Immunoprecipitation of interaction of T172 inactive mutant T172A , activation mutant T172D , double inactive mutant T172 \ T144 ( 2A ) or activation mutant ( 2D ) transiently - expressed in mitotic .

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Immunoprecipitation analysis of the interaction of the T172 inactive mutant T172A, the activation mutant T172D, and the double inactive mutant T172\T144 (2A) or activation mutant (2D) transiently-expressed in mitotic cells.


for inactive or activation mutant interactions indicated in Fig 6F . wild type BCL9 interaction is normalised to 1 , indicated by a green dashed with an arrow .

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Quantification for the inactive or activation mutant interactions indicated in Fig 6F. The wild type BCL9 interaction is normalised to 1, indicated by a green dashed line with an arrow.


Immunohistochemical of p - T172 - BCL9 expression in a human HCC tissue . MN : matched normal tissues , T : tumour tissues . Scale bar represents1000 μm .

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Immunohistochemical staining of p-T172-BCL9 expression in a human HCC tissue. MN: matched normal tissues, T: tumour tissues. Scale bar represents1000 μm.


expression scored in a tissue array of 75 pairs of human lung cancer tissues . Scale bar represents 1000 μm .

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IHC staining and the expression scored in a tissue array of 75 pairs of human lung cancer patient tissues. Scale bar represents 1000 μm.


A representative figure of p - T172 - BCL9 in HCC to indicate expression in mitotic or in interphase . Scale bar represents 100 μm .

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A representative figure of p-T172-BCL9 IHC staining in HCC to indicate the expression in mitotic or in interphase cells. Scale bar represents 100 μm.


Representative figures of p - T172 - BCL9 in interphase of breast cancer ( D ) or lung cancer ( E ) tissue . Scale bars represent 100 μm .

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Representative figures of p-T172-BCL9 IHC staining in interphase cells of breast cancer (D) or lung cancer (E) patient tissue. Scale bars represent 100 μm.


Immunoprecipitation of interaction in 5 pairs of human HCC tissues by a p - T172 - BCL9 antibody or a BCL9 antibody . CLTC relative binding with BCL9 was determined .

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Immunoprecipitation analysis of the interaction in 5 pairs of human HCC patient tissues by a p-T172-BCL9 antibody or a BCL9 antibody. CLTC relative binding with BCL9 was determined.


Log - rank ( Mantel - Cox ) survival on high low p - T172 - BCL9 expression scored by immunohistochemical in two tissue arrays with ( G ) 75 lung cancer samples or ( H ) 145 breast cancer samples .

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Log-rank (Mantel-Cox) survival analysis based on high and low p-T172-BCL9 expression scored by immunohistochemical staining in two tissue arrays with (G) 75 lung cancer samples or (H) 145 breast cancer samples.


( f ) survival of 15 d old with without SA187 under NHS , HS LAT conditions . Data information : survival measuremts after recovery at 22 ° C for 4 days . For survival , with green leaves were scored as live out of 12 for each biological repeat in different treatments . treatments are compared with direct 44 ° C heat stressed . Plots represent mean of 3 biological replicates bars representSE Asterisks indicate a statistical difference on Mann - Whitney ( * P < 0 . 05 )

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(f) Percent survival of 15 d old plants with and without SA187 under NHS, HS and LAT conditions. Data information: percent survival measuremts after recovery at 22°C for 4 days. For percent survival, plants with green leaves were scored as live plant out of 12 plants for each biological repeat in different temperature treatments. All treatments are compared with direct 44°C heat stressed plants. Plots represent the mean of 3 biological replicates bars representSE Asterisks indicate a statistical difference based on the Mann-Whitney test (* P < 0.05 )


( a , b ) Dynamics of HSFA2 HSP101 transcript in control ( NHS , NHS + 187 ) , 44 ° C heat - stressed non - colonized SA187 - colonized ( HS , HS + 187 ) , thermoprimed ( LAT ) as well as thermoprimed SA187 - colonized ( LAT + 187 ) at 1 h , 24 h , 48 h , 72 h 96 h of recovery at 22 ° C . SA187 - colonized ( HS + 187 ) thermoprimed ( LAT ) showed higher transcript in comparison to exposed at 44 ° C HS for 30 min ( HS ) after 1 h of recovery at 22 ° C . Data information : plots represent means of 3 biological replicates . Error bars represent SE . Asterisks indicate a statistical difference on Student ' s t - ( * P ≤ 0 .

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(a,b) Dynamics of HSFA2 and HSP101 transcript levels in control (NHS, NHS+187), 44°C heat-stressed non-colonized and SA187-colonized (HS, HS+187), thermoprimed (LAT) as well as thermoprimed and SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C. SA187-colonized (HS+187) and thermoprimed (LAT) plants showed higher transcript levels in comparison to plants exposed at 44°C HS for 30 min (HS) after 1 h of recovery at 22°C. Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05


( c , d ) . Transcript of heat stress memory genes HSP18 . 1 APX2 in control ( NHS , NHS + 187 ) , 44 ° C heat - stressed non - colonized SA187 - colonized ( HS , HS + 187 ) , thermoprimed ( LAT ) as well as thermoprimed SA187 - colonized ( LAT + 187 ) at 1 h , 24 h , 48 h , 72 h 96 h of recovery at 22 ° C . Transcript were normalized to tubulin as reference gene respective 22 ° C NHS were harvested at same time points . treatments are compared with direct 44 ° C HS treatment for statistical significance . Data information : plots represent means of 3 biological replicates . Error bars represent SE . Asterisks indicate a statistical difference on Student ' s t - ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 .

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(c,d). Transcript levels of heat stress memory genes HSP18.1 and APX2 in control plants (NHS, NHS+187), 44°C heat-stressed non-colonized and SA187-colonized (HS, HS+187), thermoprimed (LAT) as well as thermoprimed SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C . Transcript levels were normalized to tubulin as reference gene and the respective 22°C NHS plants were harvested at the same time points. All treatments are compared with direct 44°C HS treatment for statistical significance. Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001


( g ) Relative enrichment of H3K4me3 at APX2 HSP18 . 2 in control non - primed ( NP ) , SA187 - colonized non - primed ( NP + 187 ) , 37 ° C - primed ( P ) 37 ° C - primed SA187 - colonized ( P + 187 ) at 24 72 h after priming as determined by immunoprecipitation - qPCR for indicated regions of APX2 HSP18 . 2 . Amplification values were normalized to input H3 region 1 of non - primed ( NP ) . Data information : plots represent means of 3 biological replicates . Error bars represent SE . Asterisks indicate a statistical difference on Student ' s t - ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 for differences between NP in comparison to NP + 187 , P P + 187 treatments ) .

O 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 O O O O O 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 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

(g) Relative enrichment of H3K4me3 at APX2 and HSP18.2 in control non-primed (NP), SA187-colonized non-primed plants (NP+187), 37°C-primed (P) and 37°C-primed SA187-colonized plants (P+187) at 24 and 72 h after priming as determined by chromatin immunoprecipitation-qPCR for the indicated regions of APX2 and HSP18.2. Amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments).


( b , c ) Fresh survival of Col - 0 , hsfa2 , hsfa1q ein3 - 1 in HS , HS + 187 , LAT LAT + 187 treatments . Due to dwarf of hsfa1q mutants , LAT treatment was performed on d 18 HS at d 20 . treatments are compared with upon 44 ° C HS . Data information : plots represent means of 3 biological replicates ( n = 36 , 12 per biological repeat ) . Error bars represent SD of three biological repeats . Asterisks indicate a statistical difference on Student ' s t - ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

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(b,c) Fresh weight and percent survival of Col-0, hsfa2, hsfa1q and ein3-1 plants in HS, HS+187, LAT and LAT+187 treatments. Due to the dwarf size of hsfa1q mutants, LAT treatment was performed on d 18 and HS at d 20. All treatments are compared with plants upon 44°C HS. Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).


( e ) survival of Col - 0 with without ACC under HS , HS + ACC , LAT LAT + ACC conditions . Data information : plots represent means of 3 biological replicates ( n = 36 , 12 per biological repeat ) . Error bars represent SD of three biological repeats . Asterisks indicate a statistical difference on Student ' s t - ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

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(e) Percent survival of Col-0 plants with and without ACC under HS, HS+ACC, LAT and LAT+ACC conditions. Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).


( c ) Relative enrichment of H3K4me3 at APX2 HSP18 . 2 in control non - primed ( NP ) , SA187 - colonized non - primed ( NP + 187 ) , 37 ° C - primed ( P ) 37 ° C - primed SA187 - colonized ( P + 187 ) at 24 72 h after priming as determined by immunoprecipitation - qPCR for indicated regions of APX2 HSP18 . 2 ( Fig 3F ) . Data information : For H3K4me3 enrichment , amplification values were normalized to input H3 region 1 of non - primed ( NP ) . treatments are compared with direct 44 ° C HS treatment for statistical significance . plots represent means of 3 biological replicates . Error bars indicate SE . Asterisks indicate a statistical difference on Student ' s t - * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * P ≤ 0 . 001 for differences between NP in comparison to NP + 187 , P P + 187 treatments .

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(c) Relative enrichment of H3K4me3 at APX2 and HSP18.2 in control non-primed (NP), SA187-colonized non-primed plants (NP+187), 37°C-primed (P) and 37°C-primed SA187-colonized plants (P+187) at 24 and 72 h after priming as determined by chromatin immunoprecipitation-qPCR for the indicated regions of APX2 and HSP18.2 (Fig 3F). Data information: For H3K4me3 enrichment analysis, amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments.


( f ) Relative enrichment of H3K4me3 at APX2 HSP18 . 2 memory genes in hsfa2 mutant . Data information : For H3K4me3 enrichment , amplification values were normalized to input H3 region 1 of non - primed ( NP ) . treatments are compared with direct 44 ° C HS treatment for statistical significance . plots represent means of 3 biological replicates . Error bars indicate SE . Asterisks indicate a statistical difference on Student ' s t - * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * P ≤ 0 . 001 for differences between NP in comparison to NP + 187 , P P + 187 treatments .

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(f) Relative enrichment of H3K4me3 levels at APX2 and HSP18.2 memory genes in hsfa2 mutant. Data information: For H3K4me3 enrichment analysis, amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments.


( b ) Relative abundance of phyla in soil wheat root endosphere without ( R - mock ) with SA187 treatment ( R - 187 ) . Data information : Microbiome was performed in triplicates .

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(b) Relative percent abundance of bacterial phyla in the soil and the wheat root endosphere without (R-mock) and with SA187 treatment (R-187). Data information: Microbiome analysis was performed in triplicates.


( A ) 293T were co - transfected with expression plasmids for Cherry - STING , murine IFNβ - luciferase reporter ( IFNβ - Luc ) , a Renilla luciferase normalization control ( pRL - TK ) indicated expression plasmids or empty vector ( ev ) . were additionally co - transfected with expression plasmids for cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) . 20 hours post transfection , were lysed a dual - luciferase was performed . Data information : ( A - G ) Data is combined from three independent experiments .

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(A) 293T cells were co-transfected with expression plasmids for Cherry-STING, the murine IFNβ-luciferase reporter (IFNβ-Luc), a Renilla luciferase normalization control (pRL-TK) and the indicated expression plasmids or empty vector (ev). Cells were additionally co-transfected with expression plasmids for cGAS-GFP (stimulated) or IRES-GFP (unstimulated). 20 hours post transfection, cells were lysed and a dual-luciferase assay was performed. Data information: (A-G) Data is combined from three independent experiments.


( B ) An expression plasmid for RIG - I N ( stimulated ) or ev ( unstimulated ) was co - transfected with IFNβ - Luc , pRL - TK indicated expression plasmids in 293T analyzed as in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments .

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(B) An expression plasmid for RIG-I N (stimulated) or ev (unstimulated) was co-transfected with IFNβ-Luc, pRL-TK and the indicated expression plasmids in 293T cells and analyzed as in (A). Data information: (A-G) Data is combined from three independent experiments.


( C ) An expression plasmid for TBK1 ( stimulated ) or ev ( unstimulated ) was co - transfected together with IFNβ - Luc , pRL - TK indicated expression plasmids analyzed as in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments .

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(C) An expression plasmid for TBK1 (stimulated) or ev (unstimulated) was co-transfected together with IFNβ-Luc, pRL-TK and the indicated expression plasmids and analyzed as in (A). Data information: (A-G) Data is combined from three independent experiments.


( D ) 293T were co - transfected with a plasmid expressing constitutively active IRF3 ( IRF3 - 5D ; stimulated ) or IRES - GFP ( unstimulated ) together with IFNβ - Luc , pRL - TK indicated expression plasmids analyzed as in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments .

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(D) 293T cells were co-transfected with a plasmid expressing constitutively active IRF3 (IRF3-5D; stimulated) or IRES-GFP (unstimulated) together with IFNβ-Luc, pRL-TK and the indicated expression plasmids and analyzed as in (A). Data information: (A-G) Data is combined from three independent experiments.


( E ) ISG56 - luciferase reporter , pRL - TK indicated expression plasmids were co - transfected in 293T . 24 hours post transfection , were stimulated with 0 . 1 ng / µl human IFNβ or mock stimulated analyzed 16 hours later as described in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments .

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(E) The ISG56-luciferase reporter, pRL-TK and the indicated expression plasmids were co-transfected in 293T cells. 24 hours post transfection, cells were stimulated with 0.1 ng/µl human IFNβ or mock stimulated and analyzed 16 hours later as described in (A). Data information: (A-G) Data is combined from three independent experiments.


( F - G ) iMEFgt / gt stably expressing Cherry - STING either ev or V5 - tagged m152 were stimulated with 5 µg / ml ISD ( F ) , 10 µg / ml poly ( I : C ) ( G ) or mock stimulated with Lipofectamine . 4 hours post stimulation , was extracted to determine IFNβ mRNA transcripts by qRT - PCR . Data information : ( A - G ) Data is combined from three independent experiments .

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(F-G) iMEFgt/gt stably expressing Cherry-STING and either ev or V5-tagged m152 were stimulated with 5 µg/ml ISD (F), 10 µg/ml poly(I:C) (G) or mock stimulated with Lipofectamine. 4 hours post stimulation, RNA was extracted to determine IFNβ mRNA transcripts by qRT-PCR. Data information: (A-G) Data is combined from three independent experiments.


( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with 10 µg / ml cGAMP ( H ) , 6 ( H ) or 16 ( I - K ) hours later , secreted IFNβ ( H - J ) were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently one representative experiment is shown . Student ' s t - ( unpaired , two - ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

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(H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with 10 µg/ml cGAMP (H), 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.


( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with 5 µg / ml ISD ( I ) , 6 ( H ) or 16 ( I - K ) hours later , secreted IFNβ ( H - J ) were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently one representative experiment is shown . Student ' s t - ( unpaired , two - ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

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(H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with 5 µg/ml ISD (I), 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.


( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with Newcastle ' s Disease ( NDV ) infection ( J ) 6 ( H ) or 16 ( I - K ) hours later , secreted IFNβ ( H - J ) were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently one representative experiment is shown . Student ' s t - ( unpaired , two - ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

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(H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with Newcastle's Disease Virus (NDV) infection (J) 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.


( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with µM CpG ( K ) . 6 ( H ) or 16 ( I - K ) hours later , TNFα ( K ) were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently one representative experiment is shown . Student ' s t - ( unpaired , two - ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

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

(H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with µM CpG DNA (K). 6 (H) or 16 (I-K) hours later, TNFα (K) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.


( A ) HeLa were co - transfected with expression plasmids for Cherry - STING , V5 - tagged m152 either ev ( unstimulated ) or cGAS - GFP ( stimulated ) .

O O 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

(A) HeLa cells were co-transfected with expression plasmids for Cherry-STING, V5-tagged m152 and either ev (unstimulated) or cGAS-GFP (stimulated).


( B ) iMEFgt / gt were co - transfected with expression plasmids for V5 - tagged m152 together with either Cherry - STING or ev in combination with cGAS - GFP ( stimulated ) or ev ( unstimulated ) . 24 hours post transfection were fixed for immunolabeling with an anti - V5 antibody . boxes indicate region shown at a higher magnification . Scale bar represents 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 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 O O O O O O

(B) iMEFgt/gt were co-transfected with expression plasmids for V5-tagged m152 together with either Cherry-STING or ev in combination with cGAS-GFP (stimulated) or ev (unstimulated). 24 hours post transfection cells were fixed for immunolabeling with an anti-V5 antibody. White boxes indicate the region shown at a higher magnification. Scale bar represents 10 µm.


( C ) Lysates of Cherry - STING either CD4 - V5 or m152 - V5 expressing 293T were subjected to immunoprecipitation ( IP ) with an anti - V5 antibody . Input IP samples were analyzed by IB with indicated antibodies . Data information : IB shown are representative of three ( C ) or two ( D ) 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 O O O O O O O O O O O O O O O O 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) Lysates of Cherry-STING and either CD4-V5 or m152-V5 expressing 293T cells were subjected to immunoprecipitation (IP) with an anti-V5 antibody. Input and IP samples were analyzed by IB with the indicated antibodies. Data information: IB shown are representative of three (C) or two (D) independent experiments.


( D ) iMEF stably expressing ev or m152 - V5 were left unstimulated or stimulated with 10 µg / ml ISD lysed 90 minutes later . m152 was immunoprecipitated with an anti - V5 antibody samples were analyzed by IB with V5 , STING phospho - TBK1 ( pTBK1 ) specific antibodies . Data information : IB shown are representative of three ( C ) or two ( D ) independent experiments .

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 O O O O O O O O O O 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

(D) iMEF stably expressing ev or m152-V5 were left unstimulated or stimulated with 10 µg/ml ISD and lysed 90 minutes later. m152 was immunoprecipitated with an anti-V5 antibody and samples were analyzed by IB with V5, STING and phospho-TBK1 (pTBK1) specific antibodies. Data information: IB shown are representative of three (C) or two (D) independent experiments.


( B ) Cherry - STING , IFNβ - Luc , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) indicated expression plasmids as shown in ( A ) were transiently expressed in 293T a dual luciferase was performed . Data is combined from two out of three independent experiments shown as mean ± SD . Data information : Student ' s t - ( unpaired , two - ) , n . s . not significant , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

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

(B) Cherry-STING, IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids as shown in (A) were transiently expressed in 293T cells and a dual luciferase assay was performed. Data is combined from two out of three independent experiments and shown as mean ± SD. Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, **p<0.01, ***p<0.001, ****p<0.0001


( C ) 293T were co - transfected with expression plasmids for Cherry - STING indicated chimeras as shown in ( A ) . An anti - V5 IP was performed samples were analyzed by IB with indicated antibodies . IB shown is representative of 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 O O 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) 293T cells were co-transfected with expression plasmids for Cherry-STING and indicated chimeras as shown in (A). An anti-V5 IP was performed and samples were analyzed by IB with indicated antibodies. IB shown is representative of three independent experiments.


( D ) Cherry - STING , IFNβ - Luc , pRL - TK either CD4 , m152 , m152 - Δstalk or m152 - GSstalk were transiently expressed in 293T . For stimulation , samples were co - transfected with cGAS - GFP , unstimulated samples with IRES - GFP . Lysates were analyzed as described in ( B ) . Data is combined from two out of three independent experiments shown as mean ± SD . Data information : Student ' s t - ( unpaired , two - ) , n . s . not significant , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cherry-STING, IFNβ-Luc, pRL-TK and either CD4, m152, m152-Δstalk or m152-GSstalk were transiently expressed in 293T cells. For stimulation, samples were co-transfected with cGAS-GFP, and unstimulated samples with IRES-GFP. Lysates were analyzed as described in (B). Data is combined from two out of three independent experiments and shown as mean ± SD. Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, **p<0.01, ***p<0.001, ****p<0.0001


( E ) 293T were co - transfected with expression plasmids for Cherry - STING either CD4 , m152 , m152 - Δstalk or m152 - GSstalk . An anti - V5 IP was performed samples were analyzed by IB with indicated antibodies . Immunoblot shown is representative of three independent experiments .

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 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

(E) 293T cells were co-transfected with expression plasmids for Cherry-STING and either CD4, m152, m152-Δstalk or m152-GSstalk. An anti-V5 IP was performed and samples were analyzed by IB with indicated antibodies. Immunoblot shown is representative of three independent experiments.


( A ) 293T were co - transfected with expression plasmids for IFNβ - Luc , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) indicated expression plasmids together with murine Cherry - STING ( left panel ) or human STING ( right panel ) . Lysates were analyzed by dual - luciferase . Data is combined from two out of three independent experiments shown as mean ± SD .

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

(A) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids together with murine Cherry-STING (left panel) or human STING (right panel). Lysates were analyzed by dual-luciferase assay. Data is combined from two out of three independent experiments and shown as mean ± SD.


( C ) IFNβ - Luc , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) either LacZ or m152 together with indicated murine STING mutants described in ( B ) were transiently expressed in 293T . Luciferase activity was measured as described in ( A ) . Data information : Data in ( C ) is normalized to LacZ , combined from three independent experiments shown as mean ± SD .

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 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

(C) IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either LacZ or m152 together with the indicated murine STING mutants described in (B) were transiently expressed in 293T cells. Luciferase activity was measured as described in (A). Data information: Data in (C) is normalized to LacZ, combined from three independent experiments and shown as mean ± SD.


( D ) 293T were co - transfected with expression plasmids for either LacZ or m152 together with indicated murine STING mutants described in ( B ) . An anti - V5 IP was performed samples were analyzed by IB with indicated antibodies . IB shown in ( D ) representative of three independent experiments .

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

(D) 293T cells were co-transfected with expression plasmids for either LacZ or m152 together with the indicated murine STING mutants described in (B). An anti-V5 IP was performed and samples were analyzed by IB with the indicated antibodies. IB shown in (D) representative of three independent experiments.


( E - F ) Luciferase ( E ) were performed as described in ( C ) ( D ) , respectively , using indicated human STING mutants in place of murine STING mutants described in ( B ) . Data information : Data in ( E ) is normalized to LacZ , combined from three independent experiments shown as mean ± SD .

O O 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

(E-F) Luciferase assay (E) were performed as described in (C) and (D), respectively, using the indicated human STING mutants in place of the murine STING mutants described in (B). Data information: Data in (E) is normalized to LacZ, combined from three independent experiments and shown as mean ± SD.


( E - F ) co - IP ( F ) were performed as described in ( C ) ( D ) , respectively , using indicated human STING mutants in place of murine STING mutants described in ( B ) . Data information : IB shown in ( F ) are representative of 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 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

(E-F) co-IP (F) were performed as described in (C) and (D), respectively, using the indicated human STING mutants in place of the murine STING mutants described in (B). Data information: IB shown in (F) are representative of three independent experiments.


( B ) 293T were co - transfected with expression plasmids for IFNβ - Luc , pRL - TK either ev or m152 . were further co - transfected with either Cherry - STING WT or constitutively active Cherry - STING V154M ( stimulated ) or with IRES - GFP ( unstimulated ) . Data is combined from three independent experiments shown as mean ± SD .

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 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

(B) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK and either ev or m152. Cells were further co-transfected with either Cherry-STING WT or the constitutively active Cherry-STING V154M (stimulated) or with IRES-GFP (unstimulated). Data is combined from three independent experiments and shown as mean ± SD.


( C ) iMEF stably expressing ev or m152 - V5 were stimulated with 10 µg / ml ISD for indicated times or left unstimulated ( mock ) . lysates were analyzed by SDS - PAGE under non - reducing conditions subjected to IB with specified antibodies . Data information : IB shown in ( C ) 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) iMEF stably expressing ev or m152-V5 were stimulated with 10 µg/ml ISD for the indicated times or left unstimulated (mock). Cell lysates were analyzed by SDS-PAGE under non-reducing conditions and subjected to IB with the specified antibodies. Data information: IB shown in (C) 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


( D ) Two representative still from live imaging experiments with iMEFgt / gt stably expressing Cherry - STING transfected with ISD ( right panel ) or left unstimulated ( left panel ) . Red circles highlight representative translocated STING in ISD stimulated , which is used as an indicator of activation .

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

(D) Two representative still images from live cell imaging experiments with iMEFgt/gt stably expressing Cherry-STING transfected with ISD (right panel) or left unstimulated (left panel). Red circles highlight representative translocated STING in ISD stimulated cells, which is used as an indicator of activation.


( E ) iMEFgt / gt stably expressing Cherry - STING either ev or m152 - V5 were stimulated with ISD . 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 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 and either ev or m152-V5 were stimulated with ISD. 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 ) 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 .

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(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 .

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(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 .

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(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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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


Confocal 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 .

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


Top : Schematic of 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 .

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


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 .

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


Confocal image showing a donor ( D ) 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 ) .

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


Representative confocal 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 .

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


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 .

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


Transfer of α - syn fibrils is shown in neurons at 1 DIV . 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 .

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


A TNT - like connection containing Alexa - 488 α - syn positive 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 ) .

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


Representative confocal 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 .

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


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

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


Time lapses of neurons loaded with Alexa - 568 α - syn fibrils 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 .

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


Representative confocal 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 .

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


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

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


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

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


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

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


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

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


M Succinate dehydrogenase ( SDH ) 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 :

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


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

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


I Transmission electron microscopy 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 )

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


G Masson ' s trichrome 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 ) .

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


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

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


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

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


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

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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 ) .

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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 ) .

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


C Western blot validation of 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 .

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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 ) .

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


A Immunohistochemical 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 ) .

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


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

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


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

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


G , H Representative immunoblot 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 ) .

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


J - N Representative Western blot 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 .

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


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

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


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

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


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

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


C , D . Scatter plots showing 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 .

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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 .

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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 .

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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 .

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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 ) .

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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).