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

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

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 showing infiltration of adoptively transferred human T into tumors . T were visualized by for human CD3 ( red ) nucleus ( blue ) . q ) of CD3 + T from each treatment group is presented ( n = 5 ) .

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

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).


of Prdx4 WT KO mice over 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

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

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

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

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.


of Prdx4 WT KO mice over 48 h course of LPS ( 4 . 5 mg / kg BW ) injection ( i . p . ) 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 .

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

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

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

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.


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

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

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

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

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.


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

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 B-CONTROLLED_VAR O O O O O O

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 KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) then pulsed for indicated time points with ATP ( 5 mM ) .

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 B-CONTROLLED_VAR O O O O O

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

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

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

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

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 of caspase - 1 cleavage in supernatant of Prdx4 WT 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 lysates were analyzed for pro - caspase - 1 Gapdh .

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 B-CONTROLLED_VAR 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

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 KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) 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 .

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

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.


of by LDH - release in Prdx4 WT KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) 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 .

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

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

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

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

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

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 presence of non - reduced decameric rPrdx4 , reduced dimeric monomeric rPRDX4 , YVAD or control .

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

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

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

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

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

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 of co - immunoprecipitation using HA - magnetic beads from lysates of HEK293 transfected with HA - tagged caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants 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.


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

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

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

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

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 of Prdx4 , pro - caspase ‑ 1 , Gapdh E - Cadherin from insoluble fraction of LPS / or ATP - stimulated BMDMs or untreated controls .

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

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 in supernatants of Prdx4 WT or KO BMDMs , primed for 6 h with LPS 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 - t - ) .

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 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

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).


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

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

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).


of Prdx4 in supernatants of Prdx4 WT KO BMDMs . were primed with LPS ( 100 ng / ml ) for 6 h , followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min 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 - t - ) .

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

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 of Prdx4 secretion in response to LPS + ATP stimulation pretreatment with either Glycine , NSA or GW4869 relative of caspase - 1 secretion in response to LPS + ATP stimulation pretreatment with in response to LPS + ATP stimulation 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 - t - ) .

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

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 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 stimulated with 5 mM ATP for 4 h .

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

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 of EV 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 - t - ) .

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

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 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 - t - ) .

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

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 ( ) of EV isolates . Three representative pictures are displayed . Scale bar indicates 200 nm .

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

Transmission electron microscopy (TEM) of EV isolates. Three representative pictures are displayed. Scale bar indicates 200 nm.


of distribution of EVs . Each dot indicates diameter in nm of an vesicle . SD . * * * , p < 0 . 001 ; n . s . not significant ( one - way ANOVA , followed by a Tukey multiple comparison ) .

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

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 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 - t - ) .

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 IL - 1β 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 - t - ) .

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

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 of Prdx4 , NLRP3 , ASC , caspase ‑ 1 , IL - 1β CD63 from EV lysates ( upper panel ) or caspase - 1 after immunoprecipitation against caspase - 1 ( lower panel ) .

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

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 in supernatants of caspase - 1 - deficient BMDMs stimulated with EVs from LPS , LPS ATP or control - treated Prdx4 WT or KO BMDMs , as well as caspase - 1 - deficient BMDMs pre - treated with Anakinra stimulated with EVs from LPS ATP - treated Prdx4 WT or KO BMDMs . Each bar represents a mean of n = 3 biological with 2 technical replicates , vertical lines indicate SD .

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

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 ATP or control - treated Prdx4 WT or KO or ASC KO BMDMs . Each dot represents an mouse . Horizontal lines indicate mean .

O O O O O O O O O O O O O O B-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

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 of SARS - CoV - 2 Spike 1 - 1273 , 1 - 674 , 319 - 591 expression in HEK293T . black arrows indicate location of induced target .

O O O O O O O O O O O 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) 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 Spike S1 - Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue .

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

(C) Detection of decoy antibody ACE2-Fc and Spike S1-Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue staining.


( D ) Recognition of ACE2 - Fc Spike S1 - Fc by anti - human IgG Fc antibody in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( SDS - PAGE ) .

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

(D) 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 Spike 1 - 674 - Fc by PNGase F . PNGase F digested ACE2 - Fc ( 500 ng ) Spike 1 - 674 - Fc ( 500 ng ) were subjected to Coomassie Brilliant Blue ( E ) Western blot 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 . competition effects of ACE2 - Fc on ACE2 - Spike binding was normalized to PBS control . Error bars represent standard deviation ( SD ) , n = 2 . Statistical was performed by unpaired two tail t - . * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 - Spike on H1975 ( left panel as negative control ) or H1975 - Spike ( right panel ) by ACE2 - Fc using flow cytometry . 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 . Spikes on H1975 were stained with anti - Spike antibody 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 counterstain .

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

(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 . peptidase activity of ACE2 - Fc was measured by cleavage of 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 , mixtures were added to RAW264 . 7 for 12 hr . of TNF - α in culture medium was determined by ELISA . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 . dotted represents mean value of TNF - α in control group .

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

(E) 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 metalloprotease 17 ) phosphorylation by ACE2 - Fc . extracts from ( E ) were immunoblotted with indicated antibodies ( left panel ) . β - actin served as loading control . signal was normalized to only ( right panel ) . Data are representative of three independent experiments , values are expressed as mean ± SD ( right panel ) . Error bars represent standard deviation ( SD ) , n = 2 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 . IgG represents human normal IgG control .

O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 H1975 were transduced with full - ACE2 by lentivirus . extracts were immunoblotted with 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 full - Spike co - transfected HEK293T were used as effector ( 293 - S ) . HEK293 / ACE2 ( HEK293T - overexpressing ACE2 ) ( left panel ) or H1975 / ACE2 ( H1975 - overexpressing ACE2 ) ( right panel ) were used as target . 293 - S were preincubated with normal human IgG or ACE2 - Fc at 37 ° C for 1 hr . After that , mixtures were added to target for 4 hr ( - fusion ) or 24 hr ( syncytia formation ) . areas were measured by inverted microscopy Metamorph software ( Metamorphosis ) . Scale bars equal to 50 µm . arrows indicate - 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 ) inhibition of - fusion syncytia formation by ACE2 - Fc in HEK293 / ACE2 H1975 / ACE2 was determined with formula described in Methods section . Error bars represent standard deviation ( SD ) , n = 6 . Statistical was performed by unpaired two tail t - . * * 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 entry of Spike - expressing pseudotyped lentivirus into HEK293T - ACE2 H1975 - ACE2 . relative luciferase activities , normalized to only group , represent efficiency of 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 of airway organoids . green represents specific of indicated monoclonal antibodies Alexa Fluor 488 - conjugated secondary antibodies . DAPI was used as a counterstain . Hematoxylin eosin ( H & E ) stain : hematoxylin stained nuclei in blue color ; eosin stained 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 , human normal bronchial epithelial ( HBEpc ) , HBEpc - differentiated ( airway organoids ) were harvested for immunoblotting with 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 . entry was determined by quantifying luciferase activity in 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 plaque . Mixtures of ACE2 - Fc SARS - CoV - 2 were incubated for one hour before adding to Vero E6 for another 1 hr at 37 ° C . ACE2 - Fc SARS - CoV - 2 premixtures were removed , were washed once with PBS 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 .

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

(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 was performed to determine inhibitory effects of ACE2 - Fc on titers culture medium 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 was performed to determine inhibitory effects of ACE2 - Fc on expression . culture medium extracts were harvested 24 hours post - infection for western blot ( C ) . NP / PCNA represents relative NP expression as compared to that of PCNA , which served as a loading control . NP / PCNA numbers below panel are ratios of NP / PCNA normalized to that of human IgG control group . PCNA : Proliferating antigen .

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

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 ) lysates from pretreatment full - time experiments were harvested immunoblotted with indicated antibodies .

(E) The cell lysates from the pretreatment and full-time experiments were harvested and immunoblotted with the indicated antibodies.


( F ) SARS - CoV - 2 titer in pretreatment 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 ) ( A ) NP in lysates were determined by Western blot .

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


( B , C ) in ( B ) culture medium ( C ) lysates was quantified by real - time RT - PCR . genetic information reference numbers for 5 SARS - CoV - 2 strains were summarized in Table EV1 . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * * 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 were transduced with full - Spike by Lentiviral vector . extracts were immunoblotted with indicated antibodies . O / E represents overexpress .

(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 ) effect of ACE2 - Fc activation on degranulative capacity of NK . experiments were performed with human NK derived from three independent donors . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 ( each 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 ) CD107a , IFN - γ , TNF - α expression were determined by flow cytometry after NK H1975 - Spike coincubation in presence or absence of ACE2 - Fc or ACE2 control . Error bars represent standard deviation ( SD ) , n = 3 . Statistical was performed by unpaired two tail t - . * P < 0 . 05 . of positive in both groups were normalized to NK / H1975 - Spike only group .

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

(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 ) differential expression of degranulation markers after treatment of ACE2 - Fc or ACE2 by flow cytometry . numbers in each plot indicate percentages of positive .

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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 of biotinylated isolated as in ( A ) . Parental HeLa - T - REx or expressing H3 . 1 - BirA * - HA 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 of TAP - H3 . 1 or - CENP - A pulldown of UBR7 - LAP ( schematic , upper ) . 293T were co - transfected with UBR7 - LAP TAP - tagged constructs , which were purified on IgG - conjugated sepharose beads .

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

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 non - nucleosomal fraction of 293T .

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 of HeLa transfected with UBR7 - FL . is visualized via DAPI shown in magenta immunofluorescence for 3xFLAG is shown in green . within yellow dotted box in ( E ) is magnified 4x in ( E ' ) . Scale bar = 5 μm . of anti - FLAG immunofluorescence 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 .

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

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 of anti - FLAG pulldown of full 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 PHD ( ∆ UBR ∆ PHD , residues 216 - 425 ) , or PHD C - terminus ( ∆ PHD ∆ C - term , residues 1 - 116 ) . 293T were co - transfected with H3 . 1 - HA UBR7 - FLAG constructs ( schematic , upper ) . Asterisk represents non - specific band . Numbers below blot indicate FLAG - normalized anti - HA immunoblot signal relative to full 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 of anti - H3 . 1 - HA pulldown of full - UBR7 - LAP UBR7PHD fragment ( residues 117 - 215 ) ( schematic , upper ) . 293T 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 , image is cropped to omit irrelevant samples . Numbers below blot indicate HA - normalized anti - GFP immunoblot signal relative to full 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 of streptavidin pulldown of indicated biotin - conjugated H3 peptides following incubation with 293T - GFP - UBR7 lysate after mutation of residues highlighted in ( C ) .

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

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 for total modified histones in anti - FLAG pulldown of constructs from soluble fractions demonstrates that UBR7 UBR box PHD are necessary for binding to post - nucleosomal H3 / H4 .

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 of streptavidin pulldown from proximity biotinylation described in ( G ) . 4 . 5 h represent newly synthesized histones 48 h represent pre - existing histones . Wedge represents loading titration : 1x > 0 . 1x > 0 . 01x > 0 . 001x . other lanes are loaded at 1x . Immunoblot signals were normalized to anti - FLAG signal . of anti - FLAG ( H3 . 1 - APEX2 - 3xFLAG ) anti - UBR7 immunoblot represented in ( G - H ) . Lanes 8 11 were quantified . Error bar represents mean + / - standard deviation . A two - one - sample t - was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed ) .

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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 for total modified H3 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 - one sample t - was used to calculate p - value associated with group mean difference from a hypothetical value of 1 . 0 . Experiment was performed twice .

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

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 of HeLa transfected with sNASP - FL . is visualized via DAPI shown in magenta immunofluorescence for 3xFLAG is shown in green . Scale bar = 5 μm . of anti - FLAG immunofluorescence 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 for modified histone in anti - FLAG pulldown of UBR7 - FL ( A ) or sNASP - FL ( C ) from non - nucleosomal fractions of untreated or treated with replication inhibitors ( hydroxyurea , aphidicolin ) or transcription inhibitors ( α - amanitin , triptolide ) . B , D . Plots represent either normalized immunoblot signals or prior to fractionation . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation , with grey dotted marking mean of No Treat condition . A two - one sample t - was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 . Experiments were performed three times . numbers were counted in two of 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 of H3 , UBR7 , GAPDH in non - nucleosomal fraction of parental 293T - UBR7KO . of anti - H3 immunoblot represented in ( A ) . Immunoblot signals were normalized to anti - GAPDH signal . Error bars represent mean + / - standard deviation . A two - one - sample t - was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed ) . 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 of anti - FLAG pulldown of sNASP - FL in parental or 293T - UBR7KO . 3xFLAG tagged sNASP - FL was immunoprecipitated from non - nucleosomal fraction eluted with FLAG peptide . Experiment was performed three times . of anti - FLAG ( sNASP - FL ) , anti - histone immunoblots represented in ( C ) . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation . A two - one sample t - was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed ) . Experiments were performed two ( sNASP , H4 ) to three ( 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 or 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.


of data represented in ( C , D ) . Error bars represent mean + / - standard deviation . A two - one sample t - was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed ) . 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 , anti - H3K4me3 CUT & RUN in parental UBR7KO . Plots are centered on TSS that show changes in ATAC - seq signal in UBR7KO versus parental . Sites are clustered by unsupervised k - means . Cluster color key is defined by cluster labels .

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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 , OHCs : outer hair maximum 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 ) dual ‑ AAV ‑ Hyb ( P26 ) ( C ) vectors . eGFP otoferlin immunostainings are depicted as color lookup tables in ( A - C ) with warmer colors representing higher pixel intensities ) , maximum 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


of N ‑ C ‑ terminal otoferlin labelled IHCs in dual ‑ AAV ‑ TS ( n = 10 mice ) 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 C ‑ terminal otoferlin immunofluorescence in dual ‑ AAV transduced Otof - / - wild - type IHCs ( P23 - 30 ) . Otoferlin were normalized to immunofluorescence 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 synaptic ribbons ( CtBP2 ) from wild ‑ type ( B6 : P27 , CD1B6F1 : P29 ) , dual - AAV injected CD1B6F1 Otof - / - ( dual - AAV - TS : P26 , dual - AAV - Hyb : P28 ) , their non - injected ears . ( * ) Transduced . Maximum projections of optical confocal sections . Scale bars : 5

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 ) , non - transduced Otof - / - IHCs from injected ( - AAV injected ear , n = 65 IHCs ) non - injected ( - AAV non - injected ear , n = 46 IHCs ) ears ( P25 - 29 )

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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)


synapses labelled with CtBP2 postsynaptic marker Shank1a in B6 wild - type Otof - / - P6 P14 organs of Corti . Maximum 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 ) B6 Otof - / - ( P6 : n = 62 IHCs ; P14 : n = 65 IHCs ) mice at two different developmental stages ( P6 P14 )

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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 ) , 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 ) plasma capacitance increments ( ΔCm ) of a wild ‑ type control , transduced , non ‑ transduced Otof - / - in response to a 20 ms depolarization 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 ) 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 ) , 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 average )

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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 dual ‑ AAV ‑ Hyb injected CD1B6F1 Otof - / - . AAV2 / 6 . eGFP ( + AAV . eGFP ) dual ‑ AAV ‑ TS injected CD1B6F1 wild ‑ type , 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 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 non ‑ treated Otof - / - control . In D , two best are depicted with open circles . with thresholds exceeding maximum loudspeaker output ( arrows ) of 100 dB SPL for clicks 90 dB SPL for tone bursts were set to 110 dB SPL 100 dB SPL , respectively . Apical basal cochlear turns are indicated as Apex 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 - / - , 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 dual ‑ AAV ‑ TS treated CD1B6F1 Otof - / - ( n = 8 plotted against their full - otoferlin transduction

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 immunohistochemistry reveal that β - catenin accumulates in tumors emerging in one - year - old miR - 424 ( 322 ) / 503 - / - mice . panel is a representative image of n = 5 independent tumors . 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 miR - 424 / 503 locus have higher of β - catenin ( RPPA ) . P - value is calculated on one - sided Wilcoxon rank - sum . In box - plots in B C relative expression of β - catenin in RPPA data from TCGA is shown . yellow indicates median bars indicate 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 ) bar graph shows that human breast cancers with low expression of miR - 424 miR - 503 have higher of β - catenin . P - value is calculated on one - sided Wilcoxon rank - sum .

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 ) bar graphs show number of PROCR + in 1 - year - old WT miR - 424 ( 322 ) / 503 KO mice during diestrus . error bars show 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 ) panel shows lack of induction of bona fide WNT targets in miR - over expressing 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 TBLR1 in miR - cluster - Dox - MCF - 10A model upon upregulation of miR - cluster with 100 ng / ml of doxycycline for specified number of days . numbers below blots indicate quantitation by densitometry of expression relative to β - actin . expression level of miR - 424 miR - 503 is indicated in bar graph below blots . UN indicates samples untreated with doxycycline . error bars show standard deviation .

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 TBLR1 expression in WT miR - 424 / 503 - KO basal - derived organoids after 14 days in culture with without Wnt3a ( 100ng / ml ) . arrow shows 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 ) showing that LRP6 accumulates in epithelium of miR - 424 ( 322 ) / 503 - / - mice ( 1 - year - old ) . scale bar indicates 100 micrometers ( µM ) .

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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 putative LRP6 binding sites bound to AGO2 in miR - cluster - Dox - MCF - 10A after miR - 424 / 503 cluster is induced with 100ng / ml of dox for three days . A CDC25A - binding site is included as a positive control a region of B2M GAPDH are included as negative controls . error bars show standard deviation .

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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 ) WT - blot shows expression of LRP6 upon upregulation of miR - 424 / 503 in miR - cluster - Dox - MCF - 10A ( Dox + ) when one or both of conserved microRNA binding sites ( # 1 # 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 ) WT - blot shows expression of LRP6 β - catenin in in miR - cluster - Dox - MCF - 10A cultured in presence of Dox with without WNT3a ligand ( 100ng / ml for 6 hours ) when microRNA binding sites # 1 # 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 - of whole hair follicle units from non - lesional lesional scalp psoriasis . K15 ( green ) , c - JUN ( red A , C ) JUNB ( red B , D ) 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).


Confocal of bulge region of human psoriatic hair follicles from non - lesional lesional regions of scalp . K15 ( green ) , c - JUN ( red E , G ) JUNB ( red F , H ) DAPI ( blue ) .

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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).


of HF - SCs ( K15 + ) that express c - JUN ( I ) JUNB ( J ) in bulge region of human psoriatic hair follicles from non - lesional lesional scalp in comparison with healthy scalp . n = 2 - 6 hair follicles in anagen per group from five psoriatic two healthy .

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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.


Composite immunofluorescent showing GFP expression of whole - ear sections from DKO * - mT / mG mice at day 5 , 7 , 15 30 . Increased GFP expression is shown at day 7 followed by progressive decrease of GFP + keratinocytes ( dotted represents basal layer , red dotted represents outermost skin layer ) . n = 3 per time point . of GFP expression ( top ) 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 - relative to control group ) .

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Composite 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).


of Caspase - 3 ( cCas3 top ) Ki67 ( bottom ) in ear skin of DKO * mice at different time points during psoriasis - like disease progression . n = 3 per time point .

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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 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 .

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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.


of GFP + eliminated during intravital confocal time - lapse imaging at day 5 , 7 15 in DKO * - mT / mG mice ( Time - lapse 1 : 8h ) . Positive value : of GFP increased . Negative value : of GFP reduced .

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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.


imaging of DKO * - mT / mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP + keratinocytes reside along hair follicles . dotted separates epidermis dermis . arrows represent GFP + hair follicles .

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 ( arrows ) are maintained around hair follicles .

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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.


Ear pictures of control , DKO * , DKO * K15 mice after 15 days of induction show clear signs of inflammation psoriatic - like scales in both models .

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Ear 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 of hematoxylin eosin ( H & E ) of ear sections from control , DKO * DKO * K15 mice .

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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 * DKO * K15 mice . n > 5 per group . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two - relative to control group ) .

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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 of CD45 + from ear skin of control , DKO * 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 - relative to controls ) .

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).


of IL - 17A , IL - 6 LCN2 production in sera of control , DKO * 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 - relative to controls ) .

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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).


of total number of GFP + epidermal by FACS from DKO * - mT / mG DKO * K15 - mT / mG mice at different time points during psoriasis - like progression compared to Co - mT / mG mice . n = 3 - 6 per group time point .

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 of ear sections from CoK15 - mT / mG DKO * K15 - mT / mG mouse models after 15 days of induction . Red ( Tomato ) , green ( GFP ) 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 + . n = 3 per group . Dotted lines separate epidermis dermis . of Caspase - 3 ( cCas3 ) Ki67 in ear skin of DKO * K15 - mT / mG mice at different time points during psoriasis - like disease progression .

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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.


Average number of live dead ( TOPRO - 3 + Dapi + ) 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 , no difference in average of dead . n = 4 fields from two independent experiments . Average number of live dead ( TOPRO - 3 + DAPI + ) keratinocytes derived from basal keratinocytesGFP show a significant increase in DKO * basal live keratinocytes at 12 hours , followed by a significant increase in at 48 hours .

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Average 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 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 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 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 .

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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 bottom part of trans - well system cultures with GFP + basal keratinocytes ( from DKO * or Control mice ) cultured in 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 trans - well in comparison with control GFP + basal keratinocytes .

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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.


Heat map for specific genes relative to biological process enriched 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 HF - SCTom .

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Heat 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 subpopulations sorted at mid - term ( D7 ) or late - term ( D30 ) of psoriasis - like progression in DKO * mice .

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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.


expression of TSLP in different epidermal subpopulations sorted at mid - term ( D7 ) or late - term ( D30 ) of psoriasis - like progression in DKO * mice . n = 2 - 3 .

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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.


of TSLP production in sera of control , DKO * DKO * K15 mice at day 30 of psoriasis - like disease by ELISA . n = 4 per group .

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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.


TSLP production in conditioned media quantified by ELISA three days after infection with Ad - empty or Ad - cre . n = 3 independent experiments .

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TSLP production in conditioned media quantified by ELISA three days after infection with Ad-empty or Ad-cre. n=3 independent experiments.


of EdU + Tomato + keratinocytes after blocking with anti - TSLP . Anti - TSLP neutralizes hyper - proliferation of non - mutantTom KCs in vitro after infection with Ad - cre .

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 .

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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 of treated ears after 9 days of anti - TSLP or IgG treatment .

Representative images of treated ears after 9 days of anti-TSLP or IgG treatment.


Longitudinal of ear thickness in DKO * mice at different time points during psoriasis - like disease

Longitudinal analysis of ear thickness in DKO* mice at different time points during psoriasis-like disease progression


of EdU + epidermal ( CD45 - CD49f + ) after blocking with anti - TSLP in DKO * DKO * 15

Percentage of EdU+ epidermal cells (CD45-CD49f+) after blocking with anti-TSLP in DKO* and DKO*15 mice


Representative immune - 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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Representative 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).


Representative composite immune - of whole hair follicle units by confocal from non - lesional lesional scalp psoriasis . c - JUN ( purple ) , K - 15 ( green ) , TSLP ( red ) DAPI ( blue ) .

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Representative 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 measurement in hair follicle units epidermis by ImageJ from healthy scalp , non - lesional lesional scalp psoriasis . Representation of fold change relative to control healthy scalp . n = 3 samples .

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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 - of whole hair follicle units by confocal from non - lesional lesional scalp psoriasis ( D ) magnification of specific regions from HFs ( E ) . CD200 ( green ) , c - JUN ( ) , TSLP ( red ) , DAPI ( blue ) .

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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).


b ) affinity of UVRAG - C2 domain for phospholipids was assessed using a - lipid overlay . left panel indicates identity of lipid species on PtdIns strips . - purified GST fusion of UVRAG - C2 ( wild type , WT ) , but not GST , binds PtdIns ( 3 ) P , PtdIns ( 4 ) P PtdIns ( 5 ) P . K78A / R82A mutant of C2 is defective for phospholipid - binding , whereas K87A / N88A mutant is partially impaired . Binding of FYVE PH domains of Hrs FAPP1 , respectively , to PtdIns ( 3 ) P 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 .

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b) 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 ) relative affinity of GST - fusion for phosphoinositides was quantified by densitometric of GST immunoblots in b from five independent experiments ( n = 5 ) . Error bars represent standard deviation ( s . d . ) . A . U . , arbitrary units .

(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 - UVRAG ( untagged ) to lipid blots . same PtdIns strips ( in b ) were used immunoblotted with antibodies against IgG ( right ) UVRAG ( left ) .

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(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).