MITOL - KO MEFs was vulnerable to ER stress . Control MEFs ( MITOLF / F ) and MITOL - KO MEFs ( MITOL - / - ) were treated with DMSO as control , 0 . 8 µM thapsigargin ( Tg ) , 0 . 7 µg / mL tunicamycin ( Tu ) or 1 . 2 µg / mL brefeldin A ( Br ) for 24 hours . All experiments using Tu below were performed at concentration of 0 . 7 µg / mL . The relative cell viability shows a ratio between the survival of MEFs in the absence and presence of ER stress inducers . Viable cells were detected by cell viability assay using cell counting kit - 8 .

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MITOL-KO MEFs was vulnerable to ER stress. Control MEFs (MITOLF/F) and MITOL-KO MEFs (MITOL-/-) were treated with DMSO as control, 0.8 µM thapsigargin (Tg), 0.7 µg/mL tunicamycin (Tu) or 1.2 µg/mL brefeldin A (Br) for 24 hours. All experiments using Tu below were performed at concentration of 0.7 µg/mL. The relative cell viability shows a ratio between the survival of MEFs in the absence and presence of ER stress inducers. Viable cells were detected by cell viability assay using cell counting kit-8.


MITOL KO increased ER stress - induced apoptosis . MEFs were treated with indicated ER stress inducers at same concentration as Figure 1A for 18 hours and stained with Annexin V - FITC for the detection of apoptosis using flow cytometry .

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MITOL KO increased ER stress-induced apoptosis. MEFs were treated with indicated ER stress inducers at same concentration as Figure 1A for 18 hours and stained with Annexin V-FITC for the detection of apoptosis using flow cytometry.


MITOL re - expression in MITOL - KO MEFs rescued ER stress vulnerability . MEFs were transfected with MITOL - coding vector ( WT ) , MITOL C65 / 68S - coding vector ( CS ) , or empty vector ( Vec ) 24 hours prior to Tu treatment for 18 hours , followed by immunoblotting with indicated antibodies . Cleaved caspase - 3 ( cC3 ) and cleaved PARP ( cPARP ) were used as apoptosis markers .

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MITOL re-expression in MITOL-KO MEFs rescued ER stress vulnerability. MEFs were transfected with MITOL-coding vector (WT), MITOL C65/68S-coding vector (CS), or empty vector (Vec) 24 hours prior to Tu treatment for 18 hours, followed by immunoblotting with indicated antibodies. Cleaved caspase-3 (cC3) and cleaved PARP (cPARP) were used as apoptosis markers.


MITOL KO enhanced ER stress - induced Bax translocation and cytochrome c release . Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 hours , followed by immunoblotting with indicated antibodies .

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MITOL KO enhanced ER stress-induced Bax translocation and cytochrome c release. Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 hours, followed by immunoblotting with indicated antibodies.


MITOL KO induced excessive mitochondrial depolarization by ER stress . MEFs transfected with indicated vectors were treated with Tu for 18 hours and then stained with TMRM , followed by flow cytometric analysis .

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MITOL KO induced excessive mitochondrial depolarization by ER stress. MEFs transfected with indicated vectors were treated with Tu for 18 hours and then stained with TMRM, followed by flow cytometric analysis.


IRE1α inhibition compensated MITOL - KO MEFs for vulnerability to ER stress . MEFs were transfected with scramble or siIRE1α ( # 1 , # 2 ) . After 24 hours , these MEFs were exposed with Tu for 24 hours , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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IRE1α inhibition compensated MITOL-KO MEFs for vulnerability to ER stress. MEFs were transfected with scramble or siIRE1α (#1, #2). After 24 hours, these MEFs were exposed with Tu for 24 hours , followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


MEFs were treated with 4µ8C ( 25 µM ) for 2 hours prior to Tu treatment for 24 hours ( B ) , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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MEFs were treated with 4µ8C (25 µM) for 2 hours prior to Tu treatment for 24 hours (B), followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


MITOL KO did not affect the autophosphorylation of IRE1α . MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos - tag SDS - PAGE .

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MITOL KO did not affect the autophosphorylation of IRE1α. MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos-tag SDS-PAGE.


MITOL KO enhanced RIDD and xbp1 splicing . MEFs were incubated with Tu for 4 hours . Expression levels of various UPR associated genes were determined by qRT -

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MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR


MITOL KO enhanced RIDD and xbp1 splicing . MEFs were incubated with Tu for 4 hours . MEFs were transfected with either miR - 17 or miR - 34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours , followed by luciferase reporter assay ( F ) . Error bars represent SD ( n = 3 ) .

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MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. MEFs were transfected with either miR-17 or miR-34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours, followed by luciferase reporter assay (F). Error bars represent SD (n=3).


MEFs were incubated with Tu for 4 hours . Expression levels of various UPR associated genes were determined by qRT -

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MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR


IRE1α - mediated JNK phosphorylation was prolonged by MITOL KO . MEFs were incubated with Tu for indicated periods and phosphorylated JNK was detected by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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IRE1α-mediated JNK phosphorylation was prolonged by MITOL KO. MEFs were incubated with Tu for indicated periods and phosphorylated JNK was detected by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


JNK inhibition reduced ER stress - induced apoptosis of MITOL - KO MEFs . MEFs were treated with 20 µM JNK inhibitor - II ( JNK inh ) 2 hours prior to Tu treatment for 24 hours , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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JNK inhibition reduced ER stress-induced apoptosis of MITOL-KO MEFs. MEFs were treated with 20 µM JNK inhibitor-II (JNK inh) 2 hours prior to Tu treatment for 24 hours, followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


MITOL KO facilitated IRE1α oligomerization . MEFs were transfected with IRE1α - GFP expression vector for 24 hours prior to Tu treatment for indicated periods ( A ) . The right panels show 5 - fold magnification images of the boxed regions . Percentages of cells with IRE1α - foci were calculated from 100 cells by visual inspection in each independent experiment ( A ) . To evaluate the oligomerization level of endogenous IRE1α , cells without any transfection were solubilized and separated by sucrose density - gradient centrifugation , followed by immunoblotting with anti - IRE1α antibody ( B ) .

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MITOL KO facilitated IRE1α oligomerization. MEFs were transfected with IRE1α-GFP expression vector for 24 hours prior to Tu treatment for indicated periods (A). The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment (A). To evaluate the oligomerization level of endogenous IRE1α, cells without any transfection were solubilized and separated by sucrose density-gradient centrifugation, followed by immunoblotting with anti-IRE1α antibody (B).


IRE1α oligomers were stabilized by MITOL KO . MEFs transfected with IRE1α - GFP were treated with Tu . After 4 hours , these cells were washed with PBS and subsequently re - fed with fresh media for indicated periods . The right panels show 5 - fold magnification images of the boxed regions . Percentages of cells with IRE1α - foci were calculated from 100 cells by visual inspection at indicated periods after wash out of Tu in each independent experiment . Scale bar represents 10 µm .

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IRE1α oligomers were stabilized by MITOL KO. MEFs transfected with IRE1α-GFP were treated with Tu. After 4 hours, these cells were washed with PBS and subsequently re-fed with fresh media for indicated periods. The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection at indicated periods after wash out of Tu in each independent experiment. Scale bar represents 10 µm.


Sustained IRE1α autophosphorylation after wash out of Tu in MITOL - KO MEFs . MEFs were treated with Tu for 4 hours and washed with PBS and re - fed with fresh media for indicated periods , following by immunoblotting anti - IRE1α antibody after Phos - tag SDS - PAGE . Error bars represent SD ( n = 3 ) .

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Sustained IRE1α autophosphorylation after wash out of Tu in MITOL-KO MEFs. MEFs were treated with Tu for 4 hours and washed with PBS and re-fed with fresh media for indicated periods, following by immunoblotting anti-IRE1α antibody after Phos-tag SDS-PAGE. Error bars represent SD (n=3).


MITOL deletion promoted the interaction between IRE1α and BIM under ER stress . MITOL - depleted HEK293 cells ( KO ) were generated using CRISPR - Cas9 system . Cells were co - transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours . Cells were immunoprecipitated with anti - FLAG antibody , followed by immunoblotting with indicated antibodies .

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MITOL deletion promoted the interaction between IRE1α and BIM under ER stress. MITOL-depleted HEK293 cells (KO) were generated using CRISPR-Cas9 system. Cells were co-transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours. Cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.


Interaction between MITOL and IRE1α . Lysates of HEK293 cells were subjected to immunoprecipitation with anti - IRE1α antibody , followed by immunoblotting with indicated antibodies ( A ) .

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Interaction between MITOL and IRE1α. Lysates of HEK293 cells were subjected to immunoprecipitation with anti-IRE1α antibody, followed by immunoblotting with indicated antibodies (A).


Interaction between MITOL and IRE1α . Lysates of HEK293 cells co - transfected with indicated vectors were subjected to immunoprecipitation , followed by immunoblotting with indicated antibodies ( B ) .

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Interaction between MITOL and IRE1α. Lysates of HEK293 cells co-transfected with indicated vectors were subjected to immunoprecipitation, followed by immunoblotting with indicated antibodies (B).


Interaction between MITOL and IRE1α . GST - pull - down assay of IRE1α or MITOL was performed on lysates of MEFs , followed by immunoblotting with indicated antibodies ( C , D ) . N - ter : 1 - 90 aa of MITOL , Loop : 158 - 210 aa of MITOL , C - ter : 241 - 278 aa of MITOL , 465 - 977 aa of IRE1α .

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Interaction between MITOL and IRE1α. GST-pull-down assay of IRE1α or MITOL was performed on lysates of MEFs, followed by immunoblotting with indicated antibodies (C, D). N-ter: 1-90 aa of MITOL, Loop: 158-210 aa of MITOL, C-ter: 241-278 aa of MITOL, 465-977 aa of IRE1α.


MITOL ubiquitylated IRE1α . Lysates of HEK293 cells immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies .

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MITOL ubiquitylated IRE1α. Lysates of HEK293 cells immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.


MITOL ubiquitylated IRE1α . Lysates of HEK293 cells transfected with indicated vectors , were immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies .

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MITOL ubiquitylated IRE1α. Lysates of HEK293 cells transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.


MITOL ubiquitylated IRE1α . Lysates of MEFs ( G ) transfected with indicated vectors , were immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies .

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MITOL ubiquitylated IRE1α. Lysates of MEFs (G) transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.


MITOL adds lysine 63 - linked polyubiquitin chains to IRE1α . Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody . IRE1α ubiquitylation was

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MITOL adds lysine 63-linked polyubiquitin chains to IRE1α. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody. IRE1α ubiquitylation was detected


MITOL added polyubiquitin chains to K481 of IRE1α . Lysates of HEK293 cells transfected with each lysine mutant of IRE1α and indicated vectors were immunoprecipitated with anti - FLAG antibody , followed by immunoblotting with indicated antibodies . 481 : K481R , 545 : K545R , 568 : K568R .

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MITOL added polyubiquitin chains to K481 of IRE1α. Lysates of HEK293 cells transfected with each lysine mutant of IRE1α and indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies. 481: K481R, 545: K545R, 568: K568R.


IRE1α K481R interacted with MITOL . Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with anti - FLAG antibody , followed by immunoblotting with indicated antibodies .

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IRE1α K481R interacted with MITOL. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.


Overexpression of IRE1α K481R induced apoptosis . MEFs were transfected with indicated vectors . After 24 hours , these cells were stained with Annexin V -

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Overexpression of IRE1α K481R induced apoptosis. MEFs were transfected with indicated vectors. After 24 hours, these cells were stained with Annexin V-FITC


Overexpression of IRE1α K481R induced apoptosis . or subjected by immunoblotting with indicated antibodies ( D ) .

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Overexpression of IRE1α K481R induced apoptosis. or subjected by immunoblotting with indicated antibodies (D).


Enhanced RIDD activity in IRE1α K481R . MEFs were transfected with indicated vectors 24 hours prior to analysis ( E , F ) . The RIDD activity was measured by qRT - PCR .

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Enhanced RIDD activity in IRE1α K481R. MEFs were transfected with indicated vectors 24 hours prior to analysis (E, F). The RIDD activity was measured by qRT-PCR.


Hyper - oligomerization of IRE1α K481R . MEFs were transfected with IRE1α - GFP or IRE1α K481R - GFP . After 24 hours , these cells were exposed with Tu for indicated periods and GFP signals were observed . The right panels show 5 . 5 - fold magnification images of the boxed regions . Percentages of cells with IRE1α - foci were calculated from 100 cells by visual inspection in each independent experiment .

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Hyper-oligomerization of IRE1α K481R. MEFs were transfected with IRE1α-GFP or IRE1α K481R-GFP. After 24 hours, these cells were exposed with Tu for indicated periods and GFP signals were observed. The right panels show 5.5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment.


Increased interaction of IRE1α K481R with BIM . Cells transfected with indicated vectors 24 hours prior to immunoprecipitation , followed by immunoblotting with indicated antibodies .

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Increased interaction of IRE1α K481R with BIM. Cells transfected with indicated vectors 24 hours prior to immunoprecipitation, followed by immunoblotting with indicated antibodies.


Accumulations of MITOL and IRE1α in the MAM fraction . Each organelle fraction was isolated from MEFs by percoll density - gradient centrifugation , followed by immunoblotting with indicated antibodies . mito : pure mitochondria , micro : microsome , cyto : pure cytosol .

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Accumulations of MITOL and IRE1α in the MAM fraction. Each organelle fraction was isolated from MEFs by percoll density-gradient centrifugation, followed by immunoblotting with indicated antibodies. mito: pure mitochondria, micro: microsome, cyto: pure cytosol.


High solubility of MITOL and IRE1α in the low dose digitonin . Raft - like structure including MAM was solubilized from the crude mitochondrial fraction of MEFs with indicated concentration of digitonin . S : supernatant , P : pellet .

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High solubility of MITOL and IRE1α in the low dose digitonin. Raft-like structure including MAM was solubilized from the crude mitochondrial fraction of MEFs with indicated concentration of digitonin. S: supernatant, P: pellet.


IRE1α regulation by MITOL occurred in the MAM - rich mitochondrial fraction . Crude mitochondrial fraction containing MAM ( Mito plus MAM ) and the cytosolic fraction containing ER ( Cyto plus ER ) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the interaction between MITOL and

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IRE1α regulation by MITOL occurred in the MAM-rich mitochondrial fraction. Crude mitochondrial fraction containing MAM (Mito plus MAM) and the cytosolic fraction containing ER (Cyto plus ER) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the interaction between MITOL and IRE1α


IRE1α regulation by MITOL occurred in the MAM - rich mitochondrial fraction . Crude mitochondrial fraction containing MAM ( Mito plus MAM ) and the cytosolic fraction containing ER ( Cyto plus ER ) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the level of IRE1α ubiquitylation ( D ) .

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IRE1α regulation by MITOL occurred in the MAM-rich mitochondrial fraction. Crude mitochondrial fraction containing MAM (Mito plus MAM) and the cytosolic fraction containing ER (Cyto plus ER) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the level of IRE1α ubiquitylation (D).


Knockdown of PACS2 impaired the interaction of MITOL with IRE1α . HEK293 cells were transfected with either scramble RNA or siPACS2 and indicated vectors for 24 hours prior to immunoprecipitation assay .

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Knockdown of PACS2 impaired the interaction of MITOL with IRE1α. HEK293 cells were transfected with either scramble RNA or siPACS2 and indicated vectors for 24 hours prior to immunoprecipitation assay.


Knockdown of PACS2 inhibited IRE1α ubiquitylation by MITOL . HEK293 cells were transfected with either scramble RNA , siPACS2 and indicated vectors 24 hours prior to immunoprecipitation assay .

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Knockdown of PACS2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siPACS2 and indicated vectors 24 hours prior to immunoprecipitation assay.


Knockdown of Mfn2 inhibited IRE1α ubiquitylation by MITOL . HEK293 cells were transfected with either scramble RNA , siMfn2 and indicated vectors 24 hours prior to immunoprecipitation assay .

Knockdown of Mfn2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siMfn2 and indicated vectors 24 hours prior to immunoprecipitation assay.


MITOL ubiquitylated IRE1α in the spinal cord of mice . Lysates of the spinal cord in mice were immunoprecipitated with anti - IRE1α antibody , followed by immunoblotting with indicated antibodies . WT : MITOLF / F mice , MITOLnestin : MITOLF / F , Nestin - Cre mice .

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MITOL ubiquitylated IRE1α in the spinal cord of mice. Lysates of the spinal cord in mice were immunoprecipitated with anti-IRE1α antibody, followed by immunoblotting with indicated antibodies. WT: MITOLF/F mice, MITOLnestin: MITOLF/F, Nestin-Cre mice.


Loss of MITOL led to hyper - oligomerization of IRE1α in the spinal cord under ER stress . 3 month - old WT or MITOLnestin mice were treated with 1 mg / kg Tu for 24 hours . Spinal cord was solubilized with NP - 40 lysis buffer and separated by sucrose - density gradient centrifugation .

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Loss of MITOL led to hyper-oligomerization of IRE1α in the spinal cord under ER stress. 3 month-old WT or MITOLnestin mice were treated with 1 mg/kg Tu for 24 hours. Spinal cord was solubilized with NP-40 lysis buffer and separated by sucrose-density gradient centrifugation.


MITOL deletion resulted in hyper - activation of IRE1α RNase in ER stress . WT or MITOLnestin mice were injected with Tu for 24 hours and spinal cord was analyzed by qRT - PCR .

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MITOL deletion resulted in hyper-activation of IRE1α RNase in ER stress. WT or MITOLnestin mice were injected with Tu for 24 hours and spinal cord was analyzed by qRT-PCR.


Increased ER stress - induced apoptosis in the MITOL - KO spinal cord . 3 month - old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using TUNEL and hoechst ( F ) Arrowheads indicate the representative neurons .

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Increased ER stress-induced apoptosis in the MITOL-KO spinal cord. 3 month-old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using TUNEL and hoechst (F) Arrowheads indicate the representative neurons.


Increased ER stress - induced apoptosis in the MITOL - KO spinal cord . 3 month - old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using cresyl violet ( G ) . Arrowheads indicate the representative neurons .

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Increased ER stress-induced apoptosis in the MITOL-KO spinal cord. 3 month-old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using cresyl violet (G). Arrowheads indicate the representative neurons.


( 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 .

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


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

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


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

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


( D ) 293T 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 .

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


( E ) 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 .

O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O 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 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 .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 ) levels were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently and one representative experiment is shown . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) levels were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently and one representative experiment is shown . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O 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-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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


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

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cells were co - transfected with expression plasmids for Cherry - STING , V5 - tagged m152 and either ev ( unstimulated ) or cGAS - GFP ( stimulated ) .

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


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

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


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

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


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

O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY 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 ) 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 .

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


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

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


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

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


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

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(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 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 .

O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 ) 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 .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 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 .

O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY 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 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD 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 ) and ( D ) , respectively , using the indicated human STING mutants in place of the murine STING mutants described in ( B ) . Data information : IB shown in ( F ) are representative of three independent experiments .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O B-EXP_ASSAY 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 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 .

O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD 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 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 .

O O O O O O O O B-GENEPROD O B-GENEPROD 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O

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


( E ) iMEFgt / gt stably expressing Cherry - STING 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 .

O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY 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 and V5 - tagged m152 or corresponding ev were stimulated with 5 µg / ml ISD for the indicated time or left unstimulated ( mock ) . Lysates were subjected to IB with the specified antibodies . Data information : IB shown in ( C ) and ( F ) are representative of three and two independent experiments , respectively . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * * * p < 0 .

O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O 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-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM B-GENEPROD 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-EXP_ASSAY O B-EXP_ASSAY 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 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 .

O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY 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 ) 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 .

O O O O B-CELL_TYPE O O O O B-ORGANISM O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY 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 cell imaging was performed and STING translocation quantified 120 min and 180 min post stimulation . Data shown is one representative of two independent experiments .

O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

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


( F - G ) iMEF or iMEFgt / gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop ( MOI 0 . 01 ) . At 6 hpi total 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 .

O O O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-ORGANISM B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 RNA was extracted to determine , IFNb1 and 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 B-ORGANISM O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 cells were co - transfected with Cherry - STING , the pNF - κB luciferase reporter , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) and either ev or m152 . Cells were lysed and analyzed as described in Figure 1 . Data is combined from three independent experiments .

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O

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


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

B-GENEPROD O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O O O B-EXP_ASSAY O

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


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

O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O O O O O O B-TISSUE O O B-EXP_ASSAY O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

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


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

O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

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


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

O O O O O B-GENEPROD O O O B-ORGANISM O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O O O O O O B-TISSUE O O B-ORGANISM B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

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


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

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O O O O

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


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

O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O

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


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

O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

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


Wild - type ( WT ) RAD51 SPR curves for ssDNA dN - 50 and dsDNA dN - 50p . RAD51 was injected at 150 nM in the presence of 2 . 5 mM ATP ( pH 7 . 5 ) and 10 mM CaCl2 . The normalised mean number of WT RAD51 bound to respective DNA oligonucleotides ( N ) are plotted versus time , as measured by SPR , following the equation N = S / ( L * ( MRAD51 / MDNA ) ) , where S is the signal in RU units ( 1 RU ~ 50 pg / mm2 ) , L is the amount of DNA ligand immobilised onto the experimental flow cell ( RU ) , MRAD51 is the molecular weight ( kDa ) of RAD51 ( ~ 37 kDa ) and MDNA is the molecular weight ( kDa ) of the immobilised DNA molecule .

O O O O O O B-GENEPROD B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O

Wild-type (WT) RAD51 SPR curves for ssDNA dN-50 and dsDNA dN-50p. RAD51 was injected at 150 nM in the presence of 2.5 mM ATP (pH 7.5) and 10 mM CaCl2. The normalised mean number of WT RAD51 bound to respective DNA oligonucleotides (N) are plotted versus time, as measured by SPR, following the equation N = S / (L * (MRAD51 / MDNA)), where S is the signal in RU units (1 RU ~ 50 pg/mm2), L is the amount of DNA ligand immobilised onto the experimental flow cell (RU), MRAD51 is the molecular weight (kDa) of RAD51 (~ 37 kDa) and MDNA is the molecular weight (kDa) of the immobilised DNA molecule.


Biotinylated ssDNA oligonucleotides of indicated lengths were separately immobilised onto SPR CM5 chips via biotin - streptavidin interaction . Wild - type ( WT ) RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics .

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Biotinylated ssDNA oligonucleotides of indicated lengths were separately immobilised onto SPR CM5 chips via biotin-streptavidin interaction. Wild-type (WT) RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics.


The dotted and solid curves show the normalised mean number of RAD51 bound to the ssDNA oligonucleotides , as measured by SPR ( see Fig . 1 for equation ) and the ODE model fits ( see Fig . 3A for model description ) , respectively .

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The dotted and solid curves show the normalised mean number of RAD51 bound to the ssDNA oligonucleotides, as measured by SPR (see Fig. 1 for equation) and the ODE model fits (see Fig. 3A for model description), respectively.


As in A , except biotinylated dsDNA molecules of indicated lengths were used to measure association and dissociation kinetics .

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As in A, except biotinylated dsDNA molecules of indicated lengths were used to measure association and dissociation kinetics.


As in B , the dotted and solid curves show the mean number of RAD51 bound to the dsDNA oligonucleotides , as measured by SPR and the ODE model

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As in B, the dotted and solid curves show the mean number of RAD51 bound to the dsDNA oligonucleotides, as measured by SPR and the ODE model fits


Predicted concentrations of WT RAD51 polymers in solution at equilibrium as a function of WT RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . Predicted % WT RAD51 within each polymer state in solution at equilibrium as a function of WT RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . In panels C and D , the KD was fixed to the mean value identified in Figs . 3 A and B ( i . e . KD = 1 . 14 nM ) , and the concentration of WT RAD51 monomer concentration was varied accordingly . In panel D , the % WT RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length , and dividing each value by the total RAD51 monomer concentration ( i . e . % WT RAD51 = [ n - mer ] * n / [ WT RAD51monomer ] ) . [ WT RAD51monomer ] was calculated via coomassie staining image quantification using a BSA standard curve . It is important to note that the assumed maximum polymer length in solution ( 16 - mer ) leads to an overestimation of the concentration of each n - mer in solution , given that RAD51 is likely to form polymers of length greater than 16 at 150 nM , 3 µM and 30 µM WT RAD51 monomer concentration . However , this overestimation is likely to not alter the conclusion that WT RAD51 forms long polymers in solution at 150 nM - 30 µM concentration .

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Predicted concentrations of WT RAD51 polymers in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. Predicted % WT RAD51 within each polymer state in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. In panels C and D, the KD was fixed to the mean value identified in Figs. 3 A and B (i.e. KD = 1.14 nM), and the concentration of WT RAD51 monomer concentration was varied accordingly. In panel D, the % WT RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length, and dividing each value by the total RAD51 monomer concentration (i.e. % WT RAD51 = [n-mer] * n / [WT RAD51monomer]). [WT RAD51monomer] was calculated via coomassie staining image quantification using a BSA standard curve. It is important to note that the assumed maximum polymer length in solution (16-mer) leads to an overestimation of the concentration of each n-mer in solution, given that RAD51 is likely to form polymers of length greater than 16 at 150 nM, 3 µM and 30 µM WT RAD51 monomer concentration. However, this overestimation is likely to not alter the conclusion that WT RAD51 forms long polymers in solution at 150 nM - 30 µM concentration.


Biotinylated DNA molecules of varying flexibility were separately immobilised onto SPR CM5 chips via biotin - streptavidin interaction , and WT RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics . The flexibility of respective DNA , the expected entropic penalties upon RAD51 binding to corresponding DNA and protomer - protomer binding energy contribution of WT RAD51 are indicated in gray , green and blue boxes . H and L in each box denote high and low , respectively .

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Biotinylated DNA molecules of varying flexibility were separately immobilised onto SPR CM5 chips via biotin-streptavidin interaction, and WT RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics. The flexibility of respective DNA, the expected entropic penalties upon RAD51 binding to corresponding DNA and protomer-protomer binding energy contribution of WT RAD51 are indicated in gray, green and blue boxes. H and L in each box denote high and low, respectively.


The mean number of WT RAD51 bound to respective DNA oligonucleotides , as measured by SPR ( dotted lines ) and ODE model fits ( solid lines ) .

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The mean number of WT RAD51 bound to respective DNA oligonucleotides, as measured by SPR (dotted lines) and ODE model fits (solid lines).


Bar plots of fitted kp values ( D ) and ks values ( E ) for each SPR curves . In panel D , all parameters except kp were fixed to the mean values as identified in Fig . 3 A and B , and kp was fitted using lsqcurvefit ( MATLAB ) . In panel E , all parameters except ks and kp were fixed to the mean values as identified in Fig . 3 A and B , and ks and kp were fitted using lsqcurvefit ( MATLAB ) . Only the ks values are reported here . Mean ± 1 SD of 3 ODE model fits ( n = 3 ) . D : un - paired , one - tailed Mann - Whitney - Wilcoxon tests . E : un - paired , two - tailed Mann - Whitney - Wilcoxon tests . * p ≤ 0 . 05 ; ns = non - significant .

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Bar plots of fitted kp values (D) and ks values (E) for each SPR curves. In panel D, all parameters except kp were fixed to the mean values as identified in Fig. 3 A and B, and kp was fitted using lsqcurvefit (MATLAB). In panel E, all parameters except ks and kp were fixed to the mean values as identified in Fig. 3 A and B, and ks and kp were fitted using lsqcurvefit (MATLAB). Only the ks values are reported here. Mean ± 1 SD of 3 ODE model fits (n=3). D: un-paired, one-tailed Mann-Whitney-Wilcoxon tests. E: un-paired, two-tailed Mann-Whitney-Wilcoxon tests. * p ≤ 0.05; ns = non-significant.


As in Fig . 4B , but F86E RAD51 was injected at the indicated concentration to measure association and dissociation kinetics . The flexibility of respective DNA , the expected relative entropic penalties upon F86E RAD51 binding to each DNA molecule and protomer - protomer binding enegy contribution of F86 RAD51 are indicated in gray , green and blue boxes . H and L shown in each box indicate high and low , respectively .

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As in Fig. 4B, but F86E RAD51 was injected at the indicated concentration to measure association and dissociation kinetics. The flexibility of respective DNA, the expected relative entropic penalties upon F86E RAD51 binding to each DNA molecule and protomer-protomer binding enegy contribution of F86 RAD51 are indicated in gray, green and blue boxes. H and L shown in each box indicate high and low, respectively.


The mean number of F86E RAD51 bound to respective DNA oligonucleotides , as measured by SPR ( dotted lines ) and corresponding model fits ( solid lines ) using the model shown in panel D . Using ABC - SMC , individual forward rates ( kp ) and reverse rates ( kpr ) were fitted to each experimental curve , while a single KD was fitted to all experimental curves .

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The mean number of F86E RAD51 bound to respective DNA oligonucleotides, as measured by SPR (dotted lines) and corresponding model fits (solid lines) using the model shown in panel D. Using ABC-SMC, individual forward rates (kp) and reverse rates (kpr) were fitted to each experimental curve, while a single KD was fitted to all experimental curves.


Bar plots of fitted kp values for the SPR curves . Mean values of mode particles ± 1 SD derived from the ABC - SMC fits ( n = 3 ) . Un - paired , one - tailed Mann - Whitney - Wilcoxon tests . * p ≤ 0 . 05 ; ns = non - significant .

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Bar plots of fitted kp values for the SPR curves. Mean values of mode particles ± 1 SD derived from the ABC-SMC fits (n=3). Un-paired, one-tailed Mann-Whitney-Wilcoxon tests. * p ≤ 0.05; ns = non-significant.


Predicted concentrations of F86E RAD51 polymers in solution at equilibrium as a function of F86E RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . Predicted % F86E RAD51 within each polymer state in solution at equilibrium as a function of F86E RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . In panels F and G , the KD was fixed to the mean value identified in Fig . 5D ( i . e . KD = 10 . 2 µM ) , and the concentration of F86E RAD51 monomer concentration was varied accordingly . In panel G , the % F86E RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length , and dividing each value by the total RAD51 monomer concentration ( i . e . % F86E RAD51 = [ n - mer ] * n / [ F86E RAD51monomer ] ) . [ F86E RAD51monomer ] was calculated via coomassie staining image quantification using a BSA standard curve . Bar plots of fitted kpr values for the SPR curves . Mean values of mode particles ± 1 SD derived from the ABC - SMC fits ( n = 3 ) . Un - paired , one - tailed Mann - Whitney - Wilcoxon tests . * p ≤ 0 . 05 ; ns = non - significant .

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Predicted concentrations of F86E RAD51 polymers in solution at equilibrium as a function of F86E RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. Predicted % F86E RAD51 within each polymer state in solution at equilibrium as a function of F86E RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. In panels F and G, the KD was fixed to the mean value identified in Fig. 5D (i.e. KD = 10.2 µM), and the concentration of F86E RAD51 monomer concentration was varied accordingly. In panel G, the % F86E RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length, and dividing each value by the total RAD51 monomer concentration (i.e. % F86E RAD51 = [n-mer] * n / [F86E RAD51monomer]). [F86E RAD51monomer] was calculated via coomassie staining image quantification using a BSA standard curve. Bar plots of fitted kpr values for the SPR curves. Mean values of mode particles ± 1 SD derived from the ABC-SMC fits (n=3). Un-paired, one-tailed Mann-Whitney-Wilcoxon tests. * p ≤ 0.05; ns = non-significant.


Partial embryonic lethality after E11 . 5 . Chi - square , * * * * p < 0 . 0001 . E11 . 5 n = 114 embryos , E14 . 5 n = 70 embryos , Adult n = 314 born pups .

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Partial embryonic lethality after E11.5. Chi-square, **** p<0.0001. E11.5 n=114 embryos, E14.5 n=70 embryos, Adult n=314 born pups.


Hindbrain vessel morphology . IsolectinB4 ( IsoB4 ; white ) , ERG ( red ) and EdU incorporation ( green ) . EdU signal was masked with ERG signal . Scale bar , 50 µm .

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Hindbrain vessel morphology. IsolectinB4 (IsoB4; white), ERG (red) and EdU incorporation (green). EdU signal was masked with ERG signal. Scale bar, 50 µm.


Hindbrain vessel density . IsolectinB4 vessel area normalized to wildtype ( WT ) mean ; one dot is two fields / mouse ; error bars : SD ; unpaired T test , * p < 0 . 05 . n = 18 - 20 .

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Hindbrain vessel density. IsolectinB4 vessel area normalized to wildtype (WT) mean; one dot is two fields/mouse; error bars: SD; unpaired T test, * p <0.05. n=18-20.


Hindbrain EC proliferation . EdU incorporation in ERG - positive ECs ( double - positive cells , yellow in B ) . One dot is two fields / mouse ; error bars : SD ; unpaired T test , * p < 0 . 05 . n = 7 - 12 .

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Hindbrain EC proliferation. EdU incorporation in ERG-positive ECs (double-positive cells, yellow in B). One dot is two fields/mouse; error bars: SD; unpaired T test, *p < 0.05. n=7-12.


Reduced weight gain in the adult . Error bars : SD ; 2 - way ANOVA p = 0 . 0009 ; Sidak ' s multiple comparison test , * * p < 0 . 01 . n = 13 - 20 mice .

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Reduced weight gain in the adult. Error bars: SD; 2-way ANOVA p=0.0009; Sidak's multiple comparison test, **p <0.01. n=13-20 mice.


P6 retina vessel morphology . IsolectinB4 ( IsoB4 ) in black ; the outer 20 % of the vascularized area in pink . Scale bar , 250 µm .

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P6 retina vessel morphology. IsolectinB4 (IsoB4) in black; the outer 20% of the vascularized area in pink. Scale bar, 250 µm.


Vessel parameters in the P6 developing retina . G . Radial outgrowth ( distance between optic nerve and vascular front ) .

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Vessel parameters in the P6 developing retina. G. Radial outgrowth (distance between optic nerve and vascular front).


Vascularized area . I . Vessel area in the outer 20 % of the retinal vasculature ; pink in F ( vessel area normalized to vascularized area ) . Each dot represents one retina / mouse ; n = 3 - 4 .

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Vascularized area. I. Vessel area in the outer 20% of the retinal vasculature; pink in F (vessel area normalized to vascularized area). Each dot represents one retina/mouse; n=3-4.


EdU incorporation in ERG - positive ECs normalized to total ERG - positive ECs . Each dot represents one retina / mouse ; error bars : SD . n = 8 - 9 .

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EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents one retina/mouse; error bars: SD. n=8-9.


Body weight . P , postnatal day ; w , weeks . Error bars : SD . n = 5 - 12 mice .

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Body weight. P, postnatal day; w, weeks. Error bars: SD. n=5-12 mice.


Hindbrain vessel parameters . Vessel density ; isolectinB4 vessel area normalized to WT mean of the litter .

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Hindbrain vessel parameters. Vessel density; isolectinB4 vessel area normalized to WT mean of the litter.


Hindbrain vessel parameters . EdU incorporation in ERG - positive ECs normalized to total ERG - positive ECs .

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Hindbrain vessel parameters. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs.


P4 - P7 retina vessel morphology . IsolectinB4 ( IsoB4 ) in black ; outer 20 % of the vascularized area in pink . Scale bar , 250 µm . P6 insets ; sprout morphology . Scale bar , 50 µm .

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P4-P7 retina vessel morphology. IsolectinB4 (IsoB4) in black; outer 20% of the vascularized area in pink. Scale bar, 250 µm. P6 insets; sprout morphology. Scale bar, 50 µm.


Radial outgrowth . Distance between optic nerve and vascular front . P , postnatal day ; error bars : SD ; 2 - way ANOVA p < 0 . 0001 ; Bonferroni ' s multiple comparison test , * p < 0 . 05 . P4 n = 3 - 5 retinas , one retina / mouse , P5 n = 4 - 7 , P6 n = 9 - 9 , P7 n = 4 - 5 . Retina vascularized area . P , postnatal day ; error bars : SD ; 2 - way ANOVA p = 0 . 0013 . P4 n = 7 - 9 retinas , one retina / mouse , P5 n = 4 - 9 , P6 n = 9 - 9 , P7 n = 4 - 5 .

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Radial outgrowth. Distance between optic nerve and vascular front. P, postnatal day; error bars: SD; 2-way ANOVA p<0.0001; Bonferroni's multiple comparison test, *p<0.05. P4 n=3-5 retinas, one retina/mouse, P5 n=4-7, P6 n=9-9, P7 n=4-5. Retina vascularized area. P, postnatal day; error bars: SD; 2-way ANOVA p=0.0013. P4 n=7-9 retinas, one retina/mouse, P5 n=4-9, P6 n=9-9, P7 n=4-5.


Retina vessel density in the retinal front . Quantification of the vessel area in the outer 20 % of the vasculature ( pink in D ) ( vessel area normalized to vascularized area ) . P , postnatal day ; error bars : SD ; 2 - way ANOVA p = 0 . 0054 ; Bonferroni ' s multiple comparison test , * p < 0 . 05 , * * p < 0 . 01 . P4 n = 3 - 5 retinas , one retina / mouse , P5 n = 4 - 6 , P6 n = 3 - 5 , P7 n = 4 - 5 .

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Retina vessel density in the retinal front. Quantification of the vessel area in the outer 20% of the vasculature (pink in D) (vessel area normalized to vascularized area). P, postnatal day; error bars: SD; 2-way ANOVA p=0.0054; Bonferroni's multiple comparison test, *p<0.05, ** p <0.01. P4 n=3-5 retinas, one retina/mouse, P5 n=4-6, P6 n=3-5, P7 n=4-5.


Proliferation of retinal ECs . H . EdU incorporation in ERG - positive ECs normalized to total ERG - positive ECs , at P6 .

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Proliferation of retinal ECs. H. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs, at P6.


IsolectinB4 ( IsoB4 ; white ) , ERG ( red ) , EdU ( green ) and double - positive cells ( yellow ) . Lower panel shows ERG / EdU merged staining alone with EdU signal masked by ERG signal . Scale bar , 50 µm ; error bars : SD ; unpaired T test , * p < 0 . 05 . n = 5 - 8 retinas , one retina / mouse .

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IsolectinB4 (IsoB4; white), ERG (red), EdU (green) and double-positive cells (yellow). Lower panel shows ERG/EdU merged staining alone with EdU signal masked by ERG signal. Scale bar, 50 µm; error bars: SD; unpaired T test, *p < 0.05. n=5-8 retinas, one retina/mouse.


Retina sprout length . Sprout length from tip to base in P6 retinas ; error bars : SD ; unpaired T test , * p < 0 . 05 ; n = 3 - 5 retinas , one retina / mouse .

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Retina sprout length. Sprout length from tip to base in P6 retinas; error bars: SD; unpaired T test, *p<0.05; n=3-5 retinas, one retina/mouse.


Vessel stability . IsolectinB4 ( IsoB4 , green ) and collagen IV ( red ) in P4 wildtype ( WT ) and Vegfr2Y1212F / Y1212F retinas . Arrows ; collagen IV - positive empty sleeves . A ; artery . Scale bar , 50 µm .

B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O B-TISSUE O O O B-GENEPROD I-GENEPROD O O O O O O O B-TISSUE O O O O O O O

Vessel stability. IsolectinB4 (IsoB4, green) and collagen IV (red) in P4 wildtype (WT) and Vegfr2Y1212F /Y1212F retinas. Arrows; collagen IV-positive empty sleeves. A; artery. Scale bar, 50 µm.


Vessel stability . Collagen IV empty sleeves around main arteries in P4 - P7 retinas , three fields / retina / mouse . Error bars : SD ; 2 - way ANOVA p = 0 . 0580 ; Bonferroni ' s multiple comparison test , * * p < 0 . 01 . P4 n = 7 - 9 , P5 n = 6 - 12 , P6 n = 3 - 5 , P7 n = 4 .

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Vessel stability. Collagen IV empty sleeves around main arteries in P4-P7 retinas, three fields/retina/mouse. Error bars: SD; 2-way ANOVA p=0.0580; Bonferroni's multiple comparison test, ** p <0.01. P4 n=7-9, P5 n=6-12, P6 n=3-5, P7 n=4.


Immunoblot showing VEGFR2 phosphorylation on pY1212 , pY949 and pY1173 . A . Anti - VEGFR2 immunoprecipitates ( IP ) from lung lysates from wildtype ( WT ) and Vegfr2Y1212F / Y1212F FVB mice , tail - vein injected with PBS or VEGF for 1 minute . Each lane ( A ) represent one individual mouse .

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Immunoblot showing VEGFR2 phosphorylation on pY1212, pY949 and pY1173. A. Anti-VEGFR2 immunoprecipitates (IP) from lung lysates from wildtype (WT) and Vegfr2Y1212F /Y1212F FVB mice, tail-vein injected with PBS or VEGF for 1 minute. Each lane (A) represent one individual mouse.