Figure 7MITOL 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 . 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 . 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 .

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

Figure 7MITOL 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.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.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.


Figure 1 ( A ) 293T cells were co - transfected with expression plasmids for Cherry - STING , the murine IFNβ - luciferase reporter ( IFNβ - Luc ) , a Renilla luciferase normalization control ( pRL - TK ) and the indicated expression plasmids or empty vector ( ev ) . Cells were additionally co - transfected with expression plasmids for cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) . 20 hours post transfection , cells were lysed and a dual - luciferase assay was performed . Data information : ( A - G ) Data is combined from three independent experiments . ( B ) An expression plasmid for RIG - I N ( stimulated ) or ev ( unstimulated ) was co - transfected with IFNβ - Luc , pRL - TK and the indicated expression plasmids in 293T cells and analyzed as in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments . ( C ) An expression plasmid for TBK1 ( stimulated ) or ev ( unstimulated ) was co - transfected together with IFNβ - Luc , pRL - TK and the indicated expression plasmids and analyzed as in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments . ( D ) 293T cells were co - transfected with a plasmid expressing constitutively active IRF3 ( IRF3 - 5D ; stimulated ) or IRES - GFP ( unstimulated ) together with IFNβ - Luc , pRL - TK and the indicated expression plasmids and analyzed as in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments . ( E ) The ISG56 - luciferase reporter , pRL - TK and the indicated expression plasmids were co - transfected in 293T cells . 24 hours post transfection , cells were stimulated with 0 . 1 ng / µl human IFNβ or mock stimulated and analyzed 16 hours later as described in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments . ( F - G ) iMEFgt / gt stably expressing Cherry - STING and either ev or V5 - tagged m152 were stimulated with 5 µg / ml ISD ( F ) , 10 µg / ml poly ( I : C ) ( G ) or mock stimulated with Lipofectamine . 4 hours post stimulation , RNA was extracted to determine IFNβ mRNA transcripts by qRT - PCR . Data information : ( A - G ) Data is combined from three independent experiments . ( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with 10 µg / ml cGAMP ( H ) , 6 ( H ) or 16 ( I - K ) hours later , secreted IFNβ ( H - J ) levels were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently and one representative experiment is shown . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD . ( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with 5 µg / ml ISD ( I ) , 6 ( H ) or 16 ( I - K ) hours later , secreted IFNβ ( H - J ) levels were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently and one representative experiment is shown . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD . ( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with Newcastle ' s Disease Virus ( NDV ) infection ( J ) 6 ( H ) or 16 ( I - K ) hours later , secreted IFNβ ( H - J ) levels were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently and one representative experiment is shown . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD . ( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with µM CpG DNA ( K ) . 6 ( H ) or 16 ( I - K ) hours later , TNFα ( K ) levels were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently and one representative experiment is shown . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

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

Figure 1(A) 293T cells were co-transfected with expression plasmids for Cherry-STING, the murine IFNβ-luciferase reporter (IFNβ-Luc), a Renilla luciferase normalization control (pRL-TK) and the indicated expression plasmids or empty vector (ev). Cells were additionally co-transfected with expression plasmids for cGAS-GFP (stimulated) or IRES-GFP (unstimulated). 20 hours post transfection, cells were lysed and a dual-luciferase assay was performed. Data information: (A-G) Data is combined from three independent experiments.(B) An expression plasmid for RIG-I N (stimulated) or ev (unstimulated) was co-transfected with IFNβ-Luc, pRL-TK and the indicated expression plasmids in 293T cells and analyzed as in (A). Data information: (A-G) Data is combined from three independent experiments.(C) An expression plasmid for TBK1 (stimulated) or ev (unstimulated) was co-transfected together with IFNβ-Luc, pRL-TK and the indicated expression plasmids and analyzed as in (A). Data information: (A-G) Data is combined from three independent experiments.(D) 293T cells were co-transfected with a plasmid expressing constitutively active IRF3 (IRF3-5D; stimulated) or IRES-GFP (unstimulated) together with IFNβ-Luc, pRL-TK and the indicated expression plasmids and analyzed as in (A). Data information: (A-G) Data is combined from three independent experiments.(E) The ISG56-luciferase reporter, pRL-TK and the indicated expression plasmids were co-transfected in 293T cells. 24 hours post transfection, cells were stimulated with 0.1 ng/µl human IFNβ or mock stimulated and analyzed 16 hours later as described in (A). Data information: (A-G) Data is combined from three independent experiments.(F-G) iMEFgt/gt stably expressing Cherry-STING and either ev or V5-tagged m152 were stimulated with 5 µg/ml ISD (F), 10 µg/ml poly(I:C) (G) or mock stimulated with Lipofectamine. 4 hours post stimulation, RNA was extracted to determine IFNβ mRNA transcripts by qRT-PCR. Data information: (A-G) Data is combined from three independent experiments.(H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with 10 µg/ml cGAMP (H), 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD. (H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with 5 µg/ml ISD (I), 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD. (H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with Newcastle's Disease Virus (NDV) infection (J) 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD. (H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with µM CpG DNA (K). 6 (H) or 16 (I-K) hours later, TNFα (K) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.


Figure 2 ( A ) HeLa cells were co - transfected with expression plasmids for Cherry - STING , V5 - tagged m152 and either ev ( unstimulated ) or cGAS - GFP ( stimulated ) . ( B ) iMEFgt / gt were co - transfected with expression plasmids for V5 - tagged m152 together with either Cherry - STING or ev in combination with cGAS - GFP ( stimulated ) or ev ( unstimulated ) . 24 hours post transfection cells were fixed for immunolabeling with an anti - V5 antibody . White boxes indicate the region shown at a higher magnification . Scale bar represents 10 µm . ( C ) Lysates of Cherry - STING and either CD4 - V5 or m152 - V5 expressing 293T cells were subjected to immunoprecipitation ( IP ) with an anti - V5 antibody . Input and IP samples were analyzed by IB with the indicated antibodies . Data information : IB shown are representative of three ( C ) or two ( D ) independent experiments . ( D ) iMEF stably expressing ev or m152 - V5 were left unstimulated or stimulated with 10 µg / ml ISD and lysed 90 minutes later . m152 was immunoprecipitated with an anti - V5 antibody and samples were analyzed by IB with V5 , STING and phospho - TBK1 ( pTBK1 ) specific antibodies . Data information : IB shown are representative of three ( C ) or two ( D ) independent experiments .

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

Figure 2(A) HeLa cells were co-transfected with expression plasmids for Cherry-STING, V5-tagged m152 and either ev (unstimulated) or cGAS-GFP (stimulated).(B) iMEFgt/gt were co-transfected with expression plasmids for V5-tagged m152 together with either Cherry-STING or ev in combination with cGAS-GFP (stimulated) or ev (unstimulated). 24 hours post transfection cells were fixed for immunolabeling with an anti-V5 antibody. White boxes indicate the region shown at a higher magnification. Scale bar represents 10 µm.(C) Lysates of Cherry-STING and either CD4-V5 or m152-V5 expressing 293T cells were subjected to immunoprecipitation (IP) with an anti-V5 antibody. Input and IP samples were analyzed by IB with the indicated antibodies. Data information: IB shown are representative of three (C) or two (D) independent experiments.(D) iMEF stably expressing ev or m152-V5 were left unstimulated or stimulated with 10 µg/ml ISD and lysed 90 minutes later. m152 was immunoprecipitated with an anti-V5 antibody and samples were analyzed by IB with V5, STING and phospho-TBK1 (pTBK1) specific antibodies. Data information: IB shown are representative of three (C) or two (D) independent experiments.


Figure 3 ( B ) Cherry - STING , IFNβ - Luc , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) and indicated expression plasmids as shown in ( A ) were transiently expressed in 293T cells and a dual luciferase assay was performed . Data is combined from two out of three independent experiments and shown as mean ± SD . Data information : Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( C ) 293T cells were co - transfected with expression plasmids for Cherry - STING and indicated chimeras as shown in ( A ) . An anti - V5 IP was performed and samples were analyzed by IB with indicated antibodies . IB shown is representative of three independent experiments . ( D ) Cherry - STING , IFNβ - Luc , pRL - TK and either CD4 , m152 , m152 - Δstalk or m152 - GSstalk were transiently expressed in 293T cells . For stimulation , samples were co - transfected with cGAS - GFP , and unstimulated samples with IRES - GFP . Lysates were analyzed as described in ( B ) . Data is combined from two out of three independent experiments and shown as mean ± SD . Data information : Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( E ) 293T cells were co - transfected with expression plasmids for Cherry - STING and either CD4 , m152 , m152 - Δstalk or m152 - GSstalk . An anti - V5 IP was performed and samples were analyzed by IB with indicated antibodies . Immunoblot shown is representative of three independent experiments .

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

Figure 3(B) Cherry-STING, IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids as shown in (A) were transiently expressed in 293T cells and a dual luciferase assay was performed. Data is combined from two out of three independent experiments and shown as mean ± SD. Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, **p<0.01, ***p<0.001, ****p<0.0001(C) 293T cells were co-transfected with expression plasmids for Cherry-STING and indicated chimeras as shown in (A). An anti-V5 IP was performed and samples were analyzed by IB with indicated antibodies. IB shown is representative of three independent experiments.(D) Cherry-STING, IFNβ-Luc, pRL-TK and either CD4, m152, m152-Δstalk or m152-GSstalk were transiently expressed in 293T cells. For stimulation, samples were co-transfected with cGAS-GFP, and unstimulated samples with IRES-GFP. Lysates were analyzed as described in (B). Data is combined from two out of three independent experiments and shown as mean ± SD. Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, **p<0.01, ***p<0.001, ****p<0.0001(E) 293T cells were co-transfected with expression plasmids for Cherry-STING and either CD4, m152, m152-Δstalk or m152-GSstalk. An anti-V5 IP was performed and samples were analyzed by IB with indicated antibodies. Immunoblot shown is representative of three independent experiments.


Figure 4 ( A ) 293T cells were co - transfected with expression plasmids for IFNβ - Luc , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) and indicated expression plasmids together with murine Cherry - STING ( left panel ) or human STING ( right panel ) . Lysates were analyzed by dual - luciferase assay . Data is combined from two out of three independent experiments and shown as mean ± SD . ( C ) IFNβ - Luc , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) and either LacZ or m152 together with the indicated murine STING mutants described in ( B ) were transiently expressed in 293T cells . Luciferase activity was measured as described in ( A ) . Data information : Data in ( C ) is normalized to LacZ , combined from three independent experiments and shown as mean ± SD . ( D ) 293T cells were co - transfected with expression plasmids for either LacZ or m152 together with the indicated murine STING mutants described in ( B ) . An anti - V5 IP was performed and samples were analyzed by IB with the indicated antibodies . IB shown in ( D ) representative of three independent experiments . ( E - F ) Luciferase assay ( E ) were performed as described in ( C ) and ( D ) , respectively , using the indicated human STING mutants in place of the murine STING mutants described in ( B ) . Data information : Data in ( E ) is normalized to LacZ , combined from three independent experiments and shown as mean ± SD . ( E - F ) co - IP ( F ) were performed as described in ( C ) and ( D ) , respectively , using the indicated human STING mutants in place of the murine STING mutants described in ( B ) . Data information : IB shown in ( F ) are representative of three independent experiments .

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

Figure 4(A) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids together with murine Cherry-STING (left panel) or human STING (right panel). Lysates were analyzed by dual-luciferase assay. Data is combined from two out of three independent experiments and shown as mean ± SD.(C) IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either LacZ or m152 together with the indicated murine STING mutants described in (B) were transiently expressed in 293T cells. Luciferase activity was measured as described in (A). Data information: Data in (C) is normalized to LacZ, combined from three independent experiments and shown as mean ± SD.(D) 293T cells were co-transfected with expression plasmids for either LacZ or m152 together with the indicated murine STING mutants described in (B). An anti-V5 IP was performed and samples were analyzed by IB with the indicated antibodies. IB shown in (D) representative of three independent experiments.(E-F) Luciferase assay (E) were performed as described in (C) and (D), respectively, using the indicated human STING mutants in place of the murine STING mutants described in (B). Data information: Data in (E) is normalized to LacZ, combined from three independent experiments and shown as mean ± SD.(E-F) co-IP (F) were performed as described in (C) and (D), respectively, using the indicated human STING mutants in place of the murine STING mutants described in (B). Data information: IB shown in (F) are representative of three independent experiments.


Figure 5 ( B ) 293T cells were co - transfected with expression plasmids for IFNβ - Luc , pRL - TK and either ev or m152 . Cells were further co - transfected with either Cherry - STING WT or the constitutively active Cherry - STING V154M ( stimulated ) or with IRES - GFP ( unstimulated ) . Data is combined from three independent experiments and shown as mean ± SD . ( C ) iMEF stably expressing ev or m152 - V5 were stimulated with 10 µg / ml ISD for the indicated times or left unstimulated ( mock ) . Cell lysates were analyzed by SDS - PAGE under non - reducing conditions and subjected to IB with the specified antibodies . Data information : IB shown in ( C ) are representative of three and two independent experiments , respectively . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * * * p < 0 . 0001 ( D ) Two representative still images from live cell imaging experiments with iMEFgt / gt stably expressing Cherry - STING transfected with ISD ( right panel ) or left unstimulated ( left panel ) . Red circles highlight representative translocated STING in ISD stimulated cells , which is used as an indicator of activation . ( E ) iMEFgt / gt stably expressing Cherry - STING and either ev or m152 - V5 were stimulated with ISD . Live cell imaging was performed and STING translocation quantified 120 min and 180 min post stimulation . Data shown is one representative of two independent experiments . ( F ) iMEFgt / gt stably expressing Cherry - STING and V5 - tagged m152 or corresponding ev were stimulated with 5 µg / ml ISD for the indicated time or left unstimulated ( mock ) . Lysates were subjected to IB with the specified antibodies . Data information : IB shown in ( C ) and ( F ) are representative of three and two independent experiments , respectively . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * * * p < 0 .

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

Figure 5(B) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK and either ev or m152. Cells were further co-transfected with either Cherry-STING WT or the constitutively active Cherry-STING V154M (stimulated) or with IRES-GFP (unstimulated). Data is combined from three independent experiments and shown as mean ± SD.(C) iMEF stably expressing ev or m152-V5 were stimulated with 10 µg/ml ISD for the indicated times or left unstimulated (mock). Cell lysates were analyzed by SDS-PAGE under non-reducing conditions and subjected to IB with the specified antibodies. Data information: IB shown in (C) are representative of three and two independent experiments, respectively. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, ****p<0.0001(D) Two representative still images from live cell imaging experiments with iMEFgt/gt stably expressing Cherry-STING transfected with ISD (right panel) or left unstimulated (left panel). Red circles highlight representative translocated STING in ISD stimulated cells, which is used as an indicator of activation.(E) iMEFgt/gt stably expressing Cherry-STING and either ev or m152-V5 were stimulated with ISD. Live cell imaging was performed and STING translocation quantified 120 min and 180 min post stimulation. Data shown is one representative of two independent experiments.(F) iMEFgt/gt stably expressing Cherry-STING and V5-tagged m152 or corresponding ev were stimulated with 5 µg/ml ISD for the indicated time or left unstimulated (mock). Lysates were subjected to IB with the specified antibodies. Data information: IB shown in (C) and (F) are representative of three and two independent experiments, respectively. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, ****p<0.0001


Figure 6 ( B ) iMEF were infected by centrifugal enhancement with either parental MCMV ( par . ) or MCMV m152stop at an MOI of 0 . 5 or mock infected . 3 hours post infection ( hpi ) cells were lysed and subjected to immunoblotting ( IB ) with the specified antibodies . ( C ) iMEF and iMEFgt / gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop , or parental MCMV alone , respectively ( MOI 0 . 5 ) . 3 hpi lysates were subjected to an anti - m152 IP and samples were analyzed by IB with indicated antibodies . IB shown is representative of two independent experiments . ( D ) Primary BMDM were infected with parental MCMV ( par . ) or MCMV m152stop at an MOI of 0 . 1 or mock infected . 16 hpi secreted levels of IFNα or IFNβ were quantified by ELISA . Data is representative of two independent experiments . ( E ) iMEFgt / gt stably expressing Cherry - STING were infected with parental MCMV or MCMV m152stop ( MOI 0 . 5 ) . Live cell imaging was performed and STING translocation quantified 120 min and 180 min post stimulation . Data shown is one representative of two independent experiments . ( F - G ) iMEF or iMEFgt / gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop ( MOI 0 . 01 ) . At 6 hpi total RNA was extracted to determine MCMV IE1 and MCMV E1 ( F ) , transcripts by qRT - PCR . Data shown is combined from two out of three independent experiments . ( F - G ) iMEF or iMEFgt / gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop ( MOI 0 . 01 ) . At 6 hpi total RNA was extracted to determine , IFNb1 and IL6 ( G ) transcripts by qRT - PCR . Data shown is combined from two out of three independent experiments . ( H ) 293T cells were co - transfected with Cherry - STING , the pNF - κB luciferase reporter , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) and either ev or m152 . Cells were lysed and analyzed as described in Figure 1 . Data is combined from three independent experiments .

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

Figure 6(B) iMEF were infected by centrifugal enhancement with either parental MCMV (par.) or MCMV m152stop at an MOI of 0.5 or mock infected. 3 hours post infection (hpi) cells were lysed and subjected to immunoblotting (IB) with the specified antibodies.(C) iMEF and iMEFgt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop, or parental MCMV alone, respectively (MOI 0.5). 3 hpi lysates were subjected to an anti-m152 IP and samples were analyzed by IB with indicated antibodies. IB shown is representative of two independent experiments.(D) Primary BMDM were infected with parental MCMV (par.) or MCMV m152stop at an MOI of 0.1 or mock infected. 16 hpi secreted levels of IFNα or IFNβ were quantified by ELISA. Data is representative of two independent experiments.(E) iMEFgt/gt stably expressing Cherry-STING were infected with parental MCMV or MCMV m152stop (MOI 0.5). Live cell imaging was performed and STING translocation quantified 120 min and 180 min post stimulation. Data shown is one representative of two independent experiments.(F-G) iMEF or iMEFgt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). At 6 hpi total RNA was extracted to determine MCMV IE1 and MCMV E1 (F), transcripts by qRT-PCR. Data shown is combined from two out of three independent experiments.(F-G) iMEF or iMEFgt/gt were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). At 6 hpi total RNA was extracted to determine , IFNb1 and IL6 (G) transcripts by qRT-PCR. Data shown is combined from two out of three independent experiments.(H) 293T cells were co-transfected with Cherry-STING, the pNF-κB luciferase reporter, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either ev or m152. Cells were lysed and analyzed as described in Figure 1. Data is combined from three independent experiments.


Figure 7 ( A - C ) Type I IFN levels in spleen homogenates ( A ) and serum ( B ) of C57BL / 6J ( B6J ) or STING - / - mice following i . v . infection with 4 x 105 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA . IFNβ levels of STING - / - mice were below the detection limit ( n . d . ) . IL6 levels in spleen homogenates ( C ) of C57BL / 6J ( B6J ) or STING - / - mice following i . v . infection with 4 x 105 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA . ( D ) B6J or STING - / - mice were i . v . infected with 4 x 105 PFU parental MCMV or MCMV m152stop . 6 hpi , RNA was extracted from spleen homogenates and expression of MCMV IE1 and E1 transcripts was determined by qRT - PCR . Data were normalized to 107 cellular β - actin transcripts ( D - F ) . ( E ) B6J or STING - / - mice were i . v . infected with 4 x 105 PFU parental MCMV . 6 hpi , RNA was extracted from spleen homogenates and m152 transcript levels were determined by qRT - PCR . Data were normalized to 107 cellular β - actin transcripts ( D - F ) . ( F ) B6J or STING - / - mice were depleted of NK cells via treatment with anti - NK1 . 1 ( right panel ) or left untreated ( left panel ) . One day after NK cell depletion , B6J or STING - / - mice were i . v . infected with 4 x 105 PFU parental MCMV or MCMV m152stop . 16 hpi , RNA was extracted from spleen homogenates and MCMV E1 transcript levels were determined by qRT - PCR . Data were normalized to 107 cellular β - actin transcripts ( D - F ) .

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

Figure 7(A-C) Type I IFN levels in spleen homogenates (A) and serum (B) of C57BL/6J (B6J) or STING-/- mice following i.v. infection with 4 x 105 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA. IFNβ levels of STING-/- mice were below the detection limit (n.d.).IL6 levels in spleen homogenates (C) of C57BL/6J (B6J) or STING-/- mice following i.v. infection with 4 x 105 PFU parental MCMV or MCMV m152stop were analyzed 6 hpi by ELISA.(D) B6J or STING-/- mice were i.v. infected with 4 x 105 PFU parental MCMV or MCMV m152stop. 6 hpi, RNA was extracted from spleen homogenates and expression of MCMV IE1 and E1 transcripts was determined by qRT-PCR. Data were normalized to 107 cellular β-actin transcripts (D-F).(E) B6J or STING-/- mice were i.v. infected with 4 x 105 PFU parental MCMV. 6 hpi, RNA was extracted from spleen homogenates and m152 transcript levels were determined by qRT-PCR. Data were normalized to 107 cellular β-actin transcripts (D-F).(F) B6J or STING-/- mice were depleted of NK cells via treatment with anti-NK1.1 (right panel) or left untreated (left panel). One day after NK cell depletion, B6J or STING-/- mice were i.v. infected with 4 x 105 PFU parental MCMV or MCMV m152stop. 16 hpi, RNA was extracted from spleen homogenates and MCMV E1 transcript levels were determined by qRT-PCR. Data were normalized to 107 cellular β-actin transcripts (D-F).


Figure 8 ( A ) 293T cells were co - transfected with Cherry - STING , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) and either the pNF - κB , p55 - CIB , p125 or p125AA luciferase reporter . Data is representative of two independent experiments . ( B ) 293T cells were co - transfected with expression plasmids for cGAS - GFP ( stimulated ) or ev ( unstimulated ) together with either cherry - tagged WT STING or cherry - tagged K288R STING . Cells were fixed for imaging 24 hours post transfection . Scale bar represents 10 µm . ( C - D ) iMEFgt / gt stably expressing cherry - tagged WT STING or K288R STING and either ev or V5 - tagged m152 were stimulated with ISD ( 10 µg / ml ) or mock stimulated . At 4 hpi total RNA was extracted to determine IFNb1 ( C ) and IL6 ( D ) transcripts by qRT - PCR . Data shown is combined from three ( C ) or two ( D ) out of three independent experiments . ( E ) iMEFgt / gt ( - ) and iMEFgt / gt stably expressing either cherry - tagged WT STING or K288R STING were infected by centrifugal enhancement with parental MCMV or MCMV m152stop ( MOI 0 . 01 ) . 6 hpi total RNA was extracted to determine MCMV E1 transcripts by qRT - PCR . Data shown is combined from three independent experiments .

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

Figure 8(A) 293T cells were co-transfected with Cherry-STING, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either the pNF-κB, p55-CIB, p125 or p125AA luciferase reporter. Data is representative of two independent experiments.(B) 293T cells were co-transfected with expression plasmids for cGAS-GFP (stimulated) or ev (unstimulated) together with either cherry-tagged WT STING or cherry-tagged K288R STING. Cells were fixed for imaging 24 hours post transfection. Scale bar represents 10 µm.(C-D) iMEFgt/gt stably expressing cherry-tagged WT STING or K288R STING and either ev or V5-tagged m152 were stimulated with ISD (10 µg/ml) or mock stimulated. At 4 hpi total RNA was extracted to determine IFNb1 (C) and IL6 (D) transcripts by qRT-PCR. Data shown is combined from three (C) or two (D) out of three independent experiments.(E) iMEFgt/gt (-) and iMEFgt/gt stably expressing either cherry-tagged WT STING or K288R STING were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). 6 hpi total RNA was extracted to determine MCMV E1 transcripts by qRT-PCR. Data shown is combined from three independent experiments.


Figure 1Wild - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Wild-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.


Figure 2Biotinylated 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 . 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

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

Figure 2Biotinylated 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.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 fits


Figure 3Predicted 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 .

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

Figure 3Predicted 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.


Figure 4Biotinylated 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 ) . 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 .

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

Figure 4Biotinylated 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).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.


Figure 5As 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 . 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 .

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

Figure 5As 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.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.


Figure 1Partial 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 . 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 . 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 . 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 . EdU incorporation in ERG - positive ECs normalized to total ERG - positive ECs . Each dot represents one retina / mouse ; error bars : SD . n = 8 - 9 .

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

Figure 1Partial 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.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.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.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.EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents one retina/mouse; error bars: SD. n=8-9.


Figure 2Body 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 . 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 . 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 . 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 . 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 . 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 .

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

Figure 2Body 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.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.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.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.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.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.


Figure 3Immunoblot 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 . Quantification as percentage of phosphoprotein level normalized to total VEGFR2 . each dot ( B ) represent one individual mouse . Summary plot of 51 proteins enriched in the immobilized pY1212 peptide precipitate . 10 proteins ( red oval ) were selected for further analysis . PSM : peptide - spectrum matches . Area : average area of spectra for the three most abundant peptides in arbitrary units . Blot for peptide - enriched GRB2 and PI3Kp85 . HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 and PI3Kp85 . VEGFR2 / GRB2 and VEGFR2 / PI3Kp85 complexes in isolated ( i ) ECs . E . PLA for VEGFR2 / GRB2 and VEGFR2 / PI3Kp85 complexes ( black puncta ) in iECs from FVB wildtype ( WT ) and Vegfr2Y1212F / Y1212F lungs . Hoechst 33342 show nuclei ( grey ) . Scale bar , 20 µm . F , G . Fold - increase over PBS - treated sample ; each dot represents the mean of 6 fields . 2 - way ANOVA p = 0 . 0206 ( F ) , p = 0 . 0368 ( G ) ; Sidak ' s multiple comparison test , * p < 0 . 05 . n = 3 experiments . phosphoERK1 / 2 and phosphoAkt immunostaining ( green ) of iECs from FVB wildtype ( WT ) and Vegfr2Y1212F / Y1212F lungs . Hoechst 33342 ( blue ) shows nuclei . Arrows indicate nuclear accumulation of phosphoERK1 / 2 and phosphoAkt , respectively . Scale bar , 20 µm . Nuclear accumulation expressed as phoshoERK1 / 2 - positive nuclear area normalized to the total phosphoERK1 / 2 area by field . Each dot represents 1 field . Min , minutes of stimulation . 2 - way ANOVA p = 0 . 0248 ; Sidak ' s multiple comparison test , * p < 0 . 05 , * * p < 0 . 01 . n = 3 experiments . Nuclear accumulation expressed as fold - increase of nuclear phosphoAkt area in Vegfr2Y1212F / Y1212F over PBS - treated wildtype ( WT ) iECs , normalized to the number of nuclei . Each dot represents 1 field . Min , minutes of stimulation . Unpaired T test , * p < 0 . 05 . n = 3 experiments . Immunoblot for phosphoERK1 / 2 , total ERK1 / 2 , phosphoAkt and total Akt on lung lysates from wiltype ( WT ) and Vegfr2Y1212F / Y1212F FVB mice tail - vein injected with PBS or VEGFA followed by circulation for time points indicated . Each lane represents one individual mouse . Quantification of phosphoERK1 / 2 / total ERK1 / 2 expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0025 ; unpaired T test , * p < 0 . 05 . n = 3 experiments . Quantification of phosphoAkt / total Akt expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0003 ; unpaired T test , * p < 0 . 05 . n = 3 experiments .

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

Figure 3Immunoblot 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.Quantification as percentage of phosphoprotein level normalized to total VEGFR2. each dot (B) represent one individual mouse.Summary plot of 51 proteins enriched in the immobilized pY1212 peptide precipitate. 10 proteins (red oval) were selected for further analysis. PSM: peptide-spectrum matches. Area: average area of spectra for the three most abundant peptides in arbitrary units.Blot for peptide-enriched GRB2 and PI3Kp85. HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 and PI3Kp85.VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes in isolated (i)ECs. E. PLA for VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes (black puncta) in iECs from FVB wildtype (WT) and Vegfr2Y1212F/Y1212F lungs. Hoechst 33342 show nuclei (grey). Scale bar, 20 µm. F, G. Fold-increase over PBS-treated sample; each dot represents the mean of 6 fields. 2-way ANOVA p=0.0206 (F), p=0.0368 (G); Sidak's multiple comparison test, * p <0.05. n=3 experiments.phosphoERK1/2 and phosphoAkt immunostaining (green) of iECs from FVB wildtype (WT) and Vegfr2Y1212F/Y1212F lungs. Hoechst 33342 (blue) shows nuclei. Arrows indicate nuclear accumulation of phosphoERK1/2 and phosphoAkt, respectively. Scale bar, 20 µm.Nuclear accumulation expressed as phoshoERK1/2-positive nuclear area normalized to the total phosphoERK1/2 area by field. Each dot represents 1 field. Min, minutes of stimulation. 2-way ANOVA p=0.0248; Sidak's multiple comparison test, * p <0.05, ** p <0.01. n=3 experiments.Nuclear accumulation expressed as fold-increase of nuclear phosphoAkt area in Vegfr2Y1212F/Y1212F over PBS-treated wildtype (WT) iECs, normalized to the number of nuclei. Each dot represents 1 field. Min, minutes of stimulation. Unpaired T test, *p < 0.05. n=3 experiments.Immunoblot for phosphoERK1/2, total ERK1/2, phosphoAkt and total Akt on lung lysates from wiltype (WT) and Vegfr2Y1212F /Y1212F FVB mice tail-vein injected with PBS or VEGFA followed by circulation for time points indicated. Each lane represents one individual mouse. Quantification of phosphoERK1/2 /total ERK1/2 expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0025; unpaired T test, *p < 0.05. n=3 experiments. Quantification of phosphoAkt/total Akt expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0003; unpaired T test, *p < 0.05. n=3 experiments.


Figure 4Immunoblot for Myc on lung lysates from wiltype ( WT ) and Vegfr2Y1212F / Y1212F FVB mice tail - vein injected with PBS or VEGFA followed by circulation for time points indicated . Each lane represents one individual mouse . Quantification of Myc / Actin expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0058 ; unpaired T test , * p < 0 . 05 , * * p < 0 . 01 . n = 3 experiments . Myc protein expression . C . Myc and Actin immunoblotting on lysates of lung isolated ECs from untreated wildtype ( WT ) and mutant ( VEGFR2 Y1212F ) C57Bl / 6 and FVB mice . D . Quantification of Myc / Actin expressed as Arbitrary unit . Each lane ( C ) and each dot ( D ) represent one individual mouse . 2 - way ANOVA p < 0 . 0001 ; unpaired T test , * * p < 0 . 01 . n = 5 - 7 . Baseline transcriptional level of Myc from lung isolated ECs from untreated wildtype ( WT ) and mutant ( VEGFR2 Y1212F ) C57Bl / 6 and FVB mice . Each dot represents one individual mouse . 2 - way ANOVA p = 0 . 0041 ; Sidak ' s multiple comparison test , * p < 0 . 05 . n = 4 - 5 . Proliferation of C57Bl / 6 and FVB iECs . CD31 ( white ) , ERG ( red ) , EdU ( green ) and double - positive cells ( yellow ) in C57Bl / 6 ( F ) and FVB ( G ) iECs . EdU signal was masked with ERG signal . Proliferation of C57Bl / 6 and FVB iECs . EdU incorporation in ERG - positive ECs normalized to total ERG - positive ECs . Each dot represents 2 fields / well , one well / mouse . Scale bar , 100 µm . Error bars : SD ; 2 - way ANOVA p = 0 . 0843 ( H ) , p = 0 . 0474 ( I ) ; unpaired T test , * p < 0 . 05 ; * * p < 0 . 01 . C57Bl / 6 n = 3 - 6 mice , FVB n = 3 - 5mice . Rescue of angiogenic sprouting by Myc overexpression in E11 . 5 C57Bl / 6 Vegfr2Y1212F / Y1212F explant tissue . Representative images of E11 . 5 explants from C57Bl / 6 embryos taken from wildtype ( WT ) and Vegfr2Y1212F / Y1212F littermates with ( MycOE / Cre + ) and without ( MycOE / Cre - ) endothelial - specific overexpression of Myc . Endothelial cell marker ( CD31 ) immunostaining highlights the vasculature in white . Embryonic tissue blacked out to highlight sprouting area . Rescue of angiogenic sprouting by Myc overexpression in E11 . 5 C57Bl / 6 Vegfr2Y1212F / Y1212F explant tissue . Quantification as fold - increase of vessel sprouting area compared to WT MycOE / Cre - . Scale bar , 250 µm . Error bars : SD ; unpaired T test , * * p < 0 . 01 , * * * * p < 0 . 0001 . n = 4 - 11 explants , one explant / embryo . Rescue of Ec proliferation . Myc overexpression in E11 . 5 C57Bl / 6 Vegfr2Y1212F / Y1212F explant tissue . ERG ( red ) , EdU ( green ) and double - positive cells ( yellow ; arrows ) . Rescue of Ec proliferation . Quantification of EdU / ERG double positive ECs normalized to total ECs . Scale bar , 50 µm . Error bars : SD ; unpaired T test , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 - 7 explants , one explant / embryo .

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Figure 4Immunoblot for Myc on lung lysates from wiltype (WT) and Vegfr2Y1212F /Y1212F FVB mice tail-vein injected with PBS or VEGFA followed by circulation for time points indicated. Each lane represents one individual mouse. Quantification of Myc/Actin expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0058; unpaired T test, *p < 0.05, ** p <0.01. n=3 experiments.Myc protein expression. C. Myc and Actin immunoblotting on lysates of lung isolated ECs from untreated wildtype (WT) and mutant (VEGFR2 Y1212F) C57Bl/6 and FVB mice. D. Quantification of Myc/Actin expressed as Arbitrary unit. Each lane (C) and each dot (D) represent one individual mouse. 2-way ANOVA p <0.0001; unpaired T test, ** p <0.01. n=5-7.Baseline transcriptional level of Myc from lung isolated ECs from untreated wildtype (WT) and mutant (VEGFR2 Y1212F) C57Bl/6 and FVB mice. Each dot represents one individual mouse. 2-way ANOVA p=0.0041; Sidak's multiple comparison test, * p <0.05. n=4-5.Proliferation of C57Bl/6 and FVB iECs. CD31 (white), ERG (red), EdU (green) and double-positive cells (yellow) in C57Bl/6 (F) and FVB (G) iECs. EdU signal was masked with ERG signal.Proliferation of C57Bl/6 and FVB iECs. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents 2 fields/well, one well/mouse. Scale bar, 100 µm. Error bars: SD; 2-way ANOVA p =0.0843 (H), p=0.0474 (I); unpaired T test, *p < 0.05; **p < 0.01. C57Bl/6 n=3-6 mice, FVB n=3-5mice.Rescue of angiogenic sprouting by Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. Representative images of E11.5 explants from C57Bl/6 embryos taken from wildtype (WT) and Vegfr2Y1212F /Y1212F littermates with (MycOE/Cre +) and without (MycOE/Cre-) endothelial-specific overexpression of Myc. Endothelial cell marker (CD31) immunostaining highlights the vasculature in white. Embryonic tissue blacked out to highlight sprouting area.Rescue of angiogenic sprouting by Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. Quantification as fold-increase of vessel sprouting area compared to WT MycOE/Cre -. Scale bar, 250 µm. Error bars: SD; unpaired T test, ** p <0.01, **** p <0.0001. n=4-11 explants, one explant/embryo.Rescue of Ec proliferation. Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. ERG (red), EdU (green) and double-positive cells (yellow; arrows).Rescue of Ec proliferation. Quantification of EdU/ERG double positive ECs normalized to total ECs. Scale bar, 50 µm. Error bars: SD; unpaired T test, *** p <0.001, **** p <0.0001. n=3-7 explants, one explant/embryo.


Figure 5GSEAs showing upregulation of genes belonging to the K - Ras , Angiogenesis and mTORC1 gene sets in ( WT ) compared to VEGFR2 Y1212F isolated ECs . K - Ras and Angiogenesis gene sets derived from C57Bl / 6 iECs kept in basal condition ; mTORC1 gene set derived from C57Bl / 6 iECs treated with VEGFA for 3h . See Supplementary Table 2 . Enrichment score ( ES ) , normalized ES ( NES ) are shown . P - values < 0 . 05 were regarded as significant . Heat maps displaying gene regulation in gene sets shown in A . The color in the heatmap shows the gene expression value , expressed as a z - score across animals . Red color represents relative higher expression and blue color represents relative lower expression value . The z - score is calculated by ( x - x ' ) / s where x is individual gene expression , x ' is the mean of the gene expression across samples and s is standard deviation of the gene expression across samples . Myc - regulated gene expression downstream of VEGFR2 pY1212 . Quantitative PCR analysis of a set of known Myc - regulated transcripts in C57Bl / 6 ( C , D ) and FVB ( E , F ) adult lungs at basal ( C , E ) and at 1 hour after tail - vein administration of VEGFA ( D , F ) . FASN , fatty acid synthase ; Ldha and - b , Lactate dehydrogenase A and B ; Acod1 , Aconitate Decarboxylase 1 ; Ppat , Amidophosphoribosyltransferase ; Pde4d , cAMP - specific 3 ' , 5 ' - cyclic phosphodiesterase 4D ; Prkaca , cAMP - dependent protein kinase catalytic subunit alpha . Error bars : SD ; 2 - way ANOVA between WT and Vegfr2Y1212F / Y1212F p = 0 . 0453 ( C ) , p < 0 . 0001 ( D ) , p = 0 . 0018 ( E ) , p = 0 . 09 ( F ) ; unpaired T test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C57Bl / 6 n = 8 - 11 mice , FVB n = 9 - 14 mice .

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Figure 5GSEAs showing upregulation of genes belonging to the K-Ras, Angiogenesis and mTORC1 gene sets in (WT) compared to VEGFR2 Y1212F isolated ECs. K-Ras and Angiogenesis gene sets derived from C57Bl/6 iECs kept in basal condition; mTORC1 gene set derived from C57Bl/6 iECs treated with VEGFA for 3h. See Supplementary Table 2. Enrichment score (ES), normalized ES (NES) are shown. P-values <0.05 were regarded as significant. Heat maps displaying gene regulation in gene sets shown in A. The color in the heatmap shows the gene expression value, expressed as a z-score across animals. Red color represents relative higher expression and blue color represents relative lower expression value. The z-score is calculated by (x-x')/s where x is individual gene expression, x' is the mean of the gene expression across samples and s is standard deviation of the gene expression across samples.Myc-regulated gene expression downstream of VEGFR2 pY1212. Quantitative PCR analysis of a set of known Myc-regulated transcripts in C57Bl/6 (C, D) and FVB (E, F) adult lungs at basal (C, E) and at 1 hour after tail-vein administration of VEGFA (D, F). FASN, fatty acid synthase; Ldha and -b, Lactate dehydrogenase A and B; Acod1, Aconitate Decarboxylase 1; Ppat, Amidophosphoribosyltransferase; Pde4d, cAMP-specific 3',5'-cyclic phosphodiesterase 4D; Prkaca, cAMP-dependent protein kinase catalytic subunit alpha. Error bars: SD; 2-way ANOVA between WT and Vegfr2Y1212F /Y1212F p=0.0453 (C), p < 0.0001 (D), p=0.0018 (E), p=0.09 (F); unpaired T test, *p < 0.05, **p < 0.01, ***p < 0.001. C57Bl/6 n=8-11 mice, FVB n=9-14 mice.


Figure 6A . FGF21SS and FGF21 improve insulin resistance by countering the inflammation in 3T3 - L1 adipocytes . Cells were cultured in RAW - CM to induce inflammation and insulin resistance , while vehicle , FGF21 or FGF21SS were added simultaneously . The insulin sensitivity was indicated by insulin - induced AKT phosphorylation using western blotting analysis . Error bars show the SEM of four independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - test ) vs RAW - CM treated only group . Anti - diabetic effect evaluation in ob / ob mice . Mice were injected subcutaneously with vehicle , FGF21 or FGF21SS at indicated doses every day . Body weight ( B ) was measured Error bars show the SEM of ten independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( Student ' s t - test ) vs vehicle . Anti - diabetic effect evaluation in ob / ob mice . Mice were injected subcutaneously with vehicle , FGF21 or FGF21SS at indicated doses every day . plasma glucose ( D ) was measured every day . Plasma insulin ( C ) was analyzed at day 7 using insulin ELISA kit . Error bars show the SEM of ten independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( Student ' s t - test ) vs vehicle .

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Figure 6A. FGF21SS and FGF21 improve insulin resistance by countering the inflammation in 3T3-L1 adipocytes. Cells were cultured in RAW-CM to induce inflammation and insulin resistance, while vehicle, FGF21 or FGF21SS were added simultaneously. The insulin sensitivity was indicated by insulin-induced AKT phosphorylation using western blotting analysis. Error bars show the SEM of four independent experiments. *p < 0.05, **p < 0.01 (Student's t-test) vs RAW-CM treated only group.Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. Body weight (B) was measured Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. plasma glucose (D) was measured every day. Plasma insulin (C) was analyzed at day 7 using insulin ELISA kit. Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.


Figure 1Coomassie Blue staining of recombinant human DDX5 purified in bacteria . M denotes the molecular mass markers in kDa . R - loop unwinding assay in presence of increasing DDX5 . The top panel shows a typical image obtained after the assay . The bar graph ( bottom ) shows the quantification . The average is expressed as percentage unwinding , and standard error of the mean ( S . E . M . ) , n = 4 . S9 . 6 signal in the nucleoplasm of U2OS cells . An average from 3 independent experiments performed in triplicate . In total , ~ 90 images for each condition ( 10 images / slide and 9 slides for each condition ) were taken and two cells per image were quantified . Statistical significance was assessed using one - way ANOVA t - test . * : p < 0 . 05 ; * * * * : p < 0 . 0001 . U2OS cells transfected with siCTL , or siDDX5 were subjected to DRIP - qPCR analysis with anti - IgG , and anti - S9 . 6 antibodies with or without RNaseH treatment . The gene location and genomic qPCR amplification region are shown at the top of each panel . B , E , H , S and X denote the location of the BsrGI , EcoRI , HindIII , SspI , and XbaI . The identified R - loop peaks were extracted from the R - loop database ( R - loop DB ) for each region . The bar graphs are the average and S . E . M . from 3 independent experiments . Statistical significance was assessed using t - test . * : p < 0 . 05 .

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Figure 1Coomassie Blue staining of recombinant human DDX5 purified in bacteria. M denotes the molecular mass markers in kDa.R-loop unwinding assay in presence of increasing DDX5. The top panel shows a typical image obtained after the assay. The bar graph (bottom) shows the quantification. The average is expressed as percentage unwinding, and standard error of the mean (S.E.M.), n=4.S9.6 signal in the nucleoplasm of U2OS cells. An average from 3 independent experiments performed in triplicate. In total, ~90 images for each condition (10 images/slide and 9 slides for each condition) were taken and two cells per image were quantified. Statistical significance was assessed using one-way ANOVA t-test. *: p < 0.05; ****: p < 0.0001.U2OS cells transfected with siCTL, or siDDX5 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The gene location and genomic qPCR amplification region are shown at the top of each panel. B, E, H, S and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI. The identified R-loop peaks were extracted from the R-loop database (R-loop DB) for each region. The bar graphs are the average and S.E.M. from 3 independent experiments. Statistical significance was assessed using t-test. *: p < 0.05.


Figure 2U2OS cells were lysed and the lysates were incubated with anti - PRMT5 antibody or immunoglobulin G ( IgG ) as control followed by protein A Sepharose bead addition . The immunoprecipitated ( IP ) proteins or whole cell extract ( WCE ) were separated by SDS - PAGE and Western blotted ( WB ) with the indicated antibodies . Same as panel ( A ) except anti - DDX5 antibody was used instead of anti - PRMT5 antibody for immunoprecipitation . The mass spectrum of R502 monomethylation from Flag - DDX5 expressed in U2OS . HEK293 cells transfected with Flag - DDX5 were lysed and immunoprecipitated with anti - Flag antibodies . The bound proteins were along with WCE from untransfected ( - ) and Flag - DDX5 ( + ) transfected cells were Western blotted with anti - FLAG , anti - methylarginine antibodies ( MeR ) . The knockdown using siRNAs of the indicated gene in U2OS cells was confirmed by Western blottingThe knockdown using siRNAs of the indicated gene in U2OS cells were analyzed by immunofluorescence with S9 . 6 and anti - nucleolin antibodies . A typical image is shown .

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Figure 2U2OS cells were lysed and the lysates were incubated with anti-PRMT5 antibody or immunoglobulin G (IgG) as control followed by protein A Sepharose bead addition. The immunoprecipitated (IP) proteins or whole cell extract (WCE) were separated by SDS-PAGE and Western blotted (WB) with the indicated antibodies. Same as panel (A) except anti-DDX5 antibody was used instead of anti-PRMT5 antibody for immunoprecipitation.The mass spectrum of R502 monomethylation from Flag-DDX5 expressed in U2OS.HEK293 cells transfected with Flag-DDX5 were lysed and immunoprecipitated with anti-Flag antibodies. The bound proteins were along with WCE from untransfected (-) and Flag-DDX5 (+) transfected cells were Western blotted with anti-FLAG, anti-methylarginine antibodies (MeR).The knockdown using siRNAs of the indicated gene in U2OS cells was confirmed by Western blottingThe knockdown using siRNAs of the indicated gene in U2OS cells were analyzed by immunofluorescence with S9.6 and anti-nucleolin antibodies. A typical image is shown.


Figure 3Western blotting of PRMT5 and DDX5 knockdowns . Representative images of S9 . 6 staining in siCTL , siDDX5 , or siPRMT5 knockdown at transcription on ( TA - KR ) or off ( TetR - KR ) genomic loci in U2OS TRE cells . The nuclei are visualized by DAPI . Quantification of the S9 . 6 foci intensity in the indicated conditions . Three independent experiments were performed and 50 cells were analyzed in each experiment . Error bars represent mean of S9 . 6 foci intensity quantification with s . e . m . The statistical analysis was performed by two - tailed Student ' s t - test ( Mann - Whitney test U - test ) . * * * * , p < 0 . 0001 .

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

Figure 3Western blotting of PRMT5 and DDX5 knockdowns.Representative images of S9.6 staining in siCTL, siDDX5, or siPRMT5 knockdown at transcription on (TA-KR) or off (TetR-KR) genomic loci in U2OS TRE cells. The nuclei are visualized by DAPI.Quantification of the S9.6 foci intensity in the indicated conditions. Three independent experiments were performed and 50 cells were analyzed in each experiment. Error bars represent mean of S9.6 foci intensity quantification with s.e.m. The statistical analysis was performed by two-tailed Student's t-test (Mann-Whitney test U-test). ****, p < 0.0001.


Figure 4U2OS cells transfected with siCTL or siPRMT5 were subjected to DRIP - qPCR analysis . The average and S . E . M . from 3 independent experiments is shown . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; and * * : p < 0 . 01 . Western blotting of whole cell extracts from siCTL , siPRMT5 , siDDX5 or siDDX5 / siPRMT5 transfected cells . U2OS cells transfected with siCTL , siPRMT5 , siDDX5 or siDDX5 / siPRMT5 were subjected to DRIP - qPCR analysis . The average and S . E . M . from 3 independent experiments is shown . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; and * * : p < 0 . 01 .

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

Figure 4U2OS cells transfected with siCTL or siPRMT5 were subjected to DRIP-qPCR analysis. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; and **: p < 0.01.Western blotting of whole cell extracts from siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 transfected cells.U2OS cells transfected with siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 were subjected to DRIP-qPCR analysis. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; and **: p < 0.01.