Figure 1Normalised Wnt / STOP signalling activity by quantification of the protein expression of two Wnt / STOP target proteins , β - catenin and c - Myc , in mitotic ( M ) and asynchronised cells ( A ) after silencing 47 Wnt signalling regulators . ELISA validation of selected Wnt / STOP signalling regulators ( n = 3 ) . Immunoblotting and ( D ) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX ( n = 3 ) . Representative pictures ( left panel ) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3 - GFP biosensor following different treatments before and after metaphase ( n = 10 ) . Scale bars represent 10 μm . Myc and β - catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9 . The representative xenograft tumours ( top panel ) and tumour growth analysis ( bottom panel ) of scramble and BCL9 stable knockdown cells . LF : left flank of mouse , RF : right flank . n = 6 for shS1 vs . shB1 , n = 5 for shS1 vs . shB2 . Immunohistochemical staining of the xenograft ; the red arrows indicate the representative mitotic cells . Scale bars represent 200 μm .

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Figure 1Normalised Wnt/STOP signalling activity by quantification of the protein expression of two Wnt/STOP target proteins, β-catenin and c-Myc, in mitotic (M) and asynchronised cells (A) after silencing 47 Wnt signalling regulators.ELISA validation of selected Wnt/STOP signalling regulators (n=3).Immunoblotting and (D) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX (n=3).Representative pictures (left panel) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3-GFP biosensor following different treatments before and after metaphase (n=10). Scale bars represent 10 μm.Myc and β-catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9.The representative xenograft tumours (top panel) and tumour growth analysis (bottom panel) of scramble and BCL9 stable knockdown cells. LF: left flank of mouse, RF: right flank. n=6 for shS1 vs. shB1, n=5 for shS1 vs. shB2.Immunohistochemical staining of the xenograft; the red arrows indicate the representative mitotic cells. Scale bars represent 200 μm.


Figure 2Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells ; the black arrow indicates the delayed band of mitotic BCL9 . Immunofluorescence confocal analysis of BCL9 localization on the spindle by antibody staining . Yellow arrows indicate the spindle pole . Scale bars represent : 2 μm for interphase and anaphase ; 5 μm for prophase , metaphase and telophase . Immunofluorescence confocal analysis of BCL9 localization on the centrosome . Yellow arrows indicate the centrosome . Scale bars represent : 5 μm for control treatment , 10 μm for BCL9 peptide treatment . Confocal microscope 3D - analysis and radial histograms of the spindle angle in the cells with siRNA treatment ; yellow arrows indicate the centrosome . Scale bars represent 5 μm . Confocal ( left panel ) and live cell imaging ( right panel ) analysis of chromosome segregation in H2BGFP - cells following siRNA treatment ; yellow arrows indicate the typical abnormal chromosomes by BCL9 knockdown . 23 H2BGFP - mitotic cells were monitored for each time experiment ; data are shown from three independent experiments . Scale bars represent 5 μm . Quantification of aberrant chromosome segregation rate by live cell imaging of H2BGFP cells upon Wnt3a or DKK1 treatment . 23 H2BGFP - mitotic cells were monitored for each time experiment ; data are shown from three independent experiments . Quantification of the duration of NEB to ANA ( NEB : nuclear envelope breakdown , ANA : anaphase ) , NEB to cell death in interphase after cell division ( blue colour bar ) or in mitosis ( dark red bar ) , or growth well in 30 hours ( pink bar ) by live cell imaging analysis in monitored H2BGFP cells with different treatments .

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Figure 2Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells; the black arrow indicates the delayed band of mitotic BCL9.Immunofluorescence confocal analysis of BCL9 localization on the spindle by antibody staining. Yellow arrows indicate the spindle pole. Scale bars represent: 2 μm for interphase and anaphase; 5 μm for prophase, metaphase and telophase.Immunofluorescence confocal analysis of BCL9 localization on the centrosome. Yellow arrows indicate the centrosome. Scale bars represent: 5 μm for control treatment, 10 μm for BCL9 peptide treatment.Confocal microscope 3D-analysis and radial histograms of the spindle angle in the cells with siRNA treatment; yellow arrows indicate the centrosome. Scale bars represent 5 μm.Confocal (left panel) and live cell imaging (right panel) analysis of chromosome segregation in H2BGFP-cells following siRNA treatment; yellow arrows indicate the typical abnormal chromosomes by BCL9 knockdown. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments. Scale bars represent 5 μm. Quantification of aberrant chromosome segregation rate by live cell imaging of H2BGFP cells upon Wnt3a or DKK1 treatment. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments.Quantification of the duration of NEB to ANA (NEB: nuclear envelope breakdown, ANA: anaphase), NEB to cell death in interphase after cell division (blue colour bar) or in mitosis (dark red bar), or growth well in 30 hours (pink bar) by live cell imaging analysis in monitored H2BGFP cells with different treatments.


Figure 3Volcano plot of the BCL9 mitotic interactome by the SILAC proteomics approach . Red dashed line indicates a strict cut off by a normalised SILAC H / L ratio > 2 . An independent immunoprecipitation assay for validation of potential BCL9 interactors in both asynchronised and mitotic cells . The relative binding of CLTC with BCL9 is quantified and labelled on top of the CLTC blot . Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells , from G1S to G2M . P - H3S10 , a mitotic marker indicating mitosis .

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Figure 3Volcano plot of the BCL9 mitotic interactome by the SILAC proteomics approach. Red dashed line indicates a strict cut off by a normalised SILAC H/L ratio > 2.An independent immunoprecipitation assay for validation of potential BCL9 interactors in both asynchronised and mitotic cells. The relative binding of CLTC with BCL9 is quantified and labelled on top of the CLTC blot.Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells, from G1S to G2M. P-H3S10, a mitotic marker indicating mitosis.


Figure 4Duolink analysis ( A ) and its quantification ( B ) of the interaction by assessing the PLA complex in mitotic cells after BCL9 or Axin1 knockdown ( n = 20 ) . Scale bars represent 5 μm . Duolink analysis of the interaction of BCL9 and CLTC in mitotic cells . Scale bars represent 5 μm . Immunoprecipitation analysis of the interaction of truncated BCL9 forms in mitosis ; the purple stars indicate the domain required for clathrin complex binding , and the green stars indicate the domains for β - catenin complex binding . The domain required for both clathrin and β - catenin complexes binding is indicated by a red line . Quantification of the relative binding of the truncated forms with the clathrin complex or β - catenin complex ; the red frame indicates the common domains of BCL9 for binding of both complexes . Immunoprecipitation analysis of the BCL9 - MF interaction in CLTC knockdown mitotic cells . Immunoprecipitation analysis of the endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF - overexpressing mitotic cells .

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Figure 4Duolink analysis (A) and its quantification (B) of the interaction by assessing the PLA complex in mitotic cells after BCL9 or Axin1 knockdown (n=20). Scale bars represent 5 μm.Duolink analysis of the interaction of BCL9 and CLTC in mitotic cells. Scale bars represent 5 μm.Immunoprecipitation analysis of the interaction of truncated BCL9 forms in mitosis; the purple stars indicate the domain required for clathrin complex binding, and the green stars indicate the domains for β-catenin complex binding. The domain required for both clathrin and β-catenin complexes binding is indicated by a red line.Quantification of the relative binding of the truncated forms with the clathrin complex or β-catenin complex; the red frame indicates the common domains of BCL9 for binding of both complexes.Immunoprecipitation analysis of the BCL9-MF interaction in CLTC knockdown mitotic cells.Immunoprecipitation analysis of the endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF-overexpressing mitotic cells.


Figure 5Immunoblotting analysis of signalosome components by immunoblotting in BCL9 , CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX . Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX . Immunofluorescence confocal analysis of Ser1490 - phosphorylated LRP6 and CLTC co - localization in mitotic HeLa cells ; white arrows indicate the colocalization . Scale bars represent 5 μm . Immunofluorescence analysis of α - tubulin , Axin1 and CLTC triple - staining in mitotic cells . The white arrows indicate the colocalization . Scale bars represent 5 μm . Immunofluorescence analysis of colocalization of LRP6CAGFP and CLTC or CD63 in interphase cells . The white arrows indicate the colocalization . Scale bars represent 5 μm . Quantification of signalosome components and mitotic Wnt target expression level by immunoblotting in natural mitotic cells treated for 1 hour with DMSO or MDC , an inhibitor of clathrin - mediated - endocytosis . A representative image of soft agar oncogenic colony formation assays for stable knockdown cells . A representative image of the mouse xenograft tumours for stable knockdown cells at the end point of the experiment .

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Figure 5Immunoblotting analysis of signalosome components by immunoblotting in BCL9, CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX. Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX.Immunofluorescence confocal analysis of Ser1490-phosphorylated LRP6 and CLTC co-localization in mitotic HeLa cells; white arrows indicate the colocalization. Scale bars represent 5 μm.Immunofluorescence analysis of α-tubulin, Axin1 and CLTC triple-staining in mitotic cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.Immunofluorescence analysis of colocalization of LRP6CAGFP and CLTC or CD63 in interphase cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.Quantification of signalosome components and mitotic Wnt target expression level by immunoblotting in natural mitotic cells treated for 1 hour with DMSO or MDC, an inhibitor of clathrin-mediated-endocytosis.A representative image of soft agar oncogenic colony formation assays for stable knockdown cells.A representative image of the mouse xenograft tumours for stable knockdown cells at the end point of the experiment.


Figure 6In vitro CDK1 / cyclin B1 kinase assay for immunoprecipitation - purified BCL9 - Flag or T172A - Flag proteins . Immunoprecipitation analysis of transiently - expressed BCL9 - MF or T172A - MF interactors in synchronised cells , from G1S to G2 M phase . The relative level of BCL9 - MF or T172A - MF immunoprecipitation is determined ; red numbers indicate up - regulation , and green numbers indicate down - regulation compared to the starting time point . Immunoprecipitation analysis of the transiently - expressed BCL9 - MF interaction in mitotic cells treated with RO3306 . 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 . 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 .

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Figure 6In vitro CDK1/cyclin B1 kinase assay for immunoprecipitation-purified BCL9-Flag or T172A-Flag proteins.Immunoprecipitation analysis of transiently-expressed BCL9-MF or T172A-MF interactors in synchronised cells, from G1S to G2 M phase. The relative level of BCL9-MF or T172A-MF immunoprecipitation is determined; red numbers indicate up-regulation, and green numbers indicate down-regulation compared to the starting time point.Immunoprecipitation analysis of the transiently-expressed BCL9-MF interaction in mitotic cells treated with RO3306.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.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.


Figure 7Immunohistochemical staining of p - T172 - BCL9 expression in a human HCC tissue . MN : matched normal tissues , T : tumour tissues . Scale bar represents1000 μm . 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 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 IHC staining in interphase cells of breast cancer ( D ) or lung cancer ( E ) patient tissue . Scale bars represent 100 μm . 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 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 .

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Figure 7Immunohistochemical staining of p-T172-BCL9 expression in a human HCC tissue. MN: matched normal tissues, T: tumour tissues. Scale bar represents1000 μm.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 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 IHC staining in interphase cells of breast cancer (D) or lung cancer (E) patient tissue. Scale bars represent 100 μm.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 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.


Figure 1 ( 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 )

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Figure 1(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 )


Figure 3 ( 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 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 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 ) .

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Figure 3(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 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 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).


Figure 4 ( 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 ) 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 ) .

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Figure 4(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) 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).


Figure 5 ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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 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.


Figure 6 ( 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 .

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Figure 6(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.


Figure 1 ( 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 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 and the indicated expression plasmids and analyzed as in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments . ( 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 ) 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 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 ) 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 ) 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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 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 and the indicated expression plasmids and analyzed as in (A). Data information: (A-G) Data is combined from three independent experiments.(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) 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 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) 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) 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 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 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.


Figure 2 ( 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 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 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 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 .

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Figure 2(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 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 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 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.


Figure 3 ( 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 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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 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 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 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.


Figure 4 ( 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 ) 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 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 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 ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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) 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 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 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) 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.


Figure 5 ( 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 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 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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 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 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 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 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


Figure 6 ( 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 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 ) 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 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 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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 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) 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 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 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 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 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.


Figure 7 ( 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 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 , 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 , 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 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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 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, 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, 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 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).


Figure 8 ( 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 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 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 ( - ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(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 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 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 (-) 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.


Figure 2A . 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 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 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 .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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 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 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.


Figure 1Representative 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative 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 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 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.


Figure 2Representative 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative 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 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 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.


Figure 3Representative 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 . Percentage of TNT - connected CAD cells 24 h after transfection with indicated GFP - fusion proteins . Percentage of transfection rate of indicated GFP - fusion proteins in CAD cells . 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 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 .

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Figure 3Representative 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.Percentage of TNT-connected CAD cells 24 h after transfection with indicated GFP-fusion proteins.Percentage of transfection rate of indicated GFP-fusion proteins in CAD cells.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 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.


Figure 4Time 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Time 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 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 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 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.


Figure 5Confocal images of untransfected and GFP - βCaMKII WT transfected CAD cells ( green ) , treated or not with Wnt7a . Top : Cells were stained with phalloidin ( red ) and DAPI ( blue ) . Middle : Phalloidin staining ( white ) of the cells is shown . Bottom : Pseudocolor images of cells stained with phalloidin highlight the differences in F - actin levels among the treatments . Scale bars represent 5 μm . Anti - actin immunoblot and bar graph showing changes in the ratio between G - actin ( in supernatant fraction , s ) and F - actin ( in the pellet fraction , p ) of CAD cells untransfected or transfected with GFP - βCaMKII WT plasmid and treated or not with Wnt7a . Confocal images of CAD cells transfected with GFP - βCaMKII WT plasmid ( green ) and treated or not with Wnt7a . Cells were fixed and then stained with phalloidin ( red ) and DAPI ( blue ) . The insets ( right panels ) show a magnified image of the area depicted in the expanded field images ( left ) . 3D reconstruction shows co - localization of GFP / phalloidin signals . Scale bars represent 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Confocal images of untransfected and GFP-βCaMKII WT transfected CAD cells (green), treated or not with Wnt7a. Top: Cells were stained with phalloidin (red) and DAPI (blue). Middle: Phalloidin staining (white) of the cells is shown. Bottom: Pseudocolor images of cells stained with phalloidin highlight the differences in F-actin levels among the treatments. Scale bars represent 5 μm. Anti-actin immunoblot and bar graph showing changes in the ratio between G-actin (in supernatant fraction, s) and F-actin (in the pellet fraction, p) of CAD cells untransfected or transfected with GFP-βCaMKII WT plasmid and treated or not with Wnt7a.Confocal images of CAD cells transfected with GFP-βCaMKII WT plasmid (green) and treated or not with Wnt7a. Cells were fixed and then stained with phalloidin (red) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field images (left). 3D reconstruction shows co-localization of GFP/phalloidin signals. Scale bars represent 10 µm.


Figure 6Top : Schematic of the culture systems used to assess cell - to - cell contact - dependent or secretion - mediated transfer of vesicles in primary neurons . After dissection , neurons were labeled in suspension with DiI ( donor , D ) or CTG ( acceptor , A ) . In the co - culture system , neurons were plated in 1 : 1 ratio and cultured for 20 h and then treated with 200 ng / mL of Wnt5a or Wnt7a for 4 h . In the secretion - based system , donor neurons cultured alone for 20 h were treated with 200 ng / mL of Wnt5a or Wnt7a for 4 h , and the medium was then collected and replaced for the medium of acceptor neurons cultured separately . Acceptor neurons were further incubated with the conditioned media of donor neuron for 24 h . Bottom : Representative confocal images of co - cultured donor and acceptor neurons . The insets ( right panels ) show a magnification of the area depicted in the expanded field images ( left ) . Yellow arrows point to DiI - labeled vesicles inside acceptor neurons . Percentage of acceptor neurons containing DiI - labeled vesicles after being co - cultured with donor neurons and incubated for 4 h with the indicated treatments . Percentage of acceptor neurons containing DiI - labeled vesicles after being cultured for 4 h with the conditioned media of treated donor neurons . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Top: Schematic of the culture systems used to assess cell-to-cell contact-dependent or secretion-mediated transfer of vesicles in primary neurons. After dissection, neurons were labeled in suspension with DiI (donor, D) or CTG (acceptor, A). In the co-culture system, neurons were plated in 1:1 ratio and cultured for 20 h and then treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h. In the secretion-based system, donor neurons cultured alone for 20 h were treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h, and the medium was then collected and replaced for the medium of acceptor neurons cultured separately. Acceptor neurons were further incubated with the conditioned media of donor neuron for 24 h. Bottom: Representative confocal images of co-cultured donor and acceptor neurons. The insets (right panels) show a magnification of the area depicted in the expanded field images (left). Yellow arrows point to DiI-labeled vesicles inside acceptor neurons.Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments. Percentage of acceptor neurons containing DiI-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor neurons.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).


Figure 7Representative confocal images of co - cultured donor ( D , labeled with DiI ) and acceptor ( A , labeled with CTG ) neurons treated or not with 200 ng / mL of Wnt5a , 200 ng / mL sFRP - 2 or 200 ng / mL Wnt5a plus 200 ng / mL sFRP - 2 for 4 h . Yellow arrows point to DiI - labeled vesicles inside acceptor neurons . Percentage of acceptor neurons containing DiI - labeled vesicles after being co - cultured with donor neurons and incubated for 4 h with the indicated treatments . Primary cortical neurons at 1 DIV were fixed and labeled with MAP - 2 ( green ) , β - III - tubulin ( red ) and WGA ( white ) . The inset shows a magnified image of the area depicted in the merged panel . Yellow arrowheads point out TNT - like structures . Transfer of α - syn fibrils is shown in neurons at 1 DIV . Cells were loaded in suspension with either Alexa - 568 α - syn fibrils ( red ) or Alexa - 488 α - syn fibrils ( green ) and cultured together . The insets ( right panels ) show a magnification of the area depicted in the expanded field image ( left ) . Yellow arrows point to red and green α - syn puncta contained in the soma of a neuron . Yellow arrowhead points to a TNT - like connection . A TNT - like connection containing Alexa - 488 α - syn positive puncta is found in neurons at 1 DIV stained with phalloidin ( white ) and DAPI ( blue ) . The insets ( right panels ) show the 3D reconstruction of the area depicted in the expanded field image ( left ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative confocal images of co-cultured donor (D, labeled with DiI) and acceptor (A, labeled with CTG) neurons treated or not with 200 ng/mL of Wnt5a, 200 ng/mL sFRP-2 or 200 ng/mL Wnt5a plus 200 ng/mL sFRP-2 for 4 h. Yellow arrows point to DiI-labeled vesicles inside acceptor neurons. Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments.Primary cortical neurons at 1 DIV were fixed and labeled with MAP-2 (green), β-III-tubulin (red) and WGA (white). The inset shows a magnified image of the area depicted in the merged panel. Yellow arrowheads point out TNT-like structures.Transfer of α-syn fibrils is shown in neurons at 1 DIV. Cells were loaded in suspension with either Alexa-568 α-syn fibrils (red) or Alexa-488 α-syn fibrils (green) and cultured together. The insets (right panels) show a magnification of the area depicted in the expanded field image (left). Yellow arrows point to red and green α-syn puncta contained in the soma of a neuron. Yellow arrowhead points to a TNT-like connection.A TNT-like connection containing Alexa-488 α-syn positive puncta is found in neurons at 1 DIV stained with phalloidin (white) and DAPI (blue). The insets (right panels) show the 3D reconstruction of the area depicted in the expanded field image (left).


Figure 8Representative confocal images of 1 DIV co - cultured donor ( D , loaded with Alexa - 568 α - syn fibrils ) and acceptor ( A , labeled with CTG ) neurons . After 20 h post - plating , neurons were treated with 200 ng / mL Wnt5a for 4 h . The insets ( right panels ) show a magnification of the area depicted in the expanded field ( left ) . Yellow arrows point to α - syn puncta inside acceptor neurons . Percentage of acceptor neurons containing α - syn puncta for each of the indicated treatments . Time lapses of neurons loaded with Alexa - 568 α - syn fibrils and cultured in control conditions or treated with 200 ng / mL Wnt5a . Neuron - to - neuron transfer of α - syn puncta ( yellow arrows ) through a TNT - like structure ( yellow arrowheads ) is shown upon Wnt5a treatment . Representative confocal images of 1 DIV co - cultured donor ( D , loaded with Alexa - 568 α - syn fibrils ) and acceptor ( A , labeled with CTG ) neurons . Neurons were obtained from WT or βCaMKII K . O . mice . Yellow arrows point to α - syn puncta inside acceptor neurons in both conditions .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. After 20 h post-plating, neurons were treated with 200 ng/mL Wnt5a for 4 h. The insets (right panels) show a magnification of the area depicted in the expanded field (left). Yellow arrows point to α-syn puncta inside acceptor neurons.Percentage of acceptor neurons containing α-syn puncta for each of the indicated treatments.Time lapses of neurons loaded with Alexa-568 α-syn fibrils and cultured in control conditions or treated with 200 ng/mL Wnt5a. Neuron-to-neuron transfer of α-syn puncta (yellow arrows) through a TNT-like structure (yellow arrowheads) is shown upon Wnt5a treatment.Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. Neurons were obtained from WT or βCaMKII K.O. mice. Yellow arrows point to α-syn puncta inside acceptor neurons in both conditions.