Figure 5 : HSFA2 associates with HS memory - related genes transiently after an acclimatizing HS . Binding of HSFA2 - YFP to the promoter regions of HSP18 . 2 ( A ) , HSP22 . 0 ( B ) , APX2 ( C ) , HSP70 ( D ) and ACTIN7 ( E ) was determined by ChIP - qPCR . Quantifications represent the enrichment relative to input in % . Red - orange - yellow bars represent time points ( 0 . 5 h - 4 h - 28 h ) after a ACC and grey bars represent the respective NHS control samples . Schematics show the promoter region of the respective gene with regions analyzed ( arrow , TSS ; grey bar , 5 ' UTR ; black bar , exon ; black boxes , heat shock elements ( 5 ' - nnGnAnnTnCtn - 3 ' ) . Regions are drawn to scale . Intergenic control region 1 is 4610 bp ( HSP18 . 2 ) and 4192 bp ( HSP70 ) downstream of the 3 ' UTR and 2570 bp ( HSP22 . 0 ) or 3123 bp ( APX2 ) upstream of the TSS , respectively . One representative experiment out of at least four biological replicates is shown . Error bars are SD of three replicates .

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Figure 5: HSFA2 associates with HS memory-related genes transiently after an acclimatizing HS.Binding of HSFA2-YFP to the promoter regions of HSP18.2 (A), HSP22.0 (B), APX2 (C), HSP70 (D) and ACTIN7 (E) was determined by ChIP-qPCR. Quantifications represent the enrichment relative to input in %. Red-orange-yellow bars represent time points (0.5 h-4 h-28 h) after a ACC and grey bars represent the respective NHS control samples. Schematics show the promoter region of the respective gene with regions analyzed (arrow, TSS; grey bar, 5'UTR; black bar, exon; black boxes, heat shock elements (5'-nnGnAnnTnCtn-3'). Regions are drawn to scale. Intergenic control region 1 is 4610 bp (HSP18.2) and 4192 bp (HSP70) downstream of the 3'UTR and 2570 bp (HSP22.0) or 3123 bp (APX2) upstream of the TSS, respectively. One representative experiment out of at least four biological replicates is shown. Error bars are SD of three replicates.


Figure 6 : Expression profiles of HS memory - related genes after a recurring HS show HSFA2 - dependent transcriptional memoryResponse of selected HS - inducible loci upon recurring HS as determined by qRT - PCR . Transcript levels of APX2 ( A ) , HSP18 . 2 ( B ) , HSP22 . 0 ( C ) , and HSP70 ( D ) in Col - 0 ( blue bars ) and hsfa2 ( orange bars ) at the indicated time points after a single HS or two HS separated by 2 d . Plants were subjected to 37 ° C for 60 min on either d 4 , d 6 or on d 4 + d 6 after germination . NHS samples were harvested at the same time points as HS d 4 samples . Transcript levels of four biological replicates normalized to TUB6 are shown . Small brackets compare the two genotypes at the same condition , large brackets compare the same genotype across two treatments ( at 1 h after treatment ) with Col - 0 indicated first ( Col - 0 / hsfa2 ) . Error bars show SE over four biological replicates . * , p < 0 . 05 ; * * , p < 0 . 01 ; Student ´ s t - test .

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Figure 6: Expression profiles of HS memory-related genes after a recurring HS show HSFA2-dependent transcriptional memoryResponse of selected HS-inducible loci upon recurring HS as determined by qRT-PCR. Transcript levels of APX2 (A), HSP18.2 (B), HSP22.0 (C), and HSP70 (D) in Col-0 (blue bars) and hsfa2 (orange bars) at the indicated time points after a single HS or two HS separated by 2 d. Plants were subjected to 37°C for 60 min on either d 4, d 6 or on d 4 + d 6 after germination. NHS samples were harvested at the same time points as HS d 4 samples. Transcript levels of four biological replicates normalized to TUB6 are shown. Small brackets compare the two genotypes at the same condition, large brackets compare the same genotype across two treatments (at 1 h after treatment) with Col-0 indicated first (Col-0/ hsfa2). Error bars show SE over four biological replicates. *, p<0.05; **, p<0.01; Student´s t-test.


b ) Histograms of GFP expression ( x - axis ) are presented from A549 cells transduced with the different viral vectors .

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b) Histograms of GFP expression (x-axis) are presented from A549 cells transduced with the different viral vectors.


c ) Percentage of GFP positive cells from the experiment described in ( b ) .

c) Percentage of GFP positive cells from the experiment described in (b).


. d ) A PCR assay along with primers , as illustrated in Appendix Figure S2a , was used to detect formation of LTR junction and thereby circularization of the vector . The expected size of PCR product is 552 bp from a 1 - LTR circle and 1029 bp from a 2 - LTR circle .

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. d) A PCR assay along with primers, as illustrated in Appendix Figure S2a, was used to detect formation of LTR junction and thereby circularization of the vector. The expected size of PCR product is 552 bp from a 1-LTR circle and 1029 bp from a 2-LTR circle.


. e ) Southern Blot analysis to detect integration events in LV - and NILV - S / MAR - transduced single cell clones with DNA probed against the viral genome . A 6 . 8 kb positive control from a linearized plasmid ( lane 3 ) was included for indication of the size of DNA circles formed by non - integrated NILV - S / MAR vector .

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. e) Southern Blot analysis to detect integration events in LV- and NILV-S/MAR-transduced single cell clones with DNA probed against the viral genome. A 6.8 kb positive control from a linearized plasmid (lane 3) was included for indication of the size of DNA circles formed by non-integrated NILV-S/MAR vector.


f - h ) A padlock probe assay , as illustrated in Appendix Figure S4 , was used to demonstrate persistence of proviral DNA either in a circular or integrated form . Representative images show DNA persistence in ( f ) LV - , ( g ) NILV - or ( h ) NILV - S / MAR - transduced cells . Each red dot ( Cy3 ) represents a single DNA molecule and cell nuclei were stained with Hoechst 33342 .

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f-h) A padlock probe assay, as illustrated in Appendix Figure S4, was used to demonstrate persistence of proviral DNA either in a circular or integrated form. Representative images show DNA persistence in (f) LV-, (g) NILV- or (h) NILV-S/MAR-transduced cells. Each red dot (Cy3) represents a single DNA molecule and cell nuclei were stained with Hoechst 33342.


c ) Flow cytometry histograms of RFP expression ( x - axis ) of A549 / RFP + Luc + cells at day 20 and 27 after NILV - S / MAR ( GFP , shNT ) or NILV - S / MAR ( GFP , shRFP ) transduction .

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c) Flow cytometry histograms of RFP expression (x-axis) of A549/RFP+Luc+ cells at day 20 and 27 after NILV-S/MAR(GFP, shNT) or NILV-S/MAR(GFP, shRFP) transduction.


d ) Luciferase activity of the A549 / RFP + Luc + cells at day 20 and day 44 after NILV - S / MAR ( GFP , shNT ) - or NILV - S / MAR ( GFP , shRFP ) - transduction .

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d) Luciferase activity of the A549/RFP+Luc+ cells at day 20 and day 44 after NILV-S/MAR(GFP, shNT)- or NILV-S/MAR(GFP, shRFP)-transduction.


e - h ) Representative fluorescence microscopy images show the expression of RFP from either NILV - S / MAR - transduced or un - transduced A549 / RFP + Luc + cells with cell nuclei stained with Hoechst 33342 ( blue ) . RFP signal ( orange ) were collected with a fixed exposure time ( 300 ms ) for equal comparison .

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e-h) Representative fluorescence microscopy images show the expression of RFP from either NILV-S/MAR-transduced or un-transduced A549/RFP+Luc+ cells with cell nuclei stained with Hoechst 33342 (blue). RFP signal (orange) were collected with a fixed exposure time (300 ms) for equal comparison.


c ) Percentage of CD19CAR expression on CD3 + T cells from each independent donor . d ) Histograms of CD19 CAR expression on T cells form one representative donor before and after expansion . e ) CD107a expression on transduced T cells ( CD3 + ) after exposure to CD19 + target cells ( Daudi ) for 24 h ( n = 6 ) . f ) IFN - γ production in the supernatant was analyzed by ELISA after 24 h of exposure of transduced T cells to target cells ( n = 6 ) . g ) Relative viability of target cells ( CD19 + Daudi ) , as an indication of T cell killing efficacy , were measured at different effector T cell to target cell ratio ( E : T ratio ) and normalized to the signal from target cells alone ( n = 6 ) . h ) Transduced T cells were labeled with a fluorescent cell membrane dye and co - cultured with CD19 + Daudi cells for 4 days . Dye dilution upon T cell division was monitored by flow cytometry and representative histograms are shown .

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c) Percentage of CD19CAR expression on CD3+ T cells from each independent donor. d) Histograms of CD19 CAR expression on T cells form one representative donor before and after expansion. e) CD107a expression on transduced T cells (CD3+) after exposure to CD19+ target cells (Daudi) for 24 h (n=6). f) IFN-γ production in the supernatant was analyzed by ELISA after 24 h of exposure of transduced T cells to target cells (n=6). g) Relative viability of target cells (CD19+ Daudi), as an indication of T cell killing efficacy, were measured at different effector T cell to target cell ratio (E:T ratio) and normalized to the signal from target cells alone (n=6). h) Transduced T cells were labeled with a fluorescent cell membrane dye and co-cultured with CD19+ Daudi cells for 4 days. Dye dilution upon T cell division was monitored by flow cytometry and representative histograms are shown.


i - m ) Mice were treated by intravenous injection of NILV - S / MAR ( Mock ) , LV ( CD19CAR ) and NILV - S / MAR ( CD19CAR ) - transduced T cells ( 8x106 cells / injection ) at days 18 , 21 , 24 after tumor implantation . Tumor growth was monitored by caliper measurements . i - k ) Tumor volume of individual mice in each treatment groups are presented . l ) The average tumor volume of each treatment group is presented and the values on day 37 were statistically compared ( n = 12 in LV group , n = 9 in NILV - S / MAR group and n = 11 in Mock control group ) . m ) The survival of mice is shown as Kaplan - Meier curves .

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i-m) Mice were treated by intravenous injection of NILV-S/MAR(Mock), LV(CD19CAR) and NILV-S/MAR(CD19CAR)-transduced T cells (8x106 cells/injection) at days 18, 21, 24 after tumor implantation. Tumor growth was monitored by caliper measurements. i-k) Tumor volume of individual mice in each treatment groups are presented. l) The average tumor volume of each treatment group is presented and the values on day 37 were statistically compared (n=12 in LV group, n=9 in NILV-S/MAR group and n=11 in Mock control group). m) The survival of mice is shown as Kaplan-Meier curves.


n - p ) Representative images showing infiltration of adoptively transferred human T cells into tumors . T cells were visualized by staining for human CD3 ( red ) and nucleus ( blue ) . q ) Quantification of CD3 + T cells from each treatment group is presented ( n = 5 ) .

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n-p) Representative images showing infiltration of adoptively transferred human T cells into tumors. T cells were visualized by staining for human CD3 (red) and nucleus (blue). q) Quantification of CD3+ T cells from each treatment group is presented (n=5).


Percent body weight of male Prdx4 WT and KO mice over the 72 h course of LPS ( 4 . 5 mg / kg BW ) or PBS injection ( i . p . ) . Each circle represents a mean of n = 7 mice , vertical lines indicate

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Percent body weight of male Prdx4 WT and KO mice over the 72 h course of LPS (4.5 mg/kg BW) or PBS injection (i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM


Cytokine concentration in Prdx4 WT and KO mice in response to LPS injection . ( B ) Cxcl1 levels in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . ( C ) TNF - α levels in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . ( D ) IL - 1β levels in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . Each dot ( B - D ) represents an individual mouse . Horizontal lines indicate mean .

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Cytokine concentration in Prdx4 WT and KO mice in response to LPS injection. (B) Cxcl1 levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (C) TNF-α levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (D) IL-1β levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. Each dot (B-D) represents an individual mouse. Horizontal lines indicate mean.


Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS ( 4 . 5 mg / kg BW ) injection ( i . p . ) and treatment with IL - 1 receptor antagonist ( IL - 1RA ) Anakinra ( 200 µg / mouse ) or control . Arrows indicate time point of Anakinra injection . Each circle represents a mean of n = 5 mice , vertical lines indicate SEM .

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Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS (4.5 mg/kg BW) injection (i.p.) and treatment with IL-1 receptor antagonist (IL-1RA) Anakinra (200 µg/mouse) or control. Arrows indicate time point of Anakinra injection. Each circle represents a mean of n=5 mice, vertical lines indicate SEM.


Serum concentration of Cxcl1 , TNF - α and IL - 1β in Prdx4 WT and KO mice injected with LPS , LPS and IL - 1RA or control . Horizontal lines indicate mean .

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Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4 WT and KO mice injected with LPS, LPS and IL-1RA or control. Horizontal lines indicate mean.


Percent body weight of male Prdx4 - flox and Prdx4 - ∆ LysMCre mice over the 48 h course of 4 . 5 mg / kg BW LPS ( i . p . ) . Each circle represents a mean of n = 7 mice , vertical lines indicate SEM .

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Percent body weight of male Prdx4-flox and Prdx4-∆LysMCre mice over the 48 h course of 4.5 mg/kg BW LPS ( i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM.


Serum concentration of Cxcl1 , TNF - α and IL - 1β in Prdx4 - flox and Prdx4 - ∆ LysMCre mice injected with LPS . Each dot ( B , D ) represents an individual mouse . Horizontal lines indicate mean .

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Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4-flox and Prdx4-∆LysMCre mice injected with LPS. Each dot (B, D) represents an individual mouse. Horizontal lines indicate mean.


Concentration of Cxcl1 , TNF - α and IL - 1β in the supernatants of Prdx4 WT and KO BMDMs in response to a time course of LPS stimulation ( 100 ng / ml LPS , time points indicated ) .

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Concentration of Cxcl1, TNF-α and IL-1β in the supernatants of Prdx4 WT and KO BMDMs in response to a time course of LPS stimulation (100 ng/ml LPS, time points indicated).


IL - 1β release of Prdx4 WT and KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) and then pulsed for indicated time points with ATP ( 5 mM ) .

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IL-1β release of Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for indicated time points with ATP (5 mM).


Western blot analysis of IL - 1β in cell lysates and supernatants of Prdx4 WT and KO BMDMs , primed with LPS ( 100 ng / ml ) and pulsed with ATP ( 5 mM ) for 4 h or left untreated . Dashed line indicates vertical slice .

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Western blot analysis of IL-1β in cell lysates and supernatants of Prdx4 WT and KO BMDMs, primed with LPS (100 ng/ml) and pulsed with ATP (5 mM) for 4 h or left untreated. Dashed line indicates vertical slice.


Immunofluorescence microscopy of ASC speck formation in Prdx4 WT and KO BMDMs in response to Nigericin ( 10 µg / ml ) stimulation for 45 min of LPS - primed cells . Cells were stained with an antibody to ASC and nuclei were counterstained using DAPI . Scale bar indicates 20 µm . ASC speck - positive cells were counted and expressed as percentage of total cells . Bars represent a mean of n = 4 mice , vertical lines indicate SD . n . s . not significant ( two - tailed t - test ) .

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Immunofluorescence microscopy of ASC speck formation in Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 45 min of LPS-primed cells. Cells were stained with an antibody to ASC and nuclei were counterstained using DAPI. Scale bar indicates 20 µm. ASC speck-positive cells were counted and expressed as percentage of total cells. Bars represent a mean of n=4 mice, vertical lines indicate SD. n.s. not significant (two-tailed t-test).


Western blot analysis of caspase - 1 cleavage in the supernatant of Prdx4 WT and KO BMDMs in response to Nigericin ( 10 µg / ml ) stimulation for 1 h after priming with LPS ( 100 ng / ml ) , LPS - priming alone or without stimulation . Whole cell lysates were analyzed for pro - caspase - 1 and Gapdh levels .

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Western blot analysis of caspase-1 cleavage in the supernatant of Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 1 h after priming with LPS (100 ng/ml), LPS-priming alone or without stimulation. Whole cell lysates were analyzed for pro-caspase-1 and Gapdh levels.


IL - 1β release in Prdx4 WT and KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) and then pulsed for 3 h with ATP ( 5 mM ) or Nigericin ( 10 µg / ml ) or transfected for 3 h with poly ( dA : dT ) or Flagellin ( 1 µg / ml each ) or treated with transfection agent only .

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IL-1β release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or Flagellin (1 µg/ml each) or treated with transfection agent only.


Quantification of cell death by LDH - release in Prdx4 WT and KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) and then pulsed for 3 h with ATP ( 5 mM ) or Nigericin ( 10 µg / ml ) or transfected for 3 h with poly ( dA : dT ) or flagellin ( 1 µg / ml each ) or treated with transfection agent only .

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Quantification of cell death by LDH-release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or flagellin (1 µg/ml each) or treated with transfection agent only.


Western blot analysis of rPRDX4 , rCASP - 1 and co - incubated rPRDX4 and rCASP - 1 after non - reducing SDS - PAGE . Black arrows at 250 kDa indicate the corresponding molecular weights of rCASP - 1 and rPRDX4 oligomers and decrease of the Prdx4 band intensity upon co - incubation with rCASP - 1 . Black arrow at approx . 50 kDa indicates the appearance of an additional rPRDX4 band which corresponds to the molecular weight of a Prdx4 dimer .

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Western blot analysis of rPRDX4, rCASP-1 and co-incubated rPRDX4 and rCASP-1 after non-reducing SDS-PAGE. Black arrows at 250 kDa indicate the corresponding molecular weights of rCASP-1 and rPRDX4 oligomers and decrease of the Prdx4 band intensity upon co-incubation with rCASP-1. Black arrow at approx. 50 kDa indicates the appearance of an additional rPRDX4 band which corresponds to the molecular weight of a Prdx4 dimer.


Western blot analysis of rPRDX4 ( = P ) and rCASP - 1 ( = C ) after alkylation ( = A ) and non - reducing SDS - PAGE . Black arrows indicate the termination of the integration of rCASP - 1 into the rPRDX4 decamer / HMWC upon alkylation .

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Western blot analysis of rPRDX4 (=P) and rCASP-1 (=C) after alkylation (=A) and non-reducing SDS-PAGE. Black arrows indicate the termination of the integration of rCASP-1 into the rPRDX4 decamer/HMWC upon alkylation.


Rate of caspase - 1 activity in the presence of non - reduced decameric rPrdx4 , reduced dimeric and monomeric rPRDX4 , YVAD or control .

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Rate of caspase-1 activity in the presence of non-reduced decameric rPrdx4, reduced dimeric and monomeric rPRDX4, YVAD or control.


Foldchange in IL - 1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3 , ASC , IL - 1β and caspase - 1 WT or Cys - to - Ser mutants C362S or C397S and co - transfected with Prdx4 or control . Cells were stimulated with 2 . 5 mM ATP for 30 min before analysis .

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Foldchange in IL-1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3, ASC, IL-1β and caspase-1 WT or Cys-to-Ser mutants C362S or C397S and co-transfected with Prdx4 or control. Cells were stimulated with 2.5 mM ATP for 30 min before analysis.


Western blot analysis of non - reducing SDS - PAGE of cell lysates from HEK293 cells transfected with caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants and co - transfected with Prdx4 - GFP or GFP as control .

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Western blot analysis of non-reducing SDS-PAGE of cell lysates from HEK293 cells transfected with caspase-1 WT, C362S, C397S, or C362S plus C397S mutants and co-transfected with Prdx4-GFP or GFP as control.


Western blot analysis of co - immunoprecipitation using HA - magnetic beads from cell lysates of HEK293 cells transfected with HA - tagged caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants and co - transfected with Prdx4 - GFP or GFP as control .

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Western blot analysis of co-immunoprecipitation using HA-magnetic beads from cell lysates of HEK293 cells transfected with HA-tagged caspase-1 WT, C362S, C397S, or C362S plus C397S mutants and co-transfected with Prdx4-GFP or GFP as control.


IL - 1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3 , ASC , IL - 1β and caspase - 1 and co - transfected with Prdx4 WT or Cys - to - Ala mutants C51A , C124A , C245A or DM C124A / C245A or control . Cells were stimulated with 2 . 5 mM ATP for 30 min before analysis .

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IL-1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3, ASC, IL-1β and caspase-1 and co-transfected with Prdx4 WT or Cys-to-Ala mutants C51A, C124A, C245A or DM C124A/C245A or control. Cells were stimulated with 2.5 mM ATP for 30 min before analysis.


Western blot analysis of co - immunoprecipitation using HA - magnetic beads from cell lysates of HEK293 cells transfected with HA - tagged caspase - 1 WT or control and co - transfected with Prdx4 WT or Cys - to - Ala mutants C51A , C124A , C245A or DM C124A / C245A .

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Western blot analysis of co-immunoprecipitation using HA-magnetic beads from cell lysates of HEK293 cells transfected with HA-tagged caspase-1 WT or control and co-transfected with Prdx4 WT or Cys-to-Ala mutants C51A, C124A, C245A or DM C124A/C245A.


Western blot analysis of Prdx4 , pro - caspase ‑ 1 , Gapdh and E - Cadherin from the cytosolic and insoluble cell fraction of LPS and / or ATP - stimulated BMDMs or untreated controls .

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Western blot analysis of Prdx4, pro-caspase‑1, Gapdh and E-Cadherin from the cytosolic and insoluble cell fraction of LPS and/or ATP-stimulated BMDMs or untreated controls.


Prdx4 concentration in supernatants of Prdx4 WT or KO BMDMs , primed for 6 h with LPS and pulsed for indicated time points with 5 mM ATP . Each circle represents a mean of n = 3 mice , vertical lines indicate SD . * * , p < 0 . 01 ; n . s . not significant ( two - tailed t - test ) .

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Prdx4 concentration in supernatants of Prdx4 WT or KO BMDMs, primed for 6 h with LPS and pulsed for indicated time points with 5 mM ATP. Each circle represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test).


Concentration of Prdx4 in the serum of WT mice , injected with LPS ( 4 . 5 mg / kg BW ) for the time points indicated . Each dot represents an individual mouse . Horizontal lines indicate mean . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed t - test ) .

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Concentration of Prdx4 in the serum of WT mice, injected with LPS (4.5 mg/kg BW) for the time points indicated. Each dot represents an individual mouse. Horizontal lines indicate mean. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Concentration of Prdx4 in supernatants of Prdx4 WT and KO BMDMs . Cells were primed with LPS ( 100 ng / ml ) for 6 h , followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min and stimulated with 5 mM ATP for 4 h or no further stimulation . Each bar represents a mean of n = 3 mice , vertical lines indicate SD . * * , p < 0 . 01 ; n . s . not significant ( two - tailed t - test ) .

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Concentration of Prdx4 in supernatants of Prdx4 WT and KO BMDMs. Cells were primed with LPS (100 ng/ml) for 6 h, followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min and stimulated with 5 mM ATP for 4 h or no further stimulation. Each bar represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test).


Relative levels of Prdx4 secretion in response to LPS + ATP stimulation and pretreatment with either Glycine , NSA or GW4869 and relative levels of caspase - 1 secretion in response to LPS + ATP stimulation and pretreatment with in response to LPS + ATP stimulation and pretreatment with GW4869 . Each bar represents a mean of n = 3 biological with 2 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . not significant ( two - tailed t - test ) .

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Relative levels of Prdx4 secretion in response to LPS+ATP stimulation and pretreatment with either Glycine, NSA or GW4869 and relative levels of caspase-1 secretion in response to LPS+ATP stimulation and pretreatment with in response to LPS+ATP stimulation and pretreatment with GW4869. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; n.s. not significant (two-tailed t-test).


Western blot analysis of subcellular organelle fractions . OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS ( 100 ng / ml ) for 12 h and stimulated with 5 mM ATP for 4 h .

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Western blot analysis of subcellular organelle fractions. OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS (100 ng/ml) for 12 h and stimulated with 5 mM ATP for 4 h.


BCA 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 ) .

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BCA 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 ) .

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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 .

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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 ) .

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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 ) .

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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 ) .

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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).


Western 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 ) .

Western 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 .

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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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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.


( 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 .

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(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 .

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(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 ) .

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(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 ) .

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(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).


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 )

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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 .

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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 .

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(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 .

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(F) The SARS-CoV-2 titer in the pretreatment and full-time experiments was analyzed by real-time PCR.


( A ) The ( A ) NP proteins in the cell lysates were determined by Western blot analysis .

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(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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(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.


( A ) H1975 cells were transduced with full - length Spike by Lentiviral vector . Protein extracts were immunoblotted with the indicated antibodies . O / E represents overexpress .

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(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 ) .

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(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 .

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(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 .

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(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.


Immunoblot 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 .

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Immunoblot 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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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.


Immunoblot 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 .

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Immunoblot 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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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.


Immunoblot 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 .

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Immunoblot 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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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.


Anti - 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 ) .

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Anti-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 .

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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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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).


Immunoblot 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 .

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Immunoblot 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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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.


A , 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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A,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.


Immunoblot 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 .

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Immunoblot 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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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.


Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7 - FL ( left ) or sNASP - FL ( right ) following treatment with siCTRL , siLAMP2A , or MG - 132 .

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Immunoblot 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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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.


Heatmaps 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 .

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

Heatmaps 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.


Low 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

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Low 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 sections


High 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

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High 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 sections


Percentage 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 )

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Percentage 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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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


High 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

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

High 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 µm


Synaptic 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 )

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

Synaptic 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 )

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

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 )

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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 )

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

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)


Representative 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 -

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Representative 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-V


Average ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks /

Average ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks/s


ABR click sound ( C thresholds in otoferlin dual ‑ AAV treated Otof - / - mice compared to wild ‑ type and non ‑ treated Otof - / - control

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ABR click sound (C thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animals


ABR 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 ,

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ABR 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, respectively


Summed ABR wave I - V amplitudes at different click sound intensities in otoferlin dual ‑ AAV injected , non ‑ injected Otof - / - , and wild ‑ type control

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Summed ABR wave I-V amplitudes at different click sound intensities in otoferlin dual‑AAV injected, non‑injected Otof-/-, and wild‑type control mice


Summed 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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Summed 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


A ) 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 ) .

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A) 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 ) .

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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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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.


B ) 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 .

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B) 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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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.


D ) 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 .

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D) 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 .

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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 ) .

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

G) 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).


A ) 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 .

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

A) 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 .

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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 .

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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.


Representative 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 ) .

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Representative 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).