Figure 1E - H Whole mount stereomicrographs of carmine alum stained mammary glands either uninjected ( E ) , 2 months after intraductal injection with MCF7 , arrows point to undilated ductal tips ( F ) , MM134 , dotted line highlight the subtending duct ( G ) , or SUM44 cells ( H ) , ( n > 6 ) scale bar , 1 mm . I Ex vivo luminescence images of bones , brain , lungs and liver from MM134 engrafted mice 1 month post injection ( top ) and ovaries ( bottom ) 12 months post injection . Scale bar , 1 cm . J Dot plot showing ex vivo bioluminescence of different organs from mice xenografted with MM134 and SUM44 cells plotted over organ and time of analysis . Number of mice analyzed MM134 and SUM44 for 30 - 180 : N = 5 , 10 , 180 - 270 days : N = 9 , 5 , 270 - 360 days : N = 14 , 20 , respectively . Dashed line indicates background levels .

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

Figure 1E-H Whole mount stereomicrographs of carmine alum stained mammary glands either uninjected (E), 2 months after intraductal injection with MCF7, arrows point to undilated ductal tips (F), MM134, dotted line highlight the subtending duct (G), or SUM44 cells (H), (n>6) scale bar, 1 mm.I Ex vivo luminescence images of bones, brain, lungs and liver from MM134 engrafted mice 1 month post injection (top) and ovaries (bottom) 12 months post injection. Scale bar, 1 cm. J Dot plot showing ex vivo bioluminescence of different organs from mice xenografted with MM134 and SUM44 cells plotted over organ and time of analysis. Number of mice analyzed MM134 and SUM44 for 30-180: N=5, 10, 180-270 days: N=9, 5, 270-360 days: N=14, 20, respectively. Dashed line indicates background levels.


Figure 2E , F Representative micrographs of IHC for Ki67 , ER , and PR on histological sections of MM134 ( E ) and SUM44 ( F ) xenografts 5 months after intraductal injection counter stained with hematoxylin . Scale bars , 200 μm . Bar plots represent means ± SEM of measurements and indicate the percentage of positive cells for > 1000 cells counted on 3 glands . G Representative micrographs of PR - IHC on glands from 5 - month - old MM134 xenografts from hosts treated for 2 months with vehicle ( left ) or E2 ( right ) pellets . Scale bars , 200 μm . Right , bar plot showing percentage of PR + cells in MM134 and SUM44 xenografts with n > 1000 cells per condition on glands from 3 different mice . Data represent mean ± SEM unpaired , Student ' s t - test , two - tailed ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

Figure 2E, F Representative micrographs of IHC for Ki67, ER, and PR on histological sections of MM134 (E) and SUM44 (F) xenografts 5 months after intraductal injection counter stained with hematoxylin. Scale bars, 200 μm. Bar plots represent means ± SEM of measurements and indicate the percentage of positive cells for >1000 cells counted on 3 glands.G Representative micrographs of PR-IHC on glands from 5-month-old MM134 xenografts from hosts treated for 2 months with vehicle (left) or E2 (right) pellets. Scale bars, 200 μm. Right, bar plot showing percentage of PR+ cells in MM134 and SUM44 xenografts with n>1000 cells per condition on glands from 3 different mice. Data represent mean ± SEM unpaired, Student's t-test, two-tailed; *** p < 0.001, **** p < 0.0001.


Figure 4D Representative RNAscope image of sections from patient ILCs ( n = 5 ) stained with anti - LOXL1 probes ( red ) and counterstained with DAPI ( blue ) . Scale bars ; 50 μm . E Representative immunofluorescence image of primary ILC ( n = 3 ) stained with anti - LOXL1 ( green ) and anti - ER ( red ) antibodies counterstained with DAPI ( blue ) . Scale bars ; 50 μm and right inset 30 μm .

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

Figure 4D Representative RNAscope image of sections from patient ILCs (n=5) stained with anti-LOXL1 probes (red) and counterstained with DAPI (blue). Scale bars; 50 μm.E Representative immunofluorescence image of primary ILC (n=3) stained with anti-LOXL1 (green) and anti-ER (red) antibodies counterstained with DAPI (blue). Scale bars; 50 μm and right inset 30 μm.


Figure 5E Radiance - based tumor growth curves ( mean ± SE ) for T47D treated with vehicle ( n = 8 ) or BAPN ( n = 12 ) 8 days after intraductal injections . n . s not significant . F Radiance - based tumor growth curves for ( mean ± SE ) ILC ER + PDXs T125 and T137 treated with vehicle ( n = 6 , 7 ) or BAPN ( n = 7 , 8 ) 66 days after engraftment . * * p < 0 . 01 . G Radiance - based tumor growth curves ( mean ± SE ) for non - ILC ER + PDXs T99 and T157 treated with vehicle ( n = 7 , 7 ) or BAPN ( n = 8 , 12 ) , 8 days after intraductal injection 3 weeks after intraductal injections . n . s not significant .

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

Figure 5E Radiance-based tumor growth curves (mean ± SE) for T47D treated with vehicle (n=8) or BAPN (n=12) 8 days after intraductal injections. n.s not significant.F Radiance-based tumor growth curves for (mean ± SE) ILC ER+ PDXs T125 and T137 treated with vehicle (n=6, 7) or BAPN (n=7, 8) 66 days after engraftment. ** p < 0.01.G Radiance-based tumor growth curves (mean ± SE) for non-ILC ER+ PDXs T99 and T157 treated with vehicle (n=7, 7) or BAPN (n=8, 12), 8 days after intraductal injection 3 weeks after intraductal injections. n.s not significant.


Figure 6E Representative fluorescent stereo - micrographs of glands injected with MM134 : RFP - luc2 cells ( left , top and bottom ) infected with either GFP : sh - scramble ( top ) or GFP + shLOXL1 ( bottom ) , n = 5 and 3 . Scale bar , 1000 μm . F Barplot showing the ratio of GFP / RFP signal from fluorescent stereo - micrographs of glands engrafted with MM134 cells , n = 8 . Statistical analysis by paired Student t - test , two - tailed . * p < 0 . 05 . H Representative fluorescent stereo - micrographs of glands engrafted with SUM44 : RFPluc2 cells infected with either GFP : sh - scramble ( top ) or GFP + shLOXL1 , n = 6 . Scale bar , 1000 μm . I Barplot ( mean ± SEM ) showing quantification of the ratio GFP / RFP from fluorescent stereo - micrographs of SUM44 . Statistical analysis by unpaired Student ' s t - test , two - tailed , n = 7 sh - scramble and n = 6 for sh - LOXL1 , * * * * p < 0 . 0001 . J , K Representative photomicrographs of picrosirius red stained histological sections for sh - scramble or sh - LOXL1 MM134 ( J ) and SUM44 ( K ) xenografts . Scale bars , 100 μm .

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

Figure 6E Representative fluorescent stereo-micrographs of glands injected with MM134:RFP-luc2 cells (left, top and bottom) infected with either GFP:sh-scramble (top) or GFP+ shLOXL1 (bottom), n=5 and 3. Scale bar, 1000 μm. F Barplot showing the ratio of GFP/RFP signal from fluorescent stereo-micrographs of glands engrafted with MM134 cells, n=8. Statistical analysis by paired Student t-test, two-tailed. *p < 0.05. H Representative fluorescent stereo-micrographs of glands engrafted with SUM44:RFPluc2 cells infected with either GFP:sh-scramble (top) or GFP+ shLOXL1, n=6. Scale bar, 1000 μm. I Barplot (mean ± SEM) showing quantification of the ratio GFP/RFP from fluorescent stereo-micrographs of SUM44. Statistical analysis by unpaired Student's t-test, two-tailed, n=7 sh-scramble and n=6 for sh-LOXL1, ****p < 0.0001. J, K Representative photomicrographs of picrosirius red stained histological sections for sh-scramble or sh-LOXL1 MM134 (J) and SUM44 (K) xenografts. Scale bars, 100 μm.


Figure 1A - C . SPF C57BL / 6Jmice were co - colonized with E1 and N1 wild type , ( WT , A , n = 5 mice ) N1 T6SS mutant , ( ΔtssC , B , n = 4 ) or N1 complemented ( ΔtssC pTssC , C , n = 5 ) . FecalCFU was quantified for E1 ( closed squares ) and N1 ( open squares ) weekly . D . Four weeks post - colonization , E1 fecal recovery was compared between the N1 WT , ΔtssC , and ΔtssC pTssC groups .

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

Figure 1A-C. SPF C57BL/6Jmice were co-colonized with E1 and N1 wild type, (WT, A, n=5 mice) N1 T6SS mutant, (ΔtssC, B, n=4) or N1 complemented (ΔtssC pTssC, C, n=5). FecalCFU was quantified for E1 (closed squares) and N1 (open squares) weekly.D. Four weeks post-colonization, E1 fecal recovery was compared between the N1 WT, ΔtssC, and ΔtssC pTssC groups.


Figure 2B - E . Co - colonization of N1 WT ( B and D , n = 4 mice ) or N1 Δbte2 ( C , n = 4 ) with E1 WT ( B and C ) or E1 overexpressing Bti2a ( E1 pBti2a , D ) . FecalCFU was monitored over time ( B - D ) and E1 CFU compared to N1 WT - E1 WT group at four - weeks post co - colonization ( E ) .

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

Figure 2B-E. Co-colonization of N1 WT (B and D, n=4 mice) or N1 Δbte2 (C, n=4) with E1 WT (B and C) or E1 overexpressing Bti2a (E1 pBti2a, D). FecalCFU was monitored over time (B-D) and E1 CFU compared to N1 WT-E1 WT group at four-weeks post co-colonization (E).


Figure 3A and B . Initial colonization of gnotobiotic ( A ) or SPF ( B ) mice ( n = 4 mice per group ) with N1 followed by secondary challenge with N1 ( closed squares ) or E1 ( open squares ) . FecalCFU was determined for the primary and secondary colonization strains through four weeks post - secondary challenges .

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

Figure 3A and B. Initial colonization of gnotobiotic (A) or SPF (B) mice (n=4 mice per group) with N1 followed by secondary challenge with N1 (closed squares) or E1 (open squares). FecalCFU was determined for the primary and secondary colonization strains through four weeks post-secondary challenges.


Figure 4A - F . Primary colonization of SPF mice with N2 WT , T6SS mutant ( ΔtssC ) and complemented ( ΔtssC pTssC ) followed by secondary challenge with N1 WT ( A and B , n = 5 mice ) N2 WT ( C and D , n = 5 ) or E1 WT ( E and F , n = 4 ) was performed . A , C and E . FecalCFU for primary and secondary strains was determined for four weeks post - secondary challenge . B , D and F . Selected time points were tested for statistical difference of secondary challenge between groups . This includes four weeks post - secondary challenge ( B ) and three days post - challenge ( D and F ) .

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

Figure 4A-F. Primary colonization of SPF mice with N2 WT, T6SS mutant (ΔtssC) and complemented (ΔtssC pTssC) followed by secondary challenge with N1 WT (A and B, n=5 mice) N2 WT (C and D, n=5) or E1 WT (E and F, n=4) was performed.A, C and E. FecalCFU for primary and secondary strains was determined for four weeks post-secondary challenge.B, D and F. Selected time points were tested for statistical difference of secondary challenge between groups. This includes four weeks post-secondary challenge (B) and three days post-challenge (D and F).


Figure 5A . Mice were co - colonized with E1 and either N1 WT ( n = 4 ) or N1 ΔtssC ( n = 3 ) . Five days post - inoculation , fecal RNA was extracted and tested for BFT expression via qRT - PCR . B . Four weeks after co - colonization with E1 and either N1 WT or N1 ΔtssC ( n = 4 mice per group ) , the sera were collected , tested via ELISA for anti - BFT IgG and end point titer calculated . C - F . Mice pre - treated with DSS were inoculated with no organisms ( sham ) , E1 only , or E1 competed with N1 WT or N1 ΔtssC . Five days post - inoculation , the ceca were weighed ( C ) and fixed for histopathological examination after sham ( D ) , E1 only ( E ) and E1 - N1 WT ( F ) colonizations . Scale bars denote 100μm ( main image ) and 200μm ( inset ) .

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

Figure 5A. Mice were co-colonized with E1 and either N1 WT (n=4) or N1 ΔtssC (n=3). Five days post-inoculation, fecal RNA was extracted and tested for BFT expression via qRT-PCR.B. Four weeks after co-colonization with E1 and either N1 WT or N1 ΔtssC (n= 4 mice per group), the sera were collected, tested via ELISA for anti-BFT IgG and end point titer calculated.C-F. Mice pre-treated with DSS were inoculated with no organisms (sham), E1 only, or E1 competed with N1 WT or N1 ΔtssC. Five days post-inoculation, the ceca were weighed (C) and fixed for histopathological examination after sham (D), E1 only (E) and E1-N1 WT (F) colonizations. Scale bars denote 100μm (main image) and 200μm (inset).


Figure 1In vitro niche dependency of HGSOC tumor cells . Phase - contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth , while they do not grow at all in Wnt3a supplemented medium . Also , inhibition of BMP signaling through Noggin has strong negative effect on the initial growth . Scale bar : 500 µm . E - EGF , F - FGF10 , N - Noggin , R - R - spondin1 , B - Basic medium , P - PassageCancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines . Scale bar : 20 µm . HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization . Scale bar : 100 µm . HGSOC organoids show differential response to carboplatin treatment , confirming patient - specific sensitivity of the cultures . Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors . Data represent mean ± SD of technical triplicates .

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

Figure 1In vitro niche dependency of HGSOC tumor cells. Phase-contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth, while they do not grow at all in Wnt3a supplemented medium. Also, inhibition of BMP signaling through Noggin has strong negative effect on the initial growth. Scale bar: 500 µm. E - EGF, F - FGF10, N -Noggin, R - R-spondin1, B - Basic medium, P - PassageCancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines. Scale bar: 20 µm.HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization. Scale bar: 100 µm.HGSOC organoids show differential response to carboplatin treatment, confirming patient-specific sensitivity of the cultures. Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors. Data represent mean ±SD of technical triplicates.


Figure 2Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long - term organoid culture . Color code indicates type of mutations which were detected . Representative western blot showing loss or overexpression of p53 in individual organoid lines . Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain - of - function TP53 mutation . Scale bars : 100 µm ( Tissue ) and 50 µm ( Organoid ) . Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue . Data represent the median , quartiles , maximum , and minimum of the normalized expression from 8 different donors . Heat map of differentially expressed genes between cancer and healthy tissue / organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples . Differential expression determined by single - color microarray for 8 different patient samples was significant for all genes with p < 0 . 05 except for OVGP1 and TOP2A in organoids .

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

Figure 2Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long-term organoid culture. Color code indicates type of mutations which were detected.Representative western blot showing loss or overexpression of p53 in individual organoid lines.Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain-of-function TP53 mutation. Scale bars: 100 µm (Tissue) and 50 µm (Organoid).Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue. Data represent the median, quartiles, maximum, and minimum of the normalized expression from 8 different donors.Heat map of differentially expressed genes between cancer and healthy tissue/organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples. Differential expression determined by single-color microarray for 8 different patient samples was significant for all genes with p < 0.05 except for OVGP1 and TOP2A in organoids.


Figure 3Experimental approach for genetic manipulation of FT epithelial cells ( FTECs ) . FTECs were sequentially transduced in 2D culture with replication - deficient viruses containing specific shRNAs and different selection markers . After sorting cells were seeded in Matrigel for organoid formation . Scale bars : 2 cm ( tissue ) , 200 µm ( 2D culture ) and 500 µm ( 3D culture ) . 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 typeCyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis . Confocal images of triple KD organoids reveal increased DNA damage ( yH2AX , marked by asterisks ) , atypic nuclei ( DRAQ5 ) and loss of apicobasal polarity . Scale bar : 20µm . 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 .

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

Figure 3Experimental approach for genetic manipulation of FT epithelial cells (FTECs). FTECs were sequentially transduced in 2D culture with replication-deficient viruses containing specific shRNAs and different selection markers. After sorting cells were seeded in Matrigel for organoid formation. Scale bars: 2 cm (tissue), 200 µm (2D culture) and 500 µm (3D culture). 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 typeCyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis.Confocal images of triple KD organoids reveal increased DNA damage (yH2AX, marked by asterisks), atypic nuclei (DRAQ5) and loss of apicobasal polarity. Scale bar: 20µm.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.


Figure 4In 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 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 . 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 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 - 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 the mean ± SEM from technical triplicates for two independent knockdown cultures . 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 .

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

Figure 4In 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 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.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 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- 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 the mean ±SEM from technical triplicates for two independent knockdown cultures.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.


Figure 5The 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 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 typePhase - 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 . 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 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 .

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

Figure 5The 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 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 typePhase-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.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 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.


Figure 6Comparative 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 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 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 analysis of 3 different patient samples . Data represent the mean ± SEM of technical triplicates .

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

Figure 6Comparative 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 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 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 analysis of 3 different patient samples. Data represent the mean ±SEM of technical triplicates.


Figure 1A Co ‐ localization of endogenous TBC1D5 and VPS35 with mCherry ‐ ATG9A , in steady state and autophagy induced by mTOR inhibitor KU0063794 ( 6 h ) . B , C Quantification of co ‐ localization experiments presented in ( A ) . 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 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 and ATG9A with HA ‐ Flag ‐ TBC1D5stably expressed in 293T cells , treated with DMSO or KU0063794 ( 6 h ) . 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 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 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 .

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

Figure 1A Co‐localization of endogenous TBC1D5 and VPS35 with mCherry‐ATG9A, in steady state and autophagy induced by mTOR inhibitor KU0063794 (6 h).B, C Quantification of co‐localization experiments presented in (A).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 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 and ATG9A with HA‐Flag‐TBC1D5stably expressed in 293T cells, treated with DMSO or KU0063794 (6 h).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 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 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.


Figure 2A 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 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 Quantification of ATG9 vesicular structures in cells from ( B ) under non ‐ stimulated conditions . D Quantification of ATG9 and LAMP1 co ‐ localization in cells presented in ( B ) . 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 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 ) 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 .

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

Figure 2A 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 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 Quantification of ATG9 vesicular structures in cells from (B) under non‐stimulated conditions.D Quantification of ATG9 and LAMP1 co‐localization in cells presented in (B).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 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) 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.


Figure 3A 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 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 staining of AP2A1 in U2OS shRNA control , or shRNA # 1 TBC1D5 cells treated with KU0063974 or DMSO ( 6 h ) . 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 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 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 .

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

Figure 3A 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 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 staining of AP2A1 in U2OS shRNA control, or shRNA#1 TBC1D5 cells treated with KU0063974 or DMSO (6 h).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 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 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.


Figure 4A 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 . C Lysates of U2OS cells transfected with siRNA targeting AP2A1 , or siRNA control AllStars analyzed by SDS - PAGE . 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 .

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

Figure 4A 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.C Lysates of U2OS cells transfected with siRNA targeting AP2A1, or siRNA control AllStars analyzed by SDS-PAGE.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.


Figure 5A 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 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 ‐ line profile across the vesicle indicated with arrow in ( B ) ; red - AP2A1 ; green - ATG9A ; blue - TBC1D5 . 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 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 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 .

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

Figure 5A 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 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‐line profile across the vesicle indicated with arrow in (B); red-AP2A1; green-ATG9A; blue-TBC1D5.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 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 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.


Figure 1A , 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 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 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 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 ) .

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

Figure 1A, 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 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 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 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).


Figure 2Immunoblotting 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 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 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 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 .

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

Figure 2Immunoblotting 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 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 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 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.


Figure 3A 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 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 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 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 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 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 .

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

Figure 3A 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 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 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 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 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 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.


Figure 4B 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 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 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 ) , 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 , 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 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 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 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 .

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

Figure 4B 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 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 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), 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, 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 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 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 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.


Figure 5A 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 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 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 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 , 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 , 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 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 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 .

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

Figure 5A 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 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 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 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, 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, 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 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 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.


Figure 6B 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 ) , 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 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 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 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 , 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 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 .

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

Figure 6B 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), 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 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 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 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, 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 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.


Figure 7Wild - 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 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 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 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 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 . 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 . 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 . 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 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 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 .

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

Figure 7Wild-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 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 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 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 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. 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. 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. 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 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 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.


figf1 ( 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 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 ) 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 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 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 ) .

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

figf1(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 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) 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 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 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).