Immunostaining for mitochondria ( Tom20 , green ) mtDNA ( anti - , red ) in striatal organoids treated with DH1 or TAT at D60 . Scatterplots with bar showed of mitochondria number of mtDNA colocalized with mitochondria . data were obtained from 3 independent biological experiments . Left , n = 222 - 311 mitochondria per group ; Right , n = 14 - 20 organoids per group . were taken using Structured Illumination Microscopy ( SIM ) . scale bar represents 10 μm .

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Immunostaining for mitochondria (Tom20, green) and mtDNA (anti-DNA, red) in striatal organoids treated with DH1 or TAT at D60. Scatterplots with bar showed the length of mitochondria and the number of mtDNA colocalized with mitochondria. The data were obtained from 3 independent biological experiments. Left, n=222-311 mitochondria per group; Right, n=14-20 organoids per group. Images were taken using Structured Illumination Microscopy (SIM). The scale bar represents 10 μm.


Immunostaining for mitochondria ( Tom20 , red ) p - Drp1 S616 ( green ) in striatal treated with DH1 or TAT at D60 . Scatterplot with bar showed number of p - Drp1 S616 per in organoids . data were obtained from 3 independent biological experiments . n = 29 - 34 from at least 10 organoids . scale bar represents 5 μm .

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Immunostaining for mitochondria (Tom20, red) and p-Drp1 S616 (green) in striatal  treated with DH1 or TAT at D60. Scatterplot with bar showed the number of p-Drp1 S616 per cell in organoids. The data were obtained from 3 independent biological experiments. n=29-34 cells from at least 10 organoids. The scale bar represents 5 μm.


Scatterplot with bar showed of MSNs ( DARPP32 , green ) in striatal organoids in presence or absence of DH1 . data were obtained from 3 independent biological experiments . n = 30 organoids per group .

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Scatterplot with bar showed the ratio of MSNs (DARPP32, green) in striatal organoids in the presence or absence of DH1. The data were obtained from 3 independent biological experiments. n=30 organoids per group.


Representative tracing of DARPP32 + neurons treated with DH1 or TAT at D45 . Scatterplot with bar showed results of longest neurites . data were obtained from 3 independent biological experiments . n = 45 - 92 neurons per group .

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Representative tracing images of DARPP32+ neurons treated with DH1 or TAT at D45. Scatterplot with bar showed the results of longest neurites length. The data were obtained from 3 independent biological experiments. n=45-92 neurons per group.


Representative scatterplot with bar of fragmentation in striatal organoids treated with DH1 . arrows ( ) mark fragmented nuclei . data were obtained from 3 independent biological experiments . n = 7 - 10 organoids per group . scale bar represents 20 μm .

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Representative images and scatterplot with bar of nuclear fragmentation in striatal organoids treated with DH1. The arrows (white) mark fragmented nuclei. The data were obtained from 3 independent biological experiments. n=7-10 organoids per group. The scale bar represents 20 μm.


H . PGC1α were determined by immunoblotting . ( n = 3 biological replicates ) .

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H. PGC1α protein levels were determined by immunoblotting. (n=3 biological replicates).


I . DARPP32 were determined by immunoblotting . ( n = 3 biological replicates ) .

I. DARPP32 protein levels were determined by immunoblotting. (n=3 biological replicates).


Mouse behavior was tested after DH1 treatment . ( n = 9 - 12 mice / group ) .

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Mouse behavior was tested after DH1 treatment. (n=9-12 mice/group).


) harboring CRISPR - generated deletion alleles in y48g1c . 1 ( gg682 or gg683 ) were treated with dpy - 11 RNAi progeny , grown in absence of dpy - 11 RNAi , were scored for Dpy . of Dpy is shown . N = 3 , error bar is standard deviation , s . d .

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) Animals harboring CRISPR-generated deletion alleles in y48g1c.1 (gg682 or gg683) were treated with dpy-11 RNAi and progeny, grown in the absence of dpy-11 RNAi, were scored for Dpy. Percentage of Dpy animals is shown. N=3, error bar is standard deviation, s.d.


E ) micrograph of GFP : : ZNFX - 1 PGL - 1 : : TagRFP in harboring a CRISPR - generated deletion allele ( gg682 ) in y48g1c . 1 . Scale bar , 5μm .

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E) Fluorescence micrograph of GFP::ZNFX-1 and PGL-1::TagRFP fluorescence in animals harboring a CRISPR-generated deletion allele (gg682) in y48g1c.1. Scale bar, 5μm.


micrograph of gfp : : zsp - 1 at indicated stages using a Nikon Eclipse Ti microscope equipped with a W1 Yokogawa Spinning disc under a 63X / 1 . 4 Plan Apo oil objective . Scale bar , : 5μm , germline : 10μm . Dashed lines indicate outline of adult germline .

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Fluorescence micrograph of gfp::zsp-1 at the indicated stages using a Nikon Eclipse Ti microscope equipped with a W1 Yokogawa Spinning disc under a 63X/1.4 Plan Apo oil objective. Scale bar, embryos: 5μm, germline: 10μm. Dashed lines indicate the outline of the adult germline.


E ) Germlines dissected from expressing indicated were fixed stained with α - GFP α - HA or α - FLAG antibodies , followed by secondary antibodies conjugated with Alexa 488 CF430 , respectively . Pachytene germ - nuclei were imaged with a Leica TCS SP8 STED 3X microscope using 592nm to selectively deactivate fluorophores . Bottom panel . GFP : : ZNFX - 1 HA : : TagRFP : : ZNFX - 1 expressing were crossed germlines of trans - heterozygous progeny from this cross were fixed stained imaged by STED as described above . Scale bar , 0 . 5μm .

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E) Germlines dissected from animals expressing the indicated proteins were fixed and stained with α-GFP and α-HA or α-FLAG antibodies, followed by secondary antibodies conjugated with Alexa 488 and CF430, respectively. Pachytene germ-cell nuclei were imaged with a Leica TCS SP8 STED 3X microscope using 592nm to selectively deactivate fluorophores. Bottom panel. GFP::ZNFX-1 and HA::TagRFP::ZNFX-1 expressing animals were crossed and germlines of trans-heterozygous progeny from this cross were fixed and stained and imaged by STED as described above. Scale bar, 0.5μm.


Z - stack confocal from pachytene germ from of indicated genotypes were analysed as described in Fig . EV4B Materials Methods to determine ; of P granules ( panel B ) Granules from 9 total nuclei from 3 different ( 3 per ) were quantified for these analyses . Black lines indicate mean of data points in each group . P value , student ' s t , two .

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Z-stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described in Fig. EV4B and Materials and Methods to determine; the size of P granules (panel B) Granules from 9 total nuclei from 3 different animals (3 cells per animal) were quantified for these analyses. Black lines indicate mean of all data points in each group. P value, student's t test, two tailed.


Z - stack confocal from pachytene germ from of indicated genotypes were analysed as described to determine ; of P or Z granules ( panel C ) ; or number of P or Z granules surrounding a typical nucleus in wild - type zsp - 1 ( gg682 ) ( panel D ) . Granules from 9 total nuclei from 3 different ( 3 per ) were quantified for these analyses . Black lines indicate mean of data points in each group . P value , student ' s t , two .

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Z-stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described to determine; the size of P or Z granules (panel C); or the number of P or Z granules surrounding a typical nucleus in wild-type and zsp-1(gg682) animals (panel D). Granules from 9 total nuclei from 3 different animals (3 cells per animal) were quantified for these analyses. Black lines indicate mean of all data points in each group. P value, student's t test, two tailed.


G , H ) GFP : : ZNFX - 1 ( panel G ) or PGL - 1 : : TagRFP ( panel H ) in anterior ( A ) or posterior ( P ) section of a one . Anterior posterior regions were determined on corresponding DIC . N = 4 for G , N = 3 for H . Error bars are + / - s . d .

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G,H) GFP::ZNFX-1 (panel G) fluorescence or PGL-1::TagRFP fluorescence (panel H) in the anterior (A) or posterior (P) section of a one cell embryo. Anterior and posterior regions were determined based on corresponding DIC images. N=4 embryos for G, N=3 embryos for H. Error bars are +/- s.d.


A ) Heat map showing recovery of ZNFX - 1 after photobleaching in Z granules from pachytene stage adult germ from wild - type or zsp - 1 ( gg682 ) mutant . Scale bar , 0 . 5 μm . B ) Normalized recovery of after photobleaching of GFP : : ZNFX - 1 in wild - type or zsp - 1 ( - ) mutant over indicated time points . Bleaching event occurred at 0s . Data are represented as ± s . e . m . of n = 13 , n = 16 granules from wild - type zsp - 1 ( gg682 ) , respectively .

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A) Heat map showing recovery of ZNFX-1 fluorescence after photobleaching in Z granules from pachytene stage adult germ cells from wild-type or zsp-1(gg682) mutant animals. Scale bar, 0.5 μm. B) Normalized recovery of fluorescence after photobleaching of GFP::ZNFX-1 in wild-type or zsp-1(-) mutant animals over indicated time points. Bleaching event occurred at 0s. Data are represented as ± s.e.m. of n=13, and n=16 individual granules from wild-type and zsp-1(gg682) animals, respectively.


A ) Lineages of of indicated genotypes were grown at 25 ° C or 20 ° C for indicated generations . Each generation six were isolated in order to maintain lineage to determine numbers of progeny . Data are represented as + / - s . e . m . N = 3 for generation 8 or 9 , N = 6 for other generations .

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A) Lineages of animals of the indicated genotypes were grown at 25°C or 20°C for the indicated generations. Each generation six animals were isolated in order to maintain the lineage and to determine numbers of progeny. Data are represented as +/- s.e.m. N=3 for generation 8 or 9, N=6 for all other generations.


F ) oma - 1 ( zu405 ) is a sensitive ( ts ) lethal ( embryonic arrest at temperatures > 20 ° C ) allele of oma - 1 ( R . Lin 2003 ) . oma - 1 RNAi suppresses oma - 1 ( zu405ts ) lethality . were exposed to oma - 1 RNAi number of that hatched was scored at 20 ° C . N = 3 . Error bars are + / - s . d . harboring mutations in genes specifically required for inheritance of RNAi would respond normally to direct exposure to oma - 1 dsRNA but fail to inherit this silencing ( Wan et al . 2018 ) . zsp - 1 mutants fail to respond immediately to direct exposure to oma - 1 dsRNA , indicating that ZSP - 1 is not specific to inheritance , therefore , is generally required for RNAi ( see also Fig . EV5B - D ) .

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F) oma-1(zu405) is a temperature sensitive (ts) lethal (embryonic arrest at temperatures > 20°C) allele of oma-1 (R. Lin 2003). oma-1 RNAi suppresses oma-1(zu405ts) lethality. Animals were exposed to oma-1 RNAi and the number of embryos that hatched was scored at 20°C. N=3. Error bars are +/- s.d. Animals harboring mutations in genes specifically required for the inheritance of RNAi would respond normally to direct exposure to oma-1 dsRNA but fail to inherit this silencing (Wan et al. 2018). zsp-1 mutants fail to respond immediately to direct exposure to oma-1 dsRNA, indicating that ZSP-1 is not specific to inheritance and, therefore, is generally required for RNAi (see also Fig. EV5B-D).


Volcano plot highlighting significantly enriched ( open circles , FDR < 0 . 05 ) in eNOS - FLAG immunoprecipitates from growth factor - stimulated human endothelial ; n = 3 independent batches .

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Volcano plot highlighting proteins significantly enriched (open circles, FDR<0.05) in eNOS-FLAG immunoprecipitates from growth factor-stimulated human endothelial cells; n=3 independent cell batches.


Nitrite in supernatant of HEK293 expressing wild - type ( WT ) , Y657F ( YF ) or Y657D ( YD ) eNOS treated with solvent ( Sol ) or ionomycin ( Io , 1 µmol / L , 15 min ) ; n = 6 independent experiments ( 2 - way ANOVA Bonferroni ) .

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Nitrite in the cell supernatant of HEK293 cells expressing wild-type (WT), Y657F (YF) or Y657D (YD) eNOS and treated with solvent (Sol) or ionomycin (Io, 1 µmol/L, 15 min); n=6 independent experiments (2-way ANOVA and Bonferroni).


FMN / FAD measured in eNOS immunoprecipitates from expressing WT , YF or YD eNOS ; n = 6 independent experiments ( 1 - way ANOVA Newman - Keuls ) .

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The FMN/FAD ratio measured in eNOS immunoprecipitates from cells expressing WT, YF or YD eNOS; n=6 independent experiments (1-way ANOVA and Newman-Keuls).


Consequence of mutating Y657 on growth factor - induced formation of eNOS complexes with Hsp90 calmodulin ( CaM ) ; n = 6 - 19 independent batches ( 1 - way ANOVA Newman - Keuls ) .

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Consequence of mutating Y657 on the growth factor-induced formation of eNOS complexes with Hsp90 and calmodulin (CaM); n=6-19 independent cell batches (1-way ANOVA and Newman-Keuls).


Effect of mutating Y657 on formation of complexes between eNOS , Hsp90 PKM2 in growth factor - stimulated human endothelial . formation was assessed in eNOS - FLAG immunoprecipitates ( E ) , or PKM2 immunoprecipitates ( F ) ; n = 4 - 8 independent batches ( 1 - way ANOVA Newman - Keuls ) .

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Effect of mutating Y657 on the formation of complexes between eNOS, Hsp90 and PKM2 in growth factor-stimulated human endothelial cells. Complex formation was assessed in eNOS-FLAG immunoprecipitates (E), or PKM2 immunoprecipitates (F); n=4-8 independent cell batches (1-way ANOVA and Newman-Keuls).


Pyruvate kinase ( PK ) activity in FLAG immunoprecipitates from human endothelial expressing FLAG - tagged wild - type ( WT ) , Y657F ( YF ) or Y657D ( YD ) eNOS ; n = 6 independent batches ( 1 - way ANOVA Tukey ) .

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Pyruvate kinase (PK) activity in FLAG immunoprecipitates from human endothelial cells expressing FLAG-tagged wild-type (WT), Y657F (YF) or Y657D (YD) eNOS; n=6 independent cell batches (1-way ANOVA and Tukey).


PK activity in eNOS immunoprecipitates from HEK293 expressing WT , YF , YD or S633D ( SD ) eNOS ; n = 5 - 9 independent experiments ( 1 - way ANOVA Bonferroni ) .

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PK activity in eNOS immunoprecipitates from HEK293 cells expressing WT, YF, YD or S633D (SD) eNOS; n=5-9 independent experiments (1-way ANOVA and Bonferroni).


S - nitrosation of PKM2 in human endothelial expressing FLAG - tagged WT or YF eNOS treated with H2O2 ( 30 µmol / L ) for 15 min ; n = 8 independent batches ( Unpaired Student ' s t ) . Dithiothreitol ( DTT ) was included to demonstrate specificity of S - NO signal .

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S-nitrosation of PKM2 in human endothelial cells expressing FLAG-tagged WT or YF eNOS and treated with H2O2 (30 µmol/L) for 15 min; n=8 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) was included to demonstrate specificity of the S-NO signal.


PKM2 S - nitrosation in HEK293 expressing YF or YD eNOS either FLAG - tagged WT PKM2 or a C358S ( CS ) PKM2 mutant . SNO - FLAG - PKM2 was detected with an anti - FLAG antibody after biotin switch technique ; n = 4 independent experiments ( 2 - way ANOVA & Bonferroni ) . DTT treatment was included to demonstrate specificity of SNO signal .

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PKM2 S-nitrosation in HEK293 cells expressing YF or YD eNOS and either FLAG-tagged WT PKM2 or a C358S (CS) PKM2 mutant. SNO-FLAG-PKM2 was detected with an anti-FLAG antibody after biotin switch technique; n=4 independent experiments (2-way ANOVA & Bonferroni). DTT treatment was included to demonstrate specificity of the SNO signal.


PK activity in eNOS immunoprecipitates from HEK293 co - expressing YF or YD eNOS either WT PKM2 or CS PKM2 mutant ; n = 6 independent experiments ( 2 - way ANOVA & Bonferroni ) .

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PK activity in eNOS immunoprecipitates from HEK293 cells co-expressing YF or YD eNOS and either WT PKM2 or the CS PKM2 mutant; n=6 independent experiments (2-way ANOVA & Bonferroni).


PKM2 enzyme kinetics measured with increasing concentrations of phosphoenolpyruvate ( PEP ) in eNOS immunoprecipitates from HEK293 expressing YF or YD eNOS . data were fit with Michaelis - Menten equation to determine Vmax Km ; n = 4 independent experiments ( 2 - way ANOVA & Bonferroni ) .

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PKM2 enzyme kinetics measured with increasing concentrations of phosphoenolpyruvate (PEP) in eNOS immunoprecipitates from HEK293 cells expressing YF or YD eNOS. The data were fit with the Michaelis-Menten equation to determine Vmax and Km; n=4 independent experiments (2-way ANOVA & Bonferroni).


of pentose phosphate pathway ( PPP ) intermediates ; gluconate - 6 - P ( G6P ) , ribulose - 5 - P ( Rl5P ) , ribose - 5 - P ( R5P ) , sedoheptulose - 7 - P ( S7P ) , fructose - 6 - P ( F6P ) erythrose - 4 - P ( E4P ) in human endothelial expressing YF or YD eNOS ; n = 6 - 8 independent batches ( Unpaired Student ' s t ) .

Quantification of pentose phosphate pathway (PPP) intermediates; gluconate-6-P (G6P), ribulose-5-P (Rl5P), ribose-5-P (R5P), sedoheptulose-7-P (S7P), fructose-6-P (F6P) and erythrose-4-P (E4P) in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test).


NADPH / NADP + GSH / GSSG ratios in human endothelial expressing YF or YD eNOS ; n = 6 - 8 independent batches ( Unpaired Student ' s t ) .

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NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test).


NADPH / NADP + GSH / GSSG ratios in human endothelial treated with solvent ( Sol ) or PKM2 inhibitor shikonin ( SKN , 1 µmol / L ) for 45 minutes ; n = 4 independent batches ( Unpaired Student ' s t ) .

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NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells treated with solvent (Sol) or the PKM2 inhibitor shikonin (SKN, 1 µmol/L) for 45 minutes; n=4 independent cell batches (Unpaired Student's t test).


Systolic blood pressure ( SBP ) in wild - type ( WT ) YF - eNOS ( YF ) mice ; n = 9 per group ( Unpaired Student ' s t ) .

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Systolic blood pressure (SBP) in wild-type (WT) and YF-eNOS (YF) mice; n=9 animals per group (Unpaired Student's t test).


Phenylephrine ( PE ) - induced contraction of endothelium - intact aortic rings from WT YF mice . Experiments were performed in absence presence of L - NAME ( LN , 300 µmol / L ) ; n = 6 - 10 per group ( 2 - way ANOVA Bonferroni ) .

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Phenylephrine (PE)-induced contraction of endothelium-intact aortic rings from WT and YF mice. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=6-10 animals per group (2-way ANOVA and Bonferroni).


acetylcholine ( Ach ) - induced relaxation ( C ) of endothelium - intact aortic rings from WT YF mice . Experiments were performed in absence presence of L - NAME ( LN , 300 µmol / L ) ; n = 6 - 10 per group ( 2 - way ANOVA Bonferroni ) .

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acetylcholine (Ach)-induced relaxation (C) of endothelium-intact aortic rings from WT and YF mice. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=6-10 animals per group (2-way ANOVA and Bonferroni).


Acetylcholine ( ACh ) - induced vasodilatation of buffer - perfused hindlimb in situ . Experiments were performed in absence presence of L - NAME ( LN , 300 µmol / L ) ; n = 12 - 13 per group ( 2 - way ANOVA Bonferroni ) .

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

Acetylcholine (ACh)-induced vasodilatation of the buffer-perfused hindlimb in situ. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=12-13 animals per group (2-way ANOVA and Bonferroni).


PKM2 S - nitrosation in pulmonary endothelial from WT YF mice ; n = 5 - 6 independent batches ( Unpaired Student ' s t ) . Dithiothreitol ( DTT ) treatment was included to demonstrate specificity of SNO signal . blots show non adjacent bands cropped from same membranes .

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

PKM2 S-nitrosation in pulmonary endothelial cells from WT and YF mice; n=5-6 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) treatment was included to demonstrate specificity of the SNO signal. The blots show non adjacent bands cropped from the same membranes.


PK activity in eNOS immunoprecipitates from WT YF pulmonary endothelial ; n = 7 - 10 independent batches ( Unpaired Student ' s t ) . PK activity in eNOS immunoprecipitates from hearts from WT YF mice ; n = 6 mice per group ( Unpaired Student ' s t ) .

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 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

PK activity in eNOS immunoprecipitates from WT and YF pulmonary endothelial cells; n=7-10 independent cell batches (Unpaired Student's t test). PK activity in eNOS immunoprecipitates from hearts from WT and YF mice; n=6 mice per group (Unpaired Student's t test).


Representative of eNOS - PKM2 interaction ( PLA foci , red ) in mouse pulmonary endothelial ; Golgi apparatus endosomes were stained with Golph4 ( green ) , plasma was stained with CD144 nuclei were highlighted with DAPI ( gray ) . Only rare PLA foci were found in samples incubated with control mouse rabbit IgGs , demonstrating specificity of reaction ; n = 3 independent batches . Scale bars : 20 µm .

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 B-MEASURED_VAR 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

Representative images of eNOS-PKM2 interaction (PLA foci, red) in mouse pulmonary endothelial cells; the Golgi apparatus and endosomes were stained with Golph4 (green), the plasma membrane was stained with CD144 and nuclei were highlighted with DAPI (gray). Only rare PLA foci were found in samples incubated with control mouse and rabbit IgGs, demonstrating the specificity of the reaction; n=3 independent cell batches. Scale bars: 20 µm.


Wild - type ( WT ) YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . Phosphorylation of eNOS on Tyr656 in lung lysates from treated for 7 days , n = 6 - 7 mice per group ( Unpaired Student ' s t ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Phosphorylation of eNOS on Tyr656 in lung lysates from animals treated for 7 days, n=6-7 mice per group (Unpaired Student's t test).


Wild - type ( WT ) YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . Systolic blood pressure ( SBP ) in WT YF mice treated for 28 days , n = 10 mice per group ( Unpaired Student ' s t ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Systolic blood pressure (SBP) in WT and YF mice treated for 28 days, n=10 mice per group (Unpaired Student's t test).


Wild - type ( WT ) YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . Acetylcholine ( ACh ) - induced relaxation of aortic rings from same as in panel B , n = 8 mice per group ( 2 - way ANOVA Bonferroni ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Acetylcholine (ACh)-induced relaxation of aortic rings from the same animals as in panel B, n=8 mice per group (2-way ANOVA and Bonferroni).


Wild - type ( WT ) YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . PKM2 S - nitrosation in lungs from WT YF mice administered AII for 28 days , n = 7 - 8 mice per group ( Unpaired Student ' s t ) . DTT treatment was included to demonstrate specificity of SNO signal . blots show non adjacent lanes from same membranes .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. PKM2 S-nitrosation in lungs from WT and YF mice administered AII for 28 days, n=7-8 mice per group (Unpaired Student's t test). DTT treatment was included to demonstrate specificity of the SNO signal. The blots show non adjacent lanes from the same membranes.


Wild - type ( WT ) YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . NADPH / NADP + GSH / GSSG ratios in lungs from WT YF mice treated with AII for 28 days ; n = 8 - 9 mice per group ( Unpaired Student ' s t ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. NADPH/NADP+ and GSH/GSSG ratios in lungs from WT and YF mice treated with AII for 28 days; n=8-9 mice per group (Unpaired Student's t test).


Wild - type ( WT ) YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . 3 - Nitrotyrosine ( 3 - NT ) in lungs from WT YF mice treated with vehicle ( Veh ) or AII ( AII ) for 28 days ; n = 7 - 9 mice per group ( 2 - way ANOVA Bonferroni ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. 3-Nitrotyrosine (3-NT) levels in lungs from WT and YF mice treated with vehicle (Veh) or AII (AII) for 28 days; n=7-9 mice per group (2-way ANOVA and Bonferroni).


Wild - type ( WT ) YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . NADPH / NADP + GSH / GSSG ratios in pulmonary endothelial from WT YF mice treated with AII for 30 min ; n = 8 batches ( 2 - way ANOVA Tukey ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. NADPH/NADP+ and GSH/GSSG ratios in pulmonary endothelial cells from WT and YF mice treated with AII for 30 min; n=8 cell batches (2-way ANOVA and Tukey).


Wild - type ( WT ) YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . H2O2 in pulmonary endothelial from WT YF mice after treatment with AII ( 1 µmol / L ) for 30 min ; n = 8 batches ( 2 - way ANOVA Tukey ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. H2O2 levels in pulmonary endothelial cells from WT and YF mice after treatment with AII (1 µmol/L) for 30 min; n=8 cell batches (2-way ANOVA and Tukey).


ApoE - / - ( - / - ) ApoEYF ( YF ) mice were subjected to partial ligation of left carotid artery ( LCA ) given a cholesterol - rich diet . right , non - ligated artery ( RCA ) was used as a control . Proximity ligation assays showing S - nitrosated PKM2 ( yellow ) in RCA LCA 2 days after ligation . Phalloidin ( blue ) was included to highlight vessel wall ; n = 4 - 5 mice per group ( 2 - way ANOVA Bonferroni ) . Scale bars : 50 µm .

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

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. Proximity ligation assays showing S-nitrosated PKM2 (yellow) in the RCA and LCA 2 days after ligation. Phalloidin staining (blue) was included to highlight the vessel wall; n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


ApoE - / - ( - / - ) ApoEYF ( YF ) mice were subjected to partial ligation of left carotid artery ( LCA ) given a cholesterol - rich diet . right , non - ligated artery ( RCA ) was used as a control . GSH ( green ) in RCA LCA 2 days after ligation . CD31 ( red ) was included to label endothelial . n = 4 - 5 mice per group ( 2 - way ANOVA Bonferroni ) . Scale bars : 50 µm .

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

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. GSH (green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


ApoE - / - ( - / - ) ApoEYF ( YF ) mice were subjected to partial ligation of left carotid artery ( LCA ) given a cholesterol - rich diet . right , non - ligated artery ( RCA ) was used as a control . 3 - nitrotyrosine ( 3NT , green ) in RCA LCA 2 days after ligation . CD31 ( red ) was included to label endothelial . n = 5 mice per group ( 2 - way ANOVA Bonferroni ) . Scale bars : 50 µm .

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

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. 3-nitrotyrosine (3NT, green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


ApoE - / - ( - / - ) ApoEYF ( YF ) mice were subjected to partial ligation of left carotid artery ( LCA ) given a cholesterol - rich diet . right , non - ligated artery ( RCA ) was used as a control . Acetylcholine ( ACh ) - induced relaxation of phenylephrine contracted LCA 7 days after ligation , n = 7 - 8 mice per group ( 2 - way ANOVA Bonferroni ) . Acetylcholine ( ACh ) - induced relaxation of phenylephrine contracted aorta 7 days after ligation , n = 7 - 8 mice per group ( 2 - way ANOVA Bonferroni ) .

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

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted LCA 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni). Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted aorta 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni).


ApoE - / - mice were treated with vehicle ( Veh ) or PKM2 inhibitor shikonin ( SKN , 1 . 2 mg / kg ) , subjected to partial ligation of left carotid artery ( LCA ) given a cholesterol - rich diet . right , non - ligated artery ( RCA ) was used as a control . GSH ( green ) in RCA LCA 3 days after ligation . CD31 ( red ) was included to label endothelial . n = 5 mice per group ( 2 - way ANOVA Bonferroni ) . Scale bars : 50 µm .

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

ApoE-/- mice were treated with vehicle (Veh) or the PKM2 inhibitor shikonin (SKN, 1.2 mg/kg), subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. GSH (green) levels in the RCA and LCA 3 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


ApoE - / - mice were treated with vehicle ( Veh ) or PKM2 inhibitor shikonin ( SKN , 1 . 2 mg / kg ) , subjected to partial ligation of left carotid artery ( LCA ) given a cholesterol - rich diet . right , non - ligated artery ( RCA ) was used as a control . 3 - Nitrotyrosine ( 3NT , green ) in RCA LCA 3 days after ligation . CD31 ( red ) was included to label endothelial . n = 5 mice per group ( 2 - way ANOVA Bonferroni ) . Scale bars : 50 µm .

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

ApoE-/- mice were treated with vehicle (Veh) or the PKM2 inhibitor shikonin (SKN, 1.2 mg/kg), subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. 3-Nitrotyrosine (3NT, green) levels in the RCA and LCA 3 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


ApoE - / - ( - / - ) ApoEYF ( YF ) mice were subjected to partial ligation of left carotid artery ( LCA ) given a cholesterol - rich diet . ICAM1 VCAM1 ( green ) expression in LCA 2 days after ligation . CD31 ( red ) was included to label endothelial nuclei are highlighted with DAPI ( gray ) . Comparable were obtained in 4 additional in each group . Scale bars : 50 µm .

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

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. ICAM1 and VCAM1 (green) expression in the LCA 2 days after ligation. CD31 (red) was included to label endothelial cells and nuclei are highlighted with DAPI (gray). Comparable images were obtained in 4 additional animals in each group. Scale bars: 50 µm.


ApoE - / - ( - / - ) ApoEYF ( YF ) mice were subjected to partial ligation of left carotid artery ( LCA ) given a cholesterol - rich diet . of neutrophils monocytes infiltrated into LCA 7 days after ligation high cholesterol diet feeding ; n = 9 - 10 mice per group ( Unpaired Student ' s t ) .

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

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. Percentage of neutrophils and monocytes infiltrated into the LCA 7 days after ligation and high cholesterol diet feeding; n=9-10 mice per group (Unpaired Student's t test).


Representative of ICAM1 ( green ) VCAM1 ( red ) in human endothelial pre - treated with PKM2 inhibitor shikonin ( SKN , 1 µmol / L ) or solvent ( Sol ) for 45 minutes then stimulated with interleukin - 1β ( IL - 1 , 20 ng / ml ) or solvent ( CTL ) for 90 min . Nuclei are highlighted with DAPI ( gray ) . n = 6 independent batches ( 2 - way ANOVA Bonferroni ) . Scale bars : 20 µm .

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

Representative images and quantification of ICAM1 (green) and VCAM1 (red) staining in human endothelial cells pre-treated with the PKM2 inhibitor shikonin (SKN, 1 µmol/L) or solvent (Sol) for 45 minutes and then stimulated with interleukin-1β (IL-1, 20 ng/ml) or solvent (CTL) for 90 min. Nuclei are highlighted with DAPI (gray). n=6 independent cell batches (2-way ANOVA and Bonferroni). Scale bars: 20 µm.


of neutrophils macrophages infiltrated into LCA from ApoE - / - mice treated with Veh or SKN ( 1 . 2 mg / kg ) 7 days after ligation high cholesterol diet feeding ; n = 4 - 6 mice per group ( Unpaired Student ' s t ) .

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

Percentage of neutrophils and macrophages infiltrated into the LCA from ApoE-/- mice treated with Veh or SKN (1.2 mg/kg) 7 days after ligation and high cholesterol diet feeding; n=4-6 mice per group (Unpaired Student's t test).


Plaque burden in ligated carotid arteries from ApoE - / - ( - / - ) ApoEYF ( YF ) mice fed a high cholesterol diet for 21 days ; scale bars : 2 mm . Intimal medial thickness was determined at four specific locations ( proximal , middle 1 , middle 2 , distal ) in cryo - sectioned arteries stained with Oil Red O ; scale bars : 200 µm . n = 7 mice per group ( Unpaired Student ' s t ) .

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

Plaque burden in ligated carotid arteries from ApoE-/- (-/-) and ApoEYF (YF) mice fed a high cholesterol diet for 21 days; scale bars: 2 mm. Intimal and medial thickness was determined at four specific locations (proximal, middle 1, middle 2, distal) in cryo-sectioned arteries stained with Oil Red O; scale bars: 200 µm. n=7 mice per group (Unpaired Student's t test).


Oil Red O of plaques in aortae from mice fed a Western diet for four ( B ) or six months ( C ) ; scale bars : 3 mm . n = 6 ( B ) or n = 12 ( C ) mice per group ( Unpaired Student ' s t ) .

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

Oil Red O staining of plaques in aortae from mice fed a Western diet for four (B) or six months (C); scale bars: 3 mm. n=6 (B) or n=12 (C) mice per group (Unpaired Student's t test).


B . Instability of BU - 1 expression in timeless . FACS plots of wild type timeless ( clone 1 ) DT40 stained with anti - Bu - 1 conjugated with phycoerythrin . Each represents Bu - 1 expression profile of an clonal population . Unstained controls are shown in blue .

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

B. Instability of BU-1 expression in timeless cells. FACS plots of wild type and timeless (clone 1) DT40 cells stained with anti-Bu-1 conjugated with phycoerythrin. Each line represents the Bu-1 expression profile of an individual clonal population. Unstained controls are shown in blue.


C . Fluctuation for Bu - 1 loss in wild - type DT40 two independent timeless clones generated by CRISPR - Cas9 targeting ( clones 1 2 ; , timeless ( clone 1 ) complemented by expression of human Timeless cDNA a timeless mutant on a background in which endogenous + 3 . 5 G4 has been deleted ( ΔG4 )

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

C. Fluctuation analysis for Bu-1 loss in wild-type DT40 cells and two independent timeless clones generated by CRISPR-Cas9 targeting (clones 1 and 2; , timeless (clone 1) complemented by expression of human Timeless cDNA and a timeless mutant on a background in which the endogenous +3.5 G4 has been deleted (ΔG4)


D . Fluctuation for Bu - 1 loss in DT40 wild - type tipin .

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

D. Fluctuation analysis for Bu-1 loss in DT40 wild-type and tipin cells.


D . - binding affinity of DBD was measured by anisotropy , titrating DBD against Cy3 3 ' - labelled ssDNA , dsDNA G4 top panel shows binding curves for ss - dsDNA , bottom panel shows binding curve for G4 substrate .

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

D. The DNA-binding affinity of DBD was measured by fluorescence anisotropy, titrating the DBD protein against Cy3 3'-labelled ssDNA, dsDNA and G4 DNA The top panel shows binding curves for ss- and dsDNA, the bottom panel shows the binding curve for the G4 DNA substrate.


anisotropy was used to measure binding affinity of Timeless - Tipin for indicated sequences . A . ssG4 : G4 flanked by single - stranded ; ssHP : hairpin flanked by single - stranded ; ss : single - stranded

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Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences. A. ssG4: G4 flanked by single-stranded DNA; ssHP: hairpin flanked by single-stranded DNA; ss: single-stranded DNA


anisotropy was used to measure binding affinity of Timeless - Tipin for indicated sequences . B . Binding affinity of Timeless - Tipin for a range of G4 sequences Single - stranded ( ss20 : 5 ′ - 6FAM - ATAAGAGTGGTTAGAGTGTA ) double - stranded ( ds20 : ss20 annealed to complementary ) were also tested as controls .

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Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences. B. Binding affinity of Timeless-Tipin for a range of G4 DNA sequences Single-stranded (ss20: 5′-6FAM-ATAAGAGTGGTTAGAGTGTA) and double-stranded (ds20: ss20 annealed to complementary sequence) DNA were also tested as controls.


Fluctuation for generation of Bu - 1 loss variants . Top to bottom : wild type , timeless ( clone 1 ) , a timeless mutant ( timeless ∆ C ) generated by CRISPR - Cas9 targeting exon 16 which truncates removing CTD containing both DBD PARP binding domains . Then , complementation of timeless # 1 with human Timeless ( hTim ) , hTim ∆ 816 - 1208 ( lacking both DBD PBD ) , hTim ∆ 816 - 965 ( lacking DBD ) hTim [ 1 : 1000 ] , lacking PBD . At least two independent fluctuation analyses were performed with 24 - 36 clones each per repeat .

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Fluctuation analysis for the generation of Bu-1 loss variants. Top to bottom: wild type, timeless (clone 1), a timeless mutant (timeless ∆C) generated by CRISPR-Cas9 targeting exon 16 which truncates the protein removing the CTD containing both the DBD and the PARP binding domains. Then, complementation of timeless#1 with human Timeless (hTim), hTim∆816-1208 (lacking both the DBD and PBD), hTim∆816-965 (lacking the DBD) and hTim[1:1000], lacking the PBD. At least two independent fluctuation analyses were performed with 24-36 individual clones each cell line per repeat.


A . Enhanced recruitment of DDX11 to associated PCNA following exposure to 4 µM PDS for 24 hours . PCNA was precipitated from cross - linked immunoprecipitate blotted for FLAG - DDX11 .

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A. Enhanced recruitment of DDX11 to chromatin associated PCNA following exposure to 4 µM PDS for 24 hours. PCNA was precipitated from cross-linked chromatin and the immunoprecipitate blotted for FLAG-DDX11.


B . Fluctuation for Bu - 1a loss in wild - type DT40 , two independent ddx11 clones generated by CRISPR - Cas9 targeting ( clones 1 2 ) one ddx11 clone generated by conventional homologous recombination gene targeting ( clone 3 ) . Each symbol represents of in an clone expanded for 2 - 3 that have lost Bu - 1ahigh expression .

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B. Fluctuation analysis for Bu-1a loss in wild-type DT40 cells, two independent ddx11 clones generated by CRISPR-Cas9 targeting (clones 1 and 2) and one ddx11 clone generated by conventional homologous recombination gene targeting (clone 3). Each symbol represents the percentage of cells in an individual clone expanded for 2-3 weeks that have lost Bu-1ahigh expression.


C . Fluctuation for Bu - 1a loss variant generation in wild - type , ddx11 ( clone 1 ) , ddx11 ( clone 1 ) complemented by expression of chicken DDX11 WT cDNA , ddx11 ( clone 1 ) complemented by expression of helicase - dead form of chicken DDX11 ( K87A ) cDNA , a ddx11 clone generated in in which endogenous + 3 . 5 G4 has been deleted ( ΔG4 ) .

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C. Fluctuation analysis for Bu-1a loss variant generation in wild-type cells, ddx11 (clone 1) cells, ddx11 (clone 1) complemented by expression of chicken DDX11 WT cDNA, ddx11 (clone 1) complemented by expression of helicase-dead form of chicken DDX11 (K87A) cDNA, and a ddx11 clone generated in cells in which the endogenous +3.5 G4 has been deleted (ΔG4).


D . Fluctuation for Bu - 1 loss in two independent timeless / ddx11 double - mutant clones ( # 1 # 2 ) , ddx11 expressing DDX11KAK fancj fancj / ddx11 double - mutants . Fluctuation analyses for wild type , timeless # 1 ddx11 # 1 are shown for comparison .

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D. Fluctuation analysis for Bu-1 loss in two independent timeless/ddx11 double-mutant clones (#1 and #2), ddx11 expressing DDX11KAK and fancj and fancj/ddx11 double-mutants. Fluctuation analyses for wild type, timeless #1 and ddx11 #1 are shown for comparison.


A . Growth curves for DT40 wild type , ddx11 , timeless ddx11 / timeless , with without 4 μM pyridostatin ( PDS ) . were seeded at 5 x104 / ml on day 0 viable were counted each 24 h for 4 days . Bars represent SD of two independent experiments performed in duplicate . Doubling times ( DMSO ) : WT 13 hours , timeless 18 hours , ddx11 16 hours , ddx11 / timeless 24 hours . Doubling times ( PDS ) : WT 13 . 6 hours , timeless 27 hours , ddx11 25 . 7 hours , ddx11 / timeless 47 . 5 hours .

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A. Growth curves for DT40 wild type, ddx11, timeless and ddx11/timeless cells, with and without 4 μM pyridostatin (PDS). Cells were seeded at 5 x104 cells/ml on day 0 and the viable cells were counted each 24 h for 4 days. Bars represent SD of two independent experiments performed in duplicate. Doubling times (DMSO): WT 13 hours, timeless 18 hours, ddx11 16 hours, ddx11/timeless 24 hours. Doubling times (PDS): WT 13.6 hours, timeless 27 hours, ddx11 25.7 hours, ddx11/timeless 47.5 hours.


B . DDR signalling detected by phosphorylation of histone H2AX ( γ - H2AX ) by flow cytometry in untreated or exposed to 4 μM PDS for 3 days . Pale histogram , untreated ; histogram , treated ; black dotted , positive control treated with 0 . 1 µM cisplatin , also for 3 days . C . of γ - H2AX in DT40 wild type , ddx11 , timeless ddx11 / timeless treated with 4 μM PDS for 3 days . central band represents median , box 25th - 75th centile whiskers minimum to maximum range of three independent experiments performed in duplicate .

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B. DDR signalling detected by phosphorylation of histone H2AX (γ-H2AX) by flow cytometry in untreated cells or cells exposed to 4 μM PDS for 3 days. Pale histogram, untreated; dark histogram, treated; black dotted line, positive control cells treated with 0.1 µM cisplatin, also for 3 days. C. Quantification of γ-H2AX in DT40 wild type, ddx11, timeless and ddx11/timeless cells treated with 4 μM PDS for 3 days. The central band represents the median, the box the 25th - 75th centile and whiskers the minimum to maximum range of three independent experiments performed in duplicate.


A . Correlation of magnitude direction of change of genes dysregulated ( relative to wild type ) in timeless vs . ddx11 DT40 . rs ( Spearman rho ) is shown for each correlation .

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A. Correlation of magnitude and direction of change of genes dysregulated (relative to wild type) in timeless vs. ddx11 DT40 cells. rs (Spearman rho) is shown for each correlation.


B . Genes dysregulated in timeless , ddx11 fancj mutants have a higher density of G4s around their TSS . Cumulative fraction of genes dysregulated in timeless ( red ) , ddx11 ( blue ) fancj ( green ) containing n ( x - axis ) G4 motifs within 1 . 5kb of TSS compared with genes ( black ) .

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B. Genes dysregulated in timeless, ddx11 and fancj mutants have a higher density of G4s around their TSS. Cumulative fraction of the genes dysregulated in timeless (red), ddx11 (blue) and fancj (green) containing n (x-axis) G4 motifs within 1.5kb of the TSS compared with all genes (black).


C . Metagene showing G4 frequency around TSS of genes dysregulated ( up or down ) in timeless ( left panel ) , ddx11 ( centre panel ) fancj ( right panel ) compared with genes ( black ) . G4 frequency is calculated separately for coding ( above x - axis ) template strands ( below x - axis ) .

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C. Metagene analysis showing G4 frequency around the TSS of genes dysregulated (up or down) in timeless (left panel), ddx11 (centre panel) and fancj (right panel) compared with all genes (black line). G4 frequency is calculated separately for coding (above the x-axis) and template strands (below the x-axis).


B Confocal of HeLa transiently expressing GFP - CBPs or GFP . dotted lines show each shape . Scale bars , 10 µm .

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B Confocal images of HeLa cells transiently expressing GFP-CBPs or GFP. The dotted lines show each cell shape. Scale bars, 10 µm.


C Confocal of HeLa transiently expressing GFP - CbpCT4 mCherry - LC3 ( upper ) . intensities of GFP - CbpCT4 ( green ) mCherry - LC3 ( red ) along arrow are shown in graph at bottom .

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C Confocal images of HeLa cells transiently expressing GFP-CbpCT4 and mCherry-LC3 (upper). The fluorescence intensities of GFP-CbpCT4 (green) and mCherry-LC3 (red) along the arrow are shown in the graph at the bottom.


D Lysates from 293T transiently expressing GFP - Cbps or GFP were subjected to SDS - PAGE analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

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D Lysates from 293T cells transiently expressing GFP-Cbps or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


E Lysates from 293T transiently expressing GFP - CbpCT4 , GFP - LytR , or GFP in presence or absence of chloroquine were subjected to SDS - PAGE analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

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E Lysates from 293T cells transiently expressing GFP-CbpCT4, GFP-LytR, or GFP in the presence or absence of chloroquine were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


F MEFs were infected with S . pneumoniae R6 WT or ΔcbpF for indicated periods , intracellular survival of expressed as number of CFUs .

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F MEFs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated periods, and the intracellular survival of bacteria expressed as the number of CFUs.


G p62 - KO MEF infected with S . pneumoniae R6 ΔcbpF / pCbpFR6 - FLAG for 2 h were fixed stained with DAPI an anti - FLAG antibody , representative epifluorescence are shown . Scale bars , 10 µm .

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G p62-KO MEF cells infected with S. pneumoniae R6 ΔcbpF/pCbpFR6-FLAG for 2 h were fixed and stained with DAPI and an anti-FLAG antibody, and representative epifluorescence images are shown. Scale bars, 10 µm.


A of band intensities following streptavidin - pulldown assays using in vitro - translated biotinylated CbpCT4 FLAG - tagged autophagy - related shown in Fig EV2A .

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A Quantification of the band intensities following streptavidin-based pulldown assays using the in vitro-translated biotinylated CbpCT4 and FLAG-tagged autophagy-related proteins shown in Fig EV2A.


B of band intensities following GST - pulldown assays with 293T lysates expressing autophagy - related recombinant GST - CbpFR6 shown in Fig EV2D .

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B Quantification of the band intensities following GST-pulldown assays with 293T cell lysates expressing the autophagy-related proteins and recombinant GST-CbpFR6 shown in Fig EV2D.


C Lysates from 293T transiently expressing 3Myc - Atg14 or Atg5 - 3Myc , GFP - CbpCT4 or GFP were immunoprecipitated with GFP - Trap , bound were analyzed by immunoblotting .

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C Lysates from 293T cells transiently expressing 3Myc-Atg14 or Atg5-3Myc, and GFP-CbpCT4 or GFP were immunoprecipitated with GFP-Trap, and the bound proteins were analyzed by immunoblotting.


D Lysates from 293T transiently expressing GFP - CbpCT4 or GFP , 6Myc - Atg14 were immunoprecipitated with an anti - Myc antibody G PLUS - Agarose . bound were analyzed by immunoblotting .

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D Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP, and 6Myc-Atg14 were immunoprecipitated with an anti-Myc antibody and Protein G PLUS-Agarose. The bound proteins were analyzed by immunoblotting.


E Recombinant GST - CbpFR6 or GST , 3Myc - Atg14 were used for GST - pulldown assays . bound were analyzed by immunoblotting using an anti - Myc antibody .

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E Recombinant GST-CbpFR6 or GST, and 3Myc-Atg14 were used for GST-pulldown assays. The bound proteins were analyzed by immunoblotting using an anti-Myc antibody.


F MEFs infected with R6 ΔcbpF expressing CbpFR6 - FLAG for 2 h were subjected to IP assayed using anti - HA agarose beads . bound were analyzed by immunoblotting using an anti - FLAG antibody .

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F MEFs infected with R6 ΔcbpF expressing CbpFR6-FLAG for 2 h were subjected to IP and assayed using anti-HA agarose beads. The bound proteins were analyzed by immunoblotting using an anti-FLAG antibody.


A Lysates from MEFs stably expressing HA - Atg14 infected with S . pneumoniae TIGR4 WT or ΔcbpC for 1 , 2 , or 3 h in presence or absence of cycloheximide were subjected to SDS - PAGE analyzed by immunoblotting with indicated antibodies .

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A Lysates from MEFs stably expressing HA-Atg14 infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of cycloheximide were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.


B MEFs / HA - Atg14 were infected with S . pneumoniae TIGR4 WT or ΔcbpC for 1 , 2 , or 3 h in presence or absence of Bafilomycin A1 ( BafA1 ) . lysates were subjected to SDS - PAGE analyzed by immunoblotting with indicated antibodies .

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B MEFs/HA-Atg14 cells were infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of Bafilomycin A1 (BafA1). The lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.


( C A549 were infected with S . pneumoniae R6 WT or ΔcbpC for 2 h fixed stained with DAPI an anti - Atg14 Representative epifluorescence are shown . Scale bars , 10 µm . percentages of perinuclear - localizing Atg14 containing were quantified .

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(C A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and an anti-Atg14 Representative epifluorescence images are shown. Scale bars, 10 µm. The percentages of perinuclear-localizing Atg14 containing cells were quantified.


A549 were infected with S . pneumoniae R6 WT or ΔcbpC for 2 h fixed stained with DAPI - GM130 antibody . Representative epifluorescence are shown . Scale bars , 10 µm . percentages of intact Golgi apparatus - containing were quantified .

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A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and -GM130 antibody. Representative epifluorescence images are shown. Scale bars, 10 µm. The percentages of intact Golgi apparatus-containing cells were quantified.


G S . pneumoniae ( Sp ) invasion - permissive A549 ( marked with GFP ) Sp invasion - non - permissive ( pIgR knocked down ) A549 were co - cultured at a 1 : 3 . Atg14 - disappearance experiments were conducted , representative epifluorescence are shown . Scale bars , 10 µm . lines show each shape arrows show invasion - permissive A549 marked with GFP . H percentages of perinuclear - localizing Atg14 containing in ( G ) were quantified .

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G S. pneumoniae (Sp) invasion-permissive A549 cells (marked with GFP) and Sp invasion-non-permissive (pIgR knocked down) A549 cells were co-cultured at a 1:3 ratio. Atg14-disappearance experiments were conducted, and representative epifluorescence images are shown. Scale bars, 10 µm. The lines show each cell shape and the arrows show invasion-permissive A549 cells marked with GFP. H The percentages of perinuclear-localizing Atg14 containing cells in (G) were quantified.


I Lysates from 293T transiently expressing GFP - CbpC or GFP , HA - Atg14 were subjected to SDS - PAGE analyzed by immunoblotting using antibodies against HA or GFP .

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I Lysates from 293T cells transiently expressing GFP-CbpC or GFP, and HA-Atg14 were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against HA or GFP.


J Lysates from 293T transiently expressing GFP - CbpCT4 or GFP were immunoprecipitated using GST - GFP - Nanobody . Additionally , beads were mixed with lysates from 293T transiently expressing p62 - 3Myc HA - Atg14 , bound were analyzed by immunoblotting .

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J Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP were immunoprecipitated using GST-GFP-Nanobody. Additionally, beads were mixed with lysates from 293T cells transiently expressing p62-3Myc and HA-Atg14, and bound proteins were analyzed by immunoblotting.


K A549 treated with indicated siRNAs were infected with S . pneumoniae R6 WT or ΔcbpF for indicated durations . were fixed stained with DAPI an anti - Atg14 antibody , percentages of perinuclear - localizing Atg14 containing were quantified .

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K A549 cells treated with the indicated siRNAs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated durations. The cells were fixed and stained with DAPI and an anti-Atg14 antibody, and percentages of perinuclear-localizing Atg14 containing cells were quantified.


L Lysates from MEFs stably expressing GFP - CbpCT4 or GFP in presence or absence of rapamycin or chloroquine were subjected to SDS - PAGE immunoblotted using antibodies against LC3 or p62 .

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L Lysates from MEFs stably expressing GFP-CbpCT4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS-PAGE and immunoblotted using antibodies against LC3 or p62.


M of NanoBRET signals in 293A transiently expressing HaloTag - Stx17 Nanoluc - Atg14 in presence or absence of GFP - CbpC or GFP .

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M Quantification of NanoBRET signals in 293A cells transiently expressing HaloTag-Stx17 and Nanoluc-Atg14 in the presence or absence of GFP-CbpC or GFP.


B GST - pulldown assays performed using GST - CbpFR6 or GST lysates from 293T expressing GFP - Atg14 derivatives or GFP . bound were analyzed by immunoblotting using an anti - GFP antibody .

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B GST-pulldown assays performed using GST-CbpFR6 or GST and lysates from 293T cells expressing GFP-Atg14 derivatives or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody.


D , E GST - pulldown assays using GST - CbpFR6 derivatives lysates from 293T expressing GFP - Atg14 CCD or GFP . bound were analyzed by immunoblotting using an anti - GFP antibody . Each GST - CbpFR6 derivative - bound bead was confirmed by Coomassie brilliant blue ( CBB ) .

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D, E GST-pulldown assays using GST-CbpFR6 derivatives and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. Each GST-CbpFR6 derivative-bound bead was confirmed by Coomassie brilliant blue (CBB) staining.


H GST - pulldown assays performed using GST - CbpFR6 mutants or GST lysates from 293T expressing GFP - Atg14 CCD or GFP . bound were analyzed by immunoblotting using an anti - GFP antibody . Each GST - CbpFR6 mutant - bound bead was confirmed by CBB .

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H GST-pulldown assays performed using GST-CbpFR6 mutants or GST and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. Each GST-CbpFR6 mutant-bound bead was confirmed by CBB staining.


I Lysates of 293T transiently expressing GFP - CbpCT4 mutants or GFP were subjected to SDS - PAGE analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

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I Lysates of 293T cells transiently expressing GFP-CbpCT4 mutants or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


J Confocal microscopy of HeLa transiently expressing indicated GFP - CbpCT4 mutants . Scale bars , 10 µm .

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J Confocal microscopy images of HeLa cells transiently expressing the indicated GFP-CbpCT4 mutants. Scale bars, 10 µm.


B Lysates from 293T transiently expressing p62 - 3Myc GFP - CbpCT4 variants were immunoprecipitated using a GST - GFP - Nanobody fusion . Bound were analyzed by immunoblotting .

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B Lysates from 293T cells transiently expressing p62-3Myc and GFP-CbpCT4 variants were immunoprecipitated using a GST-GFP-Nanobody fusion protein. Bound proteins were analyzed by immunoblotting.


E Lysates from 293T transiently expressing p62 - 3Myc GFP - CbpCT4 variants were immunoprecipitated using GST - GFP - Nanobody . Bound were analyzed by immunoblotting .

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E Lysates from 293T cells transiently expressing p62-3Myc and GFP-CbpCT4 variants were immunoprecipitated using the GST-GFP-Nanobody protein. Bound proteins were analyzed by immunoblotting.


G Lysates of 293T transiently expressing GFP - CbpCT4 p62 - 3Myc variants were immunoprecipitated with GST - GFP - Nanobody . Bound were analyzed by immunoblotting using indicated antibodies .

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G Lysates of 293T cells transiently expressing GFP-CbpCT4 and p62-3Myc variants were immunoprecipitated with the GST-GFP-Nanobody protein. Bound proteins were analyzed by immunoblotting using the indicated antibodies.


H A549 infected with indicated S . pneumoniae strains for 2 h were fixed stained with DAPI an anti - Atg14 antibody , percentages of perinuclear - localizing Atg14 containing were quantified .

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H A549 cells infected with the indicated S. pneumoniae strains for 2 h were fixed and stained with DAPI and an anti-Atg14 antibody, and the percentages of perinuclear-localizing Atg14 containing cells were quantified.


I Representative epifluorescence in ( H ) are shown . Scale bars , 10 µm .

I Representative epifluorescence images in (H) are shown. Scale bars, 10 µm.


D GST - pulldown assays using indicated GST - fusion or GST lysates from 293T expressing GFP - Atg14 CCD or GFP . Bound were analyzed by immunoblotting using an anti - GFP antibody .

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D GST-pulldown assays using the indicated GST-fusion proteins or GST and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. Bound proteins were analyzed by immunoblotting using an anti-GFP antibody.


E Lysates from 293T expressing GFP - fusion or GFP were subjected to SDS - PAGE analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

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E Lysates from 293T cells expressing GFP-fusion proteins or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


F Confocal of HeLa transiently expressing GFP - fusion . Scale bars , 10 µm .

F Confocal images of HeLa cells transiently expressing GFP-fusion proteins. Scale bars, 10 µm.


H , I GST - pulldown assays using indicated GST - fused or GST lysates from 293T expressing GFP - Atg14 CCD or GFP . Bound were analyzed by immunoblotting using an anti - GFP antibody .

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H, I GST-pulldown assays using the indicated GST-fused proteins or GST and lysates from 293T expressing GFP-Atg14 CCD or GFP. Bound proteins were analyzed by immunoblotting using an anti-GFP antibody.


Partial embryonic lethality after E11 . 5 . Chi - square , * * * * p < 0 . 0001 . E11 . 5 n = 114 , E14 . 5 n = 70 , 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 . IsolectinB4 ( IsoB4 ; ) , ERG ( red ) 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 normalized to wildtype ( WT ) mean ; one dot is two fields / mouse ; error bars : SD ; unpaired T , * 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 , yellow in B ) . One dot is two fields / mouse ; error bars : SD ; unpaired T , * p < 0 . 05 . n = 7 - 12 .

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


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

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


P6 retina vessel . IsolectinB4 ( IsoB4 ) in black ; outer 20 % of vascularized 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 P6 developing retina . G . Radial outgrowth ( between optic nerve vascular front ) .

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


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

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


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

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


. P , postnatal day ; w , . 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 normalized to WT mean of 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 .

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


P4 - P7 retina vessel . IsolectinB4 ( IsoB4 ) in black ; outer 20 % of vascularized in pink . Scale bar , 250 µm . P6 insets ; sprout . 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 . between optic nerve vascular front . P , postnatal day ; error bars : SD ; 2 - way ANOVA p < 0 . 0001 ; Bonferroni ' s multiple comparison , * 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 . P , postnatal day ; error bars : SD ; 2 - way ANOVA p = 0 . 0013 . P4 n = 7 - 9 retinas , one retina / mouse , P5 n = 4 - 9 , P6 n = 9 - 9 , P7 n = 4 - 5 .

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


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

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


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

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


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

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


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

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


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

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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 , * * p < 0 . 01 . P4 n = 7 - 9 , P5 n = 6 - 12 , P6 n = 3 - 5 , P7 n = 4 .

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


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


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

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


Summary plot of 51 enriched in immobilized pY1212 peptide precipitate . 10 ( red oval ) were selected for further . PSM : peptide - spectrum matches . : average of spectra for three most abundant peptides in arbitrary units .

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 PI3Kp85 . HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 PI3Kp85 .

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Blot for peptide-enriched GRB2 and PI3Kp85. HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 and PI3Kp85.


VEGFR2 / GRB2 VEGFR2 / PI3Kp85 complexes in isolated ( i ) ECs . E . PLA for VEGFR2 / GRB2 VEGFR2 / PI3Kp85 complexes ( black ) in iECs from FVB wildtype ( WT ) Vegfr2Y1212F / Y1212F lungs . Hoechst 33342 show nuclei ( grey ) . Scale bar , 20 µm . F , G . Fold - increase over PBS - treated sample ; each dot represents mean of 6 fields . 2 - way ANOVA p = 0 . 0206 ( F ) , p = 0 . 0368 ( G ) ; Sidak ' s multiple comparison , * p < 0 . 05 . n = 3 experiments .

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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 phosphoAkt immunostaining ( green ) of iECs from FVB wildtype ( WT ) Vegfr2Y1212F / Y1212F lungs . Hoechst 33342 ( blue ) shows nuclei . Arrows indicate accumulation of phosphoERK1 / 2 phosphoAkt , respectively . Scale bar , 20 µm .

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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.


accumulation expressed as phoshoERK1 / 2 - positive normalized to total phosphoERK1 / 2 by field . Each dot represents 1 field . Min , minutes of stimulation . 2 - way ANOVA p = 0 . 0248 ; Sidak ' s multiple comparison , * p < 0 . 05 , * * p < 0 . 01 . n = 3 experiments .

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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.


accumulation expressed as fold - increase of phosphoAkt in Vegfr2Y1212F / Y1212F over PBS - treated wildtype ( WT ) iECs , normalized to number of nuclei . Each dot represents 1 field . Min , minutes of stimulation . Unpaired T , * p < 0 . 05 . n = 3 experiments .

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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 total Akt on lung lysates from wiltype ( WT ) Vegfr2Y1212F / Y1212F FVB mice tail - vein injected with PBS or VEGFA followed by circulation for time points indicated . Each lane represents one mouse . 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 , * p < 0 . 05 . n = 3 experiments . 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 , * p < 0 . 05 . n = 3 experiments .

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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 ) Vegfr2Y1212F / Y1212F FVB mice tail - vein injected with PBS or VEGFA followed by circulation for time points indicated . Each lane represents one mouse . of Myc / Actin expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0058 ; unpaired T , * p < 0 . 05 , * * p < 0 . 01 . n = 3 experiments .

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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 expression . C . Myc Actin immunoblotting on lysates of lung isolated ECs from untreated wildtype ( WT ) mutant ( VEGFR2 Y1212F ) C57Bl / 6 FVB mice . D . of Myc / Actin expressed as Arbitrary unit . Each lane ( C ) each dot ( D ) represent one mouse . 2 - way ANOVA p < 0 . 0001 ; unpaired T , * * p < 0 . 01 . n = 5 - 7 .

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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 ) mutant ( VEGFR2 Y1212F ) C57Bl / 6 FVB mice . Each dot represents one mouse . 2 - way ANOVA p = 0 . 0041 ; Sidak ' s multiple comparison , * p < 0 . 05 . n = 4 - 5 .

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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 FVB iECs . CD31 ( ) , ERG ( red ) , EdU ( green ) double - positive ( yellow ) in C57Bl / 6 ( F ) FVB ( G ) iECs . EdU signal was masked with ERG signal .

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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 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 , * p < 0 . 05 ; * * p < 0 . 01 . C57Bl / 6 n = 3 - 6 mice , FVB n = 3 - 5mice .

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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 of E11 . 5 explants from C57Bl / 6 taken from wildtype ( WT ) Vegfr2Y1212F / Y1212F with ( MycOE / Cre + ) without ( MycOE / Cre - ) endothelial - specific overexpression of Myc . Endothelial marker ( CD31 ) immunostaining highlights vasculature in . Embryonic tissue blacked out to highlight sprouting .

O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR 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 . as fold - increase of vessel sprouting compared to WT MycOE / Cre - . Scale bar , 250 µm . Error bars : SD ; unpaired T , * * p < 0 . 01 , * * * * p < 0 . 0001 . n = 4 - 11 explants , one explant / .

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

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.