( F ) iMEFgt / gt stably expressing Cherry - STING V5 - tagged m152 or corresponding ev were stimulated with 5 µg / ml ISD for indicated time or left unstimulated ( mock ) . Lysates were subjected to IB with specified antibodies . Data information : IB shown in ( C ) ( F ) are representative of three two independent experiments , respectively . Student ' s t - ( unpaired , two - ) , n . s . not significant , * p < 0 . 05 , * * * * p < 0 .

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

(F) iMEFgt/gt stably expressing Cherry-STING and V5-tagged m152 or corresponding ev were stimulated with 5 µg/ml ISD for the indicated time or left unstimulated (mock). Lysates were subjected to IB with the specified antibodies. Data information: IB shown in (C) and (F) are representative of three and two independent experiments, respectively. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, ****p<0.0001


( B ) iMEF were infected by centrifugal enhancement with either parental MCMV ( par . ) or MCMV m152stop at an MOI of 0 . 5 or mock infected . 3 hours post infection ( hpi ) were lysed subjected to immunoblotting ( IB ) with specified antibodies .

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

(B) iMEF were infected by centrifugal enhancement with either parental MCMV (par.) or MCMV m152stop at an MOI of 0.5 or mock infected. 3 hours post infection (hpi) cells were lysed and subjected to immunoblotting (IB) with the specified antibodies.


( C ) iMEF iMEFgt / gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop , or parental MCMV alone , respectively ( MOI 0 . 5 ) . 3 hpi lysates were subjected to an anti - m152 IP samples were analyzed by IB with indicated antibodies . IB shown is representative of two independent experiments .

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

(C) iMEF and iMEFgt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop, or parental MCMV alone, respectively (MOI 0.5). 3 hpi lysates were subjected to an anti-m152 IP and samples were analyzed by IB with indicated antibodies. IB shown is representative of two independent experiments.


( D ) BMDM were infected with parental MCMV ( par . ) or MCMV m152stop at an MOI of 0 . 1 or mock infected . 16 hpi secreted of IFNα or IFNβ were quantified by ELISA . Data is representative of two independent experiments .

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

(D) Primary BMDM were infected with parental MCMV (par.) or MCMV m152stop at an MOI of 0.1 or mock infected. 16 hpi secreted levels of IFNα or IFNβ were quantified by ELISA. Data is representative of two independent experiments.


( E ) iMEFgt / gt stably expressing Cherry - STING were infected with parental MCMV or MCMV m152stop ( MOI 0 . 5 ) . Live imaging was performed STING translocation quantified 120 min 180 min post stimulation . Data shown is one representative of two independent experiments .

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

(E) iMEFgt/gt stably expressing Cherry-STING were infected with parental MCMV or MCMV m152stop (MOI 0.5). Live cell imaging was performed and STING translocation quantified 120 min and 180 min post stimulation. Data shown is one representative of two independent experiments.


( F - G ) iMEF or iMEFgt / gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop ( MOI 0 . 01 ) . At 6 hpi total was extracted to determine MCMV IE1 MCMV E1 ( F ) , transcripts by qRT - PCR . Data shown is combined from two out of three independent experiments .

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 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

(F-G) iMEF or iMEFgt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). At 6 hpi total RNA was extracted to determine MCMV IE1 and MCMV E1 (F), transcripts by qRT-PCR. Data shown is combined from two out of three independent experiments.


( F - G ) iMEF or iMEFgt / gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop ( MOI 0 . 01 ) . At 6 hpi total was extracted to determine , IFNb1 IL6 ( G ) transcripts by qRT - PCR . Data shown is combined from two out of three independent experiments .

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 B-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

(F-G) iMEF or iMEFgt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). At 6 hpi total RNA was extracted to determine , IFNb1 and IL6 (G) transcripts by qRT-PCR. Data shown is combined from two out of three independent experiments.


( H ) 293T were co - transfected with Cherry - STING , pNF - κB luciferase reporter , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) either ev or m152 . were lysed analyzed as described in Figure 1 . Data is combined from three independent experiments .

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

(H) 293T cells were co-transfected with Cherry-STING, the pNF-κB luciferase reporter, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either ev or m152. Cells were lysed and analyzed as described in Figure 1. Data is combined from three independent experiments.


( A - C ) Type I IFN in spleen homogenates ( A ) serum ( B ) of C57BL / 6J ( B6J ) or STING - / - mice following i . v . infection with 4 x 105 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA . IFNβ of STING - / - mice were below detection limit ( n . d . ) .

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

(A-C) Type I IFN levels in spleen homogenates (A) and serum (B) of C57BL/6J (B6J) or STING-/- mice following i.v. infection with 4 x 105 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA. IFNβ levels of STING-/- mice were below the detection limit (n.d.).


IL6 in spleen homogenates ( C ) of C57BL / 6J ( B6J ) or STING - / - mice following i . v . infection with 4 x 105 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA .

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

IL6 levels in spleen homogenates (C) of C57BL/6J (B6J) or STING-/- mice following i.v. infection with 4 x 105 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA.


( D ) B6J or STING - / - mice were i . v . infected with 4 x 105 PFU parental MCMV or MCMV m152stop . 6 hpi , was extracted from spleen homogenates expression of MCMV IE1 E1 transcripts was determined by qRT - PCR . Data were normalized to 107 β - actin transcripts ( D - F ) .

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

(D) B6J or STING-/- mice were i.v. infected with 4 x 105 PFU parental MCMV or MCMV m152stop. 6 hpi, RNA was extracted from spleen homogenates and expression of MCMV IE1 and E1 transcripts was determined by qRT-PCR. Data were normalized to 107 cellular β-actin transcripts (D-F).


( E ) B6J or STING - / - mice were i . v . infected with 4 x 105 PFU parental MCMV . 6 hpi , was extracted from spleen homogenates m152 transcript were determined by qRT - PCR . Data were normalized to 107 β - actin transcripts ( D - F ) .

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-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

(E) B6J or STING-/- mice were i.v. infected with 4 x 105 PFU parental MCMV. 6 hpi, RNA was extracted from spleen homogenates and m152 transcript levels were determined by qRT-PCR. Data were normalized to 107 cellular β-actin transcripts (D-F).


( F ) B6J or STING - / - mice were depleted of NK via treatment with anti - NK1 . 1 ( right panel ) or left untreated ( left panel ) . One day after NK depletion , B6J or STING - / - mice were i . v . infected with 4 x 105 PFU parental MCMV or MCMV m152stop . 16 hpi , was extracted from spleen homogenates MCMV E1 transcript were determined by qRT - PCR . Data were normalized to 107 β - actin transcripts ( D - F ) .

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 B-CONTROLLED_VAR 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 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

(F) B6J or STING-/- mice were depleted of NK cells via treatment with anti-NK1.1 (right panel) or left untreated (left panel). One day after NK cell depletion, B6J or STING-/- mice were i.v. infected with 4 x 105 PFU parental MCMV or MCMV m152stop. 16 hpi, RNA was extracted from spleen homogenates and MCMV E1 transcript levels were determined by qRT-PCR. Data were normalized to 107 cellular β-actin transcripts (D-F).


( A ) 293T were co - transfected with Cherry - STING , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) either pNF - κB , p55 - CIB , p125 or p125AA luciferase reporter . Data is representative of two independent experiments .

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

(A) 293T cells were co-transfected with Cherry-STING, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either the pNF-κB, p55-CIB, p125 or p125AA luciferase reporter. Data is representative of two independent experiments.


( B ) 293T were co - transfected with expression plasmids for cGAS - GFP ( stimulated ) or ev ( unstimulated ) together with either cherry - tagged WT STING or cherry - tagged K288R STING . were fixed for imaging 24 hours post transfection . Scale bar represents 10 µm .

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 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

(B) 293T cells were co-transfected with expression plasmids for cGAS-GFP (stimulated) or ev (unstimulated) together with either cherry-tagged WT STING or cherry-tagged K288R STING. Cells were fixed for imaging 24 hours post transfection. Scale bar represents 10 µm.


( C - D ) iMEFgt / gt stably expressing cherry - tagged WT STING or K288R STING either ev or V5 - tagged m152 were stimulated with ISD ( 10 µg / ml ) or mock stimulated . At 4 hpi total was extracted to determine IFNb1 ( C ) IL6 ( D ) transcripts by qRT - PCR . Data shown is combined from three ( C ) or two ( D ) out of three independent experiments .

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 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-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

(C-D) iMEFgt/gt stably expressing cherry-tagged WT STING or K288R STING and either ev or V5-tagged m152 were stimulated with ISD (10 µg/ml) or mock stimulated. At 4 hpi total RNA was extracted to determine IFNb1 (C) and IL6 (D) transcripts by qRT-PCR. Data shown is combined from three (C) or two (D) out of three independent experiments.


( E ) iMEFgt / gt ( - ) iMEFgt / gt stably expressing either cherry - tagged WT STING or K288R STING were infected by centrifugal enhancement with parental MCMV or MCMV m152stop ( MOI 0 . 01 ) . 6 hpi total was extracted to determine MCMV E1 transcripts by qRT - PCR . Data shown is combined from three independent experiments .

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 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 O O O O O O O O

(E) iMEFgt/gt (-) and iMEFgt/gt stably expressing either cherry-tagged WT STING or K288R STING were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). 6 hpi total RNA was extracted to determine MCMV E1 transcripts by qRT-PCR. Data shown is combined from three independent experiments.


A . SDS - PAGE of purified sACES1211 showed a single band of ~ 170 kDa thus indicates extensive N - glycosylation ( - only molecular is 138 kDa ) .

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

A. SDS-PAGE analysis of purified sACES1211 showed a single band of ~170 kDa and thus indicates extensive N-glycosylation (protein-only molecular weight is 138 kDa).


B . SEC - HPLC of respective samples of purified sACES1211 , truncated N - domain , or truncated C - domain showed a single symmetric peak for each with calibrated molecular weights of 244 kDa , 147 kDa , 75 kDa , respectively . sACES1211 sample did not show any peaks corresponding to single - domain or homodimer molecular weights .

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

B. SEC-HPLC analysis of the respective samples of purified sACES1211, truncated N-domain, or truncated C-domain showed a single symmetric peak for each protein with calibrated molecular weights of 244 kDa, 147 kDa, and 75 kDa, respectively. The sACES1211 sample did not show any peaks corresponding to single-domain or homodimer molecular weights.


C . Native PAGE showed a major diffuse band of ~ 260 kDa a minor band of ~ 500 kDa for purified sACES1211 , representing monomeric dimeric forms of . Commercial gel filtration standards bovine serum albumin ( BSA ) were used to estimate molecular . Purified truncated N - domain was used as an additional molecular marker for sACES1211 showed a single diffuse band of ~ 160 kDa .

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

C. Native PAGE analysis showed a major diffuse band of ~260 kDa and a minor band of ~500 kDa for purified sACES1211, representing monomeric and dimeric forms of the protein. Commercial gel filtration standards and bovine serum albumin (BSA) were used to estimate the molecular weight. Purified truncated N-domain was used as an additional molecular weight marker for sACES1211 and showed a single diffuse band of ~160 kDa.


Representative confocal of CAD exposed for 4 h to indicated treatments . were fixed then stained with WGA ( green ) DAPI ( blue ) . of TNT - connected CAD with each of indicated treatments .

O O O O O O O O O O O O O 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 confocal images of CAD cells exposed for 4 h to the indicated treatments. Cells were fixed and then stained with WGA (green) and DAPI (blue). Percentage of TNT-connected CAD cells with each of the indicated treatments.


Top : Schematic of co - culture system used to assess vesicle transfer from donor ( labeled with DiD ) to acceptor ( labeled with CTG ) . After labeling , were detached , replated in 1 : 1 cultured in control conditions or treated with 200 µM H2O2 or 200 ng / mL Wnt7a for 4 h . Middle : Representative confocal of co - cultured donor acceptor CAD . Each image corresponds to projection of entire Z - stack . Bottom : insets correspond to projection of selected Z - stacks of marked with a yellow square in each condition to better illustrate transferred vesicles in acceptor . Yellow arrows point to DiD - labeled vesicles inside acceptor . of acceptor containing DiD - labeled vesicles in co - culture systems with indicated treatments .

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

Top: Schematic of the co-culture system used to assess vesicle transfer from donor (labeled with DiD) to acceptor (labeled with CTG) cells. After labeling, cells were detached, replated in 1:1 ratio and cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Middle: Representative confocal images of co-cultured donor and acceptor CAD cells. Each image corresponds to the projection of the entire Z-stack. Bottom: The insets correspond to the projection of selected Z-stacks of the area marked with a yellow square in each condition to better illustrate the transferred vesicles in acceptor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments.


Top : Schematic of culture system used to assess vesicle transfer by secretion . Donor ( labeled with DiD ) were cultured in control conditions or treated with 200 µM H2O2 or 200 ng / mL Wnt7a for 4 h . Then acceptor ( labeled with CTG ) were treated for 4 h with conditioned media from donor . Bottom : Representative confocal of acceptor CAD treated with conditioned media from donor . Yellow arrows point to DiD - labeled vesicles inside acceptor . of acceptor containing DiD - labeled vesicles after being cultured for 4 h with conditioned media of treated donor .

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

Top: Schematic of the culture system used to assess vesicle transfer by secretion. Donor cells (labeled with DiD) were cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Then acceptor cells (labeled with CTG) were treated for 4 h with the conditioned media from donor cells. Bottom: Representative confocal images of acceptor CAD cells treated with the conditioned media from donor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor cells.


Representative confocal of CAD cultured in control conditions or treated with 0 . 2 % DMSO , 200 µM H2O2 , 200 ng / mL Wnt7a , 10 μM KN - 93 , 200 ng / mL Wnt7a plus 10 μM KN - 93 , 1 μM TAT or 200 ng / mL Wnt7a plus 1 μM TAT for 4 h . were fixed stained with WGA ( green ) DAPI ( blue ) . of TNT - connected CAD with each of indicated treatments .

O 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 B-CONTROLLED_VAR 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

Representative confocal images of CAD cells cultured in control conditions or treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. Cells were fixed and stained with WGA (green) and DAPI (blue). Percentage of TNT-connected CAD cells with each of the indicated treatments.


Representative confocal of co - cultured donor acceptor CAD . were co - cultured in control conditions or treated with 0 . 2 % DMSO , 200 µM H2O2 , 200 ng / mL Wnt7a , 10 μM KN - 93 , 200 ng / mL Wnt7a plus 10 μM KN - 93 , 1 μM TAT or 200 ng / mL Wnt7a plus 1 μM TAT for 4 h . Yellow arrows point to DiD - labeled vesicles inside acceptor . of acceptor containing DiD - labeled vesicles in co - culture systems with indicated treatments .

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

Representative confocal images of co-cultured donor and acceptor CAD cells. Cells were co-cultured in control conditions or treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments.


Representative confocal of CAD stained for vinculin ( red ) HCS CellMask ™ Blue after being treated with 0 . 2 % DMSO , 200 µM H2O2 , 200 ng / mL Wnt7a , 10 μM KN - 93 , 200 ng / mL Wnt7a plus 10 μM KN - 93 , 1 μM TAT or 200 ng / mL Wnt7a plus 1 μM TAT for 4 h . ring circumscribed between green lines mark where vinculin were counted for each . This is defined by board of HCS CellMask ™ Blue vinculin around it . Average number of vinculin per with indicated treatments .

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

Representative confocal images of CAD cells stained for vinculin (red) and HCS CellMask™ Blue after being treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. The ring circumscribed between the green lines mark the area where the vinculin puncta were counted for each cell. This area is defined by the board of the HCS CellMask™ Blue staining and the vinculin staining around it. Average number of vinculin puncta per cell with the indicated treatments.


Representative confocal of CAD expressing indicated GFP - fusion . Top : showing WGA ( ) DAPI ( blue ) . Bottom : showing phalloidin ( red ) DAPI ( blue ) . Note that expression pattern of βCaMKII WT mutants K43R A303R , is rather punctate than diffused .

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

Representative confocal images of CAD cells expressing the indicated GFP-fusion proteins. Top: Cells showing WGA (white) and DAPI (blue) staining. Bottom: Cells showing phalloidin (red) and DAPI (blue) staining. Note that the expression pattern of βCaMKII WT and the mutants K43R and A303R, is rather punctate than diffused.


of TNT - connected CAD 24 h after transfection with indicated GFP - fusion .

Percentage of TNT-connected CAD cells 24 h after transfection with indicated GFP-fusion proteins.


of transfection rate of indicated GFP - fusion in CAD .

Percentage of transfection rate of indicated GFP-fusion proteins in CAD cells.


of acceptor containing DiD - labeled vesicles when acceptor were co - cultured with donor expressing indicated GFP - fusion .

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

Percentage of acceptor cells containing DiD-labeled vesicles when acceptor cells were co-cultured with donor cells expressing the indicated GFP-fusion proteins.


Top : Schematic of co - culture system used to assess vesicle transfer in transfected . Donor were transfected with indicated GFP - fusion then labeled with DiD . Acceptor were transfected with a H2B - mCherry plasmid . After 24 h , were detached , replated in 1 : 1 cultured for 4 h . were then fixed stained with HCS CellMask ™ Blue . Bottom : Representative confocal of co - cultured donor acceptor CAD . Yellow arrows point to DiD - labeled vesicles inside acceptor .

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

Top: Schematic of the co-culture system used to assess vesicle transfer in transfected cells. Donor cell were transfected with the indicated GFP-fusion proteins and then labeled with DiD. Acceptor cells were transfected with a H2B-mCherry plasmid. After 24 h, the cells were detached, replated in 1:1 ratio and cultured for 4 h. Cells were then fixed and stained with HCS CellMask™ Blue. Bottom: Representative confocal images of co-cultured donor and acceptor CAD cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells.


Time lapses showing a TNT connecting two transfected . Top : GFP - vector transfected . Middle : GFP - βCaMKII WT transfected . Bottom : GFP - βCaMKII T287D transfected . were stained with WGA ( ) . Yellow arrowheads point out a TNT in each condition . Scale bars represent 10 μm .

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 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

Time lapses showing a TNT connecting two transfected cells. Top: GFP-vector transfected cells. Middle: GFP-βCaMKII WT transfected cells. Bottom: GFP-βCaMKII T287D transfected cells. Cells were stained with WGA (white). Yellow arrowheads point out a TNT in each condition. Scale bars represent 10 μm.


TNT ' s average duration between GFP - vector , GFP - βCaMKII WT GFP - βCaMKII T287D transfected . Graph represents average lifetime of 15 - 20 TNTs per condition ± . Statistical significance was calculated with respect to control ( Ctrl ) ; * * p ≤ 0 . 01 , NS = not significant ( one - way ANOVA ) .

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

TNT's average duration between GFP-vector, GFP-βCaMKII WT and GFP-βCaMKII T287D transfected cells. Graph represents the average lifetime of 15-20 TNTs per condition ± SEM. Statistical significance was calculated with respect to control (Ctrl); ** p ≤ 0.01, NS = not significant (one-way ANOVA).


CODIM super - resolution ( 3 right side panels ) of TNTs shown in confocal expanded field ( left panels ) of transfected showing expression of indicated GFP - tagged ( green ) . were stained for F - actin with phalloidin ( red ) . Scale bars in expanded fields represent 5 µm 1 µm in insets .

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

CODIM super-resolution images (3 right side panels) of TNTs shown in the confocal expanded field images (left panels) of transfected cells showing expression of indicated GFP-tagged proteins (green). Cells were stained for F-actin with phalloidin (red). Scale bars in the expanded fields represent 5 µm and 1 µm in the insets.


Confocal 3D reconstruction showing intracellular localization of endogenous βCaMKII ( green ) in CAD . were stained with DAPI ( blue ) phalloidin ( red ) . Yellow arrows point to βCaMKII - positive inside a TNT . Scale bars in expanded fields represent 10 µm in 1 µm in 3D projection .

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

Confocal images and 3D reconstruction showing the intracellular localization of endogenous βCaMKII (green) in CAD cells. Cells were stained with DAPI (blue) and phalloidin (red). Yellow arrows point to βCaMKII-positive puncta inside a TNT. Scale bars in the expanded fields represent 10 µm in the micrographs and 1 µm in the 3D projection.


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

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

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

O O O O O O O B-CONTROLLED_VAR 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

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

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

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.


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

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

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

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

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 of co - cultured donor ( D , labeled with DiI ) 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 . of acceptor neurons containing DiI - labeled vesicles after being co - cultured with donor neurons incubated for 4 h with indicated treatments .

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

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.


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

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

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 . were loaded in suspension with either Alexa - 568 α - syn fibrils ( red ) or Alexa - 488 α - syn fibrils ( green ) cultured together . insets ( right panels ) show a magnification of depicted in expanded field image ( left ) . Yellow arrows point to red green α - syn contained in soma of a neuron . Yellow arrowhead points to a TNT - like connection .

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

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 is found in neurons at 1 DIV stained with phalloidin ( ) DAPI ( blue ) . insets ( right panels ) show 3D reconstruction of depicted in expanded field image ( left ) .

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

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

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.


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

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

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 cultured in control conditions or treated with 200 ng / mL Wnt5a . Neuron - to - neuron transfer of α - syn ( yellow arrows ) through a TNT - like structure ( yellow arrowheads ) is shown upon Wnt5a treatment .

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

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

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

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

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

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 for expression of LONP1 in UUO models at different time points .

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

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 . Scale bar : 50μm . G Sirius red in WT cKI mice after UUO . Scale bar : 50μm .

O O O O O O O O O O O O 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 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 densitometric for expression of FN1 , Collagen I , Collagen III TGF - β1 ( monomer ) in WT cKI mice after UUO ( n = 3 or 4 , biological replicates ) .

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

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 ) in WT 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 of mitochondria in tubular in WT cKI mice after UUO . Scale bar : 500nm .

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 . Scale bar : 50μm . B Sirius red in WT cKO mice after UUO . Scale bar :

O O O O O O O O O O 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 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 densitometric for expression of FN1 , Collagen I , Collagen III α - SMA in WT cKO mice after UUO ( n = 3 , biological replicates ) .

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

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 of mitochondria in tubular in WT 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 of WT cKI mice after 5 / 6Nx . Scale bar : 100μm .

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


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

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

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 of WT 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 of FN1 , Collagen I , Collagen III α - SMA in WT 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 ) .

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

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

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

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 of mitochondria in tubular of WT cKO mice after 5 / 6Nx . Scale bar : 500nm .

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

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


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

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


D qRT - PCR of FN1 , Collagen I , Collagen III , Collagen IV , α - SMA 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 ) .

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

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


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

O O O O O B-MEASURED_VAR I-MEASURED_VAR 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

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 up - regulated 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 of interaction between LONP1 HMGCS2 .

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

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


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

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

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

O O O B-MEASURED_VAR 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

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

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

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


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

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

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


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

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

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

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

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 in UUO model . Scale bar : 50μm . B of fibrotic in UUO model ( n = 10 in each group , biological replicates ) .

O O O O O O O O O O O O O O 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 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 quantified in UIRI model ( n = 10 in each group , biological replicates ) . Scale bar : 50μm .

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 - NMDAR - mediated EPSCs ( collected at − 60 mV + 40 mV , respectively ) in paired uninfected ( black traces ) shRNA - 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 O O O O

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 recordings of pairs of uninfected 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 . Statistical significance was calculated according to Wilcoxon signed - rank . E . of ( open circles ) average ( grey bars ) AMPA / NMDA ratios of neurons from uninfected GSK3α shRNA lentivirus infected slices , where AMPAR - mediated current is taken as peak response at − 60 mV , NMDAR - mediated current is measured at + 40 mV at a latency of 60 ms following stimulation . Error bars represent . Statistical significance was calculated according to Mann - Whitney U .

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

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

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 ( trace ) are taken between 10 0 minutes before LTD induction , responses after LTD induction ( trace ) are taken between 40 45 minutes after 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

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

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

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

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

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 B-MEASURED_VAR O B-MEASURED_VAR 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 F , showing evoked AMPAR - NMDAR - mediated EPSCs ( collected at − 60 mV + 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 SC79 . Error bars represent . Statistical significance was calculated according to Mann - Whitney U .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ( trace ) are taken between 10 0 minutes before LTD induction , responses after LTD induction ( trace ) are taken between 40 45 minutes after 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

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

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

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

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

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

O O O O O 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 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

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 from EGFP - GSK3α FRAP experiments . Upper left panel ( ' Baseline ' ) shows EGFP - GSK3α expression in a dendrite dendritic spine of organotypic slices cultured from rat hippocampus . spine was bleached ( ' Bleach ' ; yellow dashed circle ) its partially recovered 90 s later ( ' Recovery ' ) . Single dendritic spines were imaged once per second for 10 seconds prior to , 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 ) , immediately following 10 minutes of washout ( ' washout ' ; lower panels ) . Scale bars = 1 µm . B . Time course of recovery during FRAP experiments described in A . circles ( ' basal ' ) show average spine / dendrite of FRAP on EGFP - GSK3α - expressing spines in slices prior to cLTD induction . Pink circles ( ' cLTD ' ) show in spines in which cLTD had been induced using 20 µM NMDA . Green circles ( ' washout ' ) represent average of spine after NMDA washout for 10 minutes . Recovery trajectories for 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 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 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 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 . of last 10 seconds of recovery ( 90 - 100 seconds ) in B D . Bars represent mean ± . Statistical significance was calculated according to Mann - Whitney U . N = 12 , 11 11 spines for EGFP - GSK3α baseline , cLTD washout groups 20 , 11 9 spines for EGFP - GSK3β baseline , cLTD 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 B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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 ± . Statistical significance was calculated according to Mann - Whitney U .

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

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 allowed to recover in plain ACSF for 10 minutes ( ' washout ' ) . Blots were probed for pSer21 / 9 GSK3α / β , total GSK3α / β , pSer845 GluA1 , total GluA1 . H . of experiments as those shown in G . Error bars represent statistical significance was calculated according to Mann - Whitney U . N = 4 independent experiments for 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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR 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

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 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 dendritic spine . spine was bleached ( ' Bleach ' ; yellow dashed circle ) its partially recovered 90 s later ( ' Recovery ' ) . Single dendritic spines were imaged once per second for 10 seconds prior to , 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 ) , immediately following 10 minutes of washout ( ' washout ' ; lower panels ) . Scale bars = 1 µm . B . Time course of recovery during FRAP experiments described in A . circles ( ' basal ' ) show average spine / dendrite of FRAP on EGFP - GSK3α - expressing spines in wild - type mouse slices prior to cLTD induction . Pink circles ( ' cLTD ' ) show in spines in which cLTD had been induced using 20 µM NMDA . Green circles ( ' washout ' ) represent average of spine after NMDA washout for 10 minutes . Recovery trajectories for 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 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 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-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 . of last 10 seconds of recovery ( 90 - 100 seconds ) in B D . Error bars represent statistical significance was calculated according to Mann - Whitney U . N = 13 , 11 12 spines for wt baseline , cLTD recovery 9 , 7 5 spines for tauKO baseline , cLTD 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 B-CONTROLLED_VAR 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 statistical significance was calculated according to Mann - Whitney U .

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

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 - NMDAR - mediated EPSCs ( collected at − 60 mV + 40 mV , respectively ) in paired uninfected ( black traces ) 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 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 recordings of pairs of uninfected 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 averages of pairs in a given plot , n denotes number of pairs . In cases , error bars represent statistical significance was calculated according to Mann - Whitney U .

O O O O O O O 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

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

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

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

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


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

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

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 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 . lysates were immunoblotted with anti - ABRO1 anti - GAPDH antibodies .

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 B-MEASURED_VAR O O O O O

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

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 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 O O O O

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

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

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

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

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 + / + Abro1 − / − BMDMs left untreated or treated with nigericin or poly ( dA : dT ) . Scale bars , 10 μm . of ASC specks from ( H ) was performed by counting in five random areas of each image in triplicate experiments described as a of ASC specks for total nuclei . At least 100 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 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 O O B-MEASURED_VAR 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 + / + Abro1 − / − BMDMs were left untreated or treated with ATP . Immunoblot of NLRP3 ASC in lysates immunoprecipitated with anti - ASC antibody .

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

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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

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

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

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

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

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

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 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

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

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

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

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

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


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

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

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 were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 Myc - tagged NLRP3 , ASC or pro - casapase - 1 ( Pro - Casp - 1 ) . Immunoblot of Myc - Flag - tagged in 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 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

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 of ABRO1 NLRP3 in lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 Myc - tagged NLRP3 or NLRP3 deletion mutants as indicated in ( F ) . Immunoblot of Myc - Flag - tagged in 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 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

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 were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 Flag - tagged ABRO1 or ABRO1 deletion mutants as indicated in ( H ) . Immunoblot of Myc - Flag - tagged in 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 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

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 were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 Flag - tagged NLRP3 ( WT ) , NLRP3 ( S194D ) , or NLRP3 ( S194A ) . Immunoblot of Myc - Flag - tagged in 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 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

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 of NLRP3 ABRO1 in 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 B-MEASURED_VAR I-MEASURED_VAR 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 then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot of NLRP3 ABRO1 in 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 B-MEASURED_VAR I-MEASURED_VAR 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 transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194A ) were treated with or without 5 μM Anisomycin for 2 h . Immunoblot of Myc - Flag - tagged in lysates immunoprecipitated with anti - Flag M2 beads .

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 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

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 transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194D ) were treated with or without 100 nM SP600125 for 2 h . Immunoblot of Myc - Flag - tagged in 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 Nlrp3S194A / S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot of NLRP3 ABRO1 in lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

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

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

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

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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR 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 of NLRP3 NLRP3 ( A350V ) ubiquitination ( detected by mouse anti - ubiquitin antibody ) in 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 B-MEASURED_VAR I-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

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 were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 ( A350V ) HA - ubiquitin ( HA - Ub ) . Immunoblot of NLRP3 ( A350V ) ubiquitination ( detected by anti - HA antibody ) in 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 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

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 of ABRO1 NLRP3 in lysates immunoprecipitated with anti - ABRO1 antibody .

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

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.