( I - J ) Relative abundances for Siglec - 8 in ( H ) hippocampus ( I ) posterior cingulate cortex post - mortem tissue . Linear regression R2 values are shown on plots .

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(I-J) Relative RNA abundances for Siglec-8 in (H) hippocampus and (I) posterior cingulate cortex post-mortem patient tissue. Linear regression R2 values are shown on plots.


( K ) IF showing Siglec - 8 Iba1 , MHC - II in cortical matter tissue from a with late - onset AD ( 20x wide - field ; Case ID : 01 - 43 ) . Scale bars = 50 μm . are 60x super - resolution max z - stack projections .

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(K) IF staining showing Siglec-8 and Iba1, and MHC-II in cortical white matter tissue from a patient with late-onset AD (20x wide-field; Case ID: 01-43). Scale bars = 50 μm. Images are 60x super-resolution max z-stack projections.


( L - M ) coverage of Siglec - 8 localized to Iba1 or MHC in individuals with non - AD or AD . In ( K ) each point represents one WM or GM region average from each . In ( L ) values statistics are shown for each image analyzed . Boolean mask operators indicate : ° = mask dilation , ⋂ = intersection , ⋃ = union , NOT = inverse mask . Box indicates quartiles whiskers indicate last datum within 1 . 5 inter - quartile range ; n = 8 - 43 ( n = 1 ND , 3 early - onset AD , 4 late - onset AD ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided .

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(L-M) Percent area coverage of Siglec-8 localized to Iba1 or MHC in individuals with non-AD or AD. In (K) each point represents one WM or GM region average from each individual. In (L) values and statistics are shown for each image analyzed. Boolean mask operators indicate: ° = mask dilation, ⋂ = intersection, ⋃ = union, NOT = inverse mask. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 8-43 images (n = 1 ND, 3 early-onset AD, 4 late-onset AD); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( A ) Flow cytometry of BV - 2 Siglec - F expression . were cotreated with 0 - 10 ng / mL IFNγ 0 - 10 μM tofacitinib . Values are geometric mean intensities ( gMFI ) . Bars indicate mean ± 95 % CI ; n = 2 - 4 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted indicates mean of untreated group .

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(A) Flow cytometry quantification of BV-2 Siglec-F expression levels. Cells were cotreated with 0-10 ng/mL IFNγ and 0-10 μM tofacitinib. Values are geometric mean fluorescence intensities (gMFI). Bars indicate mean ± 95% CI; n = 2-4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.


( B ) IF of iMGLs treated with IFNγ . Siglec - 8 is only shown on non - ( Hoechst 33342 - ) regions . are 60x super - resolution max z - stack projections . Colors are : Red = Iba1 , Green = Siglec - 8 , Blue = 33342 . Scale bars = 20 μm .

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(B) IF staining of iMGLs treated with IFNγ. Siglec-8 fluorescence is only shown on non-nuclear (Hoechst 33342-) regions. Images are 60x super-resolution max z-stack projections. Colors are: Red = Iba1, Green = Siglec-8, Blue = 33342. Scale bars = 20 μm.


( C ) of Siglec - 8 on iMGLs treated with PBS or 25 ng / mL IFNα , IFNβ , or IFNγ . Values are mean Siglec - 8 values from Iba1 + ; 33342 - regions . Bars indicate mean ± 95 % CI ; n = 8 ( 2 replicates ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted indicates mean of untreated group .

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(C) Quantification of Siglec-8 on iMGLs treated with PBS or 25 ng/mL IFNα, IFNβ, or IFNγ. Values are mean Siglec-8 intensity values from Iba1+;33342- regions. Bars indicate mean ± 95% CI; n = 8 images (2 replicates); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.


( D ) of Siglec - 8 on iMGLs treated ± 25 ng / mL IFNγ 5 μM tofacitinib . Legend is same as ( C ) . Bars indicate mean ± 95 % CI ; n = 5 - 20 ( 2 replicates ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted indicates mean of untreated group .

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(D) Quantification of Siglec-8 on iMGLs treated ± 25 ng/mL IFNγ and 5 μM tofacitinib. Legend is same as (C). Bars indicate mean ± 95% CI; n = 5-20 images (2 replicates); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.


( A ) Confluency doubling times quantified from BV - 2 with stable retroviral expression of Siglec - F related human Siglec constructs . Plots indicating doubling time ( normalized to 2xY - > F constructs ) estimated from Incucyte bright - field . grown in media alone or + 50 mU sialidase ( + SA ) . Bars indicate mean ± 95 % CI ; n = 3 - 12 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted indicates mean of 2xY - > F groups .

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(A) Confluency doubling times quantified from BV-2 cells with stable retroviral expression of Siglec-F and related human Siglec constructs. Plots indicating doubling time (normalized to 2xY->F constructs) estimated from Incucyte bright-field images. Cells grown in media alone or + 50 mU sialidase (+SA). Bars indicate mean ± 95% CI; n = 3-12 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F groups.


( B ) Flow cytometry of live / dead markers Annexin - V propidium iodide on BV - 2 with stable retrovirus expression of Siglec - F constructs . % parent values are shown for early apoptotic ( Annexin - V + ; PI ‑ ) , late apoptotic ( Annexin - V + ; PI ‑ ) , necrotic ( Annexin - V - ; PI + ) populations . Bars indicate mean ± SD ; n = 8 replicates .

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(B) Flow cytometry quantification of cell live / dead markers Annexin-V and propidium iodide on BV-2 cells with stable retrovirus expression of Siglec-F constructs. % parent values are shown for early apoptotic (Annexin-V+;PI‑), late apoptotic (Annexin-V+;PI‑), and necrotic (Annexin-V-;PI+) populations. Bars indicate mean ± SD; n = 8 replicates.


( C ) Confluency doubling times quantified from BV - 2 with dox - inducible expression of Siglec - F constructs . Plots indicating doubling time ( normalized to 2xY - > F constructs ) estimated from Incucyte bright - field . Bars indicate mean ± SD ; n = 8 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted indicates mean of 2xY - > F group .

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(C) Confluency doubling times quantified from BV-2 cells with dox-inducible expression of Siglec-F constructs. Plots indicating doubling time (normalized to 2xY->F constructs) estimated from Incucyte bright-field images. Bars indicate mean ± SD; n = 8 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


( D ) Selected gene expression changes ( top ) enriched gene sets ( bottom ) taken from - seq of BV - 2 with stable retroviral expression of Siglec constructs , 2xY - > F , empty vector controls . Heatmaps share a colormap range of 0 . 25 to 4 for z - score values ( top ) , normalized enrichment scores ( NES , bottom ) .

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(D) Selected gene expression changes (top) and enriched gene sets (bottom) taken from RNA-seq analysis of BV-2 cells with stable retroviral expression of Siglec constructs, 2xY->F, and empty vector controls. Heatmaps share a colormap range of 0.25 to 4 for z-score values (top), normalized enrichment scores (NES, bottom).


( F ) Expression of C1qa , IL - 1β , β - actin by qPCR in BV - 2 with inducible Siglec - F expression after 48 hours of treatment with 500 ng / mL doxycycline . Fold Change values are equal to 2ΔΔCq normalized relative to GADPH mRNA . Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted indicates mean of 2xY - > F group .

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(F) Expression levels of C1qa, IL-1β, and β-actin by qPCR analysis in BV-2 cells with inducible Siglec-F expression after 48 hours of treatment with 500 ng/mL doxycycline. Fold Change values are equal to 2ΔΔCq normalized relative to GADPH mRNA levels. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


( G ) Western blot showing pro - IL - 1β β - tubulin in BV - 2 induced to express Siglec - F at 0 48 hours .

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(G) Western blot showing pro-IL-1β and β-tubulin in BV-2 cells induced to express Siglec-F at 0 and 48 hours.


( B - C ) Volcano plots showing phosphotyrosine peptides that were ( B ) increased or ( C ) decreased after Siglec - F expression . Plots show Siglec - F compared to 2xY - > F mutant data integrated across three runs . names are shown for altered peptides . Labels are only shown for peptides with maximum directional change from each . Labels colored according to GO terms : magenta = signal transduction , red = endosome , cyan = adhesion , yellow = cytoskeleton , green = metabolism .

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(B-C) Volcano plots showing phosphotyrosine peptides that were (B) increased or (C) decreased after Siglec-F expression. Plots show Siglec-F compared to 2xY->F mutant data integrated across all three runs. Protein names are shown for altered peptides. Labels are only shown for peptides with maximum directional change from each protein. Labels colored according to GO terms: magenta = signal transduction, red = endosome, cyan = cell adhesion, yellow = cytoskeleton, green = metabolism.


( D ) showing phosphosites in CK - p25 which overlapped in their directional change with BV - 2 Siglec - F - associated sites . Colors indicate fold change relative to 2xY - > F controls .

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(D) Heatmap showing phosphosites in CK-p25 which overlapped in their directional change with BV-2 Siglec-F-associated sites. Colors indicate fold change relative to 2xY->F controls.


showing phosphosites from CD33 , Siglec - 5 , Siglec - 8 overexpression that were increased with Siglec expression shared their directional change with Siglec - F . Siglec - F column indicates average of untreated Siglec - F replicates . Color scale is same as ( D ) .

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Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were increased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D).


showing phosphosites from CD33 , Siglec - 5 , Siglec - 8 overexpression that were decreased with Siglec expression shared their directional change with Siglec - F . Siglec - F column indicates average of untreated Siglec - F replicates . Color scale is same as ( D ) .

Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were decreased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D).


( G ) Western blot of myc , SHP - 2 , SHP - 1 from myc co - IP eluates . Co - IP lysates were prepared from BV - 2 stably expressing Siglec - F optionally treated with 1 μM SHP099 .

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(G) Western blot quantification of myc, SHP-2, and SHP-1 from myc co-IP eluates. Co-IP lysates were prepared from BV-2 cells stably expressing Siglec-F and optionally treated with 1 μM SHP099.


( H ) showing phosphosites that were perturbed by SHP099 treatment . Color scale is same as ( D ) .

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(H) Heatmap showing phosphosites that were perturbed by SHP099 treatment. Color scale is same as (D).


( B - C ) Flow cytometry of monomeric Aβ uptake Siglec - F expression for ( B ) Siglec - F ( C ) Siglec - F 2xY - > F . Siglec - expressing were plated with empty vector control in same well alongside substrates .

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(B-C) Flow cytometry quantification of fluorescent monomeric Aβ uptake and Siglec-F expression for (B) Siglec-F and (C) Siglec-F 2xY->F. Siglec-expressing cells were plated with empty vector control cells in the same well alongside fluorescent substrates.


of relative mean intensities ( MFI ) between empty vector Siglec - expressing populations . Relative values are shown for each mouse human Siglec construct for uptake of monomeric Aβ , Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted indicates mean of 2xY - > F group .

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Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of monomeric Aβ, Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


of relative mean intensities ( MFI ) between empty vector Siglec - expressing populations . Relative values are shown for each mouse human Siglec construct for uptake of 10 , 000 MW Dextran Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted indicates mean of 2xY - > F group .

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Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 10,000 MW Dextran Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


of relative mean intensities ( MFI ) between empty vector Siglec - expressing populations . Relative values are shown for each mouse human Siglec construct for uptake of 1 μm FluoSpheres . Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted indicates mean of 2xY - > F group .

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Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 1 μm FluoSpheres. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


( G ) pHrodo Dextran uptake in BV - 2 with stable expression of Siglec - F estimated by Incucyte measurements . Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided .

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(G) pHrodo Dextran uptake in BV-2 cells with stable expression of Siglec-F estimated by Incucyte measurements. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( H ) pHrodo dextran uptake in BV - 2 stably expressing Siglec - F with 0 - 2 . 5 μM SHP099 treatment . Data is from same experiment as ( G ) .

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(H) pHrodo dextran uptake in BV-2 cells stably expressing Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G).


( I ) Confluency doubling times for BV - 2 with stable expression of Siglec - F with 0 - 2 . 5 μM SHP099 treatment . Data is from same experiment as ( G ) . Bars indicate mean ± 95 % CI ; n = 4 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted indicates mean of untreated 2xY - > F group .

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(I) Confluency doubling times for BV-2 cells with stable expression of Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G). Bars indicate mean ± 95% CI; n = 4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated 2xY->F group.


Multiple tissue ( eye , brain , adrenal , submandibular , oesophagus , lung , heart , liver , kidney , spleen , pancreas , stomach , duodenum , jejunum , ileum , caecum , colon , spinal cord , dorsal root ganglia , skeletal muscle , testis , epididymis , gonad , skull ) northern blot for CAVII . In P56 mouse , CAVII is prominently expressed in central nervous system including brain spinal cord . Gapdh served as loading control .

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Multiple tissue (eye, brain, adrenal gland, submandibular gland, oesophagus, lung, heart, liver, kidney, spleen, pancreas, stomach, duodenum, jejunum, ileum, caecum, colon, spinal cord, dorsal root ganglia, skeletal muscle, testis, epididymis, female gonad, skull) northern blot for CAVII. In the P56 mouse, CAVII is prominently expressed in the central nervous system including brain and spinal cord. Gapdh served as the loading control.


( C ) Developmental profile of CAVII expression . Western blot of lysates of whole hippocampus shows increasing CAVII from P3 to P30 . band of appropriate ( ∼ 30 kDa ) was absent in lysates from Car7 KO tissue . beta Actin served as loading control in C D .

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(C) Developmental profile of CAVII protein expression. Western blot analysis of protein lysates of whole hippocampus shows increasing CAVII levels from P3 to P30. The band of the appropriate size (∼30 kDa) was absent in protein lysates from Car7 KO tissue.beta Actin served as the loading control in C and D.


( D ) In western blots from lysates of cultured , CAVII was detected in mixed glial / neuron cultures but not in glia cultures . presence of astrocytes in cultures was confirmed by detection of GFAP . Actin served as loading control in C D .

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(D) In western blots from protein lysates of cultured cells, CAVII was detected in mixed glial/neuron cultures but not in glia cell cultures. The presence of astrocytes in all cultures was confirmed by detection of GFAP. Actin served as the loading control in C and D.


Original single - pHi traces their time derivatives from P5 P14 WT P14 CA VII KO neurons ( baseline pHi 7 . 21 , 7 . 13 7 . 11 , respectively ) . Superfusion with CO2 / HCO3 − - free HEPES solution ( upper horizontal bars ) evoked an intracellular alkalinization , which in P14 WT was large suppressed by 100 μM acetazolamide ( AZ , lower horizontal bar ) , indicating presence of CA activity ( see Supplementary Materials methods ) . possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide ( a poorly permeant CA inhibitor ) . Inset shows an overlay of BCECF signal Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT ( scale bar 10 μm ) .

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

Original single-cell pHi traces and their time derivatives from P5 and P14 WT and P14 CA VII KO neurons (baseline pHi 7.21, 7.13 and 7.11, respectively). Superfusion with CO2/HCO3−-free HEPES solution (upper horizontal bars) evoked an intracellular alkalinization, which in P14 WT was large and suppressed by 100 μM acetazolamide (AZ, lower horizontal bar), indicating the presence of CA activity (see Supplementary Materials and methods). The possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide (a poorly permeant CA inhibitor). Inset shows an overlay of the BCECF fluorescence signal and Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT (scale bar 10 μm).


Summary of results obtained using CA activity detection method shown in A quantified as of showing CA activity . Data from CA II KO CA II / VII KO neurons were obtained only at > P35 . number of tested is indicated for each bar . numbers for WT mice at different points was n = 3 ( P5 - 8 ) , n = 2 ( P10 - 11 ) , n = 3 ( P14 - 16 ) , n = 2 ( P17 - 18 ) , n = 2 ( P20 - 30 ) n = 2 ( > P35 ) ; for CAVII KO mice numbers were n = 3 ( P5 - 8 ) , n = 2 ( P10 - 11 ) , n = 5 ( P14 - 16 ) , n = 3 ( P17 - 18 ) , n = 4 ( P20 - 30 ) n = 2 ( > P35 ) ; for CA II KO CA II / CAVII KO mice number was n = 2 ( > P35 ) .

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

Summary of the results obtained using the cytosolic CA activity detection method shown in A and quantified as the percentage of cells showing cytosolic CA activity. Data from CA II KO and CA II/VII KO neurons were obtained only at >P35. The number of cells tested is indicated for each bar. The animal numbers for WT mice at the different age points was n=3 (P5-8), n=2 (P10-11), n=3 (P14-16), n=2 (P17-18), n=2 (P20-30) and n=2 (>P35); for CAVII KO mice the numbers were n=3 (P5-8), n=2 (P10-11), n=5 (P14-16), n=3 (P17-18), n=4 (P20-30) and n=2 (>P35); and for CA II KO and CA II/CAVII KO mice the number was n=2 (>P35).


Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2 / HCO3 − ( horizontal bar , HEPES ) in slices from > P35 WT , CAVII KO CA II / VII KO mice . rate of rise amplitude of alkaline shift are identical in WT ( P39 , baseline pHi 7 . 01 ) CAVII KO neurons ( P40 , 7 . 06 ) , whereas alkalinization develops much more slowly in CA II / VII KO ( P46 , 7 . 04 )

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

Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2/HCO3− (horizontal bar, HEPES) in slices from >P35 WT, CAVII KO and CA II/VII KO mice. The rate of rise and the amplitude of the alkaline shift are identical in WT (P39, baseline pHi 7.01) and CAVII KO neurons (P40, 7.06), whereas alkalinization develops much more slowly in the CA II/VII KO (P46, 7.04)


Excitatory GABAergic response in WT , but not in CAVII KO neurons . Picrotoxin blocked biphasic response ( PiTX 90 μM ; n = 3 / genotype ) . mean initial slope of depolarizing shift was faster in WT than in CAVII KO ( 13 . 53 ± 1 . 27 versus 6 . 39 ± 0 . 96 mV / s , P < 0 . 001 , Student ' s t - ) . resting potential of WT CAVII KO neurons did not differ ( − 71 . 0 ± 1 . 1 versus − 72 . 3 ± 1 . 4 mV , respectively ; n = 15 neurons for both genotypes , P = 0 . 47 , Student ' s t - ) . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 . Error bars denote s . e . m . n is number of slices ( C ) or tested ( one per slice ) ( A , B D )

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

Excitatory GABAergic response in WT, but not in CAVII KO neurons. Picrotoxin blocked the biphasic response (PiTX 90 μM; n=3/genotype). The mean initial slope of the depolarizing shift was faster in WT than in CAVII KO (13.53±1.27 versus 6.39±0.96 mV/s, P<0.001, Student's t-test). The resting membrane potential of WT and CAVII KO neurons did not differ (−71.0±1.1 versus −72.3±1.4 mV, respectively; n=15 neurons for both genotypes, P=0.47, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)


Microinjection of GABA ( 5 mM for 100 ms , sr / slm border in presence of CGP55845 / TTX , arrowheads ) induced a pronounced depolarization in WT neurons ( black ) that was abolished in absence of CO2 / HCO3 − ( grey ) . depolarization was smaller in CA VII KO ( red ) . Bar diagram illustrates GABAergic mean peak depolarization in P12 - 16 WT CAVII KO ( 13 . 3 ± 0 . 5 versus 8 . 3 ± 0 . 6 mV , P < 0 . 001 , Student ' s t - ) . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 . Error bars denote s . e . m . n is number of slices ( C ) or tested ( one per slice ) ( A , B D ) .

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

Microinjection of GABA (5 mM for 100 ms, sr/slm border in the presence of CGP55845/TTX, arrowheads) induced a pronounced depolarization in WT neurons (black) that was abolished in the absence of CO2/HCO3− (grey). The depolarization was smaller in CA VII KO (red). Bar diagram illustrates GABAergic mean peak depolarization in P12-16 WT and CAVII KO (13.3±0.5 versus 8.3±0.6 mV, P<0.001, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D).


In presence of AP5 / CNQX / CGP55845 , microinjection of GABA ( 5 mM , sr / slm border ) triggered SR 95531 - sensitive field potential spikes in WT slices already upon 8 - ms injection , while even 16 - ms injection evoked hardly any spikes in CAVII KO ( left ) . Mean number of spikes was plotted against GABA puff duration in P12 - 16 WT CAVII KO ( P < 0 . 01 P < 0 . 001 , Student ' s t - ; right ) . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 . Error bars denote s . e . m . n is number of slices ( C ) or tested ( one per slice ) ( A , B D )

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

In the presence of AP5/CNQX/CGP55845, microinjection of GABA (5 mM, sr/slm border) triggered SR 95531-sensitive field potential spikes in WT slices already upon 8-ms injection, while even 16-ms injection evoked hardly any spikes in CAVII KO (left). Mean number of spikes was plotted against GABA puff duration in P12-16 WT and CAVII KO (P<0.01 and P<0.001, Student's t-test; right). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)


After P35 , depolarizations upon GABA microinjection ( arrowheads ) in presence of CGP55845 / TTX were indistinguishable in WT ( black ) , CA VII KO ( red ) CA II KO ( blue ) but smaller in CA II / VII KO ( green ) . depolarization in WT was abolished in absence of CO2 / HCO3 − ( grey , n = 5 ) . Bar diagram summarizes mean peak depolarization for each genotype ( right ; ANOVA , Bonferroni ) . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 . Error bars denote s . e . m . n is number of slices ( C ) or tested ( one per slice ) ( A , B D )

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

After P35, depolarizations upon GABA microinjection (arrowheads) in the presence of CGP55845/TTX were indistinguishable in WT (black), CA VII KO (red) and CA II KO (blue) but smaller in CA II/VII KO (green). The depolarization in WT was abolished in the absence of CO2/HCO3− (grey, n=5). Bar diagram summarizes mean peak depolarization for each genotype (right; ANOVA, Bonferroni). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)


Representative raw traces of epidural EEG recordings during hyperthermia ( HT ) from a WT a CA VII KO mouse . Seizures were reliably induced in WT ( n = 9 ) but not in CA VII KO ( n = 7 ) . Error bars denote s . e . m . , P - values are on Student ' s t - ( * * * P < 0 . 001 ) , number is given in bar diagrams . Rectal temperatures are given above traces .

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

Representative raw traces of epidural EEG recordings during hyperthermia (HT) from a WT and a CA VII KO mouse. Seizures were reliably induced in WT (n=9) but not in CA VII KO (n=7). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Specimen recordings of breath rate from a WT a CA VII KO mouse . baseline breath rate ( WT 153 ± 11 breaths / min CA VII KO 157 ± 8 breaths / min ) was increased during HT in both genotypes ( WT 272 ± 28 breaths / min CA VII KO 279 ± 12 breaths / min ) . bar diagram summarizes mean increase in breath rate in WT ( 177 ± 12 % ) CA VII KO mice ( 178 ± 6 % , P = 0 . 98 ) . Error bars denote s . e . m . , P - values are on Student ' s t - ( * * * P < 0 . 001 ) , number is given in bar diagrams . Rectal temperatures are given above traces .

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

Specimen recordings of breath rate from a WT and a CA VII KO mouse. The baseline breath rate (WT 153±11 breaths/min and CA VII KO 157±8 breaths/min) was increased during HT in both genotypes (WT 272±28 breaths/min and CA VII KO 279±12 breaths/min). The bar diagram summarizes the mean increase in breath rate in WT (177±12%) and CA VII KO mice (178±6%, P=0.98).Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Hyperthermia caused a comparable alkalosis in blood pH in both WT CA VII KO mice . blood pH did not differ between two genotypes under control conditions ( P = 0 . 37 ) or during HT ( P = 0 . 44 ) . Error bars denote s . e . m . , P - values are on Student ' s t - ( * * * P < 0 . 001 ) , number is given in bar diagrams . Rectal temperatures are given above traces .

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

Hyperthermia caused a comparable alkalosis in blood pH in both WT and CA VII KO mice. The blood pH did not differ between the two genotypes under control conditions (P=0.37) or during HT (P=0.44). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Electrographic seizures were not detected in WT mice when hyperthermia was induced in continuous presence of 5 % CO2 ( n = 5 ) . Error bars denote s . e . m . , P - values are on Student ' s t - ( * * * P < 0 . 001 ) , number is given in bar diagrams . Rectal temperatures are given above traces .

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

Electrographic seizures were not detected in WT mice when hyperthermia was induced in the continuous presence of 5% CO2 (n=5). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Diazepam ( DZP , 150 μg / kg ) reduced latency to seizure onset in P14 rat pups ( P < 0 . 05 ) . There was no statistical difference between 50 μg / kg saline - injected . DZP 2 . 5 mg / kg completely blocked seizures . DZP was given intraperitoneally 15 min before hyperthermia ( HT ) onset .

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

Diazepam (DZP, 150 μg/kg) reduced the latency to seizure onset in P14 rat pups (P<0.05). There was no statistical difference between 50 μg/kg and saline-injected animals. DZP 2.5 mg/kg completely blocked the seizures. DZP was given intraperitoneally 15 min before hyperthermia (HT) onset.


Diazepam given at 150 μg / kg did not affect baseline breath rate , HT induced an analogous increase in breath rate in saline - diazepam - injected rats ( P > 0 . 05 ) . At a higher ( 2 . 5 mg / kg ) , DZP suppressed breathing significantly ( P < 0 . 01 ) HT caused only a small increase in breath rate .

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

Diazepam given at 150 μg/kg did not affect the baseline breath rate, and HT induced an analogous increase in the breath rate in saline- and diazepam-injected rats (P>0.05). At a higher concentration (2.5 mg/kg), DZP suppressed breathing significantly (P<0.01) and HT caused only a small increase in the breath rate.


In slice preparation , DZP ( 1 μM ) potentiated high - frequency stimulation ( 40 pulses at 100 Hz , horizontal bar ) - evoked GABAAR - mediated depolarization . Sample trace from a slice from a P14 rat pup where stimulation to border of stratum radiatum stratum lacunosum - moleculare was given in presence of AP5 , CNQX CGP 55845 . bar diagrams summarize effects of DZP on duration of depolarization ( time to half - maximum ) number of action potentials associated with depolarizing phase . P - values ( * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ) are on ANOVA , Bonferroni ( A , B ) or Student ' s t - ( C ) , error bars denote s . e . m . number of ( A , B ) or ( C ) is indicated in bar diagrams .

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

In slice preparation, DZP (1 μM) potentiated the high-frequency stimulation (40 pulses at 100 Hz, horizontal bar) -evoked GABAAR-mediated depolarization. Sample trace from a slice from a P14 rat pup where stimulation to the border of stratum radiatum and stratum lacunosum-moleculare was given in the presence of AP5, CNQX and CGP 55845. The bar diagrams summarize the effects of DZP on the duration of the depolarization (time to half-maximum) and the number of action potentials associated with the depolarizing phase. P-values (*P<0.05, **P<0.01 and ***P<0.001) are based on ANOVA, Bonferroni (A, B) or Student's t-test (C), and error bars denote s.e.m. The number of animals (A, B) or cells (C) is indicated in bar diagrams.


CA VII expression during human brain development . plots show log2 - transformed exon array signal in neocortex ( NCX ) hippocampal formation ( HIP ) from early period to late adulthood . solid between periods 7 8 separates prenatal from postnatal period .

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

CA VII expression during human brain development. Line plots show the log2-transformed exon array signal intensity in the neocortex (NCX) and hippocampal formation (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.


CA VII expression during human brain development . plots show log . plots show normalized of absolute copy number of CA VII to Gapdh by droplet digital PCR ( ddPCR , right ) in neocortex ( NCX ) hippocampus ( HIP ) from early period to late adulthood . solid between periods 7 8 separates prenatal from postnatal period .

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

CA VII expression during human brain development. Line plots show the log. Line plots show the normalized ratio of absolute copy number of CA VII to Gapdh by droplet digital PCR (ddPCR, right) in the neocortex (NCX) and hippocampus (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.


Normalised Wnt / STOP signalling activity by of expression of two Wnt / STOP target , β - catenin c - Myc , in mitotic ( M ) asynchronised ( A ) after silencing 47 Wnt signalling regulators .

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

Normalised 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 ) .

ELISA validation of selected Wnt/STOP signalling regulators (n=3).


Immunoblotting ( D ) of stability of four STOP target in both G1S natural mitotic treated with BCL9 siGenome pools CHX ( n = 3 ) .

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

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 ) normalised biosensor on live imaging of with a GSK3 - GFP biosensor following different treatments before after metaphase ( n = 10 ) . Scale bars represent 10 μm .

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

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 β - catenin target gene enrichment on transcriptome targets regulated by BCL9 .

Myc and β-catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9.


representative xenograft tumours ( top panel ) tumour growth ( bottom panel ) of scramble BCL9 stable knockdown . LF : left flank of mouse , RF : right flank . n = 6 for shS1 vs . shB1 , n = 5 for shS1 vs . shB2 .

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

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 of xenograft ; red arrows indicate representative mitotic . Scale bars represent 200 μm .

Immunohistochemical staining of the xenograft; the red arrows indicate the representative mitotic cells. Scale bars represent 200 μm.


Immunoblotting of BCL9 cycle expression in double thymidine synchronised ; black arrow indicates delayed band of mitotic BCL9 .

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

Immunoblotting 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 of BCL9 localization on spindle by antibody . Yellow arrows indicate spindle pole . Scale bars represent : 2 μm for interphase anaphase ; 5 μm for prophase , metaphase telophase .

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

Immunofluorescence 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 of BCL9 localization on centrosome . Yellow arrows indicate centrosome . Scale bars represent : 5 μm for control treatment , 10 μm for BCL9 peptide treatment .

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

Immunofluorescence 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 - radial histograms of spindle angle in with siRNA treatment ; yellow arrows indicate centrosome . Scale bars represent 5 μm .

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

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 ) live imaging ( right panel ) of chromosome segregation in H2BGFP - following siRNA treatment ; yellow arrows indicate typical abnormal chromosomes by BCL9 knockdown . 23 H2BGFP - mitotic were monitored for each time experiment ; data are shown from three independent experiments . Scale bars represent 5 μm . of aberrant chromosome segregation rate by live imaging of H2BGFP upon Wnt3a or DKK1 treatment . 23 H2BGFP - mitotic were monitored for each time experiment ; data are shown from three independent experiments .

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

Confocal (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.


of duration of NEB to ANA ( NEB : envelope breakdown , ANA : anaphase ) , NEB to in interphase after division ( blue colour bar ) or in mitosis ( red bar ) , or growth well in 30 hours ( pink bar ) by live imaging in monitored H2BGFP with different treatments .

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

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.


Volcano plot of BCL9 mitotic interactome by SILAC proteomics approach . Red dashed indicates a strict cut off by a normalised SILAC H / L > 2 .

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

Volcano 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 for validation of potential BCL9 interactors in both asynchronised mitotic . relative binding of CLTC with BCL9 is quantified labelled on top of CLTC blot .

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

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 of endogenous BCL9 Axin1 interaction in synchronised HeLa , from G1S to G2M . P - H3S10 , a mitotic marker indicating mitosis .

Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells, from G1S to G2M. P-H3S10, a mitotic marker indicating mitosis.


Duolink ( A ) its ( B ) of interaction by assessing PLA in mitotic after BCL9 or Axin1 knockdown ( n = 20 ) . Scale bars represent 5 μm .

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

Duolink 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 of interaction of BCL9 CLTC in mitotic . Scale bars represent 5 μm .

Duolink analysis of the interaction of BCL9 and CLTC in mitotic cells. Scale bars represent 5 μm.


Immunoprecipitation of interaction of truncated BCL9 forms in mitosis ; purple stars indicate domain required for clathrin binding , green stars indicate domains for β - catenin binding . domain required for both clathrin β - catenin complexes binding is indicated by a red .

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

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.


of relative binding of truncated forms with clathrin or β - catenin ; red frame indicates common domains of BCL9 for binding of both complexes .

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

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 of BCL9 - MF interaction in CLTC knockdown mitotic .

Immunoprecipitation analysis of the BCL9-MF interaction in CLTC knockdown mitotic cells.


Immunoprecipitation of endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF - overexpressing mitotic .

Immunoprecipitation analysis of the endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF-overexpressing mitotic cells.


Immunoblotting of signalosome components by immunoblotting in BCL9 , CLTC or in both BCL9 CLTC knockdown mitotic with CHX . of stability of several LRP6 signalosome components by immunoblotting of mitotic with CHX .

Immunoblotting 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 of Ser1490 - phosphorylated LRP6 CLTC co - localization in mitotic HeLa ; arrows indicate colocalization . Scale bars represent 5 μm .

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 of α - tubulin , Axin1 CLTC triple - in mitotic . arrows indicate colocalization . Scale bars represent 5 μm .

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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 of colocalization of LRP6CAGFP CLTC or CD63 in interphase . arrows indicate colocalization . Scale bars represent 5 μm .

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

Immunofluorescence analysis of colocalization of LRP6CAGFP and CLTC or CD63 in interphase cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.


of signalosome components mitotic Wnt target expression level by immunoblotting in natural mitotic treated for 1 hour with DMSO or MDC , an inhibitor of clathrin - mediated - endocytosis .

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

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 .

A representative image of soft agar oncogenic colony formation assays for stable knockdown cells.


A representative image of mouse xenograft tumours for stable knockdown at end point of experiment .

A representative image of the mouse xenograft tumours for stable knockdown cells at the end point of the experiment.


In vitro CDK1 / cyclin B1 kinase for immunoprecipitation - purified BCL9 - Flag or T172A - Flag .

In vitro CDK1/cyclin B1 kinase assay for immunoprecipitation-purified BCL9-Flag or T172A-Flag proteins.


Immunoprecipitation of transiently - expressed BCL9 - MF or T172A - MF interactors in synchronised , from G1S to G2 M phase . relative level of BCL9 - MF or T172A - MF immunoprecipitation is determined ; red numbers indicate up - regulation , green numbers indicate down - regulation compared to starting time point .

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 of transiently - expressed BCL9 - MF interaction in mitotic treated with RO3306 .

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

Immunoprecipitation analysis of the transiently-expressed BCL9-MF interaction in mitotic cells treated with RO3306.


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


( C ) iMEF stably expressing ev or m152 - V5 were stimulated with 10 µg / ml ISD for indicated times or left unstimulated ( mock ) . lysates were analyzed by SDS - PAGE under non - reducing conditions subjected to IB with specified antibodies . Data information : IB shown in ( C ) are representative of three two independent experiments , respectively . Student ' s t - ( unpaired , two - ) , n . s . not significant , * p < 0 . 05 , * * * * p < 0 .

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

(C) iMEF stably expressing ev or m152-V5 were stimulated with 10 µg/ml ISD for the indicated times or left unstimulated (mock). Cell lysates were analyzed by SDS-PAGE under non-reducing conditions and subjected to IB with the specified antibodies. Data information: IB shown in (C) are representative of three and two independent experiments, respectively. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, ****p<0.0001


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

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


( E ) iMEFgt / gt stably expressing Cherry - STING either ev or m152 - V5 were stimulated with ISD . Live imaging was performed STING translocation quantified 120 min 180 min post stimulation . Data shown is one representative of two independent experiments .

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

(E) iMEFgt/gt stably expressing Cherry-STING and either ev or m152-V5 were stimulated with ISD. Live cell imaging was performed and STING translocation quantified 120 min and 180 min post stimulation. Data shown is one representative of two independent experiments.


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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

(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.