Figure 6BCA analysis of EV protein concentration in EVs isolates . Each bar represents a mean of n = 3 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed t - test ) . Exocet quantification assay of EVs particle numbers . Each bar represents a mean of n = 3 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed t - test ) . Transmission electron microscopy ( TEM ) of EV isolates . Three representative pictures are displayed . Scale bar indicates 200 nm . Analysis of size distribution of EVs . Each dot indicates the diameter in nm of an individual vesicle . SD . * * * , p < 0 . 001 ; n . s . not significant ( one - way ANOVA , followed by a Tukey multiple comparison test ) . Prdx4 concentration in EV lysates . Each bar represents a mean of n = 2 technical replicates , vertical lines indicate SD . * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed t - test ) . Caspase - 1 and IL - 1β concentration in EV lysates . Each bar represents a mean of n = 2 technical replicates , vertical lines indicate SD . * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 6BCA analysis of EV protein concentration in EVs isolates. Each bar represents a mean of n=3 technical replicates, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).Exocet quantification assay of EVs particle numbers. Each bar represents a mean of n=3 technical replicates, vertical lines indicate SD. *, p < 0.05; ***, p < 0.001; n.s. not significant (two-tailed t-test).Transmission electron microscopy (TEM) of EV isolates. Three representative pictures are displayed. Scale bar indicates 200 nm.Analysis of size distribution of EVs. Each dot indicates the diameter in nm of an individual vesicle. SD. ***, p < 0.001; n.s. not significant (one-way ANOVA, followed by a Tukey multiple comparison test).Prdx4 concentration in EV lysates. Each bar represents a mean of n= 2 technical replicates, vertical lines indicate SD. **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).Caspase-1 and IL-1β concentration in EV lysates. Each bar represents a mean of n= 2 technical replicates, vertical lines indicate SD. **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Figure 7Western blot analysis of Prdx4 , NLRP3 , ASC , caspase ‑ 1 , IL - 1β and CD63 from EV lysates ( upper panel ) or caspase - 1 after immunoprecipitation against caspase - 1 ( lower panel ) . Cxcl1 concentration in supernatants of caspase - 1 - deficient BMDMs stimulated with EVs from LPS , LPS and ATP or control - treated Prdx4 WT or KO BMDMs , as well as caspase - 1 - deficient BMDMs pre - treated with Anakinra and stimulated with EVs from LPS and ATP - treated Prdx4 WT or KO BMDMs . Each bar represents a mean of n = 3 biological with 2 technical replicates , vertical lines indicate SD . Serum Cxcl1 in C57Bl6 / N mice injected with either PBS or EVs from LPS and ATP or control - treated Prdx4 WT or KO or ASC KO BMDMs . Each dot represents an individual mouse . Horizontal lines indicate mean .

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Figure 7Western blot analysis of Prdx4, NLRP3, ASC, caspase‑1, IL-1β and CD63 from EV lysates (upper panel) or caspase-1 after immunoprecipitation against caspase-1 (lower panel).Cxcl1 concentration in supernatants of caspase-1-deficient BMDMs stimulated with EVs from LPS, LPS and ATP or control-treated Prdx4 WT or KO BMDMs, as well as caspase-1-deficient BMDMs pre-treated with Anakinra and stimulated with EVs from LPS and ATP-treated Prdx4 WT or KO BMDMs. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD.Serum Cxcl1 in C57Bl6/N mice injected with either PBS or EVs from LPS and ATP or control-treated Prdx4 WT or KO or ASC KO BMDMs. Each dot represents an individual mouse. Horizontal lines indicate mean.


Figure 1 ( B ) Western blot analysis of SARS - CoV - 2 Spike 1 - 1273 , 1 - 674 , and 319 - 591 expression in HEK293T cells . The black arrows indicate the location of the induced target proteins . ( C ) Detection of decoy antibody ACE2 - Fc and Spike S1 - Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue staining . ( D ) Recognition of the ACE2 - Fc and Spike S1 - Fc by anti - human IgG Fc antibody in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( SDS - PAGE ) . ( E , F ) Deglycosylation of ACE2 - Fc and Spike 1 - 674 - Fc by PNGase F . PNGase F digested ACE2 - Fc ( 500 ng ) and Spike 1 - 674 - Fc ( 500 ng ) were subjected to Coomassie Brilliant Blue staining ( E ) and Western blot analysis by anti - human IgG Fc antibody ( F ) .

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

Figure 1(B) Western blot analysis of SARS-CoV-2 Spike 1-1273, 1-674, and 319-591 expression in HEK293T cells. The black arrows indicate the location of the induced target proteins.(C) Detection of decoy antibody ACE2-Fc and Spike S1-Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue staining.(D) Recognition of the ACE2-Fc and Spike S1-Fc by anti-human IgG Fc antibody in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).(E, F) Deglycosylation of ACE2-Fc and Spike 1-674-Fc by PNGase F. PNGase F digested ACE2-Fc (500 ng) and Spike 1-674-Fc (500 ng) were subjected to Coomassie Brilliant Blue staining (E) and Western blot analysis by anti-human IgG Fc antibody (F).


Figure 2 ( A ) Competitive ELISA . ACE2 - Fc can compete with ACE2 - Fc - Biotin for Spike 1 - 674 binding in a dose - dependent manner . The competition effects of ACE2 - Fc on ACE2 - Spike protein binding was normalized to PBS control . Error bars represent the standard deviation ( SD ) , n = 2 . Statistical analysis was performed by unpaired two tail t - test . * * P < 0 . 01 , * * * P < 0 . 001 . ( B ) Recognition of full - length Spike on H1975 ( left panel as negative control ) or H1975 - Spike ( right panel ) cells by ACE2 - Fc using flow cytometry analysis . Isotype control : 40 μg / mL mouse IgG - FITC . ( C ) Confocal microscopy of H1975 - Spike - overexpressing cells . Spikes on H1975 cells were stained with anti - Spike antibody and Alexa Fluor ® 594 - conjugated secondary antibody on ice for 1 hr . After that , ACE2 - Fc - FITC was incubated for another 1 hr . Scale bars equal to 20 µm . DAPI was used as a nuclear counterstain . ( D ) Preservation of ACE2 - Fc peptidase activity . The peptidase activity of ACE2 - Fc was measured by cleavage of the fluorescent peptide substrates . ( E ) Inhibition of angiotensin II ( Ang II ) - induced TNF - α production by ACE2 - Fc . Ang II was preincubated with indicated amounts of ACE2 - Fc or IgG for 30 minutes . After that , the mixtures were added to RAW264 . 7 cells for 12 hr . The concentration of TNF - α in the culture medium was determined by ELISA . Error bars represent the standard deviation ( SD ) , n = 3 . Statistical analysis was performed by unpaired two tail t - test . * P < 0 . 05 . The dotted line represents the mean value of TNF - α in control group . ( F ) Inhibition of Ang II - induced ADAM17 ( a disintegrin and metalloprotease 17 ) phosphorylation by ACE2 - Fc . The protein extracts from ( E ) were immunoblotted with the indicated antibodies ( left panel ) . β - actin served as the loading control . The signal intensity was normalized to cells only ( right panel ) . Data are representative of three independent experiments , and the values are expressed as the mean ± SD ( right panel ) . Error bars represent the standard deviation ( SD ) , n = 2 . Statistical analysis was performed by unpaired two tail t - test . * P < 0 . 05 . IgG represents the human normal IgG control .

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

Figure 2(A) Competitive ELISA. ACE2-Fc can compete with ACE2-Fc-Biotin for Spike 1-674 binding in a dose-dependent manner. The competition effects of ACE2-Fc on ACE2-Spike protein binding was normalized to PBS control. Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. **P < 0.01, ***P < 0.001.(B) Recognition of full-length Spike on H1975 (left panel as negative control) or H1975-Spike (right panel) cells by ACE2-Fc using flow cytometry analysis. Isotype control: 40 μg/mL mouse IgG-FITC.(C) Confocal microscopy of H1975-Spike-overexpressing cells. Spikes on H1975 cells were stained with anti-Spike antibody and Alexa Fluor® 594-conjugated secondary antibody on ice for 1 hr. After that, ACE2-Fc-FITC was incubated for another 1 hr. Scale bars equal to 20 µm. DAPI was used as a nuclear counterstain.(D) Preservation of ACE2-Fc peptidase activity. The peptidase activity of ACE2-Fc was measured by cleavage of the fluorescent peptide substrates.(E) Inhibition of angiotensin II (Ang II)-induced TNF-α production by ACE2-Fc. Ang II was preincubated with indicated amounts of ACE2-Fc or IgG for 30 minutes. After that, the mixtures were added to RAW264.7 cells for 12 hr. The concentration of TNF-α in the culture medium was determined by ELISA. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. *P < 0.05. The dotted line represents the mean value of TNF-α in control group.(F) Inhibition of Ang II-induced ADAM17 (a disintegrin and metalloprotease 17) phosphorylation by ACE2-Fc. The protein extracts from (E) were immunoblotted with the indicated antibodies (left panel). β-actin served as the loading control. The signal intensity was normalized to cells only (right panel). Data are representative of three independent experiments, and the values are expressed as the mean ± SD (right panel). Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. *P < 0.05. IgG represents the human normal IgG control.


Figure 3 ( B ) HEK293 and H1975 cells were transduced with full - length ACE2 by lentivirus . Protein extracts were immunoblotted with the indicated antibodies . O / E represents overexpression . ( C ) GFP and full - length Spike co - transfected HEK293T cells were used as effector cells ( 293 - S ) . HEK293 / ACE2 ( HEK293T - overexpressing ACE2 ) ( left panel ) or H1975 / ACE2 ( H1975 - overexpressing ACE2 ) ( right panel ) cells were used as target cells . The 293 - S cells were preincubated with normal human IgG or ACE2 - Fc at 37 ° C for 1 hr . After that , the mixtures were added to target cells for 4 hr ( cell - cell fusion ) or 24 hr ( syncytia formation ) . The fluorescent areas were measured by inverted fluorescence microscopy and Metamorph software ( Metamorphosis ) . Scale bars equal to 50 µm . The white arrows indicate the cell - cell fusion or syncytia formation . ( D ) The inhibition of cell - cell fusion and syncytia formation by ACE2 - Fc in HEK293 / ACE2 and H1975 / ACE2 was determined with the formula described in the Methods section . Error bars represent the standard deviation ( SD ) , n = 6 . Statistical analysis was performed by unpaired two tail t - test . * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 3(B) HEK293 and H1975 cells were transduced with full-length ACE2 by lentivirus. Protein extracts were immunoblotted with the indicated antibodies. O/E represents overexpression.(C) GFP and full-length Spike co-transfected HEK293T cells were used as effector cells (293-S). HEK293/ACE2 (HEK293T-overexpressing ACE2) (left panel) or H1975/ACE2 (H1975-overexpressing ACE2) (right panel) cells were used as target cells. The 293-S cells were preincubated with normal human IgG or ACE2-Fc at 37°C for 1 hr. After that, the mixtures were added to target cells for 4 hr (cell-cell fusion) or 24 hr (syncytia formation). The fluorescent areas were measured by inverted fluorescence microscopy and Metamorph software (Metamorphosis). Scale bars equal to 50 µm. The white arrows indicate the cell-cell fusion or syncytia formation.(D) The inhibition of cell-cell fusion and syncytia formation by ACE2-Fc in HEK293/ACE2 and H1975/ACE2 was determined with the formula described in the Methods section. Error bars represent the standard deviation (SD), n=6. Statistical analysis was performed by unpaired two tail t-test. ** P < 0.01, *** P < 0.001.


Figure 4 ( A ) ACE2 - Fc blocked the entry of Spike - expressing pseudotyped lentivirus into HEK293T - ACE2 and H1975 - ACE2 cells . The relative luciferase activities , normalized to the only virus group , represent the efficiency of virus entry . MOI : Multiplicity of infection . ( B ) Confocal microscopy images of the airway organoids . The green fluorescence represents the specific staining of indicated monoclonal antibodies and Alexa Fluor 488 - conjugated secondary antibodies . DAPI was used as a nuclear counterstain . Hematoxylin and eosin ( H & E ) stain : hematoxylin stained the nuclei in blue color ; eosin stained the cytoplasm in pink color . Scale bars equal to 20 µm . ( C ) Lung cancer A549 cells , human normal bronchial epithelial cells ( HBEpc ) , and HBEpc - differentiated cells ( airway organoids ) were harvested for immunoblotting with the indicated antibodies . ( D ) Blockage of pseudovirus entry into airway organoids by ACE2 - Fc . Mixtures of pseudotyped lentivirus with or without ACE2 - Fc were cocultured with airway organoids for 72 hr . The virus entry was determined by quantifying the luciferase activity in the cell lysates .

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 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 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

Figure 4(A) ACE2-Fc blocked the entry of Spike-expressing pseudotyped lentivirus into HEK293T-ACE2 and H1975-ACE2 cells. The relative luciferase activities, normalized to the only virus group, represent the efficiency of virus entry. MOI: Multiplicity of infection.(B) Confocal microscopy images of the airway organoids. The green fluorescence represents the specific staining of indicated monoclonal antibodies and Alexa Fluor 488-conjugated secondary antibodies. DAPI was used as a nuclear counterstain. Hematoxylin and eosin (H&E) stain: hematoxylin stained the nuclei in blue color; eosin stained the cytoplasm in pink color. Scale bars equal to 20 µm.(C) Lung cancer A549 cells, human normal bronchial epithelial cells (HBEpc), and HBEpc-differentiated cells (airway organoids) were harvested for immunoblotting with the indicated antibodies.(D) Blockage of pseudovirus entry into airway organoids by ACE2-Fc. Mixtures of pseudotyped lentivirus with or without ACE2-Fc were cocultured with airway organoids for 72 hr. The virus entry was determined by quantifying the luciferase activity in the cell lysates.


Figure 5 ( A ) Inhibition of SARS - CoV - 2 infection by ACE2 - Fc in the plaque assay . Mixtures of ACE2 - Fc and SARS - CoV - 2 were incubated for one hour before adding to Vero E6 cells for another 1 hr at 37 ° C . The ACE2 - Fc and SARS - CoV - 2 premixtures were removed , and the cells were washed once with PBS and overlaid with methylcellulose with 2 % FBS for 5 - 7 days before being stained with crystal violet . Those results showed increase of plaque formation was regarded as no inhibition of plaque formation . TPCK - treated trypsin : N - tosyl - L - phenylalanine chloromethyl ketone - treated trypsin . Yield reduction assay was performed to determine the inhibitory effects of ACE2 - Fc on virus titers The culture medium and cell extracts were harvested 24 hours post - infection for real - time PCR ( B ) Yield reduction assay was performed to determine the inhibitory effects of ACE2 - Fc on protein expression . The culture medium and cell extracts were harvested 24 hours post - infection for western blot ( C ) . The NP / PCNA represents the relative NP expression as compared to that of PCNA , which served as a loading control . The NP / PCNA numbers below the panel are the ratios of NP / PCNA normalized to that of the human IgG control group . PCNA : Proliferating cell nuclear antigen . ( E ) The cell lysates from the pretreatment and full - time experiments were harvested and immunoblotted with the indicated antibodies . ( F ) The SARS - CoV - 2 titer in the pretreatment and full - time experiments was analyzed by real - time PCR .

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 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 O O O O O O O O O O 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

Figure 5(A) Inhibition of SARS-CoV-2 infection by ACE2-Fc in the plaque assay. Mixtures of ACE2-Fc and SARS-CoV-2 were incubated for one hour before adding to Vero E6 cells for another 1 hr at 37°C. The ACE2-Fc and SARS-CoV-2 premixtures were removed, and the cells were washed once with PBS and overlaid with methylcellulose with 2% FBS for 5-7 days before being stained with crystal violet. Those results showed increase of plaque formation was regarded as no inhibition of plaque formation. TPCK-treated trypsin: N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin.Yield reduction assay was performed to determine the inhibitory effects of ACE2-Fc on virus titers The culture medium and cell extracts were harvested 24 hours post-infection for real-time PCR (B)Yield reduction assay was performed to determine the inhibitory effects of ACE2-Fc on protein expression. The culture medium and cell extracts were harvested 24 hours post-infection for western blot (C). The NP/PCNA represents the relative NP expression as compared to that of PCNA, which served as a loading control. The NP/PCNA numbers below the panel are the ratios of NP/PCNA normalized to that of the human IgG control group. PCNA: Proliferating cell nuclear antigen.(E) The cell lysates from the pretreatment and full-time experiments were harvested and immunoblotted with the indicated antibodies.(F) The SARS-CoV-2 titer in the pretreatment and full-time experiments was analyzed by real-time PCR.


Figure 6 ( A ) The ( A ) NP proteins in the cell lysates were determined by Western blot analysis . ( B , C ) The virus RNA in the ( B ) culture medium and ( C ) cell lysates was quantified by real - time RT - PCR . The genetic information and sequence reference numbers for the 5 SARS - CoV - 2 strains were summarized in Table EV1 . Error bars represent the standard deviation ( SD ) , n = 3 . Statistical analysis was performed by unpaired two tail t - test . * * P < 0 . 01 , * * * P < 0 . 001 . Experiments were performed at least three times with similar results .

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Figure 6(A) The (A) NP proteins in the cell lysates were determined by Western blot analysis.(B, C) The virus RNA in the (B) culture medium and (C) cell lysates was quantified by real-time RT-PCR. The genetic information and sequence reference numbers for the 5 SARS-CoV-2 strains were summarized in Table EV1. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. ** P < 0.01, *** P < 0.001. Experiments were performed at least three times with similar results.


Figure 7 ( A ) H1975 cells were transduced with full - length Spike by Lentiviral vector . Protein extracts were immunoblotted with the indicated antibodies . O / E represents overexpress . ( B ) The effect of ACE2 - Fc activation on degranulative capacity of NK cells . The experiments were performed with the primary human NK cells derived from three independent donors . Error bars represent the standard deviation ( SD ) , n = 3 . Statistical analysis was performed by unpaired two tail t - test . * P < 0 . 05 ( each concentration of ACE2 - Fc vs control ) . ( C ) The CD107a , IFN - γ , and TNF - α expression levels were determined by flow cytometry after the NK cells and H1975 - Spike cells coincubation in the presence or absence of ACE2 - Fc or ACE2 control . Error bars represent the standard deviation ( SD ) , n = 3 . Statistical analysis was performed by unpaired two tail t - test . * P < 0 . 05 . The percentage of positive cells in both groups were normalized to the NK / H1975 - Spike only group . ( D ) The differential expression of degranulation markers after the treatment of ACE2 - Fc or ACE2 by the flow cytometry . The numbers in each plot indicate the percentages of positive cells .

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

Figure 7(A) H1975 cells were transduced with full-length Spike by Lentiviral vector. Protein extracts were immunoblotted with the indicated antibodies. O/E represents overexpress.(B) The effect of ACE2-Fc activation on degranulative capacity of NK cells. The experiments were performed with the primary human NK cells derived from three independent donors. Error bars represent the standard deviation (SD), n= 3. Statistical analysis was performed by unpaired two tail t-test. * P < 0.05 (each concentration of ACE2-Fc vs control).(C) The CD107a, IFN-γ, and TNF-α expression levels were determined by flow cytometry after the NK cells and H1975-Spike cells coincubation in the presence or absence of ACE2-Fc or ACE2 control. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. * P < 0.05. The percentage of positive cells in both groups were normalized to the NK/H1975-Spike only group.(D) The differential expression of degranulation markers after the treatment of ACE2-Fc or ACE2 by the flow cytometry. The numbers in each plot indicate the percentages of positive cells.


Figure 1Immunoblot analysis of proteins biotinylated and isolated as in ( A ) . Parental HeLa - T - REx cells or cells expressing H3 . 1 - BirA * - HA and treated with biotin for indicated times . Immunoblot analysis of TAP - H3 . 1 or - CENP - A pulldown of UBR7 - LAP ( schematic , upper ) . 293T cells were co - transfected with UBR7 - LAP and TAP - tagged constructs , which were purified on IgG - conjugated sepharose beads .

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Figure 1Immunoblot analysis of proteins biotinylated and isolated as in (A). Parental HeLa-T-REx cells or cells expressing H3.1-BirA*-HA and treated with biotin for indicated times.Immunoblot analysis of TAP-H3.1 or -CENP-A pulldown of UBR7-LAP (schematic, upper). 293T cells were co-transfected with UBR7-LAP and TAP-tagged constructs, which were purified on IgG-conjugated sepharose beads.


Figure 2Immunoblot of anti - FLAG IP of 3x - FLAG tagged GFP , or FLAG - HA tagged H3 . 1 or H2B , from the non - nucleosomal fraction of 293T cells . Representative images of HeLa cells transfected with UBR7 - FL . DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green . The area within yellow dotted box in ( E ) is magnified 4x in ( E ' ) . Scale bar = 5 μm . Quantification of the nuclear anti - FLAG immunofluorescence intensity in ( E ) . Error bars represent mean + / - standard deviation . One way ANOVA was used to calculate p - values ( italics ) associated with differences between treatments . Experiment in ( E - F ) was performed twice .

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Figure 2Immunoblot of anti-FLAG IP of 3x-FLAG tagged GFP, or FLAG-HA tagged H3.1 or H2B, from the non-nucleosomal fraction of 293T cells.Representative images of HeLa cells transfected with UBR7-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. The area within yellow dotted box in (E) is magnified 4x in (E'). Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (E). Error bars represent mean +/- standard deviation. One way ANOVA was used to calculate p-values (italics) associated with differences between treatments. Experiment in (E-F) was performed twice.


Figure 3Immunoblot analysis of anti - FLAG pulldown of full length UBR7 - FLAG ( residues 1 - 425 ) mutant UBR7 bearing deletion of UBR7 C - terminus ( ∆ C - term , residues 1 - 215 ) , UBR box ( ∆ UBR , residues 117 - 425 ) , UBR box and PHD ( ∆ UBR ∆ PHD , residues 216 - 425 ) , or PHD and C - terminus ( ∆ PHD ∆ C - term , residues 1 - 116 ) . 293T cells were co - transfected with H3 . 1 - HA and UBR7 - FLAG constructs ( schematic , upper ) . Asterisk represents non - specific band . Numbers below blot indicate FLAG - normalized anti - HA immunoblot signal relative to full length UBR7 - FLAG . Immunoblot analysis of anti - H3 . 1 - HA pulldown of full - length UBR7 - LAP and UBR7PHD fragment ( residues 117 - 215 ) ( schematic , upper ) . 293T cells were transfected as in ( A ) . Values below pulldown represent H3 . 1 - HA - normalized GFP - UBR7 immunoblot signal relative to UBR7 - FL . Data were acquired from a continuous membrane , and the image is cropped to omit irrelevant samples . Numbers below blot indicate HA - normalized anti - GFP immunoblot signal relative to full length UBR7 - LAP .

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Figure 3Immunoblot analysis of anti-FLAG pulldown of full length UBR7-FLAG (residues 1-425) mutant UBR7 bearing deletion of UBR7 C-terminus (∆C-term, residues 1-215), UBR box (∆UBR, residues 117-425), UBR box and PHD (∆UBR∆PHD, residues 216-425), or PHD and C-terminus (∆PHD∆C-term, residues 1-116). 293T cells were co-transfected with H3.1-HA and UBR7-FLAG constructs (schematic, upper). Asterisk represents non-specific band. Numbers below blot indicate FLAG-normalized anti-HA immunoblot signal relative to full length UBR7-FLAG.Immunoblot analysis of anti-H3.1-HA pulldown of full-length UBR7-LAP and UBR7PHD fragment (residues 117-215) (schematic, upper). 293T cells were transfected as in (A). Values below pulldown represent H3.1-HA-normalized GFP-UBR7 immunoblot signal relative to UBR7-FL. Data were acquired from a continuous membrane, and the image is cropped to omit irrelevant samples. Numbers below blot indicate HA-normalized anti-GFP immunoblot signal relative to full length UBR7-LAP.


Figure 4Anti - GFP immunoblot analysis of streptavidin pulldown of the indicated biotin - conjugated H3 peptides following incubation with 293T - GFP - UBR7 cell lysate after mutation of residues highlighted in ( C ) . Immunoblot analysis for total and modified histones in anti - FLAG pulldown of constructs from soluble cellular fractions demonstrates that UBR7 UBR box and PHD are necessary for binding to post - nucleosomal H3 / H4 . Immunoblot analysis of streptavidin pulldown from proximity biotinylation described in ( G ) . 4 . 5 h represent newly synthesized histones and 48 h represent pre - existing histones . Wedge represents loading titration : 1x > 0 . 1x > 0 . 01x > 0 . 001x . All other lanes are loaded at 1x concentration . Immunoblot signals were normalized to anti - FLAG signal . Quantification of anti - FLAG ( H3 . 1 - APEX2 - 3xFLAG ) and anti - UBR7 immunoblot represented in ( G - H ) . Lanes 8 and 11 were quantified . Error bar represents mean + / - standard deviation . A two - tailed one - sample t - test was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed line ) .

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Figure 4Anti-GFP immunoblot analysis of streptavidin pulldown of the indicated biotin-conjugated H3 peptides following incubation with 293T-GFP-UBR7 cell lysate after mutation of residues highlighted in (C).Immunoblot analysis for total and modified histones in anti-FLAG pulldown of constructs from soluble cellular fractions demonstrates that UBR7 UBR box and PHD are necessary for binding to post-nucleosomal H3/H4.Immunoblot analysis of streptavidin pulldown from proximity biotinylation described in (G). 4.5 h represent newly synthesized histones and 48 h represent pre-existing histones. Wedge represents loading titration: 1x > 0.1x > 0.01x > 0.001x. All other lanes are loaded at 1x concentration. Immunoblot signals were normalized to anti-FLAG signal.   Quantification of anti-FLAG (H3.1-APEX2-3xFLAG) and anti-UBR7 immunoblot represented in (G-H). Lanes 8 and 11 were quantified. Error bar represents mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line).


Figure 5Immunoblot analysis for total and modified H3 and H4 in anti - FLAG IP . Experiment was performed as in ( D ) . Dot plot error bars represent mean + / - standard deviation . Immunoblot signals were normalized to anti - H4 signal . A two - tailed one sample t - test was used to calculate p - value associated with group mean difference from a hypothetical value of 1 . 0 . Experiment was performed twice . Representative images of HeLa cells transfected with sNASP - FL . DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green . Scale bar = 5 μm . Quantification of the nuclear anti - FLAG immunofluorescence intensity in ( G ) demonstrating that sNASP - FL is highly susceptible to pre - extraction with Triton X - 100 . One way ANOVA was used to determine p - values ( italics ) associated with differences between treatments . Error bars represent mean + / - standard deviation . Experiment in ( G - H ) was performed twice .

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Figure 5Immunoblot analysis for total and modified H3 and H4 in anti-FLAG IP. Experiment was performed as in (D). Dot plot error bars represent mean +/- standard deviation. Immunoblot signals were normalized to anti-H4 signal. A two-tailed one sample t-test was used to calculate p-value associated with group mean difference from a hypothetical value of 1.0. Experiment was performed twice.Representative images of HeLa cells transfected with sNASP-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (G) demonstrating that sNASP-FL is highly susceptible to pre-extraction with Triton X-100. One way ANOVA was used to determine p-values (italics) associated with differences between treatments. Error bars represent mean +/- standard deviation. Experiment in (G-H) was performed twice.


Figure 6A , C . Immunoblot analysis for modified histone in anti - FLAG pulldown of UBR7 - FL ( A ) or sNASP - FL ( C ) from non - nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors ( hydroxyurea , aphidicolin ) or transcription inhibitors ( α - amanitin , triptolide ) . B , D . Plots represent either normalized immunoblot signals or cell counts prior to fractionation . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation , with grey dotted line marking mean of No Treat condition . A two - tailed one sample t - test was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 . Experiments were performed three times . Cells numbers were counted in two of the three experiments .

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Figure 6A,C. Immunoblot analysis for modified histone in anti-FLAG pulldown of UBR7-FL (A) or sNASP-FL (C) from non-nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors (hydroxyurea, aphidicolin) or transcription inhibitors (α-amanitin, triptolide). B,D. Plots represent either normalized immunoblot signals or cell counts prior to fractionation. Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation, with grey dotted line marking mean of No Treat condition. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0. Experiments were performed three times. Cells numbers were counted in two of the three experiments.


Figure 7Immunoblot analysis of H3 , UBR7 , and GAPDH in the non - nucleosomal fraction of parental and 293T - UBR7KO cells . Quantification of anti - H3 immunoblot represented in ( A ) . Immunoblot signals were normalized to anti - GAPDH signal . Error bars represent mean + / - standard deviation . A two - tailed one - sample t - test was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed line ) . Experiment in ( A - B ) was performed in three times . Immunoblot analysis of anti - FLAG pulldown of sNASP - FL in parental or 293T - UBR7KO cells . 3xFLAG tagged sNASP - FL was immunoprecipitated from non - nucleosomal fraction and eluted with FLAG peptide . Experiment was performed three times . Quantification of anti - FLAG ( sNASP - FL ) , and anti - histone immunoblots represented in ( C ) . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation . A two - tailed one sample t - test was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed line ) . Experiments were performed two ( sNASP , H4 ) to three ( all other targets ) times .

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Figure 7Immunoblot analysis of H3, UBR7, and GAPDH in the non-nucleosomal fraction of parental and 293T-UBR7KO cells.   Quantification of anti-H3 immunoblot represented in (A). Immunoblot signals were normalized to anti-GAPDH signal. Error bars represent mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment in (A-B) was performed in three times.  Immunoblot analysis of anti-FLAG pulldown of sNASP-FL in parental or 293T-UBR7KO cells. 3xFLAG tagged sNASP-FL was immunoprecipitated from non-nucleosomal fraction and eluted with FLAG peptide. Experiment was performed three times.   Quantification of anti-FLAG (sNASP-FL), and anti-histone immunoblots represented in (C). Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments were performed two (sNASP, H4) to three (all other targets) times.


Figure 8Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7 - FL ( left ) or sNASP - FL ( right ) following treatment with siCTRL , siLAMP2A , or MG - 132 . Quantification of data represented in ( C , D ) . Error bars represent mean + / - standard deviation . A two - tailed one sample t - test was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed line ) . Experiments in ( C - E ) were performed three times .

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Figure 8Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7-FL (left) or sNASP-FL (right) following treatment with siCTRL, siLAMP2A, or MG-132.Quantification of data represented in (C, D). Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments in (C-E) were performed three times.


Figure 9Heatmaps of ATAC - seq , anti - GFP ( GFP - UBR7 ) ChIP - seq , and anti - H3K4me3 CUT & RUN in parental and UBR7KO cells . Plots are centered on TSS that show changes in ATAC - seq signal in UBR7KO versus parental cells . Sites are clustered by unsupervised k - means analysis . Cluster color key is defined by cluster labels .

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Figure 9Heatmaps of ATAC-seq, anti-GFP (GFP-UBR7) ChIP-seq, and anti-H3K4me3 CUT&RUN in parental and UBR7KO cells. Plots are centered on TSS that show changes in ATAC-seq signal in UBR7KO versus parental cells. Sites are clustered by unsupervised k-means analysis. Cluster color key is defined by cluster labels.


Figure 1Low magnification views of a CD1 Otof - / - organ of Corti ( P23 ) transduced with otoferlin dual ‑ AAV ‑ TS vectors . IHCs : inner hair cells , OHCs : outer hair cells maximum intensity projections of confocal optical sectionsHigh magnification views of CD1B6F1 Otof - / - IHCs transduced with otoferlin dual ‑ AAV ‑ TS ( P26 ) ( B ) and dual ‑ AAV ‑ Hyb ( P26 ) ( C ) vectors . Individual eGFP and otoferlin immunostainings are depicted as color lookup tables in ( A - C ) with warmer colors representing higher pixel intensities ) , maximum intensity projections of confocal optical sectionsPercentage of N ‑ and C ‑ terminal otoferlin labelled IHCs in dual ‑ AAV ‑ TS ( n = 10 mice ) and dual ‑ AAV ‑ Hyb ( n = 9 mice ) injected CD1B6F1 Otof - / - mice ( P18 ‑ 30 ) Average N - terminal and C ‑ terminal otoferlin immunofluorescence levels in dual ‑ AAV transduced Otof - / - and wild - type IHCs ( P23 - 30 ) . Otoferlin levels were normalized to immunofluorescence levels in non - transduced B6 wild - type IHCs for each antibody

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Figure 1Low magnification views of a CD1 Otof-/- organ of Corti (P23) transduced with otoferlin dual‑AAV‑TS vectors. IHCs: inner hair cells, OHCs: outer hair cells maximum intensity projections of confocal optical sectionsHigh magnification views of CD1B6F1 Otof-/- IHCs transduced with otoferlin dual‑AAV‑TS (P26) (B) and dual‑AAV‑Hyb (P26) (C) vectors. Individual eGFP and otoferlin immunostainings are depicted as color lookup tables in (A-C) with warmer colors representing higher pixel intensities ), maximum intensity projections of confocal optical sectionsPercentage of N‑ and C‑terminal otoferlin labelled IHCs in dual‑AAV‑TS (n=10 mice) and dual‑AAV‑Hyb (n=9 mice) injected CD1B6F1 Otof-/- mice (P18‑30)Average N-terminal and C‑terminal otoferlin immunofluorescence levels in dual‑AAV transduced Otof-/- and wild-type IHCs (P23-30). Otoferlin levels were normalized to immunofluorescence levels in non-transduced B6 wild-type IHCs for each antibody separately


Figure 2High magnification views of IHCs immunolabeled for otoferlin and synaptic ribbons ( CtBP2 ) from wild ‑ type ( B6 : P27 , CD1B6F1 : P29 ) , dual - AAV injected CD1B6F1 Otof - / - ( dual - AAV - TS : P26 , dual - AAV - Hyb : P28 ) , and their contralateral non - injected ears . ( * ) Transduced cells . Maximum intensity projections of optical confocal sections . Scale bars : 5 µmSynaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild ‑ type ( B6 : n = 48 IHCs , CD1B6F1 : n = 108 IHCs ) , transduced Otof - / - ( dual ‑ AAV ‑ TS : n = 59 IHCs , dual - AAV - Hyb : n = 37 IHCs ) , and non - transduced Otof - / - IHCs from injected ( - AAV injected ear , n = 65 IHCs ) and contralateral non - injected ( - AAV non - injected ear , n = 46 IHCs ) ears ( P25 - 29 ) IHC synapses labelled with CtBP2 and the postsynaptic marker Shank1a in B6 wild - type and Otof - / - P6 and P14 organs of Corti . Maximum intensity projections of optical confocal sections . Scale bars : 5 µm . D Synapse numbers quantified from IHCs in apical cochlear turns ( C ) of B6 wild - type ( P6 : n = 53 IHCs ; P14 : n = 73 IHCs ) and B6 Otof - / - ( P6 : n = 62 IHCs ; P14 : n = 65 IHCs ) mice at two different developmental stages ( P6 and P14 ) Ca2 + ‑ current - voltage relationship of control CD1B6F1 wild ‑ type ( n = 6 IHCs ) , dual - AAV - TS transduced ( n = 8 IHCs ) , and non ‑ transduced CD1B6F1 Otof - / - ( n = 10 IHCs ) IHCs ( P14 ‑ 18 ) Representative Ca2 + ‑ currents ( Ica ) and IHC plasma membrane capacitance increments ( ΔCm ) of a wild ‑ type control , transduced , and non ‑ transduced Otof - / - IHC in response to a 20 ms depolarization pulse at maximum Ca2 + ‑ current potentials ( typically - 14 mV ) Average exocytosis level measured as ΔCm ( G ) and corresponding Ca2 + ‑ current integrals ( QCa2 + ) ( H ) in wild ‑ type ( CD1B6F1 : n = 6 IHCs ; B6 : n = 11 IHCs ( B6 data replotted from Strenzke et al , 2016 ) ) , dual - AAV - TS transduced Otof - / - ( n = 8 IHCs ) , and non ‑ transduced ( n = 11 IHCs ) Otof - / - IHCs . Transduced IHCs that showed eGFP expression , but had almost no exocytosis are depicted as dashed lines ( not included into the average )

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

Figure 2High magnification views of IHCs immunolabeled for otoferlin and synaptic ribbons (CtBP2) from wild‑type (B6: P27, CD1B6F1: P29), dual-AAV injected CD1B6F1 Otof-/- (dual-AAV-TS: P26, dual-AAV-Hyb: P28), and their contralateral non-injected ears. (*) Transduced cells. Maximum intensity projections of optical confocal sections. Scale bars: 5 µmSynaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild‑type (B6: n=48 IHCs, CD1B6F1: n=108 IHCs), transduced Otof-/- (dual‑AAV‑TS: n=59 IHCs, dual-AAV-Hyb: n=37 IHCs), and non-transduced Otof-/- IHCs from injected (-AAV injected ear, n=65 IHCs) and contralateral non-injected (-AAV non-injected ear, n=46 IHCs) ears (P25-29)IHC synapses labelled with CtBP2 and the postsynaptic marker Shank1a in B6 wild-type and Otof-/- P6 and P14 organs of Corti. Maximum intensity projections of optical confocal sections. Scale bars: 5 µm. D Synapse numbers quantified from IHCs in apical cochlear turns (C) of B6 wild-type (P6: n= 53 IHCs; P14: n=73 IHCs) and B6 Otof-/- (P6: n=62 IHCs; P14: n=65 IHCs) mice at two different developmental stages (P6 and P14)Ca2+‑current-voltage relationship of control CD1B6F1 wild‑type (n=6 IHCs), dual-AAV-TS transduced (n=8 IHCs), and non‑transduced CD1B6F1 Otof-/- (n=10 IHCs) IHCs (P14‑18)Representative Ca2+‑currents (Ica) and IHC plasma membrane capacitance increments (ΔCm) of a wild‑type control, transduced, and non‑transduced Otof-/- IHC in response to a 20 ms depolarization pulse at maximum Ca2+‑current potentials (typically -14 mV)Average exocytosis level measured as ΔCm (G) and corresponding Ca2+‑current integrals (QCa2+) (H) in wild‑type (CD1B6F1: n=6 IHCs; B6: n=11 IHCs (B6 data replotted from Strenzke et al, 2016)), dual-AAV-TS transduced Otof-/- (n=8 IHCs), and non‑transduced (n=11 IHCs) Otof-/- IHCs. Transduced IHCs that showed eGFP expression, but had almost no exocytosis are depicted as dashed lines (not included into the average)


Figure 3Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual ‑ AAV ‑ TS and dual ‑ AAV ‑ Hyb injected CD1B6F1 Otof - / - animals . AAV2 / 6 . eGFP ( + AAV . eGFP ) and dual ‑ AAV ‑ TS injected CD1B6F1 wild ‑ type , and non ‑ injected control Otof - / - littermate ( - AAV ) mice served as controls . SP : summating potential ; ABR waves are indicated from I - VAverage ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks / sABR click sound ( C thresholds in otoferlin dual ‑ AAV treated Otof - / - mice compared to wild ‑ type and non ‑ treated Otof - / - control animalsABR tone burst ( D ) thresholds in otoferlin dual ‑ AAV treated Otof - / - mice compared to wild ‑ type and non ‑ treated Otof - / - control animals . In D , the two best animals are depicted with open circles . Animals with thresholds exceeding the maximum loudspeaker output ( arrows ) of 100 dB SPL for clicks and 90 dB SPL for tone bursts were set to 110 dB SPL and 100 dB SPL , respectively . Apical and basal cochlear turns are indicated as Apex and Base , respectivelySummed ABR wave I - V amplitudes at different click sound intensities in otoferlin dual ‑ AAV injected , non ‑ injected Otof - / - , and wild ‑ type control miceSummed ABR wave I ‑ V amplitudes of individual dual ‑ AAV ‑ TS treated CD1B6F1 Otof - / - animals ( n = 8 animals plotted against their full - length otoferlin IHC transduction

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Figure 3Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual‑AAV‑TS and dual‑AAV‑Hyb injected CD1B6F1 Otof-/- animals. AAV2/6.eGFP (+AAV.eGFP) and dual‑AAV‑TS injected CD1B6F1 wild‑type, and non‑injected control Otof-/- littermate (-AAV) mice served as controls. SP: summating potential; ABR waves are indicated from I-VAverage ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks/sABR click sound (C thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animalsABR tone burst (D) thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animals. In D, the two best animals are depicted with open circles. Animals with thresholds exceeding the maximum loudspeaker output (arrows) of 100 dB SPL for clicks and 90 dB SPL for tone bursts were set to 110 dB SPL and 100 dB SPL, respectively. Apical and basal cochlear turns are indicated as Apex and Base, respectivelySummed ABR wave I-V amplitudes at different click sound intensities in otoferlin dual‑AAV injected, non‑injected Otof-/-, and wild‑type control miceSummed ABR wave I‑V amplitudes of individual dual‑AAV‑TS treated CD1B6F1 Otof-/- animals (n=8 animals plotted against their full-length otoferlin IHC transduction rate


Figure 1A ) H & E and immunohistochemistry reveal that β - catenin accumulates in tumors emerging in one - year - old female miR - 424 ( 322 ) / 503 - / - mice . The panel is a representative image of n = 5 independent tumors . The scale bar indicates 100 micrometers ( µM ) . B ) Bar graphic showing that human breast cancers with deletion of the miR - 424 / 503 locus have higher protein levels of β - catenin ( RPPA ) . P - value is calculated based on one - sided Wilcoxon rank - sum test . In the box - plots in B and C the relative expression of β - catenin in the RPPA data from TCGA is shown . The yellow line indicates the median and the bars indicate the 75th percentile ( Q3 ) + 1 . 5 interquartile range ( IQR ) . C ) The bar graph shows that human breast cancers with low expression levels of miR - 424 and miR - 503 have higher protein levels of β - catenin . P - value is calculated based on one - sided Wilcoxon rank - sum test .

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Figure 1A) H&E and immunohistochemistry reveal that β-catenin accumulates in tumors emerging in one-year-old female miR-424(322)/503-/- mice. The panel is a representative image of n=5 independent tumors. The scale bar indicates 100 micrometers (µM).B) Bar graphic showing that human breast cancers with deletion of the miR-424/503 locus have higher protein levels of β-catenin (RPPA). P-value is calculated based on one-sided Wilcoxon rank-sum test. In the box-plots in B and C the relative expression of β-catenin in the RPPA data from TCGA is shown. The yellow line indicates the median and the bars indicate the 75th percentile (Q3)+1.5 interquartile range (IQR).C) The bar graph shows that human breast cancers with low expression levels of miR-424 and miR-503 have higher protein levels of β-catenin. P-value is calculated based on one-sided Wilcoxon rank-sum test.


Figure 2B ) The bar graphs show the number of PROCR + in 1 - year - old female WT and miR - 424 ( 322 ) / 503 KO mice during diestrus . The error bars show the standard deviation . E ) The panel shows the lack of induction of bona fide WNT targets in miR - over expressing cells from panel C compared with EV control . UT indicates samples untreated with WNT3 .

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Figure 2B) The bar graphs show the number of PROCR+ in 1-year-old female WT and miR-424(322)/503 KO mice during diestrus. The error bars show the standard deviation.E) The panel shows the lack of induction of bona fide WNT targets in miR-over expressing cells from panel C compared with EV control. UT indicates samples untreated with WNT3.


Figure 3D ) Western blot of LRP6 and TBLR1 in the miR - cluster - Dox - MCF - 10A model upon upregulation of miR - cluster with 100 ng / ml of doxycycline for the specified number of days . The numbers below the blots indicate the quantitation by densitometry of protein expression relative to β - actin . The expression level of miR - 424 and miR - 503 is indicated in the bar graph below the blots . UN indicates samples untreated with doxycycline . The error bars show the standard deviation . F ) Western blot of LRP6 and TBLR1 expression in WT and miR - 424 / 503 - KO basal cell - derived organoids after 14 days in culture with and without Wnt3a ( 100ng / ml ) . The arrow shows the correct TBLR1 band . G ) IHC staining showing that LRP6 accumulates in the epithelium of female miR - 424 ( 322 ) / 503 - / - mice ( 1 - year - old ) . The scale bar indicates 100 micrometers ( µM ) .

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Figure 3D) Western blot of LRP6 and TBLR1 in the miR-cluster-Dox-MCF-10A model upon upregulation of miR-cluster with 100 ng/ml of doxycycline for the specified number of days. The numbers below the blots indicate the quantitation by densitometry of protein expression relative to β-actin. The expression level of miR-424 and miR-503 is indicated in the bar graph below the blots. UN indicates samples untreated with doxycycline. The error bars show the standard deviation.F) Western blot of LRP6 and TBLR1 expression in WT and miR-424/503-KO basal cell-derived organoids after 14 days in culture with and without Wnt3a (100ng/ml). The arrow shows the correct TBLR1 band.G) IHC staining showing that LRP6 accumulates in the epithelium of female miR-424(322)/503-/- mice (1-year-old). The scale bar indicates 100 micrometers (µM).


Figure 4A ) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR - cluster - Dox - MCF - 10A cells after the miR - 424 / 503 cluster is induced with 100ng / ml of dox for three days . A CDC25A - binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls . The error bars show the standard deviation . B ) The WT - blot shows the expression of LRP6 upon upregulation of miR - 424 / 503 in miR - cluster - Dox - MCF - 10A cells ( Dox + ) when one or both of the conserved microRNA binding sites ( # 1 and # 3 ) are blocked by target protection . C ) The WT - blot shows the expression of LRP6 and β - catenin in cells in miR - cluster - Dox - MCF - 10A cells cultured in the presence of Dox and with and without WNT3a ligand ( 100ng / ml for 6 hours ) when the microRNA binding sites # 1 and # 3 are blocked by target protection .

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 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

Figure 4A) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR-cluster-Dox-MCF-10A cells after the miR-424/503 cluster is induced with 100ng/ml of dox for three days. A CDC25A-binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls. The error bars show the standard deviation.B) The WT-blot shows the expression of LRP6 upon upregulation of miR-424/503 in miR-cluster-Dox-MCF-10A cells (Dox+) when one or both of the conserved microRNA binding sites (#1 and #3) are blocked by target protection.C) The WT-blot shows the expression of LRP6 and β-catenin in cells in miR-cluster-Dox-MCF-10A cells cultured in the presence of Dox and with and without WNT3a ligand (100ng/ml for 6 hours) when the microRNA binding sites #1 and #3 are blocked by target protection.


Figure 1Representative composite immune - fluorescent images of whole hair follicle units from non - lesional and lesional scalp psoriasis patients . K15 ( green ) , c - JUN ( red A , C ) and JUNB ( red B , D ) and DAPI ( blue ) . Confocal images of the bulge region of human psoriatic hair follicles from non - lesional and lesional regions of the scalp . K15 ( green ) , c - JUN ( red E , G ) and JUNB ( red F , H ) and DAPI ( blue ) . Percentage of HF - SCs ( K15 + ) that express c - JUN ( I ) and JUNB ( J ) in the bulge region of human psoriatic hair follicles from non - lesional and lesional scalp in comparison with healthy scalp . n = 2 - 6 hair follicles in anagen per group from five psoriatic patients and two healthy patients .

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Figure 1Representative composite immune-fluorescent images of whole hair follicle units from non-lesional and lesional scalp psoriasis patients. K15 (green), c-JUN (red A, C) and JUNB (red B, D) and DAPI (blue).Confocal images of the bulge region of human psoriatic hair follicles from non-lesional and lesional regions of the scalp. K15 (green), c-JUN (red E, G) and JUNB (red F, H) and DAPI (blue).Percentage of HF-SCs (K15+) that express c-JUN (I) and JUNB (J) in the bulge region of human psoriatic hair follicles from non-lesional and lesional scalp in comparison with healthy scalp. n=2-6 hair follicles in anagen per group from five psoriatic patients and two healthy patients.


Figure 2Composite immunofluorescent images showing GFP expression of whole - ear sections from DKO * - mT / mG mice at day 5 , 7 , 15 and 30 . Increased GFP expression is shown at day 7 followed by progressive decrease of GFP + keratinocytes ( white dotted line represents basal layer , and red dotted line represents outermost skin layer ) . n = 3 per time point . Quantification analysis of GFP expression ( top ) and epidermal thickness ( bottom ) of DKO * mice at different time points during psoriasis - like disease progression . n = 6 per time point . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two tailed - test relative to control group ) . Quantification analysis of cleaved Caspase - 3 ( cCas3 top ) and Ki67 ( bottom ) in ear skin of DKO * mice at different time points during psoriasis - like disease progression . n = 3 per time point . Intravital confocal time - lapse imaging of non - mutant Tomato keratinocytes and mutant GFP keratinocytes of DKO * - mT / mG at day 7 after first tamoxifen injection . Dotted circles emphasize areas in which mutant GFP + keratinocytes are replaced by non - mutant Tomato + keratinocytes . Percentage of GFP + area eliminated during intravital confocal time - lapse imaging at day 5 , 7 and 15 in DKO * - mT / mG mice ( Time - lapse 1 : 8h ) . Positive value : Area of GFP increased . Negative value : Area of GFP reduced . Fluorescence imaging of DKO * - mT / mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP + keratinocytes reside along the hair follicles . White dotted line separates epidermis and dermis . White arrows represent GFP + hair follicles . Intravital confocal time - lapse imaging of DKO * - mT / mG at day 15 after first tamoxifen injection . Mutant GFP + Keratinocytes ( white arrows ) are maintained around hair follicles .

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Figure 2Composite immunofluorescent images showing GFP expression of whole-ear sections from DKO*-mT/mG mice at day 5, 7, 15 and 30. Increased GFP expression is shown at day 7 followed by progressive decrease of GFP+ keratinocytes (white dotted line represents basal layer, and red dotted line represents outermost skin layer). n=3 per time point. Quantification analysis of GFP expression (top) and epidermal thickness (bottom) of DKO* mice at different time points during psoriasis-like disease progression. n=6 per time point. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to control group).Quantification analysis of cleaved Caspase-3 (cCas3 top) and Ki67 (bottom) in ear skin of DKO* mice at different time points during psoriasis-like disease progression. n=3 per time point.Intravital confocal time-lapse imaging of non-mutant Tomato keratinocytes and mutant GFP keratinocytes of DKO*-mT/mG at day 7 after first tamoxifen injection. Dotted circles emphasize areas in which mutant GFP+ keratinocytes are replaced by non-mutant Tomato+ keratinocytes.Percentage of GFP+ area eliminated during intravital confocal time-lapse imaging at day 5, 7 and 15 in DKO*-mT/mG mice (Time-lapse 1:8h). Positive value: Area of GFP increased. Negative value: Area of GFP reduced.Fluorescence imaging of DKO*-mT/mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP+ keratinocytes reside along the hair follicles. White dotted line separates epidermis and dermis. White arrows represent GFP+ hair follicles.Intravital confocal time-lapse imaging of DKO*-mT/mG at day 15 after first tamoxifen injection. Mutant GFP+ Keratinocytes (white arrows) are maintained around hair follicles.


Figure 3Ear pictures of control , DKO * , and DKO * K15 mice after 15 days of induction show clear signs of inflammation and psoriatic - like scales in both models . Representative images of hematoxylin and eosin ( H & E ) staining of ear sections from control , DKO * and DKO * K15 mice . Ear thickness measurement at different time points during psoriasis - like development in control , DKO * and DKO * K15 mice . n > 5 per group . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two tailed - test relative to control group ) . FACS quantification of CD45 + cells from ear skin of control , DKO * and DKO * K15 mice at different time points during psoriasis - like development . n > 5 per group . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two tailed - test relative to controls ) . Quantification of IL - 17A , IL - 6 and LCN2 production in sera of control , DKO * and DKO * K15 mice at day 30 of psoriasis - like disease by ELISA . n > 4 per group . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two tailed - test relative to controls ) . Quantification of total number of GFP + epidermal cells by FACS analysis from DKO * - mT / mG and DKO * K15 - mT / mG mice at different time points during psoriasis - like progression compared to Co - mT / mG mice . n = 3 - 6 per group and time point . Representative immunofluorescence images of ear sections from CoK15 - mT / mG and DKO * K15 - mT / mG mouse models after 15 days of induction . Red ( Tomato ) , green ( GFP ) and blue ( DAPI ) . Hyperplasia of Tomato + keratinocytes in psoriatic - like DKO * K15 - mT / mG mouse with clusters of GFP + keratinocytes are derived from hair follicle K15 + GFP + stem cells . n = 3 per group . Dotted lines separate epidermis and dermis . Quantification analysis of cleaved Caspase - 3 ( cCas3 ) and Ki67 in ear skin of DKO * K15 - mT / mG mice at different time points during psoriasis - like disease progression .

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

Figure 3Ear pictures of control, DKO*, and DKO*K15 mice after 15 days of induction show clear signs of inflammation and psoriatic-like scales in both models.Representative images of hematoxylin and eosin (H&E) staining of ear sections from control, DKO* and DKO*K15 mice.Ear thickness measurement at different time points during psoriasis-like development in control, DKO* and DKO*K15 mice. n > 5 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to control group).FACS quantification of CD45+ cells from ear skin of control, DKO* and DKO*K15 mice at different time points during psoriasis-like development. n > 5 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to controls).Quantification of IL-17A, IL-6 and LCN2 production in sera of control, DKO* and DKO*K15 mice at day 30 of psoriasis-like disease by ELISA. n > 4 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to controls).Quantification of total number of GFP+ epidermal cells by FACS analysis from DKO*-mT/mG and DKO*K15-mT/mG mice at different time points during psoriasis-like progression compared to Co-mT/mG mice. n = 3-6 per group and time point.Representative immunofluorescence images of ear sections from CoK15-mT/mG and DKO*K15-mT/mG mouse models after 15 days of induction. Red (Tomato), green (GFP) and blue (DAPI). Hyperplasia of Tomato+ keratinocytes in psoriatic-like DKO*K15-mT/mG mouse with clusters of GFP+ keratinocytes are derived from hair follicle K15+ GFP+ stem cells. n=3 per group. Dotted lines separate epidermis and dermis. Quantification analysis of cleaved Caspase-3 (cCas3) and Ki67 in ear skin of DKO*K15-mT/mG mice at different time points during psoriasis-like disease progression.


Figure 4Average number of live and dead ( TOPRO - 3 + Dapi + ) primary keratinocytes derived from HF - SCGFP at different time points of time lapse capture during 48 hours . There is a slight increase over time of live HF - SCs from DKO * mice when compared to control cells , and no difference in average of dead cells . n = 4 fields from two independent experiments . Average number of live and dead ( TOPRO - 3 + DAPI + ) primary keratinocytes derived from basal keratinocytesGFP show a significant increase in DKO * basal live keratinocytes at 12 hours , followed by a significant increase in cell death at 48 hours . Average number of live primary keratinocytes from co - cultures of GFP + with Tomato + bulge HF - SCs at different time points of time lapse capture during 48 hours . Co - culture of HF - SCGFP with HF - SCTom from DKO * - mT / mG mice shows a significant increase of HF - SCTom , while the HF - SCTom from DKO * - mT / mG mice show a normal growth when they are co - culture with HF - SCGFP from control mice . Average number of live primary keratinocytes from co - cultures of GFP + with Tomato + basal keratinocytes at different time points of time lapse capture during 48 hours . Co - culture of b - KCGFP with b - KCTom from DKO * - mT / mG mice show significant increase of b - KCTom , while these b - KCTom from DKO * - mT / mG mice show a normal growth when they are co - culture with b - KCGFP from control mice . Average total number of live Tomato + basal keratinocytes ( from DKO * or control mice ) at different time points cultured in the bottom part of trans - well system cultures with GFP + basal keratinocytes ( from DKO * or Control mice ) cultured in the upper trans - well ( Tw ) . n = 5 fields from three independent experiments . Tom + basal keratinocytes significantly increased when these were cultured with mutant GFP + basal keratinocytes in the trans - well in comparison with control GFP + basal keratinocytes .

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Figure 4Average number of live and dead (TOPRO-3+ Dapi+) primary keratinocytes derived from HF-SCGFP at different time points of time lapse capture during 48 hours. There is a slight increase over time of live HF-SCs from DKO* mice when compared to control cells, and no difference in average of dead cells. n = 4 fields from two independent experiments. Average number of live and dead (TOPRO-3+ DAPI+) primary keratinocytes derived from basal keratinocytesGFP show a significant increase in DKO* basal live keratinocytes at 12 hours, followed by a significant increase in cell death at 48 hours.Average number of live primary keratinocytes from co-cultures of GFP+ with Tomato+ bulge HF-SCs at different time points of time lapse capture during 48 hours. Co-culture of HF-SCGFP with HF-SCTom from DKO*-mT/mG mice shows a significant increase of HF-SCTom, while the HF-SCTom from DKO*-mT/mG mice show a normal growth when they are co-culture with HF-SCGFP from control mice. Average number of live primary keratinocytes from co-cultures of GFP+ with Tomato+ basal keratinocytes at different time points of time lapse capture during 48 hours. Co-culture of b-KCGFP with b-KCTom from DKO*-mT/mG mice show significant increase of b-KCTom, while these b-KCTom from DKO*-mT/mG mice show a normal growth when they are co-culture with b-KCGFP from control mice.Average total number of live Tomato+ basal keratinocytes (from DKO* or control mice) at different time points cultured in the bottom part of trans-well system cultures with GFP+ basal keratinocytes (from DKO* or Control mice) cultured in the upper trans-well (Tw). n = 5 fields from three independent experiments. Tom+ basal keratinocytes significantly increased when these were cultured with mutant GFP+ basal keratinocytes in the trans-well in comparison with control GFP+ basal keratinocytes.


Figure 5Heat map for specific genes relative to biological process enriched based on Gene Ontology IDs for DEGs that are : i ) exclusively up - regulated in HF - SCGFP ; ii ) exclusively up - regulated in HF - SCTom ; iii ) commonly up - regulated in HF - SCGFP and HF - SCTom . Gene expression of TSLP in different epidermal cell subpopulations sorted at mid - term ( D7 ) or late - term ( D30 ) of psoriasis - like progression in DKO * mice . protein expression of TSLP in different epidermal cell subpopulations sorted at mid - term ( D7 ) or late - term ( D30 ) of psoriasis - like progression in DKO * mice . n = 2 - 3 . Quantification of TSLP production in sera of control , DKO * and DKO * K15 mice at day 30 of psoriasis - like disease by ELISA . n = 4 per group .

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Figure 5Heat map for specific genes relative to biological process enriched based on Gene Ontology IDs for DEGs that are: i) exclusively up-regulated in HF-SCGFP; ii) exclusively up-regulated in HF-SCTom; iii) commonly up-regulated in HF-SCGFP and HF-SCTom.Gene expression of TSLP in different epidermal cell subpopulations sorted at mid-term (D7) or late-term (D30) of psoriasis-like progression in DKO* mice.protein expression of TSLP in different epidermal cell subpopulations sorted at mid-term (D7) or late-term (D30) of psoriasis-like progression in DKO* mice. n=2-3.Quantification of TSLP production in sera of control, DKO* and DKO*K15 mice at day 30 of psoriasis-like disease by ELISA. n = 4 per group.


Figure 6TSLP production in conditioned media quantified by ELISA three days after infection with Ad - empty or Ad - cre . n = 3 independent experiments . Percentage of EdU + Tomato + keratinocytes after blocking with anti - TSLP . Anti - TSLP neutralizes the hyper - proliferation of non - mutantTom KCs in vitro after infection with Ad - cre . Gene expression of Tslpr after TSLP neutralization is reduced . Gene expression of IL - 7ra after TSLP neutralization is increased . Gene expression of VEGFα after TSLP neutralization is reduced . Representative images of treated ears after 9 days of anti - TSLP or IgG treatment . Longitudinal analysis of ear thickness in DKO * mice at different time points during psoriasis - like disease progressionPercentage of EdU + epidermal cells ( CD45 - CD49f + ) after blocking with anti - TSLP in DKO * and DKO * 15 miceRepresentative immune - fluorescence images of ear sections from control or DKO * mice treated with IgG or anti - TSLP ) stained for Ki67 . Ki67 expression was reduced in mice treated with anti - TSLP ( n = 3 ) .

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Figure 6TSLP production in conditioned media quantified by ELISA three days after infection with Ad-empty or Ad-cre. n=3 independent experiments.Percentage of EdU+ Tomato+ keratinocytes after blocking with anti-TSLP. Anti-TSLP neutralizes the hyper-proliferation of non-mutantTom KCs in vitro after infection with Ad-cre.Gene expression of Tslpr after TSLP neutralization is reduced. Gene expression of IL-7ra after TSLP neutralization is increased. Gene expression of VEGFα after TSLP neutralization is reduced.Representative images of treated ears after 9 days of anti-TSLP or IgG treatment.Longitudinal analysis of ear thickness in DKO* mice at different time points during psoriasis-like disease progressionPercentage of EdU+ epidermal cells (CD45-CD49f+) after blocking with anti-TSLP in DKO* and DKO*15 miceRepresentative immune-fluorescence images of ear sections from control or DKO* mice treated with IgG or anti-TSLP) stained for Ki67. Ki67 expression was reduced in mice treated with anti-TSLP (n =3).


Figure 7Representative composite immune - fluorescent images of whole hair follicle units by confocal from non - lesional and lesional scalp psoriasis patients . c - JUN ( purple ) , K - 15 ( green ) , TSLP ( red ) and DAPI ( blue ) . TSLP intensity measurement in hair follicle units and epidermis by ImageJ from healthy scalp , non - lesional and lesional scalp psoriasis patients . Representation of fold change relative to control healthy scalp . n = 3 samples . Representative composite immune - fluorescent images of whole hair follicle units by confocal from non - lesional and lesional scalp psoriasis patients ( D ) and magnification images of specific regions from the HFs ( E ) . CD200 ( green ) , c - JUN ( white ) , TSLP ( red ) , DAPI ( blue ) .

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Figure 7Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients. c-JUN (purple), K-15 (green), TSLP (red) and DAPI (blue).TSLP intensity measurement in hair follicle units and epidermis by ImageJ from healthy scalp, non-lesional and lesional scalp psoriasis patients. Representation of fold change relative to control healthy scalp. n= 3 samples.Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients (D) and magnification images of specific regions from the HFs (E). CD200 (green), c-JUN (white), TSLP (red), DAPI (blue).


figf1b ) The affinity of the UVRAG - C2 domain for phospholipids was assessed using a protein - lipid overlay assay . The left panel indicates the identity of lipid species on PtdIns strips . Bacterial - purified GST fusion of UVRAG - C2 ( wild type , WT ) , but not GST , binds PtdIns ( 3 ) P , PtdIns ( 4 ) P and PtdIns ( 5 ) P . The K78A / R82A mutant of C2 is defective for phospholipid - binding , whereas the K87A / N88A mutant is partially impaired . Binding of the FYVE and PH domains of Hrs and FAPP1 , respectively , to PtdIns ( 3 ) P and PtdIns ( 4 ) P served as quality controls ( right ) . PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; LPA , lysophosphatidic acid ; LPC , lysophosphatidylcholine ; S1P , sphingosine 1 - phosphate ; PA , phosphatidic acid ; PS , phosphatidylserine . ( c ) The relative affinity of the GST - fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments ( n = 5 ) . Error bars represent standard deviation ( s . d . ) . A . U . , arbitrary units . ( d ) Binding of full - length UVRAG ( untagged ) to lipid blots . The same PtdIns strips ( in b ) were used and immunoblotted with antibodies against IgG ( right ) and UVRAG ( left ) . ( g ) Pulldown of UVRAG - C2 domain GST - fusion proteins by liposomes bearing the indicated phosphoinositides . GST - Hrs - FYVE and GST - FAPP1 - PH are also shown .

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figf1b) The affinity of the UVRAG-C2 domain for phospholipids was assessed using a protein-lipid overlay assay. The left panel indicates the identity of lipid species on PtdIns strips. Bacterial-purified GST fusion of UVRAG-C2 (wild type, WT), but not GST, binds PtdIns(3)P, PtdIns(4)P and PtdIns(5)P. The K78A/R82A mutant of C2 is defective for phospholipid-binding, whereas the K87A/N88A mutant is partially impaired. Binding of the FYVE and PH domains of Hrs and FAPP1, respectively, to PtdIns(3)P and PtdIns(4)P served as quality controls (right). PE, phosphatidylethanolamine; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.(c) The relative affinity of the GST-fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments (n = 5). Error bars represent standard deviation (s.d.). A.U., arbitrary units.(d) Binding of full-length UVRAG (untagged) to lipid blots. The same PtdIns strips (in b) were used and immunoblotted with antibodies against IgG (right) and UVRAG (left).(g) Pulldown of UVRAG-C2 domain GST-fusion proteins by liposomes bearing the indicated phosphoinositides. GST-Hrs-FYVE and GST-FAPP1-PH are also shown.


figf2 ( a - k ) Confocal analyses of the subcellular co - localization of endogenous UVRAG with ER markers , including endogenous calnexin ( red , a ) , endogenous PDI ( red , b ) , and overexpressed DSred - ER ( red , c ) ; with Golgi markers , including endogenous GM130 and p115 ( red , d , e , cis - Golgi ) , overexpressed GFP - Man II ( green , f , medial - Golgi ) , endogenous TGN46 ( red , g , trans - Golgi ) , and overexpressed PtdIns ( 4 ) P probe , GFP - FAPP1 - PH ( green , h ) ; with endogenous coatomer proteins , including Sec31 ( red , i , COPII - related ) and β ′ - COP ( red , j , COPI - related ) ; and with the endosome marker GFP - p40phox ( PX ) ( green , k ) in HeLa cells . The insets show a high magnification of the selected areas . Scale bars , 10 μm . ( l ) Confocal microscopic quantification of co - localization of UVRAG with the indicated markers ( data are mean ± s . d . , n = 150 cells obtained by gathering data from three independent experiments ) ; * * P0 . 01 ; * * * P0 . 001 .

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figf2(a-k) Confocal analyses of the subcellular co-localization of endogenous UVRAG with ER markers, including endogenous calnexin (red, a), endogenous PDI (red, b), and overexpressed DSred-ER (red, c); with Golgi markers, including endogenous GM130 and p115 (red, d,e, cis-Golgi), overexpressed GFP-Man II (green, f, medial-Golgi), endogenous TGN46 (red, g, trans-Golgi), and overexpressed PtdIns(4)P probe, GFP-FAPP1-PH (green, h); with endogenous coatomer proteins, including Sec31 (red, i, COPII-related) and β′-COP (red, j, COPI-related); and with the endosome marker GFP-p40phox(PX) (green, k) in HeLa cells. The insets show a high magnification of the selected areas. Scale bars, 10 μm. (l) Confocal microscopic quantification of co-localization of UVRAG with the indicated markers (data are mean±s.d., n = 150 cells obtained by gathering data from three independent experiments); **P0.01; ***P0.001.


figf3a ) UVRAG is released from the ER membrane in cells overexpressing MTMR3 . HeLa cells transfected with MTMR3 - mStrawberry or the catalytically inactive mutant MTMR3C413S were stained with anti - UVRAG antibody and processed for confocal microscopy . Asterisks indicate MTMR3 - transfected cells ( left ) . The percentage of transfected cells with diffused staining of UVRAG was quantified ( middle ) . Data are mean ± s . d . ; n = 240 cells collected from four independent experiments ; * * P0 . 01 . Western blot analysis shows the levels of endogenous UVRAG in the presence or absence of MTMR3 ( right ) . ( b ) Wortmannin treatment releases UVRAG from the ER . HeLa cells were treated with 100 nM wortmannin for 1h , and processed for confocal microscopy using anti - UVRAG and anti - PDI antibodies ( top panel ) . UT , untreated . Insets highlight the relative localization of UVRAG at the ER . Confocal co - localization is indicated ( bottom left panel ) . Data are mean ± s . d . ; n = 75 cells for each group from three independent experiments ; * * * P0 . 001 . Endogenous UVRAG and PDI expression before and after wortmannin treatment were confirmed by immunoblotting ( bottom right panel ) . ( c ) Mutation of the C2 domain leaves UVRAG unable to associate with the ER . HeLa cells expressing Flag - UVRAG ( WT ) or its K78A / R82A mutant were fixed and processed for confocal microscopy for co - localization with the ER marker PDI ( top panel ) . Insets highlight the relative distribution of UVRAG at the ER . UVRAG and endogenous PDI expression were confirmed by immunoblotting ( bottom panel ) . See Supplementary Fig . S9 for uncropped data . Scale bars , 10 μm .

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figf3a) UVRAG is released from the ER membrane in cells overexpressing MTMR3. HeLa cells transfected with MTMR3-mStrawberry or the catalytically inactive mutant MTMR3C413S were stained with anti-UVRAG antibody and processed for confocal microscopy. Asterisks indicate MTMR3-transfected cells (left). The percentage of transfected cells with diffused staining of UVRAG was quantified (middle). Data are mean±s.d.; n = 240 cells collected from four independent experiments; **P0.01. Western blot analysis shows the levels of endogenous UVRAG in the presence or absence of MTMR3 (right).(b) Wortmannin treatment releases UVRAG from the ER. HeLa cells were treated with 100 nM wortmannin for 1h, and processed for confocal microscopy using anti-UVRAG and anti-PDI antibodies (top panel). UT, untreated. Insets highlight the relative localization of UVRAG at the ER. Confocal co-localization is indicated (bottom left panel). Data are mean±s.d.; n = 75 cells for each group from three independent experiments; ***P0.001. Endogenous UVRAG and PDI expression before and after wortmannin treatment were confirmed by immunoblotting (bottom right panel).(c) Mutation of the C2 domain leaves UVRAG unable to associate with the ER. HeLa cells expressing Flag-UVRAG (WT) or its K78A/R82A mutant were fixed and processed for confocal microscopy for co-localization with the ER marker PDI (top panel). Insets highlight the relative distribution of UVRAG at the ER. UVRAG and endogenous PDI expression were confirmed by immunoblotting (bottom panel). See Supplementary Fig. S9 for uncropped data. Scale bars, 10 μm.


figf4 ( a ) The UVRAG C - terminal 270 - 442 region interacts with RINT - 1 . Cells ( HEK293T ) were co - transfected with HA - RINT - 1 , together with Flag - UVRAG or its mutant derivatives , and whole - cell lysates ( WCLs ) were immunoprecipitated ( IP ) with anti - Flag , followed by immunoblotting ( IB ) with an anti - HA antibody . c ) Interaction between endogenous UVRAG and RINT - 1 . WCLs of HEK293T cells were used for immunoprecipitation with control serum ( control ) or an anti - UVRAG ( left ) or anti - RINT - 1 antibody ( right ) , followed by immunoblotting with the indicated antibodies . The bottom panel shows endogenous protein expression . d ) ER localization of RINT - 1 , ZW10 and UVRAG . HeLa cells were stained with anti - UVRAG ( green ) , anti - PDI ( ER marker , red ) , and anti - ZW10 or anti - RINT - 1 ( blue ) , followed by confocal microscopy . Insets highlight UVRAG co - localization with RINT - 1 and ZW10 at the ER . Scale bars , 10 μm . ( e ) Interaction between endogenous UVRAG and the RINT - 1 - ZW10 - NAG tethering complex proteins . WCLs of HEK293T cells were used for immunoprecipitation with control serum ( IgG ) or an anti - UVRAG ( first panel ) , anti - RINT - 1 ( second panel ) , anti - ZW10 ( third panel ) and anti - NAG ( fourth panel ) , followed by immunoblotting with the indicated antibodies . As input , 10 % of the WCLs was used . Syn 18 , syntaxin 18 ; Syn 6 , syntaxin 6 . See Supplementary Fig . S9 for uncropped data .

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figf4(a) The UVRAG C-terminal 270-442 region interacts with RINT-1. Cells (HEK293T) were co-transfected with HA-RINT-1, together with Flag-UVRAG or its mutant derivatives, and whole-cell lysates (WCLs) were immunoprecipitated (IP) with anti-Flag, followed by immunoblotting (IB) with an anti-HA antibody.c) Interaction between endogenous UVRAG and RINT-1. WCLs of HEK293T cells were used for immunoprecipitation with control serum (control) or an anti-UVRAG (left) or anti-RINT-1 antibody (right), followed by immunoblotting with the indicated antibodies. The bottom panel shows endogenous protein expression.d) ER localization of RINT-1, ZW10 and UVRAG. HeLa cells were stained with anti-UVRAG (green), anti-PDI (ER marker, red), and anti-ZW10 or anti-RINT-1 (blue), followed by confocal microscopy. Insets highlight UVRAG co-localization with RINT-1 and ZW10 at the ER. Scale bars, 10 μm.(e) Interaction between endogenous UVRAG and the RINT-1-ZW10-NAG tethering complex proteins. WCLs of HEK293T cells were used for immunoprecipitation with control serum (IgG) or an anti-UVRAG (first panel), anti-RINT-1 (second panel), anti-ZW10 (third panel) and anti-NAG (fourth panel), followed by immunoblotting with the indicated antibodies. As input, 10% of the WCLs was used. Syn 18, syntaxin 18; Syn 6, syntaxin 6. See Supplementary Fig. S9 for uncropped data.