Confocal images of untransfected and GFP - βCaMKII WT transfected CAD cells ( green ) , treated or not with Wnt7a . Top : Cells were stained with phalloidin ( red ) and DAPI ( blue ) . Middle : Phalloidin staining ( white ) of the cells is shown . Bottom : Pseudocolor images of cells stained with phalloidin highlight the differences in F - actin levels among the treatments . Scale bars represent 5 μm . Anti - actin immunoblot and bar graph showing changes in the ratio between G - actin ( in supernatant fraction , s ) and F - actin ( in the pellet fraction , p ) of CAD cells untransfected or transfected with GFP - βCaMKII WT plasmid and treated or not with Wnt7a .

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Confocal images of untransfected and GFP-βCaMKII WT transfected CAD cells (green), treated or not with Wnt7a. Top: Cells were stained with phalloidin (red) and DAPI (blue). Middle: Phalloidin staining (white) of the cells is shown. Bottom: Pseudocolor images of cells stained with phalloidin highlight the differences in F-actin levels among the treatments. Scale bars represent 5 μm. Anti-actin immunoblot and bar graph showing changes in the ratio between G-actin (in supernatant fraction, s) and F-actin (in the pellet fraction, p) of CAD cells untransfected or transfected with GFP-βCaMKII WT plasmid and treated or not with Wnt7a.


Confocal images of CAD cells transfected with GFP - βCaMKII WT plasmid ( green ) and treated or not with Wnt7a . Cells were fixed and then stained with phalloidin ( red ) and DAPI ( blue ) . The insets ( right panels ) show a magnified image of the area depicted in the expanded field images ( left ) . 3D reconstruction shows co - localization of GFP / phalloidin signals . Scale bars represent 10 µm .

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Confocal images of CAD cells transfected with GFP-βCaMKII WT plasmid (green) and treated or not with Wnt7a. Cells were fixed and then stained with phalloidin (red) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field images (left). 3D reconstruction shows co-localization of GFP/phalloidin signals. Scale bars represent 10 µm.


Top : Schematic of the culture systems used to assess cell - to - cell contact - dependent or secretion - mediated transfer of vesicles in primary neurons . After dissection , neurons were labeled in suspension with DiI ( donor , D ) or CTG ( acceptor , A ) . In the co - culture system , neurons were plated in 1 : 1 ratio and cultured for 20 h and then treated with 200 ng / mL of Wnt5a or Wnt7a for 4 h . In the secretion - based system , donor neurons cultured alone for 20 h were treated with 200 ng / mL of Wnt5a or Wnt7a for 4 h , and the medium was then collected and replaced for the medium of acceptor neurons cultured separately . Acceptor neurons were further incubated with the conditioned media of donor neuron for 24 h . Bottom : Representative confocal images of co - cultured donor and acceptor neurons . The insets ( right panels ) show a magnification of the area depicted in the expanded field images ( left ) . Yellow arrows point to DiI - labeled vesicles inside acceptor neurons .

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Top: Schematic of the culture systems used to assess cell-to-cell contact-dependent or secretion-mediated transfer of vesicles in primary neurons. After dissection, neurons were labeled in suspension with DiI (donor, D) or CTG (acceptor, A). In the co-culture system, neurons were plated in 1:1 ratio and cultured for 20 h and then treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h. In the secretion-based system, donor neurons cultured alone for 20 h were treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h, and the medium was then collected and replaced for the medium of acceptor neurons cultured separately. Acceptor neurons were further incubated with the conditioned media of donor neuron for 24 h. Bottom: Representative confocal images of co-cultured donor and acceptor neurons. The insets (right panels) show a magnification of the area depicted in the expanded field images (left). Yellow arrows point to DiI-labeled vesicles inside acceptor neurons.


Percentage of acceptor neurons containing DiI - labeled vesicles after being co - cultured with donor neurons and incubated for 4 h with the indicated treatments . Percentage of acceptor neurons containing DiI - labeled vesicles after being cultured for 4 h with the conditioned media of treated donor neurons .

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Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments. Percentage of acceptor neurons containing DiI-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor neurons.


Confocal image showing a donor ( D ) and an acceptor ( A ) neuron connected by TNT - like structures ( yellow arrowhead in the merged panel ) . Neurons were stained with WGA ( white ) and DAPI ( blue ) . The insets ( right panels ) show a magnified image of the area depicted in the expanded field ( left ) .

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Confocal image showing a donor (D) and an acceptor (A) neuron connected by TNT-like structures (yellow arrowhead in the merged panel). Neurons were stained with WGA (white) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field (left).


Representative confocal images of co - cultured donor ( D , labeled with DiI ) and acceptor ( A , labeled with CTG ) neurons treated or not with 200 ng / mL of Wnt5a , 200 ng / mL sFRP - 2 or 200 ng / mL Wnt5a plus 200 ng / mL sFRP - 2 for 4 h . Yellow arrows point to DiI - labeled vesicles inside acceptor neurons . Percentage of acceptor neurons containing DiI - labeled vesicles after being co - cultured with donor neurons and incubated for 4 h with the indicated treatments .

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Representative confocal images of co-cultured donor (D, labeled with DiI) and acceptor (A, labeled with CTG) neurons treated or not with 200 ng/mL of Wnt5a, 200 ng/mL sFRP-2 or 200 ng/mL Wnt5a plus 200 ng/mL sFRP-2 for 4 h. Yellow arrows point to DiI-labeled vesicles inside acceptor neurons. Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments.


Primary cortical neurons at 1 DIV were fixed and labeled with MAP - 2 ( green ) , β - III - tubulin ( red ) and WGA ( white ) . The inset shows a magnified image of the area depicted in the merged panel . Yellow arrowheads point out TNT - like structures .

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Primary cortical neurons at 1 DIV were fixed and labeled with MAP-2 (green), β-III-tubulin (red) and WGA (white). The inset shows a magnified image of the area depicted in the merged panel. Yellow arrowheads point out TNT-like structures.


Transfer of α - syn fibrils is shown in neurons at 1 DIV . Cells were loaded in suspension with either Alexa - 568 α - syn fibrils ( red ) or Alexa - 488 α - syn fibrils ( green ) and cultured together . The insets ( right panels ) show a magnification of the area depicted in the expanded field image ( left ) . Yellow arrows point to red and green α - syn puncta contained in the soma of a neuron . Yellow arrowhead points to a TNT - like connection .

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Transfer of α-syn fibrils is shown in neurons at 1 DIV. Cells were loaded in suspension with either Alexa-568 α-syn fibrils (red) or Alexa-488 α-syn fibrils (green) and cultured together. The insets (right panels) show a magnification of the area depicted in the expanded field image (left). Yellow arrows point to red and green α-syn puncta contained in the soma of a neuron. Yellow arrowhead points to a TNT-like connection.


A TNT - like connection containing Alexa - 488 α - syn positive puncta is found in neurons at 1 DIV stained with phalloidin ( white ) and DAPI ( blue ) . The insets ( right panels ) show the 3D reconstruction of the area depicted in the expanded field image ( left ) .

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A TNT-like connection containing Alexa-488 α-syn positive puncta is found in neurons at 1 DIV stained with phalloidin (white) and DAPI (blue). The insets (right panels) show the 3D reconstruction of the area depicted in the expanded field image (left).


Representative confocal images of 1 DIV co - cultured donor ( D , loaded with Alexa - 568 α - syn fibrils ) and acceptor ( A , labeled with CTG ) neurons . After 20 h post - plating , neurons were treated with 200 ng / mL Wnt5a for 4 h . The insets ( right panels ) show a magnification of the area depicted in the expanded field ( left ) . Yellow arrows point to α - syn puncta inside acceptor neurons .

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Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. After 20 h post-plating, neurons were treated with 200 ng/mL Wnt5a for 4 h. The insets (right panels) show a magnification of the area depicted in the expanded field (left). Yellow arrows point to α-syn puncta inside acceptor neurons.


Percentage of acceptor neurons containing α - syn puncta for each of the indicated treatments .

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Percentage of acceptor neurons containing α-syn puncta for each of the indicated treatments.


Time lapses of neurons loaded with Alexa - 568 α - syn fibrils and cultured in control conditions or treated with 200 ng / mL Wnt5a . Neuron - to - neuron transfer of α - syn puncta ( yellow arrows ) through a TNT - like structure ( yellow arrowheads ) is shown upon Wnt5a treatment .

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Time lapses of neurons loaded with Alexa-568 α-syn fibrils and cultured in control conditions or treated with 200 ng/mL Wnt5a. Neuron-to-neuron transfer of α-syn puncta (yellow arrows) through a TNT-like structure (yellow arrowheads) is shown upon Wnt5a treatment.


Representative confocal images of 1 DIV co - cultured donor ( D , loaded with Alexa - 568 α - syn fibrils ) and acceptor ( A , labeled with CTG ) neurons . Neurons were obtained from WT or βCaMKII K . O . mice . Yellow arrows point to α - syn puncta inside acceptor neurons in both conditions .

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Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. Neurons were obtained from WT or βCaMKII K.O. mice. Yellow arrows point to α-syn puncta inside acceptor neurons in both conditions.


A Immunohistochemical analysis of LONP1 expression in CKD children with mild ( n = 11 ) , moderate ( n = 10 ) , or severe fibrosis ( n = 9 ) . N = 4 in Normal group .

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A Immunohistochemical analysis of LONP1 expression in CKD children with mild (n=11), moderate (n=10), or severe fibrosis (n=9). N=4 in Normal group.


D Western blot analysis for the expression of LONP1 in UUO models at different time points .

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D Western blot analysis for the expression of LONP1 in UUO models at different time points.


F Deposition of total fibrosis in kidney tissues was determined by Masson ' s trichrome staining . Scale bar : 50μm . G Sirius red staining in WT and cKI mice after UUO . Scale bar : 50μm .

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F Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm. G Sirius red staining in WT and cKI mice after UUO. Scale bar: 50μm.


J , K Western blot and densitometric analysis for the expression of FN1 , Collagen I , Collagen III and TGF - β1 ( monomer ) in WT and cKI mice after UUO ( n = 3 or 4 , biological replicates ) .

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J, K Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and TGF-β1 (monomer) in WT and cKI mice after UUO (n=3 or 4, biological replicates).


M Succinate dehydrogenase ( SDH ) staining in WT and cKI mice after UUO . Scale bar : 50μm .

M Succinate dehydrogenase (SDH) staining in WT and cKI mice after UUO. Scale bar: 50μm.


N Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKI mice after UUO . Scale bar : 500nm .

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N Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKI mice after UUO. Scale bar: 500nm.


A Deposition of total fibrosis in kidney tissues was determined by Masson ' s trichrome staining . Scale bar : 50μm . B Sirius red staining in WT and cKO mice after UUO . Scale bar :

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A Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm. B Sirius red staining in WT and cKO mice after UUO. Scale bar: 50μ


D , E Western blot and densitometric analysis for the expression of FN1 , Collagen I , Collagen III and α - SMA in WT and cKO mice after UUO ( n = 3 , biological replicates ) .

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D, E Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after UUO (n=3, biological replicates).


I Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKO mice after UUO . Scale bar : 500nm .

I Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKO mice after UUO. Scale bar: 500nm.


C Masson ' s trichrome staining of WT and cKI mice after 5 / 6Nx . Scale bar : 100μm .

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C Masson's trichrome staining of WT and cKI mice after 5/6Nx. Scale bar: 100μm.


D qRT - PCR analysis of FN1 , Collagen I and Collagen III in WT and cKI mice after 5 / 6Nx ( n = 6 in cKI + Sham group , n = 5 in the other three groups , biological replicates )

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D qRT-PCR analysis of FN1, Collagen I and Collagen III in WT and cKI mice after 5/6Nx (n=6 in cKI+Sham group, n=5 in the other three groups, biological replicates)


G Masson ' s trichrome staining of WT and cKO mice after 5 / 6Nx . Scale bar : 100μm .

G Masson's trichrome staining of WT and cKO mice after 5/6Nx. Scale bar: 100μm.


H qRT - PCR analysis of FN1 , Collagen I , Collagen III and α - SMA in WT and cKO mice after 5 / 6Nx ( n = 5 in WT + Sham group , n = 7 in cKO + Sham group , n = 6 in 5 / 6Nx groups , biological replicates ) .

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H qRT-PCR analysis of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after 5/6Nx (n=5 in WT+Sham group, n=7 in cKO+Sham group, n=6 in 5/6Nx groups, biological replicates).


I , J Western blot and densitometric analysis for the expression of Collagen I and α - SMA in WT and cKO mice after 5 / 6Nx ( n = 4 , biological replicates ) .

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I, J Western blot and densitometric analysis for the expression of Collagen I and α-SMA in WT and cKO mice after 5/6Nx (n=4, biological replicates).


L Transmission electron microscopy images of the mitochondria in tubular cells of WT and cKO mice after 5 / 6Nx . Scale bar : 500nm .

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L Transmission electron microscopy images of the mitochondria in tubular cells of WT and cKO mice after 5/6Nx. Scale bar: 500nm.


A Quantification of mitochondrial ROS production ( n = 4 , biological replicates ) .

A Quantification of mitochondrial ROS production (n=4, biological replicates).


D qRT - PCR analysis of FN1 , Collagen I , Collagen III , Collagen IV , α - SMA and Vimentin ( n = 3 , biological replicates ) .

D qRT-PCR analysis of FN1, Collagen I, Collagen III, Collagen IV, α-SMA and Vimentin (n=3, biological replicates).


Measurement of OCR using an XF96 Extracellular Flux Analyzer ( n = 8 or 10 in each group , biological replicates ) .

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Measurement of OCR using an XF96 Extracellular Flux Analyzer (n=8 or 10 in each group, biological replicates).


Western blot analysis for the expression of Collagen I , α - SMA , and Vimentin ( n = 3 , two independent experiments were carried out ) .

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Western blot analysis for the expression of Collagen I, α-SMA, and Vimentin (n=3, two independent experiments were carried out).


C Western blot validation of the up - regulated proteins in mitochondria ( n = 2 , biological replicates ) .

C Western blot validation of the up-regulated proteins in mitochondria (n=2, biological replicates).


E GST pull down analysis of interaction between LONP1 and HMGCS2 .

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E GST pull down analysis of interaction between LONP1 and HMGCS2.


G Immunofluorescence co - localization analysis of LONP1 , HMGCS2 and mitochondria in mPTC ( up , scale bar : 20μm ) and human kidney tissue ( down , scale bar : 200μm ) .

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G Immunofluorescence co-localization analysis of LONP1, HMGCS2 and mitochondria in mPTC (up, scale bar: 20μm) and human kidney tissue (down, scale bar: 200μm).


A Immunohistochemical analysis of HMGCS2 expression in CKD children . Scale bar : 50μm . B Immunohistochemical semi - quantitative IOD analysis of HMGCS2 ( n = 8 in Control group , n = 15 in CKD group ) .

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A Immunohistochemical analysis of HMGCS2 expression in CKD children. Scale bar: 50μm. B Immunohistochemical semi-quantitative IOD analysis of HMGCS2 (n=8 in Control group, n=15 in CKD group).


Western blot analysis of HMGCS2 in WT and cKI mice of UUO model ( n = 3 or 4 , biological replicates ) .

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Western blot analysis of HMGCS2 in WT and cKI mice of UUO model (n=3 or 4, biological replicates).


western blotting analysis of Hmgcs2 expression in WT and Hmgcs2 + / - mice ( n = 3 , biological replicates ) .

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western blotting analysis of Hmgcs2 expression in WT and Hmgcs2+/- mice (n=3, biological replicates).


G , H Representative immunoblot and densitometric analysis of HMGCS2 , FN1 , Collagen III , α - SMA and Vimentin in mPTCs treated as indicated . Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel ( n = 3 in each group ) .

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G, H Representative immunoblot and densitometric analysis of HMGCS2, FN1, Collagen III, α-SMA and Vimentin in mPTCs treated as indicated. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group).


J - N Representative Western blot and densitometric analysis of LONP1 , FN1 , α - SMA and Vimentin of each group was shown . Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel ( n = 3 in each group ) . mPTCs were transfected with siNC or siLONP1 , and then pre - treated with 84 - B10 ( 10 μM ) 2h before TGF - β1 ( 10ng / mL ) treatment for another 24h .

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J-N Representative Western blot and densitometric analysis of LONP1, FN1, α-SMA and Vimentin of each group was shown. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group). mPTCs were transfected with siNC or siLONP1, and then pre-treated with 84-B10 (10 μM) 2h before TGF-β1 (10ng/mL) treatment for another 24h.


A Fibrosis deposition of kidney tissues was determined by Sirius red staining in UUO model . Scale bar : 50μm . B Quantification of fibrotic area in UUO model ( n = 10 in each group , biological replicates ) .

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A Fibrosis deposition of kidney tissues was determined by Sirius red staining in UUO model. Scale bar: 50μm. B Quantification of fibrotic area in UUO model (n=10 in each group, biological replicates).


G , H Fibrosis deposition of kidney tissues was determined by Sirius red staining and quantified in UIRI model ( n = 10 in each group , biological replicates ) . Scale bar : 50μm .

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G, H Fibrosis deposition of kidney tissues was determined by Sirius red staining and quantified in UIRI model (n=10 in each group, biological replicates). Scale bar: 50μm.


B . Representative traces for C - H , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in paired uninfected ( black traces ) and shRNA - expressing neurons ( green traces ) .

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B. Representative traces for C-H, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and shRNA-expressing neurons (green traces).


C , D . Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices expressing shRNA against GSK3α at − 60 mV ( AMPAR - mediated EPSCs ; C ) or at + 40 mV ( NMDAR - mediated EPSCs ; D ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . E . Quantification of individual ( open circles ) and average ( grey bars ) AMPA / NMDA ratios of neurons from uninfected and GSK3α shRNA lentivirus infected slices , where AMPAR - mediated current is taken as the peak response at − 60 mV , and NMDAR - mediated current is measured at + 40 mV at a latency of 60 ms following stimulation . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test .

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

C, D. Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices expressing shRNA against GSK3α at −60 mV (AMPAR-mediated EPSCs; C) or at +40 mV (NMDAR-mediated EPSCs; D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. E. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from uninfected and GSK3α shRNA lentivirus infected slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.


Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices at − 60 mV ( AMPAR - mediated EPSCs ; C ) or at + 40 mV ( NMDAR - mediated EPSCs ; D ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . Quantification of individual ( open circles ) and average ( grey bars ) AMPA / NMDA ratios of neurons from uninfected and infected slices , where AMPAR - mediated current is taken as the peak response at − 60 mV , and NMDAR - mediated current is measured at + 40 mV at a latency of 60 ms following stimulation . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test . F , G . As C , D , for GSK3β . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . H . As E , for GSK3β . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test .

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

Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices at −60 mV (AMPAR-mediated EPSCs; C) or at +40 mV (NMDAR-mediated EPSCs; D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from uninfected and infected slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. F, G. As C, D, for GSK3β. Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. H. As E, for GSK3β. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.


B . Representative traces for C , where baseline responses ( dark trace ) are taken between 10 and 0 minutes before LTD induction , and responses after LTD induction ( light trace ) are taken between 40 and 45 minutes after the end of LTD induction .

B. Representative traces for C, where baseline responses (dark trace) are taken between 10 and 0 minutes before LTD induction, and responses after LTD induction (light trace) are taken between 40 and 45 minutes after the end of LTD induction.


C . Time course of the peak AMPAR - mediated synaptic response to Schaffer collateral stimulation before , during , and after the induction of LTD by LFS ( 300 pulses at 1 Hz ) , in control ( uninfected ) and GSK3α ( blue ) or GSK3β ( orange ) shRNA - expressing neurons . Error bars represent SEM . D . Quantification of average synaptic depression from the last 5 minutes of the time course shown in C . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test . E . Cumulative frequency distribution of the ESPC values plotted in D . Statistical significance was calculated according to the Kolmogorov - Smirnov test .

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

C. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of LTD by LFS (300 pulses at 1 Hz), in control (uninfected) and GSK3α (blue) or GSK3β (orange) shRNA-expressing neurons. Error bars represent SEM. D. Quantification of average synaptic depression from the last 5 minutes of the time course shown in C. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. E. Cumulative frequency distribution of the ESPC values plotted in D. Statistical significance was calculated according to the Kolmogorov-Smirnov test.


A . Representative traces for B and C , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in neurons from inactive control - , GSK3α or GSK3β inhibitor - treated slices ( all at 10 µM ) . Slices were incubated with each compound for 1 hour before recording , and during whole - cell recording itself . Compounds were also included in the internal solution of the recording pipette ( all at 10 µM ) .

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

A. Representative traces for B and C, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in neurons from inactive control-, GSK3α or GSK3β inhibitor-treated slices (all at 10 µM). Slices were incubated with each compound for 1 hour before recording, and during whole-cell recording itself. Compounds were also included in the internal solution of the recording pipette (all at 10 µM).


B . Quantification of individual ( open circles ) and average ( grey bars ) AMPA / NMDA ratios of neurons from inactive control , GSK3α or GSK3β inhibitor - treated slices , where AMPAR - mediated current is taken as the peak response at − 60 mV , and NMDAR - mediated current is measured at + 40 mV at a latency of 60 ms following stimulation . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test . C . Cumulative frequency distribution of the AMPA / NMDA ratios plotted in B . Statistical significance was calculated using the Kolmogorov - Smirnov test .

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

B. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from inactive control, GSK3α or GSK3β inhibitor-treated slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. C. Cumulative frequency distribution of the AMPA/NMDA ratios plotted in B. Statistical significance was calculated using the Kolmogorov-Smirnov test.


D . Representative traces for E and F , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in neurons from hippocampal slices treated with DMSO ( vehicle control ) , AR - 18 ( 10 µM ) , CHIR ( 1 µM ) or SC79 ( 20 µM ) . E , F . As B , C , for DMSO , AR - 18 , CHIR and SC79 . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative traces for E and F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in neurons from hippocampal slices treated with DMSO (vehicle control), AR-18 (10 µM), CHIR (1 µM) or SC79 (20 µM). E, F. As B, C, for DMSO, AR-18, CHIR and SC79. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.


B . Representative traces for C , D , where baseline responses ( dark trace ) are taken between 10 and 0 minutes before LTD induction , and responses after LTD induction ( light trace ) are taken between 40 and 45 minutes after the end of LTD induction .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative traces for C, D, where baseline responses (dark trace) are taken between 10 and 0 minutes before LTD induction, and responses after LTD induction (light trace) are taken between 40 and 45 minutes after the end of LTD induction.


C . Time course of the peak AMPAR - mediated synaptic response to Schaffer collateral stimulation before , during , and after the induction of LTD by LFS ( 300 pulses at 1 Hz ) , in control ( inactive control ) , GSK3α ( blue ) and GSK3β ( orange ) inhibitor , and AR - 18 - treated neurons ( yellow ) . Drug treatment was for 1 hour before recording at 10 µM for all compounds . Error bars represent SEM . D . Quantification of average synaptic depression from the last 5 minutes of the time course shown in C . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test .

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

C. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of LTD by LFS (300 pulses at 1 Hz), in control (inactive control), GSK3α (blue) and GSK3β (orange) inhibitor, and AR-18-treated neurons (yellow). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Error bars represent SEM. D. Quantification of average synaptic depression from the last 5 minutes of the time course shown in C. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.


E . As B , for F and G . F . Time course of the peak AMPAR - mediated synaptic response to Schaffer collateral stimulation before , during , and after the induction of mGluR - dependent LTD by PP - LFS ( 900 paired pulses , separated by 50 ms , at 1 Hz ) , in control ( inactive control ) and GSK3α inhibitor - treated neurons ( blue ) . Drug treatment was for 1 hour before recording at 10 µM for all compounds . Representative traces for synaptic responses before ( dark trace ) and after ( light trace ) LTD induction are shown above the time course . Error bars represent SEM . G . Quantification of average synaptic depression from the last 5 minutes of the time course shown in F . Error bars represent SEM . No statistically significant difference was found , according to the Mann - Whitney U test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O 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. As B, for F and G. F. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of mGluR-dependent LTD by PP-LFS (900 paired pulses, separated by 50 ms, at 1 Hz), in control (inactive control) and GSK3α inhibitor-treated neurons (blue). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Representative traces for synaptic responses before (dark trace) and after (light trace) LTD induction are shown above the time course. Error bars represent SEM. G. Quantification of average synaptic depression from the last 5 minutes of the time course shown in F. Error bars represent SEM. No statistically significant difference was found, according to the Mann-Whitney U test.


B . Representative traces for C - F , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in paired uninfected ( black traces ) and EGFP - GSK3α - or EGFP - GSK3β - expressing neurons ( green traces ) .

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

B. Representative traces for C-F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and EGFP-GSK3α- or EGFP-GSK3β -expressing neurons (green traces).


C , D . Scatter plots showing individual recordings of pairs of uninfected and infected neurons at − 60 mV ( AMPAR - mediated EPSCs ) in slices expressing EGFP - GSK3α ( C ) or EGFP - GSK3β ( D ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . E , F . Scatter plots showing individual recordings of pairs of uninfected and infected neurons at + 40 mV ( NMDAR - mediated EPSCs ) in slices expressing EGFP - GSK3α ( E ) or EGFP - GSK3β ( F ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test .

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

C, D. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at −60 mV (AMPAR-mediated EPSCs) in slices expressing EGFP-GSK3α (C) or EGFP-GSK3β (D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. E, F. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at +40 mV (NMDAR-mediated EPSCs) in slices expressing EGFP-GSK3α (E) or EGFP-GSK3β (F). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test.


A . Representative confocal images from EGFP - GSK3α FRAP experiments . Upper left panel ( ' Baseline ' ) shows EGFP - GSK3α expression in a dendrite and dendritic spine of organotypic slices cultured from rat hippocampus . The spine was bleached ( ' Bleach ' ; yellow dashed circle ) and its fluorescence partially recovered 90 s later ( ' Recovery ' ) . Single dendritic spines were imaged once per second for 10 seconds prior to , and for 90 seconds following , spine photobleaching . Such single FRAP experiments were performed on different spines during basal conditions ( ' basal ' ; upper panels ) , immediately after cLTD induction with 20 µM NMDA for 5 minutes ( ' cLTD ' ; middle panels ) , and immediately following 10 minutes of washout ( ' washout ' ; lower panels ) . Scale bars = 1 µm . B . Time course of fluorescence recovery during FRAP experiments described in A . White circles ( ' basal ' ) show the average spine / dendrite fluorescence of FRAP on EGFP - GSK3α - expressing spines in slices prior to cLTD induction . Pink circles ( ' cLTD ' ) show fluorescence in spines in which cLTD had been induced using 20 µM NMDA . Green circles ( ' washout ' ) represent the average of spine fluorescence after NMDA washout for 10 minutes . Recovery trajectories for the three conditions are fitted with two - phase exponential curves ( red lines ) . C , D . As A , B but for spines expressing EGFP - GSK3β . Scale bars = 1 µm .

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

A. Representative confocal images from EGFP-GSK3α FRAP experiments. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine of organotypic slices cultured from rat hippocampus. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but for spines expressing EGFP-GSK3β. Scale bars = 1 µm.


E . Quantification of last 10 seconds of fluorescence recovery ( 90 - 100 seconds ) in B and D . Bars represent mean ± SEM . Statistical significance was calculated according to the Mann - Whitney U test . N = 12 , 11 and 11 spines for EGFP - GSK3α baseline , cLTD and washout groups and 20 , 11 and 9 spines for EGFP - GSK3β baseline , cLTD and washout groups , respectively .

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

E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Bars represent mean ± SEM. Statistical significance was calculated according to the Mann-Whitney U test. N = 12, 11 and 11 spines for EGFP-GSK3α baseline, cLTD and washout groups and 20, 11 and 9 spines for EGFP-GSK3β baseline, cLTD and washout groups, respectively.


F . Spine / dendrite ratios showing enrichment of EGFP - GSK3α or EGFP - GSK3β in ' baseline ' , ' cLTD ' or ' recovery ' conditions . Bars represent mean ± SEM . Statistical significance was calculated according to the Mann - Whitney U test .

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

F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α or EGFP-GSK3β in 'baseline', 'cLTD' or 'recovery' conditions. Bars represent mean ± SEM. Statistical significance was calculated according to the Mann-Whitney U test.


G . Representative western blots from whole extracts of organotypic slices incubated in ACSF ( untreated ; ' baseline ' ) , treated with 20 µM NMDA for 5 minutes ( ' cLTD ' ) or treated with 20 µM NMDA for 5 minutes and allowed to recover in plain ACSF for 10 minutes ( ' washout ' ) . Blots were probed for pSer21 / 9 GSK3α / β , total GSK3α / β , pSer845 GluA1 , and total GluA1 . H . Quantification of experiments as those shown in G . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test . N = 4 independent experiments for all conditions . Asterisks ( * * ) denote P < 0 . 01 .

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

G. Representative western blots from whole extracts of organotypic slices incubated in ACSF (untreated; 'baseline'), treated with 20 µM NMDA for 5 minutes ('cLTD') or treated with 20 µM NMDA for 5 minutes and allowed to recover in plain ACSF for 10 minutes ('washout'). Blots were probed for pSer21/9 GSK3α/β, total GSK3α/β, pSer845 GluA1, and total GluA1. H. Quantification of experiments as those shown in G. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 4 independent experiments for all conditions. Asterisks (**) denote P < 0.01.


A . Representative confocal images from EGFP - GSK3α FRAP experiments in organotypic slices cultured from C57 wild - type mice . Upper left panel ( ' Baseline ' ) shows EGFP - GSK3α expression in a dendrite and dendritic spine . The spine was bleached ( ' Bleach ' ; yellow dashed circle ) and its fluorescence partially recovered 90 s later ( ' Recovery ' ) . Single dendritic spines were imaged once per second for 10 seconds prior to , and for 90 seconds following , spine photobleaching . Such single FRAP experiments were performed on different spines during basal conditions ( ' basal ' ; upper panels ) , immediately after cLTD induction with 20 µM NMDA for 5 minutes ( ' cLTD ' ; middle panels ) , and immediately following 10 minutes of washout ( ' washout ' ; lower panels ) . Scale bars = 1 µm . B . Time course of fluorescence recovery during FRAP experiments described in A . White circles ( ' basal ' ) show the average spine / dendrite fluorescence of FRAP on EGFP - GSK3α - expressing spines in wild - type mouse slices prior to cLTD induction . Pink circles ( ' cLTD ' ) show fluorescence in spines in which cLTD had been induced using 20 µM NMDA . Green circles ( ' washout ' ) represent the average of spine fluorescence after NMDA washout for 10 minutes . Recovery trajectories for the three conditions are fitted with two - phase exponential curves ( red lines ) . C , D . As A , B but in slices cultured from C57 tauKO mice . Scale bars = 1 µm .

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

A. Representative confocal images from EGFP-GSK3α FRAP experiments in organotypic slices cultured from C57 wild-type mice. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in wild-type mouse slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout ') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but in slices cultured from C57 tauKO mice. Scale bars = 1 µm.


E . Quantification of last 10 seconds of fluorescence recovery ( 90 - 100 seconds ) in B and D . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test . N = 13 , 11 and 12 spines for wt baseline , cLTD and recovery and 9 , 7 and 5 spines for tauKO baseline , cLTD and recovery groups , respectively .

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

E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 13, 11 and 12 spines for wt baseline, cLTD and recovery and 9, 7 and 5 spines for tauKO baseline, cLTD and recovery groups, respectively.


F . Spine / dendrite ratios showing enrichment of EGFP - GSK3α in wild - type or tauKO slices in ' baseline ' , ' cLTD ' or ' recovery ' conditions . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test .

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

F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α in wild-type or tauKO slices in 'baseline', 'cLTD' or 'recovery' conditions. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.


B . Representative traces for C - F , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in paired uninfected ( black traces ) and EGFP - GSK3α - expressing neurons ( green traces ) in C57 wt or tauKO slices .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative traces for C-F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and EGFP-GSK3α-expressing neurons (green traces) in C57 wt or tauKO slices.


C - F . Scatter plots showing individual recordings of pairs of uninfected and infected neurons at − 60 mV ( AMPAR - mediated EPSCs ) in C57 wt ( C ) or tauKO slices ( D ) , or at + 40 mV ( NMDAR - mediated EPSCs ) in wt ( E ) or tauKO slices ( F ) . Red circles represent the averages of all pairs in a given plot , and n denotes the number of cell pairs . In all cases , error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test .

O O O O O O O O O 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 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

C-F. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at −60 mV (AMPAR-mediated EPSCs) in C57 wt (C) or tauKO slices (D), or at +40 mV (NMDAR-mediated EPSCs) in wt (E) or tauKO slices (F). Red circles represent the averages of all pairs in a given plot, and n denotes the number of cell pairs. In all cases, error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were treated with various doses of ATP . CBA analysis of IL - 1β ( A ) in the culture supernatants .

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LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. CBA analysis of IL-1β (A) in the culture supernatants.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were treated with various doses of ATP . ELISA of IL - 18 ( B ) in the culture supernatants .

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LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. ELISA of IL-18 (B) in the culture supernatants.


CBA analysis of IL - 1β secretion from Abro1 + / + and Abro1 − / − BMDMs transduced with GFP - or ABRO1 - GFP - expressing lentiviruses prior to stimulation with LPS and ATP . Cell lysates were immunoblotted with anti - ABRO1 antibody .

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CBA analysis of IL-1β secretion from Abro1+/+ and Abro1−/− BMDMs transduced with GFP- or ABRO1-GFP-expressing lentiviruses prior to stimulation with LPS and ATP. Cell lysates were immunoblotted with anti-ABRO1 antibody.


ELISA analysis of IL - 1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium ( Con ) , LPS or LPS plus ATP . Cell lysates were immunoblotted with anti - ABRO1 and anti - GAPDH antibodies .

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ELISA analysis of IL-1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium (Con), LPS or LPS plus ATP. Cell lysates were immunoblotted with anti-ABRO1 and anti-GAPDH antibodies.


Abro1 + / + and Abro1 − / − BMDMs were left untreated ( Con ) , treated with LPS alone ( LPS ) or pretreated with LPS and then stimulated with nigericin , silica , MSU , MDP , Alum , poly ( dA : dT ) , flagellin or LTx ( Anthrax Lethal Factor ) . CBA analysis of IL - 1β secretion in the culture supernatants .

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Abro1+/+ and Abro1−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with nigericin, silica, MSU, MDP, Alum, poly (dA: dT), flagellin or LTx (Anthrax Lethal Factor). CBA analysis of IL-1β secretion in the culture supernatants.


Representative immunoblot analysis of cleaved caspase - 1 and IL - 1β in culture supernatants ( SN ) of LPS - primed Abro1 + / + and Abro1 − / − BMDMs treated with ATP , nigericin , MSU , flagellin or poly ( dA : dT ) .

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Representative immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Abro1+/+ and Abro1−/− BMDMs treated with ATP, nigericin, MSU, flagellin or poly (dA: dT).


Flow cytometric analysis of caspase - 1 activity in LPS - primed Abro1 + / + and Abro1 − / − BMDMs stimulated with nigericin for indicated times .

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Flow cytometric analysis of caspase-1 activity in LPS-primed Abro1+/+ and Abro1−/− BMDMs stimulated with nigericin for indicated times.


Immunostaining of endogenous ASC specks in LPS - primed Abro1 + / + and Abro1 − / − BMDMs left untreated or treated with nigericin or poly ( dA : dT ) . Scale bars , 10 μm . Quantification of ASC specks from ( H ) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei . At least 100 cells from each treatment condition were quantified . UD , undetectable .

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

Immunostaining of endogenous ASC specks in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with nigericin or poly (dA:dT). Scale bars, 10 μm. Quantification of ASC specks from (H) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei. At least 100 cells from each treatment condition were quantified. UD, undetectable.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were left untreated or treated with ATP . Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti - ASC antibody .

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

LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti-ASC antibody.


WT , Abro1 − / − and Nlrp3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β and TNF - α in the peritoneal lavage fluid ( A ) . n = 6 for PBS groups and n = 12 for MSU groups .

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WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (A). n = 6 for PBS groups and n = 12 for MSU groups.


WT , Abro1 − / − and Nlrp3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . Flow cytometric analysis of peritoneal cell exudates ( B ) . n = 6 for PBS groups and n = 12 for MSU groups .

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WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. Flow cytometric analysis of peritoneal cell exudates (B). n = 6 for PBS groups and n = 12 for MSU groups.


Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) . Flow cytometric analysis of caspase - 1 activity in peritoneal exudate monocytes and neutrophils . n = 12 per group .

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Abro1+/+ and Abro1−/− mice were intraperitoneally injected with MSU (1 mg per mouse). Flow cytometric analysis of caspase-1 activity in peritoneal exudate monocytes and neutrophils. n=12 per group.


Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with Alum ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β in the peritoneal lavage fluid ( D ) n = 6 for PBS groups and n = 12 for Alum groups .

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Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β in the peritoneal lavage fluid (D) n = 6 for PBS groups and n = 12 for Alum groups.


Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with Alum ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . flow cytometric analysis of peritoneal cell exudates ( E ) . n = 6 for PBS groups and n = 12 for Alum groups .

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Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (E). n = 6 for PBS groups and n = 12 for Alum groups.


Survival of Abro1 + / + and Abro1 − / − mice subjected to LPS ( 15 mg / kg ) . n = 20 per group .

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Survival of Abro1+/+ and Abro1−/− mice subjected to LPS (15 mg/kg). n=20 per group.


ELISA of serum IL - 1β from Abro1 + / + and Abro1 − / − mice 3 h after intraperitoneal injection of LPS ( 15mg / kg ) or vehicle ( PBS ) . n = 6 for PBS groups and n = 12 for LPS groups .

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ELISA of serum IL-1β from Abro1+/+ and Abro1−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 and Myc - tagged NLRP3 , ASC or pro - casapase - 1 ( Pro - Casp - 1 ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3, ASC or pro-casapase-1 (Pro-Casp-1). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.


BMDMs were treated with indicated stimuli . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 and Myc - tagged NLRP3 or NLRP3 deletion mutants as indicated in ( F ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - c - Myc agarose .

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 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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3 or NLRP3 deletion mutants as indicated in (F). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 and Flag - tagged ABRO1 or ABRO1 deletion mutants as indicated in ( H ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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 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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3 and Flag-tagged ABRO1 or ABRO1 deletion mutants as indicated in (H). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - tagged NLRP3 ( WT ) , NLRP3 ( S194D ) , or NLRP3 ( S194A ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-tagged NLRP3 (WT), NLRP3 (S194D), or NLRP3 (S194A). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


WT BMDMs were treated with or without 5 μM Anisomycin for 2 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT BMDMs were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194A ) were treated with or without 5 μM Anisomycin for 2 h . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194A) were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194D ) were treated with or without 100 nM SP600125 for 2 h . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194D) were treated with or without 100 nM SP600125 for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


WT and Nlrp3S194A / S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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WT and Nlrp3S194A/S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 , Flag - ABRO1 and HA - ubiquitin ( HA - Ub ) , HA - Ub K48 only or K63 only . Before collection , cells were treated with MG132 ( 10 μM ) for 6 h . Immunoblot analysis of NLRP3 ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3, Flag-ABRO1 and HA-ubiquitin (HA-Ub), HA-Ub K48 only or K63 only. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding HA - Ub , Flag - ABRO1 , and NLRP3 truncations as indicated . Before collection , cells were treated with MG132 ( 10 μM ) for 6 h . Immunoblot analysis of ubiquitination of NLRP3 truncations ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding HA-Ub, Flag-ABRO1, and NLRP3 truncations as indicated. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of ubiquitination of NLRP3 truncations (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were left untreated or treated with ATP ( left ) or nigericin ( right ) . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP (left) or nigericin (right). Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


Nlrp3 - / - BMDMs transduced with lentiviruses expressing Flag - NLRP3 ( WT ) or Flag - NLRP3 ( A350V ) were left unstimulated or stimulated with LPS or LPS plus ATP . Immunoblot analysis of NLRP3 and NLRP3 ( A350V ) ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were left unstimulated or stimulated with LPS or LPS plus ATP. Immunoblot analysis of NLRP3 and NLRP3 (A350V) ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 ( A350V ) and HA - ubiquitin ( HA - Ub ) . Immunoblot analysis of NLRP3 ( A350V ) ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3 (A350V) and HA-ubiquitin (HA-Ub). Immunoblot analysis of NLRP3 (A350V) ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


Nlrp3 - / - BMDMs transduced with lentiviruses expressing Flag - NLRP3 ( WT ) or Flag - NLRP3 ( A350V ) were stimulated with or without 100ng / ml LPS for 1 h . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti - ABRO1 antibody .

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Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were stimulated with or without 100ng/ml LPS for 1 h. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 , and HA - ubiquitin ( HA - Ub ) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h . Immunoblot analysis of NLRP3 ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3, and HA-ubiquitin (HA-Ub) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


LPS - primed WT and Nlrp3S194A / S194A BMDMs were left untreated or treated with ATP . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed WT and Nlrp3S194A/S194A BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


WT BMDMs were stimulated with LPS for 1 h . Immunoblot analysis of NLRP3 , ABRO1 , and BRCC3 from the cell lysates immunoprecipitated with control IgG , anti - ABRO1 , anti - BRCC3 or anti - NLRP3 antibody .

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WT BMDMs were stimulated with LPS for 1 h. Immunoblot analysis of NLRP3, ABRO1, and BRCC3 from the cell lysates immunoprecipitated with control IgG, anti-ABRO1, anti-BRCC3 or anti-NLRP3 antibody.


Brcc3 + / + and Brcc3 − / − BMDMs were treated with or without LPS for 1 h . Before LPS treatment , Brcc3 − / − BMDMs were pretreated with MG132 ( 10 μM ) for 6 h to rescue the expression of ABRO1 . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti - ABRO1 antibody .

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Brcc3+/+ and Brcc3−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Brcc3−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of ABRO1. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.


Abro1 + / + and Abro1 − / − BMDMs were treated with or without LPS for 1 h . Before LPS treatment , Abro1 − / − BMDMs were pretreated with MG132 ( 10 μM ) for 6 h to rescue the expression of BRCC3 . Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti - BRCC3 antibody .

Abro1+/+ and Abro1−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Abro1−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of BRCC3. Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti-BRCC3 antibody.


A constant amount of Myc - NLRP3 plasmid together with an increasing amount of Myc - ABRO1 plasmid were co - transfected in HEK - 293T cells . Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti - BRCC3 antibody .

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A constant amount of Myc-NLRP3 plasmid together with an increasing amount of Myc-ABRO1 plasmid were co-transfected in HEK-293T cells. Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti-BRCC3 antibody.


Brcc3 + / + and Brcc3 − / − BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP were treated with LPS and nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - NLRP3 antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3

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

Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody


Abro1 + / + , Abro1 − / − , BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP were treated with LPS and nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - NLRP3 antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3

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

Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody


Brcc3 + / + and Brcc3 − / − BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP CBA analysis of IL - 1β in culture

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Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants


Abro1 + / + , Abro1 − / − , BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP CBA analysis of IL - 1β in culture

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Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants


Brcc3 + / + and Brcc3 − / − BMDMs were left untreated ( Con ) , treated with LPS alone ( LPS ) or pretreated with LPS and then stimulated with ATP , nigericin , MSU , Alum , poly ( dA : dT ) or flagellin . CBA analysis of IL - 1β secretion in the culture supernatants .

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Brcc3+/+ and Brcc3−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with ATP, nigericin, MSU, Alum, poly (dA:dT) or flagellin. CBA analysis of IL-1β secretion in the culture supernatants.


Immunoblot analysis of cleaved caspase - 1 and IL - 1β in culture supernatants ( SN ) of LPS - primed Brcc3 + / + and Brcc3 − / − BMDMs treated with ATP , nigericin , MSU , flagellin , and poly ( dA : dT ) .

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Immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Brcc3+/+ and Brcc3−/− BMDMs treated with ATP, nigericin, MSU, flagellin, and poly (dA: dT).


Immunostaining of endogenous ASC specks in LPS - primed Brcc3 + / + and Brcc3 - / - BMDMs left untreated or treated with nigericin or poly ( dA : dT ) . ASC speck positive cells were counted and analyzed as described in Fig . 1I . UD , undetectable .

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

Immunostaining of endogenous ASC specks in LPS-primed Brcc3+/+ and Brcc3-/- BMDMs left untreated or treated with nigericin or poly (dA:dT). ASC speck positive cells were counted and analyzed as described in Fig. 1I. UD, undetectable.


LPS - primed Brcc3 + / + and Brcc3 − / − BMDMs were left unstimulated or stimulated with nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed Brcc3+/+ and Brcc3−/− BMDMs were left unstimulated or stimulated with nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


Survival of Brcc3 + / + and Brcc3 − / − mice subjected to LPS ( 15 mg / kg ) . n = 20 per group .

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Survival of Brcc3+/+ and Brcc3−/− mice subjected to LPS (15 mg/kg). n=20 per group.


ELISA of serum IL - 1β from Brcc3 + / + and Brcc3 − / − mice 3 h after intraperitoneal injection of LPS ( 15mg / kg ) or vehicle ( PBS ) . n = 6 for PBS groups and n = 12 for LPS groups .

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ELISA of serum IL-1β from Brcc3+/+ and Brcc3−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.


Brcc3 + / + and Brcc3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β and TNF - α in the peritoneal lavage fluid ( G )

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Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (G)


Brcc3 + / + and Brcc3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . flow cytometric analysis of peritoneal cell exudates ( H ) . n = 6 for PBS groups and n = 12 for MSU groups .

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Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (H). n = 6 for PBS groups and n = 12 for MSU groups.


b ) Box plot showing the distribution of log2 ( FC ) of all or selected miR - 29a targets in the Data ref : Mazziotti et al , 2017 dataset of age - regulated genes in the visual cortex . Notches represent confidence intervals for the median . Whiskers indicate 95th and 5th percentile , the box marks the 25th and 75th percentile . Significance vs . all detected genes was assessed by Wilcoxon ' s test ( miR - 29a targets p = 4 . 9x10 - 14 , top 200 miR - 29a targets p = 3 . 7x10 - 38 ) .

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b) Box plot showing the distribution of log2(FC) of all or selected miR-29a targets in the Data ref: Mazziotti et al, 2017 dataset of age-regulated genes in the visual cortex. Notches represent confidence intervals for the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance vs. all detected genes was assessed by Wilcoxon's test (miR-29a targets p=4.9x10-14, top 200 miR-29a targets p=3.7x10-38).


( d , e ) Mature miR - 29a ( n = 3 / 4 mice for each age ; one - way ANOVA , p < 0 . 0001 ; post hoc Tukey ' s test * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns not significant ) and Dnmt3a expression level ( normalized to P10 ) at different ages measured by qPCR ( n = 3 / 4 mice for each age ; one - way ANOVA , p < 0 . 0001 ; post hoc Tukey ' s test : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns not significant ) . One way ANOVA p - values are reported . Error bars represent ± SEM . f ) Scatter plot showing correlation of miR - 29a and Dnmt3a levels in the visual cortex display a strong negative correlation ( Pearson correlation : r = - 0 . 817 , p < 0 . 001 ) . Each circle represents data from a single animal .

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

(d,e) Mature miR-29a (n=3/4 mice for each age; one-way ANOVA, p<0.0001; post hoc Tukey's test ***p<0.001, ****p<0.0001, ns not significant) and Dnmt3a expression level (normalized to P10) at different ages measured by qPCR (n=3/4 mice for each age; one-way ANOVA, p<0.0001; post hoc Tukey's test: *p<0.05, **p<0.01, ****p<0.0001, ns not significant). One way ANOVA p-values are reported. Error bars represent ±SEM. f) Scatter plot showing correlation of miR-29a and Dnmt3a levels in the visual cortex display a strong negative correlation (Pearson correlation: r=-0.817, p<0.001). Each circle represents data from a single animal.


g ) Representative examples of in situ hybridization on visual cortex ( left P25 , right P120 ) using a LNA probe against miR - 29a . Scale bar 180 µm . Cortical layers are delimited by roman numerals and

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g) Representative examples of in situ hybridization on visual cortex (left P25, right P120) using a LNA probe against miR-29a. Scale bar 180 µm. Cortical layers are delimited by roman numerals and brackets


h ) Example of co - staining for PV ( green ) , miR - 29a ( red ) and nuclear marker ( blue ) in a P120 mouse . White triangles represent PV + / miR - 29a + cells . Scale bar 20 µm .

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h) Example of co-staining for PV (green), miR-29a (red) and nuclear marker (blue) in a P120 mouse. White triangles represent PV+/miR-29a+ cells. Scale bar 20 µm.


Mature miR - 29a expression ( miR29 ) in visually deprived mice : a ) normal rearing ( NR , N = 5 ) vs dark rearing ( DRb , N = 5 ) ; t - test , p = 0 . 19 , b ) NR ( N = 4 ) vs dark rearing from P10 ( DRp10 , N = 5 ; t - test , p = 0 . 70 . In both experiments , analysis was performed at

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Mature miR-29a expression (miR29) in visually deprived mice: a) normal rearing (NR, N=5) vs dark rearing (DRb, N=5); t-test, p=0.19, b) NR (N=4) vs dark rearing from P10 (DRp10, N=5; t-test, p=0.70. In both experiments, analysis was performed at P25


Mature miR - 29a expression ( miR29 ) in visually deprived mice : c ) one week period of dark rearing beginning at P30 ( DRw ) followed by a 4 hour exposure to light ( DRw + 4h light ) vs control animals ( ctr , N = 4 for each group ; one - way ANOVA , p = 0 . 68 )

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Mature miR-29a expression (miR29) in visually deprived mice: c) one week period of dark rearing beginning at P30 (DRw) followed by a 4 hour exposure to light (DRw+4h light) vs control animals (ctr, N=4 for each group; one-way ANOVA, p=0.68)


Mature miR - 29a expression ( miR29 ) in visually deprived mice : d ) days of MD beginning at P25 , contralateral cortex vs ipsilateral cortex to deprived eye , N = 5 ; paired t test , p = 0 . 52 ) . Error bars represent ± SEM , symbols represent individual cases .

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Mature miR-29a expression (miR29) in visually deprived mice: d) days of MD beginning at P25, contralateral cortex vs ipsilateral cortex to deprived eye, N=5; paired t test, p=0.52). Error bars represent ±SEM, symbols represent individual cases.


b ) MiR - 29a and Dnmt3a expression in the cortex treated with scrambled or miR - 29a mimic ( N = 3 mice ) . Data were normalized to the untreated cortex of the same animal . Mir - 29a : scr vs mim29a , t - test , p = 0 . 005 ; Dnmt3a scr vs mim29a t - test , p < 0 . 0001 .

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b) MiR-29a and Dnmt3a expression in the cortex treated with scrambled or miR-29a mimic (N=3 mice). Data were normalized to the untreated cortex of the same animal. Mir-29a: scr vs mim29a, t-test, p=0.005; Dnmt3a scr vs mim29a t-test, p<0.0001.


c ) IOS analysis . Ocular dominance index ( ODI ) of deprived animals treated with scrambled ( scr , N = 6 black lines ) or miR - 29a mimic ( mimic29 , N = 8 orange lines ) , or left untreated ( MD no treat , N = 5 grey lines ) before and after 3 days of MD . Dashed lines represent single animals , continuous lines the group average . two - way ANOVA RM , treatment X time , p = 0 . 028 ; post hoc Sidak ' s comparison , post - MD mim29a vs scr p < 0 . 05 , mim29a vs MD no treat p < 0 . 01 ; pre - MD scr vs post - MD scr p < 0 . 001 ; pre - MD MD no treat vs post - MD MD no treat p < 0 . 01 ; other comparisons not significant ) .

O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IOS analysis. Ocular dominance index (ODI) of deprived animals treated with scrambled (scr, N=6 black lines) or miR-29a mimic (mimic29, N=8 orange lines), or left untreated (MD no treat, N=5 grey lines) before and after 3 days of MD. Dashed lines represent single animals, continuous lines the group average. two-way ANOVA RM, treatment X time, p= 0.028; post hoc Sidak's comparison, post-MD mim29a vs scr p<0.05, mim29a vs MD no treat p<0.01; pre-MD scr vs post-MD scr p<0.001; pre-MD MD no treat vs post-MD MD no treat p<0.01; other comparisons not significant).


d ) The effects of MD on expression of Npas IV and Bdnf exIV is reported as ratio between expression level of deprived and nondeprived cortex for animals treated with scrambled ( scr + MD ) and miR - 29a mimic ( mim29a + MD ) . Each symbol represents the result of a single mouse . ( MD + scr vs MD + mim29a ; N = 4 ; t - test , Npas4 : p = 0 . 038 ; Bdnf exIV : p = 0 . 049 ) .

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

d) The effects of MD on expression of Npas IV and Bdnf exIV is reported as ratio between expression level of deprived and nondeprived cortex for animals treated with scrambled (scr+MD) and miR-29a mimic (mim29a+MD). Each symbol represents the result of a single mouse. (MD+scr vs MD+mim29a; N=4; t-test, Npas4: p=0.038; Bdnf exIV: p=0.049).


b ) Effects of amiR - 29a on the expression of miR - 29a and Dnmt3a . Fold change values with respect to the contralateral untreated cortex are reported ( ± SEM ) for mice treated with amiR - 29a or scr . scr vs amiR - 29a ; N = 3 t - test , miR - 29a * * p = 0 . 0010 ; Dnmt3a * * * * p = 0 . 0019 .

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

b) Effects of amiR-29a on the expression of miR-29a and Dnmt3a. Fold change values with respect to the contralateral untreated cortex are reported (±SEM) for mice treated with amiR-29a or scr. scr vs amiR-29a; N=3 t-test, miR-29a **p=0.0010; Dnmt3a ****p=0.0019.