .

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(a) LC-MS/MS analysis showing the level of Glc-Cer in control, GBA-PD, isogenic controls and GD iPSC-derived neurons. Data are represented as mean+s.d.; n=3, *P0.01, One-way ANOVA.


( b ) Control iPSCs ( C1 - 1 ) were differentiated for 65 days exposed to GCase inhibitor CBE ( 25 μM ) for 48 h . Representative western blot showing increased of α - syn in CBE - treated iPSC - derived neurons compared to untreated neurons .

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(b) Control iPSCs (line C1-1) were differentiated for 65 days and exposed to the GCase inhibitor CBE (25 μM) for 48 h. Representative western blot showing increased levels of α-syn in CBE-treated iPSC-derived neurons compared to untreated neurons.


( c , d ) Representative western blots showing of α - syn in control , GBA - PD , gene - corrected isogenic controls GD iPSC - derived neurons .

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(c,d) Representative western blots showing the protein levels of α-syn in control, GBA-PD, gene-corrected isogenic controls and GD iPSC-derived neurons.


( e ) Optical densities of α - syn bands normalized by averaged value of GAPDH expressed as a of control ( CTRL ) value . Data are represented as mean + s . d . ; experiments were independently repeated five times . * P0 . 01 , one - way ANOVA . ( f ) Average α - syn normalized by averaged value of GAPDH expressed as a of respective isogenic mutant lines . Data are represented as mean + s . d . ; experiments were independently repeated five times . * P0 . 01 , Student ' s t - .

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(e) Optical densities of α-syn bands normalized by the averaged value of GAPDH and expressed as a percentage of the control (CTRL) line value. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, one-way ANOVA. (f) Average α-syn protein levels normalized by the averaged value of GAPDH and expressed as a percentage of the respective isogenic mutant lines. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, Student's t-test.


( a ) Glycohydrolase enzyme activities ( GCase , GBA2 , β - galactosidase , β - hexosaminidase ) were measured in enriched neurons from control , GBA - PD , isogenic gene - corrected control GD iPSC - derived neurons at DIV65 . Enzyme activities are expressed as pmoles mg − 1 h − 1 . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 compared with controls , one - way ANOVA .

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(a) Glycohydrolase enzyme activities (GCase, GBA2, β-galactosidase, β-hexosaminidase) were measured in enriched neurons from control, GBA-PD, isogenic gene-corrected control and GD iPSC-derived neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01; ***P0.001 compared with controls, one-way ANOVA.


( b , c ) Representative western blots of GCase in control , GBA - PD , corresponding gene - corrected GD iPSC - derived neurons . Data are represented as mean + s . d . ; experiments were independently repeated three times . * P0 . 05 ; * * P0 . 01 , one - way ANOVA Student ' s t - .

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(b,c) Representative western blots of GCase protein levels in control, GBA-PD, corresponding gene-corrected and GD iPSC-derived neurons. Data are represented as mean+s.d.; experiments were independently repeated three times. *P0.05; **P0.01, one-way ANOVA and Student's t-test.


CSF glycohydrolase activities were measured in with idiopathic PD ( iPD ) , GBA - PD - matched unaffected individuals ( CTRL ) . Enzyme activities were normalized to total concentrations expressed as pmoles mg − 1 h − 1 . Data are represented as mean + s . e . m . ; CTRL , n = 14 ; iPD , n = 15 ; GBA - PD , n = 15 . Experiments were performed in triplicate . * P0 . 05 ; * * P0 . 01 , Mann - Whitney U .

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CSF glycohydrolase activities were measured in patients with idiopathic PD (iPD), GBA-PD patients and age-matched unaffected individuals (CTRL). Enzyme activities were normalized to total protein concentrations and expressed as pmoles mg−1 h−1. Data are represented as mean+s.e.m.; CTRL, n=14; iPD, n=15; GBA-PD, n=15. Experiments were performed in triplicate. *P0.05; **P0.01, Mann-Whitney U test.


( a ) Immunostaining of differentiated iPSC cultures at DIV65 under basal conditions . were stained for TH ( green ) , β - TubIII ( magenta ) LAMP1 ( red ) . nuclei were counterstained with Hoechst ( blue ) . High magnification insets are shown in right lane . ( Bars , 20 μm 10 μm in insets . )

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(a) Immunostaining of differentiated iPSC cultures at DIV65 under basal conditions. Cells were stained for TH (green), β-TubIII (magenta) and LAMP1 (red). Cell nuclei were counterstained with Hoechst (blue). High magnification insets are shown in the right lane. (Bars, 20 μm and 10 μm in the insets.)


( b ) Average number of LAMP1 + particles in TH + neurons were quantified by ImageJ . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 , * * P0 . 001 , one - way ANOVA .

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(b) Average size and number of LAMP1+ particles in TH+ neurons were quantified by ImageJ. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, **P0.001, one-way ANOVA.


( c ) Western blot for LC3 in iPSC - derived neuronal cultures at DIV65 , untreated ( − ) or treated with 200 μM leupeptin 20 mM NH4Cl for 4 h ( + ) .

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(c) Western blot analysis for LC3 in iPSC-derived neuronal cultures at DIV65, untreated (−) or treated with 200 μM leupeptin and 20 mM NH4Cl for 4 h (+).


( d ) of basal of LC3 - II relative to β - actin . Data are represented as mean + s . e . m . ; experiments were independently repeated three times . * P0 . 01 ; * * P0 . 001 , one - way ANOVA .

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(d) Quantification of the basal levels of LC3-II relative to β-actin. Data are represented as mean+s.e.m.; experiments were independently repeated three times. *P0.01; **P0.001, one-way ANOVA.


( e ) of LC3 flux normalized to actin . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; * * P0 . 001 , one - way ANOVA .

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(e) Quantification of LC3 flux normalized to actin. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, one-way ANOVA.


( a ) calcium were evaluated in iPSC - derived neurons at basal conditions by measuring 340 / 380 nm of Fura - 2 acetoxymethyl ester . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; Student ' s t - . ( Number of analysed : CTRL , n = 58 ; RecNcil / wt , n = 36 ; RecNcil / wt GC , n = 107 ; L444P / wt , n = 82 ; L444P / wt GC , n = 33 ; L444P / L444P , n = 62 ) . ( b ) Caffeine ( 10 mM ) was applied to iPSC - derived neurons , peak calcium was measured as a change in Fura - 2 . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; * * P0 . 001 , Student ' s t - . ( Number of analysed : CTRL , n = 58 ; RecNcil / wt , n = 36 ; RecNcil / wt GC , n = 107 ; L444P / wt , n = 82 ; L444P / wt GC , n = 33 ; L444P / L444P , n = 62 ) .

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(a) Cytosolic calcium levels were evaluated in iPSC-derived neurons at basal conditions by measuring the 340/380 nm ratio of Fura-2 acetoxymethyl ester. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62). (b) Caffeine (10 mM) was applied to iPSC-derived neurons, and the peak cytosolic calcium concentration was measured as a change in Fura-2 fluorescence. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, Student's t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62).


( c ) iPSC - derived neurons were treated with A23187 ( 0 - 5 μM ) for 4 h cytotoxicity was evaluated by released LDH activity . LDH release values were calculated as of untreated lysed by incubation with Triton X - 100 . Data are represented as mean + s . e . m . ; experiments were independently repeated four times in triplicate . * P0 . 05 , one - way ANOVA , control lines versus mutated lines ; δP0 . 05 , one - way ANOVA , mutated lines versus isogenic gene corrected controls .

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(c) iPSC-derived neurons were treated with A23187 (0-5 μM) for 4 h and cytotoxicity was evaluated by released LDH activity. LDH release values were calculated as the percentage of untreated cells lysed by incubation with Triton X-100. Data are represented as mean+s.e.m.; experiments were independently repeated four times in triplicate. *P0.05, one-way ANOVA, control lines versus mutated lines; δP0.05, one-way ANOVA, mutated lines versus isogenic gene corrected controls.


( d ) of TH + treated with A23187 ( 1 μM ) for 4 h expressed as a of DMSO - treated controls . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 01 , one - way ANOVA .

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(d) Quantification of TH+ cells treated with A23187 (1 μM) for 4 h expressed as a percentage of DMSO-treated controls. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01, one-way ANOVA.


( e ) Knockdown efficiency of NECAB2 determined by qRT - PCR normalized to scrambled non - targeting shRNA . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * * * P0 . 0001 , Student ' s t - .

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(e) Knockdown efficiency of NECAB2 determined by qRT-PCR and normalized to scrambled non-targeting shRNA. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. ***P0.0001, Student's t-test.


( f ) Representative western blot for NECAB2 showing knockdown efficiency . 1 , scrambled non - targeting shRNA ; 2 , NECAB2 shRNA .

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(f) Representative western blot for NECAB2 showing knockdown efficiency. 1, scrambled non-targeting shRNA; 2, NECAB2 shRNA.


( g ) of TH + in GBA - PD GD iPSC - derived neuronal cultures infected with scrambled non - targeting shRNA or NECAB2 shRNA lentivirus treated with A23187 ( 1 μM for 4 h ) . Values were calculated as a of scrambled shRNA - treated . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 , Student ' s t - .

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(g) Quantification of TH+ cells in GBA-PD and GD iPSC-derived neuronal cultures infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Values were calculated as a percentage of scrambled shRNA-treated cells. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, Student's t-test.


( h ) Representative showing GBA - PD iPSC - derived DA neurons infected with scrambled non - targeting shRNA or NECAB2 shRNA lentivirus treated with A23187 ( 1 μM for 4 h ) . were stained for β - TubIII ( green ) TH ( red ) . Nuclei were counterstained with Hoechst ( blue ) . ( Bar , 20 μm . )

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(h) Representative images showing GBA-PD iPSC-derived DA neurons infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Cells were stained for β-TubIII (green) and TH (red). Nuclei were counterstained with Hoechst (blue). (Bar, 20 μm.)


( B ) Serum CRAMP determination by ELISA ( n = 8 ) . Data information : Data in B were representative were mean ± SD from three independent experiments . P values were calculated by unpaired two - t - for comparison of two

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(B) Serum CRAMP determination by ELISA (n = 8). Data information: Data in B were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups


( E ) Localization expression of CRAMP ( red ) , E - cadherin ( green ) , F4 / 80 ( green ) Ly6G ( green ) in duodenum by immunofluorescent . Representative photomicrographs of merged were shown . Nuclei were stained with DAPI ( blue ) . Scale bar : 50 μm . Data information : Data were representative from three independent experiments .

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(E) Localization and expression of CRAMP (red), E-cadherin (green), F4/80 (green) and Ly6G (green) in duodenum by immunofluorescent staining. Representative photomicrographs of individual and merged staining were shown. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Data information: Data were representative from three independent experiments.


( C ) Left , representative of duodenal damage by H & E . Scale bar : 200 μm . Right , graph depicted of morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons . .

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(C) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..


( D ) Western blot densitometry analyses of duodenal tight junction ( claudin - 1 , occludin , ZO - 1 ZO - 2 ; n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons . .

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(D) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..


( E ) Western blot densitometry of duodenal zonulin ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons . .

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(E) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..


( D ) Left , representative of duodenal damage by H & E . Scale bar : 200 μm . Right , graph depicted of morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(D) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( E ) Western blot densitometry analyses of duodenal tight junction ( claudin - 1 , occludin , ZO - 1 ZO - 2 ; n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(E) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( F ) Western blot densitometry of duodenal zonulin ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(F) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( A ) Western blot densitometry analyses of gliadin - triggered inflammatory signaling [ p - EGFR ( Tyr1068 ) , p - Akt , Akt , MyD88 , TRAF6 , p - p65 p65 ] in epithelial ex vivo ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(A) Western blot and densitometry analyses of gliadin-triggered inflammatory signaling [p-EGFR (Tyr1068), p-Akt, Akt, MyD88, TRAF6, p-p65 and p65] in epithelial cells ex vivo (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( E - G ) percentages of ( E ) Foxp3 + Treg , ( F ) T - bet + Th1 ( G ) RORγt + Th17 among CD3 + CD4 + population in lamina propria were shown by flow cytometry ( n = 8 ) . Data information : Data were representative were median ± interquartile range from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(E-G) The percentages of (E) Foxp3+ Treg, (F) T-bet+ Th1 and (G) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( D ) Duodenal colonizing P . aeruginosa A . muciniphila as determined by RT - qPCR using - specific species gene primers ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by unpaired two - t - for comparison of two

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(D) Duodenal colonizing P. aeruginosa and A. muciniphila as determined by RT-qPCR using bacterial-specific species gene primers (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups


( E ) production of LasB ( a protease of P . aeruginosa ) was examined in duodenum ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by unpaired two - t - for comparison of two

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(E) The production of LasB (a protease of P. aeruginosa) was examined in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups


( H ) Duodenal colonizing PA14 PAO1 as determined by RT - qPCR using P . aeruginosa - specific species gene primer ( n = 9 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple

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(H) Duodenal colonizing PA14 and PAO1 as determined by RT-qPCR using P. aeruginosa-specific species gene primer (n = 9). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons


( C ) of CRAMP in duodenum ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(C) The levels of CRAMP in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( D ) Representative of intestinal damage by H & E . Scale bar : 200 μm . graph depicted of morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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

(D) Representative images of intestinal damage by H&E staining. Scale bar: 200 μm. The graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( E ) Western blot densitometry analyses of tight junction ( claudin - 1 occludin ) zonulin in duodenum ( n = 6 ) . Data information : Data were representative were mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(E) Western blot and densitometry analyses of tight junction proteins (claudin-1 and occludin) and zonulin in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( I ) percentages of three subsets of CD45 + CD11c + DCs ( n = 8 ) . ( J - L ) percentages of ( J ) Foxp3 + Treg , ( K ) T - bet + Th1 ( L ) RORγt + Th17 among CD3 + CD4 + population in lamina propria were shown by flow cytometry ( n = 8 ) . Data information : Data were representative were median ± interquartile range from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc for multiple comparisons .

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(I) The percentages of three subsets of CD45+CD11c+ DCs (n = 8). (J-L) The percentages of (J) Foxp3+ Treg, (K) T-bet+ Th1 and (L) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


A . Representative photomicrographs of mouse liver sections following a two - thirds PH of Ki67 positive hepatocytes ( brown ) visualized by . are representative of 3 independent per time point .

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A. Representative photomicrographs of mouse liver sections following a two-thirds PH of Ki67 positive hepatocytes (brown) visualized by IHC. Images are representative of 3 independent animals per time point.


B . Western blot detection of p - MST1 , MST1 , p - LATS1 , LATS1 from isolated from homogenised liver tissue at several time points . TBP was used as a loading control . Results of a single experiment with n = 3 per group . Each band is from an independent . were normalized to TBP mean value of of p - MST to total MST p - LATS to total LATS is plotted .

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B. Western blot detection of p-MST1, MST1, p-LATS1, LATS1 from proteins isolated from homogenised liver tissue at several time points. TBP was used as a loading control. Results of a single experiment with n = 3 animals per group. Each band is from an independent animal. Protein levels were normalized to TBP and mean value of the ratio of p-MST to total MST and p-LATS to total LATS is plotted.


C . enriched fractions obtained from sham - operated livers liver 48 h post PH were analyzed by Western blot for YAP expression . Sp1 was used as a loading control . Image is representative of a single experiment with n = 3 per group .

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C. Nuclear enriched protein fractions obtained from sham-operated livers and liver 48 h post PH were analyzed by Western blot for YAP expression. Sp1 was used as a loading control. Image is representative of a single experiment with n = 3 animals per group.


D . Photomicrograph of YAP1 expression in sham regenerating liver 48 h post PH visualized by immunoflourescence microscopy . are representative of 3 independent per condition .

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D. Photomicrograph of YAP1 expression in sham and regenerating liver 48 h post PH visualized by immunoflourescence microscopy. Images are representative of 3 independent animals per condition.