( 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 .

(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

(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 .

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


( H ) 293T 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 .

(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 . ) .

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(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 .

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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 ) .

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(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 ) .

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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(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 .

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


( C - D ) iMEFgt / gt stably expressing cherry - tagged WT STING or K288R STING 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 .

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


( E ) iMEFgt / gt ( - ) 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 .

(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 O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

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 O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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 .

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

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 .

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

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 ) .

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

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 .

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

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

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 .

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 .

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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.


Quantification as percentage of phosphoprotein level normalized to total VEGFR2 . each dot ( B ) represent one individual mouse .

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

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 .

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

Summary 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 .

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 .

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

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 .

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

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 .

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

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 .

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

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

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.


Immunoblot 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 .

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

Immunoblot 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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

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 .

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

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.


GSEAs 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 .

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

GSEAs 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 .

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

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.


A . 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 .

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

A. 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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Coomassie Blue staining of recombinant human DDX5 purified in bacteria . M denotes the molecular mass markers in kDa .

Coomassie 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 .

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 .

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

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 .

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

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.


U2OS 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 .

U2OS 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 .

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 ) .

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

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 blotting


The 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 .

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

The 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.


Western blotting of PRMT5 and DDX5 knockdowns .

Western 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 .

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

Representative 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the 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.


U2OS 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 .

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

U2OS 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 .

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

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.


U2OS cells were transfected with a Flag - DDX5 expression vector in the presence of siCTL or siPRMT5 . Lysates were immunoprecipitated with IgG or anti - Flag antibodies as indicated . The WCE and the bound proteins were Western blotted with anti - symmetrical dimethylarginine antibody or anti - Flag antibodies .

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

U2OS cells were transfected with a Flag-DDX5 expression vector in the presence of siCTL or siPRMT5. Lysates were immunoprecipitated with IgG or anti-Flag antibodies as indicated. The WCE and the bound proteins were Western blotted with anti-symmetrical dimethylarginine antibody or anti-Flag antibodies.


Coomassie blue ( left panel ) and in vitro methylation assay ( right panel ) of indicated GST - DDX5 fragments and the GST - DDX5 - RK mutant .

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

Coomassie blue (left panel) and in vitro methylation assay (right panel) of indicated GST-DDX5 fragments and the GST-DDX5-RK mutant.


Immunofluorescence analysis with S9 . 6 and anti - Flag antibodies of U2OS cells transfected with Flag - tagged DDX5 , DDX5 - RK or DDX5 - XD ( helicase dead ) . Nuclear S9 . 6 signal was counted in both Flag - negative and Flag - positive cells . The Flag - negative cells were considered as untransfected cells . The graphs shown represent the quantification with the S . E . M from 3 independent experiments . Statistical significance was assessed using one - way ANOVA t - test . * * * * , p < 0 . 0001 .

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

Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with Flag-tagged DDX5, DDX5-RK or DDX5-XD (helicase dead). Nuclear S9.6 signal was counted in both Flag-negative and Flag-positive cells. The Flag-negative cells were considered as untransfected cells. The graphs shown represent the quantification with the S.E.M from 3 independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001.


Immunofluorescence analysis with S9 . 6 and anti - Flag antibodies of U2OS cells transfected with siCTL or siDDX5 - 1 and Flag - tagged DDX5 ( + WT ) or DDX5 - RK ( + RK ) as indicated . The graphs show the average and S . E . M . from three independent experiments . Statistical significance was assessed using one - way ANOVA t - test . * * * * , p < 0 . 0001 .

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

Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with siCTL or siDDX5-1 and Flag-tagged DDX5 (+WT) or DDX5-RK (+RK) as indicated. The graphs show the average and S.E.M. from three independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001.


HEK293 cells were transfected with siCTL , or siDDX5 . The next day , the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector ( - ) , expression vectors encoding Flag - DDX5 ( WT ) , or Flag - DDX5 - RK ( RK ) . Forty eight hours later , all the cells were subjected to DRIP - qPCR analysis . The average with the S . E . M . from 3 independent experiments is shown . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 and n . s . : not significant .

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

HEK293 cells were transfected with siCTL, or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). Forty eight hours later, all the cells were subjected to DRIP-qPCR analysis. The average with the S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and n.s.: not significant.


HEK293 cells were transfected with empty pcDNA3 vector ( - ) or Flag - DDX5 ( + ) . Whole cell lysates ( WCE ) and anti - Flag immunoprecipitations ( IP ) were immunoblotted with anti - Flag and anti - XRN2 antibodies .

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

HEK293 cells were transfected with empty pcDNA3 vector (-) or Flag-DDX5 (+). Whole cell lysates (WCE) and anti-Flag immunoprecipitations (IP) were immunoblotted with anti-Flag and anti-XRN2 antibodies.


Untransfected U2OS cells were lyzed and subjected to immunoprecipitation with control immunoglobulin G ( IgG ) or anti - XRN2 antibodies . The bound proteins were separated by SDS - PAGE followed by Western blotting with anti - DDX5 or anti - XRN2 antibodies . The migration of DDX5 and XRN2 is depicted with an arrow .

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

Untransfected U2OS cells were lyzed and subjected to immunoprecipitation with control immunoglobulin G (IgG) or anti-XRN2 antibodies. The bound proteins were separated by SDS-PAGE followed by Western blotting with anti-DDX5 or anti-XRN2 antibodies. The migration of DDX5 and XRN2 is depicted with an arrow.


U2OS cells were transfected with empty pcDNA3 vector ( pcDNA3 ) or expression vectors encoding Flag - DDX5 WT ( 1 - 614 ) or truncated Flag - DDX5 proteins ( 60 - 614 ; 1 - 554 ; 1 - 435 ) . WCE of the transfected cells ( left panel ) were Western blotted with anti - Flag and anti - XRN2 antibodies to confirm equivalent expression . The transfected cells were lysed and anti - Flag immunoprecipitations ( right panel ) performed for the presence of co - immunoprecipitating endogenous XRN2 by Western blotting with anti - XRN2 antibodies . M denotes molecular mass markers in kDa .

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

U2OS cells were transfected with empty pcDNA3 vector (pcDNA3) or expression vectors encoding Flag-DDX5 WT (1-614) or truncated Flag-DDX5 proteins (60-614; 1-554; 1-435). WCE of the transfected cells (left panel) were Western blotted with anti-Flag and anti-XRN2 antibodies to confirm equivalent expression. The transfected cells were lysed and anti-Flag immunoprecipitations (right panel) performed for the presence of co-immunoprecipitating endogenous XRN2 by Western blotting with anti-XRN2 antibodies. M denotes molecular mass markers in kDa.


Untransfected and U2OS stably expressing Flag - DDX5 WT or Flag - DDX5 - RK were subjected to immunoprecipitation with anti - Flag antibody . The WCE and the anti - Flag immunoprecipitated proteins were Western blotted with anti - monomethylarginine ( MeR ) , anti - Flag and anti - XRN2 antibodies , respectively . M denotes molecular mass markers in kDa .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

Untransfected and U2OS stably expressing Flag-DDX5 WT or Flag-DDX5-RK were subjected to immunoprecipitation with anti-Flag antibody. The WCE and the anti-Flag immunoprecipitated proteins were Western blotted with anti-monomethylarginine (MeR), anti-Flag and anti-XRN2 antibodies, respectively. M denotes molecular mass markers in kDa.


U2OS cells transfected with siCTL , siDDX5 , or siXRN2 were subjected to DRIP - qPCR analysis with anti - IgG , and anti - S9 . 6 antibodies with or without RNaseH treatment . The average and S . E . M . from 3 independent experiments is shown . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 .

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U2OS cells transfected with siCTL, siDDX5, or siXRN2 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.


HEK293 cells were transfected with siCTL , siDDX5 , or siXRN2 . The gene location and qPCR amplification region are shown at the top of each panel . The identified R - loop peaks were extracted from R - loop database ( R - loop DB ) for each gene region . R - loop : DRIP denotes anti - S9 . 6 DRIP - qPCR and Pol II ChIP denotes RNA pol II ChIP . The bar graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 and * * * * : p < 0 . 0001 . B , E , H , S and X denote the location of the BsrGI , EcoRI , HindIII , SspI , and XbaI .

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HEK293 cells were transfected with siCTL, siDDX5, or siXRN2. The gene location and qPCR amplification region are shown at the top of each panel. The identified R-loop peaks were extracted from R-loop database (R-loop DB) for each gene region. R-loop: DRIP denotes anti-S9.6 DRIP-qPCR and Pol II ChIP denotes RNA pol II ChIP. The bar graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001. B, E, H, S and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI.


Schematic representation of the β - actin locus and the location of the genomic PCR primers used for ChIP - qPCR and DRIP - qPCR . Prom ( promoter ) , RB ( region B ) , poly ( A ) and RD ( region D ) indicate the regions analyzed by qPCR .

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Schematic representation of the β-actin locus and the location of the genomic PCR primers used for ChIP-qPCR and DRIP-qPCR. Prom (promoter), RB (region B), poly(A) and RD (region D) indicate the regions analyzed by qPCR.


HEK293 cells were transfected with siCTL or siDDX5 and XRN2 ChIP assays were performed . The XRN2 ChIP signal was normalized to the Pol II ChIP signal . WCE were immunoblotted with anti - DDX5 and α - tubulin antibodies , the latter was used as a loading control . The graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 .

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HEK293 cells were transfected with siCTL or siDDX5 and XRN2 ChIP assays were performed. The XRN2 ChIP signal was normalized to the Pol II ChIP signal. WCE were immunoblotted with anti-DDX5 and α-tubulin antibodies, the latter was used as a loading control. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.


HEK293 cells were transfected with siCTL , siDDX5 or siXRN2 . The cells were subjected to Western blotting , RNA Pol II ChIP and DRIP analysis of β - actin gene . The graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 and * * * * : p < 0 . 0001 .

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HEK293 cells were transfected with siCTL, siDDX5 or siXRN2. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.


HEK293 cells were transfected with siCTL or siDDX5 . The next day , the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector ( - ) , expression vectors encoding Flag - DDX5 ( WT ) , or Flag - DDX5 - RK ( RK ) . The cells were subjected to Western blotting to visualize Flag - DDX WT and RK expression . Pol II ChIP and DRIP analysis of β - actin gene were performed . For the ChIP analysis , the Y - axis shows the signal - to - noise ratio of RNA Pol II IP relative to control IgG IP . The graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 and * * * * : p < 0 . 0001 .

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HEK293 cells were transfected with siCTL or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). The cells were subjected to Western blotting to visualize Flag-DDX WT and RK expression. Pol II ChIP and DRIP analysis of β-actin gene were performed. For the ChIP analysis, the Y-axis shows the signal-to-noise ratio of RNA Pol II IP relative to control IgG IP. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.


HEK293 cells were transfected with siCTL or siPRMT5 . The cells were subjected to Western blotting , RNA Pol II ChIP and DRIP analysis of β - actin gene . The graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 .

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HEK293 cells were transfected with siCTL or siPRMT5. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.


HEK293 cells were transfected with empty plasmid ( - ) , wild type Flag - DDX5 ( WT ) or its RK mutant ( RK ) and subjected to western blot and Flag - DDX5 ChIP - qPCR analysis of β - actin gene . The graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 and * * * * : p < 0 . 0001 ; n . s . not significant .

HEK293 cells were transfected with empty plasmid (-), wild type Flag-DDX5 (WT) or its RK mutant (RK) and subjected to western blot and Flag-DDX5 ChIP-qPCR analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001; n.s. not significant.


In vitro niche dependency of HGSOC tumor cells . Phase - contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth , while they do not grow at all in Wnt3a supplemented medium . Also , inhibition of BMP signaling through Noggin has strong negative effect on the initial growth . Scale bar : 500 µm . E - EGF , F - FGF10 , N - Noggin , R - R - spondin1 , B - Basic medium , P -

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In vitro niche dependency of HGSOC tumor cells. Phase-contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth, while they do not grow at all in Wnt3a supplemented medium. Also, inhibition of BMP signaling through Noggin has strong negative effect on the initial growth. Scale bar: 500 µm. E - EGF, F - FGF10, N -Noggin, R - R-spondin1, B - Basic medium, P - Passage


Cancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines . Scale bar : 20 µm .

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Cancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines. Scale bar: 20 µm.


HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization . Scale bar : 100 µm .

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HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization. Scale bar: 100 µm.


HGSOC organoids show differential response to carboplatin treatment , confirming patient - specific sensitivity of the cultures . Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors . Data represent mean ± SD of technical triplicates .

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HGSOC organoids show differential response to carboplatin treatment, confirming patient-specific sensitivity of the cultures. Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors. Data represent mean ±SD of technical triplicates.


Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long - term organoid culture . Color code indicates type of mutations which were detected .

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Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long-term organoid culture. Color code indicates type of mutations which were detected.


Representative western blot showing loss or overexpression of p53 in individual organoid lines .

Representative western blot showing loss or overexpression of p53 in individual organoid lines.


Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain - of - function TP53 mutation . Scale bars : 100 µm ( Tissue ) and 50 µm ( Organoid ) .

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Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain-of-function TP53 mutation. Scale bars: 100 µm (Tissue) and 50 µm (Organoid).


Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue . Data represent the median , quartiles , maximum , and minimum of the normalized expression from 8 different donors .

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Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue. Data represent the median, quartiles, maximum, and minimum of the normalized expression from 8 different donors.


Heat map of differentially expressed genes between cancer and healthy tissue / organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples . Differential expression determined by single - color microarray for 8 different patient samples was significant for all genes with p < 0 . 05 except for OVGP1 and TOP2A in organoids .

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Heat map of differentially expressed genes between cancer and healthy tissue/organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples. Differential expression determined by single-color microarray for 8 different patient samples was significant for all genes with p < 0.05 except for OVGP1 and TOP2A in organoids.


Experimental approach for genetic manipulation of FT epithelial cells ( FTECs ) . FTECs were sequentially transduced in 2D culture with replication - deficient viruses containing specific shRNAs and different selection markers . After sorting cells were seeded in Matrigel for organoid formation . Scale bars : 2 cm ( tissue ) , 200 µm ( 2D culture ) and 500 µm ( 3D culture ) . Confirmation of robust knockdowns of p53 , PTEN and RB in FT organoids on RNA and protein level . Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ± SEM from 3 different donors . A representative western blot from one donor is depicted . RNA as well as protein samples were taken at passage 1 or 2 of each organoid line . KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild

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Experimental approach for genetic manipulation of FT epithelial cells (FTECs). FTECs were sequentially transduced in 2D culture with replication-deficient viruses containing specific shRNAs and different selection markers. After sorting cells were seeded in Matrigel for organoid formation. Scale bars: 2 cm (tissue), 200 µm (2D culture) and 500 µm (3D culture). Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type


Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis .

Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis.


Confocal images of triple KD organoids reveal increased DNA damage ( yH2AX , marked by asterisks ) , atypic nuclei ( DRAQ5 ) and loss of apicobasal polarity . Scale bar : 20µm .

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Confocal images of triple KD organoids reveal increased DNA damage (yH2AX, marked by asterisks), atypic nuclei (DRAQ5) and loss of apicobasal polarity. Scale bar: 20µm.


Confirmation of robust knockdowns of p53 , PTEN and RB in FT organoids on RNA and protein level . Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ± SEM from 3 different donors . A representative western blot from one donor is depicted . RNA as well as protein samples were taken at passage 1 or 2 of each organoid line . KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis . Presence of ciliated cells ( arrowheads ) , as revealed by immunostaining against detyrosinated Tubulin ( dtyrTub ) in triple KD organoids , proves capacity for terminal differentiation . Scale bar : 50µm .

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Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis. Presence of ciliated cells (arrowheads), as revealed by immunostaining against detyrosinated Tubulin (dtyrTub) in triple KD organoids, proves capacity for terminal differentiation. Scale bar: 50µm.


In long - term 3D culture triple KD organoids show loss of growth capacity and premature growth arrest compared to the controls as indicated by the representative growth curves of FT234 . Scale bar : 500 µm .

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In long-term 3D culture triple KD organoids show loss of growth capacity and premature growth arrest compared to the controls as indicated by the representative growth curves of FT234. Scale bar: 500 µm.


Growth curves and phase - contrast images of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long - term growth capacity . Scale bar : 500 µm .

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Growth curves and phase-contrast images of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long-term growth capacity. Scale bar: 500 µm.


Levels of DNA damage ( yH2AX ) and apoptosis ( cleaved caspase - 3 , PARP1 ) do not differ among the different media as shown by representative western blots comparing triple KD organoids and their controls .

Levels of DNA damage (yH2AX) and apoptosis (cleaved caspase-3, PARP1) do not differ among the different media as shown by representative western blots comparing triple KD organoids and their controls.


Triple KD ( p53 / PTEN / RB ) organoids are characterized by a HGSOC gene expression signature as revealed by microarray analysis comparing WT and KD organoids grown in either OCM or FTM . Differential expression determined by dual - color microarray for 2 biological replicates was significant for all genes with p < 0 . 00005 .

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Triple KD (p53/PTEN/RB) organoids are characterized by a HGSOC gene expression signature as revealed by microarray analysis comparing WT and KD organoids grown in either OCM or FTM. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.00005.


Differential expression of stemness - and differentiation - related genes of triple KD organoids grown in OCM vs . FTM . Fold - changes were derived from microarray data and indicate and an increase in stemness ( CD133 , SOX2 , MYCN ) and a drop in differentiation ( OVGP1 , PGR , FOXJ1 ) under OCM conditions . Differential expression determined by dual - color microarray for 2 biological replicates was significant for all genes with p < 0 . 05 .

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Differential expression of stemness- and differentiation-related genes of triple KD organoids grown in OCM vs. FTM. Fold-changes were derived from microarray data and indicate and an increase in stemness (CD133, SOX2, MYCN) and a drop in differentiation (OVGP1, PGR, FOXJ1) under OCM conditions. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.05.


FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM , suggesting a decrease in differentiation capacity . Data represent the mean ± SEM from technical triplicates for two independent knockdown cultures .

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FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM, suggesting a decrease in differentiation capacity. Data represent the mean ±SEM from technical triplicates for two independent knockdown cultures.


Proportion of ciliated cells ( detyrosinated Tubulin ; dtyrTub ) is significantly reduced in KD organoids under OCM growth conditions , confirming inhibition of differentiation as illustrated by confocal images ( Scale bar : 50 µm ) . The quantification plot depicts the mean ± SEM from 3 independent biological replicates , based on quantification of the average number of ciliated cells per counted nuclei . 1000 cells were counted per individual experiment . * p < 0 . 05 , two - sided Student ' s t - test .

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Proportion of ciliated cells (detyrosinated Tubulin; dtyrTub) is significantly reduced in KD organoids under OCM growth conditions, confirming inhibition of differentiation as illustrated by confocal images (Scale bar: 50 µm). The quantification plot depicts the mean ±SEM from 3 independent biological replicates, based on quantification of the average number of ciliated cells per counted nuclei. 1000 cells were counted per individual experiment. *p < 0.05, two-sided Student's t-test.


The proportion of CD133 + cells was determined by FACS analysis after growing triple KD and control cells in FTM vs . OCM for two passages . The graph is representative of two biological replicates .

The proportion of CD133+ cells was determined by FACS analysis after growing triple KD and control cells in FTM vs. OCM for two passages. The graph is representative of two biological replicates.


Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable cell number ( representative graph of 3 independent experiments ) . The luminescent cell viability assay was performed in triplicate and the bar plot depicts the mean ± SEM . * p < 0 . 05 , two - sided Student ` s t - test ; KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild

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Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable cell number (representative graph of 3 independent experiments). The luminescent cell viability assay was performed in triplicate and the bar plot depicts the mean ±SEM. * p < 0.05, two-sided Student`s t-test; KD - Knockdown, Vec Ctrl - Vector Control, WT - Wild type