Confirmation of robust knockdowns of p53 , PTEN RB in FT organoids on level . Relative mRNA were normalized to WT mRNA level are given as mean ± from 3 different donors . A representative western blot from one donor is depicted . as well as samples were taken at passage 1 or 2 of each organoid . KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot . Presence of ciliated ( arrowheads ) , as revealed by immunostaining against detyrosinated Tubulin ( dtyrTub ) in triple KD organoids , proves capacity for terminal differentiation . Scale bar : 50µm .

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Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis. Presence of ciliated cells (arrowheads), as revealed by immunostaining against detyrosinated Tubulin (dtyrTub) in triple KD organoids, proves capacity for terminal differentiation. Scale bar: 50µm.


In long - term 3D culture triple KD organoids show loss of growth capacity premature growth arrest compared to controls as indicated by representative growth curves of FT234 . Scale bar : 500 µm .

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In long-term 3D culture triple KD organoids show loss of growth capacity and premature growth arrest compared to the controls as indicated by the representative growth curves of FT234. Scale bar: 500 µm.


Growth curves phase - contrast of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long - term growth capacity . Scale bar : 500 µm .

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Growth curves and phase-contrast images of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long-term growth capacity. Scale bar: 500 µm.


of damage ( yH2AX ) apoptosis ( caspase - 3 , PARP1 ) do not differ among different media as shown by representative western blots comparing triple KD organoids their controls .

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Levels of DNA damage (yH2AX) and apoptosis (cleaved caspase-3, PARP1) do not differ among the different media as shown by representative western blots comparing triple KD organoids and their controls.


Triple KD ( p53 / PTEN / RB ) organoids are characterized by a HGSOC gene expression signature as revealed by microarray comparing WT KD organoids grown in either OCM or FTM . Differential expression determined by dual - color microarray for 2 biological replicates was significant for genes with p < 0 . 00005 .

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Triple KD (p53/PTEN/RB) organoids are characterized by a HGSOC gene expression signature as revealed by microarray analysis comparing WT and KD organoids grown in either OCM or FTM. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.00005.


Differential expression of stemness - differentiation - related genes of triple KD organoids grown in OCM vs . FTM . Fold - changes were derived from microarray data indicate an increase in stemness ( CD133 , SOX2 , MYCN ) a drop in differentiation ( OVGP1 , PGR , FOXJ1 ) under OCM conditions . Differential expression determined by dual - color microarray for 2 biological replicates was significant for genes with p < 0 . 05 .

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Differential expression of stemness- and differentiation-related genes of triple KD organoids grown in OCM vs. FTM. Fold-changes were derived from microarray data and indicate and an increase in stemness (CD133, SOX2, MYCN) and a drop in differentiation (OVGP1, PGR, FOXJ1) under OCM conditions. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.05.


FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM , suggesting a decrease in differentiation capacity . Data represent mean ± from technical triplicates for two independent knockdown cultures .

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FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM, suggesting a decrease in differentiation capacity. Data represent the mean ±SEM from technical triplicates for two independent knockdown cultures.


Proportion of ciliated ( detyrosinated Tubulin ; dtyrTub ) is significantly reduced in KD organoids under OCM growth conditions , confirming inhibition of differentiation as illustrated by confocal ( Scale bar : 50 µm ) . plot depicts mean ± from 3 independent biological replicates , on of average number of ciliated per counted nuclei . 1000 were counted per experiment . * p < 0 . 05 , two - sided Student ' s t - .

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Proportion of ciliated cells (detyrosinated Tubulin; dtyrTub) is significantly reduced in KD organoids under OCM growth conditions, confirming inhibition of differentiation as illustrated by confocal images (Scale bar: 50 µm). The quantification plot depicts the mean ±SEM from 3 independent biological replicates, based on quantification of the average number of ciliated cells per counted nuclei. 1000 cells were counted per individual experiment. *p < 0.05, two-sided Student's t-test.


proportion of CD133 + was determined by FACS after growing triple KD control in FTM vs . OCM for two passages . graph is representative of two biological replicates .

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The proportion of CD133+ cells was determined by FACS analysis after growing triple KD and control cells in FTM vs. OCM for two passages. The graph is representative of two biological replicates.


Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable number ( representative graph of 3 independent experiments ) . luminescent viability was performed in triplicate bar plot depicts mean ± . * p < 0 . 05 , two - sided Student ` s t - ; KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild

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Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable cell number (representative graph of 3 independent experiments). The luminescent cell viability assay was performed in triplicate and the bar plot depicts the mean ±SEM. * p < 0.05, two-sided Student`s t-test; KD - Knockdown, Vec Ctrl - Vector Control, WT - Wild type


Phase - contrast of cancer organoids which entered growth arrest upon treatment with Wnt agonists ( Wnt3a / RSPO1 ) over 2 passages ( Scale bar : 500µm ) . Differences in number were confirmed by respective viability assays ( performed in technical triplicates ) . Data represent mean ± for 5 different OC organoid lines ( n = 5 ) . * * * p < 0 . 001 , two - sided Student ` s t - .

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Phase-contrast images of cancer organoids which entered growth arrest upon treatment with Wnt agonists (Wnt3a/RSPO1) over 2 passages (Scale bar: 500µm). Differences in cell number were confirmed by the respective cell viability assays (performed in technical triplicates). Data represent the mean ±SEM for 5 different OC organoid lines (n=5). *** p < 0.001, two-sided Student`s t-test.


number of CD133 + determined by FACS for OC7 OC4 organoids grown in different media with or without Wnt3a / RSPO1 with or without Noggin over 2 passages , confirms a sharp drop in stemness in presence of Wnt agonists .

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The number of CD133+ cells determined by FACS for OC7 and OC4 organoids grown in different media with or without Wnt3a/RSPO1 and with or without Noggin over 2 passages, confirms a sharp drop in stemness in the presence of Wnt agonists.


Addition of conditioned medium collected from parental Wnt3a did not suppress growth of cancer organoids , proving that growth inhibitory effect is due to presence of Wnt agonists . bar plot represents mean ± of triplicates performed with a luminescent viability . * p < 0 . 05 , * * * p < 0 . 001 , two - sided Student ` s t - .

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Addition of conditioned medium collected from the parental Wnt3a cell line did not suppress growth of cancer organoids, proving that the growth inhibitory effect is due to the presence of Wnt agonists. The bar plot represents the mean ±SEM of triplicates performed with a luminescent cell viability assay. * p < 0.05, *** p < 0.001, two-sided Student`s t-test.


Comparative of microarray data revealed consistent up - regulation of MYCN , canonical Wnt inhibitors KREMEN2 DKK3 as well as differentiation marker FOXJ1 , AR PGR in HGSOC cancer organoids as well as triple KD FT organoids . Differential expression was determined either by single - color microarray for cancer samples ( 8 replicates ) or dual - color microarray for knockdowns ( 2 replicates ) is significant for genes with p < 0 . 05 .

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Comparative analysis of microarray data revealed consistent up-regulation of MYCN, the canonical Wnt inhibitors KREMEN2 and DKK3 as well as differentiation marker FOXJ1, AR and PGR in HGSOC cancer organoids as well as triple KD FT organoids. Differential expression was determined either by single-color microarray for the cancer samples (8 replicates) or dual-color microarray for the knockdowns (2 replicates) and is significant for all genes with p < 0.05.


Transcriptional induction of MYCN is confirmed by qPCR for three between cancer organoids normal FT organoids as well as triple KD organoids grown in OCM vs . FTM conditions . Depicted is mean ± of normalized MYCN mRNA expression level of three biological replicates ( n = 3 ) . * * p < 0 . 01 , * * * p < 0 . 001 , two - sided Student ` s t - .

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Transcriptional induction of MYCN is confirmed by qPCR for three patients between cancer organoids and normal FT organoids as well as triple KD organoids grown in OCM vs. FTM conditions. Depicted is the mean ±SEM of the normalized MYCN mRNA expression level of three biological replicates (n=3). ** p < 0.01, *** p < 0.001, two-sided Student`s t-test.


Microarray data revealed that expression of Wnt target genes are downregulated in triple KD compared to WT control FT organoids . Differential expression determined by dual - color microarray for 2 biological replicates was significant for genes with p ≤ 0 . 005 .

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Microarray data revealed that expression levels of Wnt target genes are downregulated in triple KD compared to WT control FT organoids. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p ≤ 0.005.


FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR of 3 different samples . Data represent mean ± of technical triplicates .

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FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR analysis of 3 different patient samples. Data represent the mean ±SEM of technical triplicates.


A Co ‐ localization of endogenous TBC1D5 VPS35 with mCherry ‐ ATG9A , in steady state autophagy induced by mTOR inhibitor KU0063794 ( 6 h ) .

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A Co‐localization of endogenous TBC1D5 and VPS35 with mCherry‐ATG9A, in steady state and autophagy induced by mTOR inhibitor KU0063794 (6 h).


B , C of co ‐ localization experiments presented in ( A ) .

B, C Quantification of co‐localization experiments presented in (A).


D Co ‐ immunoprecipitation of HA ‐ Flag ‐ TBC1D5 with myc ‐ VPS29 , transiently overexpressed in 293T , DMSO or KU0063794 ( 6 h ) treated .

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D Co‐immunoprecipitation of HA‐Flag‐TBC1D5 with myc‐VPS29, transiently overexpressed in 293T cells, DMSO or KU0063794 (6 h) treated.


E , F Co ‐ immunoprecipitation of endogenous TBC1D5 ATG9A with myc ‐ VPS29 ( E ) transiently overexpressed in 293T or with HA ‐ Flag ‐ VPS35 ( F ) transiently overexpressed in 293T , treated with DMSO or KU0063794 ( 6 h ) .

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E, F Co‐immunoprecipitation of endogenous TBC1D5 and ATG9A with myc‐VPS29(E) transiently overexpressed in 293T cells or with HA‐Flag‐VPS35 (F) transiently overexpressed in 293T cells, treated with DMSO or KU0063794 (6 h).


G Co ‐ immunoprecipitation of endogenous VPS29 ATG9A with HA ‐ Flag ‐ TBC1D5stably expressed in 293T , treated with DMSO or KU0063794 ( 6 h ) .

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G Co‐immunoprecipitation of endogenous VPS29 and ATG9A with HA‐Flag‐TBC1D5stably expressed in 293T cells, treated with DMSO or KU0063794 (6 h).


H Co ‐ immunoprecipitation of endogenous ULK1 pATG13 ( Ser318 ) with HA ‐ Flag ‐ TBC1D5 , stably expressed in T ‐ REx 293T , induced by doxycycline ( 50 ng / ml ) for 20 h prior to DMSO or KU0063794 ( 6 h ) treatment .

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H Co‐immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA‐Flag‐TBC1D5, stably expressed in T‐REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.


I Magnified still extracted from Supplementary Movie S2 . mCherry ‐ TBC1D5 co ‐ localizes with GFP ‐ ATG9 upon autophagy induction by mTOR inhibitor , KU0063794 .

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I Magnified still images extracted from Supplementary Movie S2. mCherry‐TBC1D5 co‐localizes with GFP‐ATG9 upon autophagy induction by mTOR inhibitor, KU0063794.


H Co - immunoprecipitation of endogenous ULK1 pATG13 ( Ser318 ) with HA - Flag - TBC1D5 , stably expressed in T - REx 293T , induced by doxycycline ( 50 ng / ml ) for 20 h prior to DMSO or KU0063794 ( 6 h ) treatment .

H Co-immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA-Flag-TBC1D5, stably expressed in T-REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.


A Lysates of U2OS stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5 . were treated with DMSO or KU0063794 ( 6 h ) , lysed in RIPA buffer ( 1 % SDS ) lysates were subjected to SDS - PAGE .

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A Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5. Cells were treated with DMSO or KU0063794 (6 h), lysed in RIPA buffer (1% SDS) and lysates were subjected to SDS-PAGE.


B U2OS stably expressing mCherry ‐ ATG9 were depleted of VPS29 TBC1D5 using shRNAs , treated with DMSO or KU0063794 ( 6 h ) , fixed with 2 % PFA , immunostained with anti ‐ LAMP1 antibody .

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B U2OS cells stably expressing mCherry‐ATG9 were depleted of VPS29 and TBC1D5 using shRNAs, treated with DMSO or KU0063794 (6 h), fixed with 2% PFA, and immunostained with anti‐LAMP1 antibody.


C of ATG9 vesicular structures in from ( B ) under non ‐ stimulated conditions .

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C Quantification of ATG9 vesicular structures in cells from (B) under non‐stimulated conditions.


D of ATG9 LAMP1 co ‐ localization in presented in ( B ) .

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D Quantification of ATG9 and LAMP1 co‐localization in cells presented in (B).


E of ATG9 LAMP1 co − localization in stably depleted for TBC1D5 ( shRNA # 1 ) or VPS29 ( shRNA # 2 ) , transiently transfected with myc − TBC1D5 plasmid resistant to TBC1D5 shRNA # 1 . were treated with KU0063794 ( 6 h ) , 20 h post − transfection , fixed , immunostained with anti − LAMP1 anti − myc antibodies .

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E Quantification of ATG9 and LAMP1 co−localization in cells stably depleted for TBC1D5 (shRNA#1) or VPS29 (shRNA#2), transiently transfected with myc−TBC1D5 plasmid resistant to TBC1D5 shRNA#1. Cells were treated with KU0063794 (6 h), 20 h post−transfection, fixed, and immunostained with anti−LAMP1 and anti−myc antibodies.


F Control shRNA U2OS lysates depleted for TBC1D5 ( shRNA # 1 ) , transfected with empty plasmid or with shRNA ‐ resistant myc ‐ TBC1D5 .

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F Control shRNA and U2OS cell lysates depleted for TBC1D5 (shRNA#1), transfected with empty plasmid or with shRNA‐resistant myc‐TBC1D5.


G Immunofluorescence of TBC1D5 ( shRNA # 1 ) transiently transfected with shRNA ‐ resistant myc ‐ TBC1D5 , treated with KU0063794 ( 6 h ) . 20 h post ‐ transfection were treated , subsequently fixed , immunostained with anti ‐ myc anti ‐ LAMP1 antibodies .

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G Immunofluorescence of TBC1D5 (shRNA#1) cells transiently transfected with shRNA‐resistant myc‐TBC1D5, treated with KU0063794 (6 h). 20 h post‐transfection cells were treated, subsequently fixed, and immunostained with anti‐myc and anti‐LAMP1 antibodies.


A HA ‐ Flag ‐ TBC1D5 expression was induced in T ‐ REx HeLa for 20 h , lysates were subjected to GSTpull ‐ down , − AP2M1 . Co ‐ precipitatedTBC1D5 was detected with anti − Flag antibody .

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A HA‐Flag‐TBC1D5 expression was induced in T‐REx HeLa cells for 20 h, lysates were subjected to GSTpull‐down, −AP2M1. Co‐precipitatedTBC1D5 was detected with anti−Flag antibody.


B Control TBC1D5 ( shRNA # 1 ) U2OS lysates subjected to SDS - PAGE , subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 AP2M1 .

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B Control cells and TBC1D5 (shRNA#1) U2OS cell lysates subjected to SDS-PAGE, subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 and AP2M1.


C Immunofluorescent of AP2A1 in U2OS shRNA control , or shRNA # 1 TBC1D5 treated with KU0063974 or DMSO ( 6 h ) .

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C Immunofluorescent staining of AP2A1 in U2OS shRNA control, or shRNA#1 TBC1D5 cells treated with KU0063974 or DMSO (6 h).


D U2OS stably expressing HA ‐ Flag ‐ ATG9A were fixed stained for endogenous AP2A1 TBC1D5 . Regions of co ‐ localization are indicated with arrows .

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D U2OS cells stably expressing HA‐Flag‐ATG9A were fixed and stained for endogenous AP2A1 and TBC1D5. Regions of co‐localization are indicated with arrows.


E U2OS stably expressing HA ‐ Flag ‐ ATG9A were starved in EBSS ( 4 h ) or treated with KU0063794 ( 6 h ) , subsequently fixed stained with anti ‐ AP2A1 antibody .

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E U2OS cells stably expressing HA‐Flag‐ATG9A were starved in EBSS (4 h) or treated with KU0063794 (6 h), subsequently fixed and stained with anti‐AP2A1 antibody.


F U2OS stably expressing mCherry ‐ ATG9A were treated with KU0063794 ( 6 h ) , lysed in co ‐ immunoprecipitation buffer . Lysates from DMSO ‐ or KU0063794 ‐ treated were split equal volumes were incubated with RFP ‐ Trap beads or GFP ‐ Trap beads , or agarose as a negative control , overnight at 4 ° C . Precipitated were analyzed by SDS - PAGE .

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F U2OS cells stably expressing mCherry‐ATG9A were treated with KU0063794 (6 h), lysed in co‐immunoprecipitation buffer. Lysates from DMSO‐ or KU0063794‐treated cells were split and equal volumes were incubated with RFP‐Trap beads or GFP‐Trap beads, or agarose as a negative control, overnight at 4°C. Precipitated proteins were analyzed by SDS-PAGE.


A U2OS stably expressing HA ‐ Flag ‐ ATG9A transfected with siRNA targeting AP2A1 ( 40 nM ) , or siRNA AllStar control ( 40 nM ) were treated with KU0063794 or DMSO ( 6 h ) 4 days post ‐ transfection , fixed , stained with anti ‐ HA antibody .

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A U2OS cells stably expressing HA‐Flag‐ATG9A transfected with siRNA targeting AP2A1 (40 nM), or siRNA AllStar control (40 nM) were treated with KU0063794 or DMSO (6 h) 4 days post‐transfection, fixed, and stained with anti‐HA antibody.


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 .

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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 .

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

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 .

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

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 O 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 .

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

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 .

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

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

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 .

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

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.