Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( E ) Nrg1 - tg mice treated with spironolactone spent more time in compartment during - ( interaction gene × treatment F ( 1 , 41 ) = 4 . 90 ; p = 0 . 0324 ; 2 - way ANOVA , p = 0 . 0219 , Bonferroni post - ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(E) Nrg1-tg mice treated with spironolactone spent more time in the light compartment during the light-dark test (interaction gene × treatment F (1,41) =4.90; p=0.0324; 2-way ANOVA, and p=0.0219, Bonferroni post-test).


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( F ) In Y - maze , transgenic mice performed less alterations ( effect of genotype F ( 1 , 44 ) = 11 . 50 ; p = 0 . 0015 ; 2 - way ANOVA ) . Bonferroni confirmed this phenotype in vehicle - treated groups ( p = 0 . 0011 ) , but not in spironolactone - treated ( p = 0 . 5950 ) . Spironolactone treatment had a significant effect on number of alterations ( F ( 1 , 44 ) = 4 . 12 ; p = 0 . 0484 ; 2 - way ANOVA ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice. (F) In the Y-maze test, transgenic mice performed less alterations (effect of genotype F (1,44) =11.50; p=0.0015; 2-way ANOVA). The Bonferroni test confirmed this phenotype in vehicle-treated groups (p=0.0011), but not in spironolactone-treated animals (p=0.5950). Spironolactone treatment had a significant effect on the number of alterations (F (1,44) =4.12; p=0.0484; 2-way ANOVA).


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( G ) Spironolactone treatment significantly enhanced PPI in Nrg1 - tg mice ( effect of treatment F ( 1 , 21 ) = 5 . 07 ; p = 0 . 0325 ; 2 - way repeated measures ANOVA ) . Data are shown as mean , error bars represent . * , p < 0 . 05 , * * , p < 0 . 01 * * * , p < 0 . 001 , Bonferroni following 2 - way ANOVA ; Spiro , spironolactone ; Veh , vehicle . n = 12 per genotype treatment with an exception of ( E ) ( Nrg1 - tg vehicle , n = 11 ; Nrg1 - tg Spiro , n = 10 ; wt vehicle , n = 12 ; wt Spiro , n = 12 ) ( G ) ( Nrg1 - tg vehicle , n = 11 ; Nrg1 - tg Spiro , n = 12 ; wt vehicle , n = 12 ; wt Spiro , n = 12 ) .

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

Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(G) Spironolactone treatment significantly enhanced PPI in Nrg1-tg mice (effect of treatment F (1,21) =5.07; p=0.0325; 2-way repeated measures ANOVA). Data are shown as mean, error bars represent SEM. *, p<0.05, **, p<0.01 and ***, p<0.001, Bonferroni test following 2-way ANOVA; Spiro, spironolactone; Veh, vehicle. n=12 per genotype and treatment with an exception of (E) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=10; wt vehicle, n=12; wt Spiro, n=12) and (G) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=12; wt vehicle, n=12; wt Spiro, n=12).


Autophagy is activated in dying nurse during late oogenesis in D . melanogaster . ( A - C ) Confocal of egg chambers of flies expressing UASp - GFP - mCherry - DrAtg8a transgene exclusively in germline ( genotype : UASp - GFP - mCherry - DrAtg8a / + ; nanos - VP - 16 Gal4 / + ) . Expression of UASp - GFP - mCherry - DrAtg8a exhibits punctate pattern ( arrows ) . Note that during developmental stages early 12 ( A ) late 12 ( B ) , punctate dots are yellow ( merge ) . In late stage 13 ( C ) , large red dots are evident , indicating presence of acidic compartments ( autolysosomes ; arrows ) . arrowheads in A point to physical connection between oocyte nurse during early stage 12 ( not observed during late stage 12 ) .

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Autophagy is activated in dying nurse cells during late oogenesis in D. melanogaster. (A-C) Confocal micrographs of egg chambers of flies expressing the UASp-GFP-mCherry-DrAtg8a transgene exclusively in the germline (genotype: UASp-GFP-mCherry-DrAtg8a/+; nanos-VP-16 Gal4/+). Expression of UASp-GFP-mCherry-DrAtg8a exhibits punctate staining pattern (arrows). Note that during developmental stages early 12 (A) and late 12 (B), the punctate dots are yellow (merge). In late stage 13 (C), large red dots are evident, indicating the presence of acidic compartments (autolysosomes; arrows). The arrowheads in A point to the physical connection between the oocyte and the nurse cells during early stage 12 (not observed during late stage 12).


( D E ) Transmission electron of nurse in stage 12 ( D ) in stage 13 ( E ) egg chambers . Autophagosomes ( inset in D ) autolysosomes ( arrow in E ) are evident in remaining nurse cytoplasm ( arrow in D indicates actin bundles ) . Note that autolysosome shown in E contains condensed material resembling material of condensed fragmented nurse nucleus ( NCN ) , providing evidence for

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

(D and E) Transmission electron micrographs of nurse cells in stage 12 (D) and in stage 13 (E) egg chambers. Autophagosomes (inset in D) and autolysosomes (arrow in E) are evident in the remaining nurse cell cytoplasm (arrow in D indicates actin bundles). Note that the autolysosome shown in E contains condensed material resembling the material of the condensed and fragmented nurse cell nucleus (NCN), providing evidence for nuclear autophagy


( F ) Nurse of a late stage 13 egg chamber expressing UASp - mCherry - DrAtg8a exclusively in germline ( genotype : UASp - mCherry - DrAtg8a / + ; nanos - VP - 16 Gal4 / + ) . mCherry - DrAtg8a are attached to or located adjacent to fragmented nucleus ( arrows insets ) . Hoechst ( blue ) was performed to visualize nuclei . Nurse are outlined with a . FC , follicle ; FCN , follicle nucleus ; OC , oocyte . Bars : ( A - C ) 10 µm ; ( D - F ) 1 µm .

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(F) Nurse cell of a late stage 13 egg chamber expressing UASp-mCherry-DrAtg8a exclusively in the germline (genotype: UASp-mCherry-DrAtg8a/+; nanos-VP-16 Gal4/+). mCherry-DrAtg8a puncta are attached to or located adjacent to the fragmented nucleus (arrows and insets). Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. FC, follicle cell; FCN, follicle cell nucleus; OC, oocyte. Bars: (A-C) 10 µm; (D-F) 1 µm.


Genetic inhibition of autophagy in germline prevents fragmentation . Confocal of stage 13 / 14 egg chambers after TUNEL . ( A ) Wild - type ( wt ) stage 13 egg chamber contains nurse nuclei exhibiting fragmented ( positive TUNEL ; arrows ) . ( B ) Wild - type stage 14 egg chambers do not contain nurse . Arrows point to respiratory appendages ( RA ) . ( C - E ) Stage 14 atg1 − / − ( C ) , atg13 − / − ( D ) , vps34 − / − ( E ) germline mutant chambers have persisting nurse nuclei that do not contain fragmented ( negative TUNEL ; arrows ) . Nurse are outlined with a . Draq5 / Hoechst ( blue ) was performed to visualize nuclei . PhC , phase contrast . Bars , 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 O O O O O O 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-CONTROLLED_VAR 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

Genetic inhibition of autophagy in the germline prevents DNA fragmentation. Confocal micrographs of stage 13/14 egg chambers after TUNEL staining. (A) Wild-type (wt) stage 13 egg chamber contains nurse cell nuclei exhibiting fragmented DNA (positive TUNEL staining; arrows). (B) Wild-type stage 14 egg chambers do not contain nurse cells. Arrows point to the respiratory appendages (RA). (C-E) Stage 14 atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant chambers have persisting nurse cell nuclei that do not contain fragmented DNA (negative TUNEL staining; arrows). Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.


Germline autophagy mutant egg chambers exhibit reduced expression of caspase - 3 . Confocal of stage 13 / 14 egg chambers stained for caspase - 3 ( casp - 3 ) . ( A ) Wild - type ( wt ) late stage 13 egg chamber showing caspase - 3 in degenerating nurse cluster . ( B ) In wild - type stage 14 egg chamber , nurse are completely degraded ( arrows ) . ( C - E ) atg1 − / − ( C ) , atg13 − / − ( D ) , vps34 − / − ( E ) germline mutant stage 14 egg chambers exhibit significantly reduced for caspase - 3 contain persisting nurse nuclei . Nurse are outlined with a . Draq5 / Hoechst ( blue ) was performed to visualize nuclei . PhC , phase contrast . ( F ) of mean of caspase - 3 observed in nurse of stage 12 - 14 egg chambers in wild - type germline autophagy mutant egg chambers . Wild type ( WT ) : three independent experiments , n = 30 egg chambers ; atg1 − / − GLCs : four independent experiments , n = 30 egg chambers ; atg13 − / − GLCs : four independent experiments , n = 30 egg chambers ; vps34 − / − GLCs : three independent experiments , n = 30 egg chambers . Data are presented as mean ± SD . Difference was significant with P < 0 . 001 for values versus wild type . Bars , 20 µm .

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

Germline autophagy mutant egg chambers exhibit reduced expression of cleaved caspase-3. Confocal micrographs of stage 13/14 egg chambers stained for cleaved caspase-3(casp-3). (A) Wild-type (wt) late stage 13 egg chamber showing cleaved caspase-3 staining in the degenerating nurse cell cluster. (B) In wild-type stage 14 egg chamber, nurse cells are completely degraded (arrows). (C-E) atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant stage 14 egg chambers exhibit significantly reduced staining for cleaved caspase-3 and contain persisting nurse cell nuclei. Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. (F) Quantification of mean intensity of cleaved caspase-3 staining observed in the nurse cells of stage 12-14 egg chambers in wild-type and germline autophagy mutant egg chambers. Wild type (WT): three independent experiments, n = 30 egg chambers; atg1−/− GLCs: four independent experiments, n = 30 egg chambers; atg13−/− GLCs: four independent experiments, n = 30 egg chambers; vps34−/− GLCs: three independent experiments, n = 30 egg chambers. Data are presented as mean ± SD. Difference was significant with P < 0.001 for all values versus wild type. Bars, 20 µm.


dBruce colocalizes with autophagic marker Atg8a - GFP in nurse during late oogenesis is accumulated in autophagy germline mutants . Confocal of late stage egg chambers expressing Atg8a - GFP stained for dBruce . ( A - C ) Stage 10 ( A ) , early stage 12 ( B ) , late stage 12 ( C ) . dBruce colocalizes with Atg8a - GFP in punctuate structures during stages 12 13 ( insets in B insets arrows in C ) not during stage 10 ( insets in A ) .

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 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-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

dBruce colocalizes with the autophagic marker Atg8a-GFP in the nurse cells during late oogenesis and is accumulated in autophagy germline mutants. Confocal micrographs of late stage egg chambers expressing Atg8a-GFP and stained for dBruce. (A-C) Stage 10 (A), early stage 12 (B), and late stage 12 (C). dBruce colocalizes with Atg8a-GFP in punctuate structures during stages 12 and 13 (insets in B and insets and arrows in C) and not during stage 10 (insets in A).


( D - G ) Confocal of stage 14 wild - type ( D ) autophagy mutant egg chambers ( E - G ) stained for dBruce . dBruce is accumulated in atg1 , atg13 , vps34 mutant nurse ( arrows ) . Draq5 / Hoechst ( blue ) was performed to visualize nuclei . Nurse are outlined with a . OC , oocyte ; PhC , phase contrast . Bars , 10 µm .

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

(D-G) Confocal micrographs of stage 14 wild-type (D) and autophagy mutant egg chambers (E-G) stained for dBruce. dBruce is accumulated in atg1, atg13, and vps34 mutant nurse cells (arrows). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. OC, oocyte; PhC, phase contrast. Bars, 10 µm.


dBruce is required for nurse survival by controlling fragmentation during oogenesis . ( A ) Confocal of dBrucee00984 mutant ovarioles stained for TUNEL . Arrows point to degenerating stage 8 / 9 egg chambers that are TUNEL positive .

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dBruce is required for nurse cell survival by controlling DNA fragmentation during oogenesis. (A) Confocal micrographs of dBrucee00984 mutant ovarioles stained for TUNEL and DNA. Arrows point to degenerating stage 8/9 egg chambers that are TUNEL positive.


( B C ) Confocal of vps34 / dBruceE81 atg , dBruceE81 double mutant egg chambers stained for TUNEL ( green red , respectively ) . ( B ) vps34 − / − GLCs ; dBruceE81 / dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL - positive nurse nuclei ( outlined ; arrow ) . ( C ) atg1 − / − GLCs , dBruceE81 / dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL - positive nurse nuclei . atg1 mutant nurse are identified by lack of GFP ( outlined ; arrow ) . Draq5 / Hoechst ( blue ) was performed to visualize nuclei . PhC , phase contrast . Bars , 50 µm .

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(B and C) Confocal micrographs of vps34/dBruceE81 and atg, dBruceE81 double mutant egg chambers stained for TUNEL (green and red, respectively) and DNA. (B) vps34−/−GLCs; dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei (outlined; arrow). (C) atg1−/−GLCs, dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei. atg1 mutant nurse cells are identified by the lack of GFP staining (outlined; arrow). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.


Labelling scheme representative from U2OS transfected with siLuc or siChk1 .

Labelling scheme and representative DNA fibers from U2OS cells transfected with siLuc or siChk1.


Western Blot of Chk1 in U2OS , 48 h after transfection with siRNA . Actin was used as loading control .

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Western Blot of Chk1 in U2OS cells, 48 h after transfection with siRNA. Actin was used as loading control.


Control ( siLuc ) Chk1 - depleted ( siChk1 ) U20S were labeled with CldU IdU for 10 30 min , respectively . Mean ( + ) track lengths ( top ) CldU / IdU ratios ( bottom ) are shown . Data come from 6 independent experiments a total of 579 ( siLuc ) 590 ( siChk1 ) were scored .

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Control (siLuc) and Chk1-depleted (siChk1) U20S cells were labeled with CldU and IdU for 10 and 30 min, respectively. Mean (+SEM) track lengths (top) and CldU/IdU ratios (bottom) are shown. Data come from 6 independent experiments and a total of 579 (siLuc) and 590 (siChk1) fibers were scored.


Western Blot of phospho - serine - 296 - Chk1 ( p - Chk1 ) in U2OS . were treated 1 . 5 h with Chk1 inhibitor Gö6976 ( Chk1i ) or DMSO , irradiated with 100J / m2 UV to induce high of p - Chk1 , collected 1 . 5 h afterwards . Actin was used as a loading control .

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Western Blot of phospho-serine-296-Chk1 (p-Chk1) in U2OS cells. Cells were treated 1.5 h with the Chk1 inhibitor Gö6976 (Chk1i) or DMSO, irradiated with 100J/m2 UV to induce high levels of p-Chk1, and collected 1.5 h afterwards. Actin was used as a loading control.


U2OS treated with DMSO ( control ) or Chk1i were labeled with CldU IdU for 10 30 min , respectively . Data come from 3 independent experiments a total of 304 ( DMSO ) 312 ( Chk1i ) were scored .

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U2OS cells treated with DMSO (control) or Chk1i were labeled with CldU and IdU for 10 and 30 min, respectively. Data come from 3 independent experiments and a total of 304 (DMSO) and 312 (Chk1i) fibers were scored.


U2OS were labeled with CldU for 10 min , followed by increasing IdU labelling times subjected to stretching . ( A ) Labelling scheme representative . Scale bars : 5 μm .

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U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. (A) Labelling scheme and representative DNA fibers. Scale bars: 5 μm.


U2OS were labeled with CldU for 10 min , followed by increasing IdU labelling times subjected to stretching . IdU track after 10 - 80 min . Theoretical IdU track lengths are shown in red ; theoretical 10 - min points were considered equal to experimental values obtained for siLuc or siChk1 , following theoretical points were calculated proportionally . Data were calculated as mean ( + SD ) of mean IdU track lengths ( B ) n = 4 for IdU times 20 - 60 min or n = 3 for IdU times 10 80 min ; for each experiment condition 60 - 100 were scored .

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U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. IdU track length after 10-80 min. Theoretical IdU track lengths are shown in red; the theoretical 10-min points were considered equal to the experimental values obtained for siLuc or siChk1, and the following theoretical points were calculated proportionally. Data were calculated as the mean (+SD) of mean IdU track lengths (B) n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored.


U2OS were labeled with CldU for 10 min , followed by increasing IdU labelling times subjected to stretching . Fork speed for each IdU labelling time . Data were calculated as mean ( + SD ) of mean fork speeds ( C ) . n = 4 for IdU times 20 - 60 min or n = 3 for IdU times 10 80 min ; for each experiment condition 60 - 100 were scored .

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U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. Fork speed for each IdU labelling time. Data were calculated as the mean (+SD) of mean fork speeds (C). n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored.


combing from HCT116 labeled with CldU IdU for 20 min . upper left panel shows labelling scheme . upper right panel shows representative sister replication forks that emanate from same origin ( ORI = origin of replication ) ; a pair of sister forks is considered asymmetric if their lengths ( CldU + IdU ) differ in more than 33 % . of asymmetric forks ( lower left panel ; a Western Blot of Chk1 Ku80 , loading control , performed in parallel to combing experiments , is shown ) lengths of each pair of forks ( lower right panel ; points outside of dotted lines correspond to asymmetric forks ) are shown . Data ( siLuc , n = 131 ; siChk1 , n = 117 ) come from 2 independent experiments ; numbers indicate mean ( + SD ) of asymmetric forks .

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

DNA combing from HCT116 cells labeled with CldU and IdU for 20 min. The upper left panel shows the labelling scheme. The upper right panel shows representative sister replication forks that emanate from the same origin (ORI=origin of replication); a pair of sister forks is considered asymmetric if their lengths (CldU+IdU) differ in more than 33%. The percentage of asymmetric forks (lower left panel; a Western Blot of Chk1 and Ku80, loading control, performed in parallel to the combing experiments, is shown) and the lengths of each pair of forks (lower right panel; points outside of the dotted lines correspond to asymmetric forks) are shown. Data (siLuc, n=131; siChk1, n=117) come from 2 independent experiments; the numbers indicate the mean (+SD) percentage of asymmetric forks.


Labelling protocol used here in figures that follow , except otherwise indicated . lower panels show representative schemes of an ongoing fork of origins that fired during CldU ( 1st ) or IdU ( 2nd ) pulses . Gray dotted lines represent unlabeled . of origins was calculated as : ( 1st + 2nd origins ) / total number of .

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Labelling protocol used here and in all figures that follow, except otherwise indicated. The lower panels show representative images and schemes of an ongoing fork and of origins that fired during the CldU (1st) or IdU (2nd) pulses. Gray dotted lines represent unlabeled DNA. Percentage of origins was calculated as: (1st+2nd pulse origins)/total number of fibers.


IdU track lengths from U2OS ( Rosc , roscovitine ) . 180 - 200 obtained from 2 independent experiments were measured for each condition . upper panel shows a Western Blot of Polη , 48 h after transfection with siRNA . Actin was used as a loading control .

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IdU track lengths from U2OS cells (Rosc, roscovitine). 180-200 DNA fibers obtained from 2 independent experiments were measured for each condition. The upper panel shows a Western Blot of Polη, 48 h after transfection with siRNA. Actin was used as a loading control.


of origin firing ( mean + SD ) from in ( A ) ( n = 2 ) . dotted represents mean of origin firing in control samples .

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Percentage of origin firing (mean+SD) from DNA fibers in (A) (n=2). The dotted line represents the mean percentage of origin firing in control samples.


Representative of siChk1 - depleted U2OS showing localization of GFP - Polη - WT / S687A / S687D . Numbers indicate of showing GFP - Polη ( mean + SD , n = 2 ) . Scale bar : 100 μm .

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Representative images of siChk1-depleted U2OS cells showing nuclear localization of GFP-Polη-WT/S687A/S687D. Numbers indicate percentage of cells showing nuclear GFP-Polη (mean+SD, n=2). Scale bar: 100 μm.


Western Blot of GFP - Polη in U2OS , 48 / 24 h after transfection with siRNA / GFP ( - Polη ) . Actin was used as loading control .

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Western Blot of GFP-Polη in U2OS cells, 48/24 h after transfection with siRNA/GFP(-Polη). Actin was used as loading control.


IdU track lengths from U2OS transfected with GFP ( - ) or GFP - Polη - WT / S687A / S687D . > 180 obtained from 2 independent experiments were measured for each condition .

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IdU track lengths from U2OS cells transfected with GFP (-) or GFP-Polη-WT/S687A/S687D. >180 fibers obtained from 2 independent experiments were measured for each condition.


Number of foci per after CSK extraction in siLuc siChk1 - depleted U2OS transfected with GFP - Polη - WT / S687A / S687D . > 150 / sample were analyzed in 4 independent experiments . p < 0 . 05 was considered significant .

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Number of foci per cell after CSK extraction in siLuc and siChk1-depleted U2OS cells transfected with GFP-Polη-WT/S687A/S687D. >150 cells/sample were analyzed in 4 independent experiments. p<0.05 was considered significant.


Representative of with GFP - Polη foci used for in ( E ) . number of foci per for each representative nucleus is indicated . Scale bar : 10 μm .

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Representative images of cells with GFP-Polη foci used for quantification in (E). The number of foci per cell for each representative nucleus is indicated. Scale bar: 10 μm.


IdU track lengths of origin firing ( mean + SD ) from U2OS treated or not with a CDC7 inhibitor ( CDC7i ) . > 380 ( track ) > 600 ( origin frequency ) obtained from 4 ( track ) or 3 ( origin frequency ) independent experiments were measured for each condition .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells treated or not with a CDC7 inhibitor (CDC7i). >380 (track length) and >600 (origin frequency) fibers obtained from 4 (track length) or 3 (origin frequency) independent experiments were measured for each condition.


IdU track lengths of origin firing ( mean + SD ) from U2OS transfected or not with siCDT1 . Track lengths from > 290 obtained from 3 independent experiments were measured for each condition . To calculate origin frequency > 700 from 3 independent experiments were scored .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with siCDT1. Track lengths from >290 fibers obtained from 3 independent experiments were measured for each condition. To calculate origin frequency >700 DNA fibers from 3 independent experiments were scored.


IdU track lengths of origin firing ( mean + SD ) from U2OS supplemented or not with nucleosides ( NS ) . > 195 ( track ) > 350 ( origin frequency ) obtained from 2 independent experiments were measured for each condition .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells supplemented or not with nucleosides (NS). >195 (track length) and >350 (origin frequency) fibers obtained from 2 independent experiments were measured for each condition.


IdU track lengths of origin firing ( mean + SD ) from U2OS transfected or not with GFP - Polη . > 180 ( track ) > 400 ( origin frequency ) obtained from 2 independent experiments were measured for each condition .

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IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with GFP-Polη. >180 (track length) and >400 (origin frequency) DNA fibers obtained from 2 independent experiments were measured for each condition.


of γH2AX - positive U2OS ( mean + SD ) after 5 h treatment with Chk1 inhibitor Gö6976 ( Chk1i ) , in presence of 5 μM NS , 1 , 25 μM CDC7i or 10 μM roscovitine . > 900 ( DMSO ) or > 2000 ( Chk1i ) / sample were analyzed in 2 independent experiments . Total γH2AX was used to perform statistics . Representative of data shown in ( B ) . Scale bar : 50 μm .

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Percentage of γH2AX-positive U2OS cells (mean+SD) after 5 h treatment with the Chk1 inhibitor Gö6976 (Chk1i), in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >900 (DMSO) or >2000 (Chk1i) cells/sample were analyzed in 2 independent experiments. Total γH2AX was used to perform the statistics. Representative images of data shown in (B). Scale bar: 50 μm.


DSB accumulation measured by neutral comet after 8 h treatment with Chk1i , in presence of 5 μM NS , siCDC7 or 10 μM roscovitine . 85 - 170 / sample were analyzed in 2 independent experiments . Representative of data shown in ( D ) . Scale bar : 25 μm .

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DSB accumulation measured by neutral comet assay after 8 h treatment with Chk1i, in the presence of 5 μM NS, siCDC7 or 10 μM roscovitine. 85-170 cells/sample were analyzed in 2 independent experiments. Representative images of data shown in (D). Scale bar: 25 μm.


of parental ssDNA - positive U2OS ( mean + SD ) after 5 h treatment with Chk1i , in presence of 5 μM NS , 1 , 25 μM CDC7i or 10 μM roscovitine . > 450 / sample were analyzed in 2 independent experiments . induction of ssDNA on control samples was null . Representative of data shown in ( F ) . Scale bar : 25 μm .

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Percentage of parental ssDNA-positive U2OS cells (mean+SD) after 5 h treatment with Chk1i, in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >450 cells/sample were analyzed in 2 independent experiments. The induction of ssDNA on control samples was null. Representative images of data shown in (F). Scale bar: 25 μm.


Sensitivity of U2OS to Chk1 inhibition , roscovitine ( 5 μM ) Polη depletion . number was determined 4 days after a 24 h treatment ( 5 days in total ) with Chk1i ± Rosc . Data represent mean ( + SD ) of 3 independent experiments .

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Sensitivity of U2OS cells to Chk1 inhibition, roscovitine (5 μM) and Polη depletion. Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 3 independent experiments.


Sensitivity of U2OS transduced with shRNA targeting Chk1 ( shChk1 ) or non - targeting shRNA ( shScr ) Lenti - GFP - Polη or Lenti - GFP ( - ) . number was determined 7 days after transduction ( 5 days after maximal downregulation ) . Data represent mean ( + SD ) of 3 independent experiments .

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Sensitivity of U2OS cells transduced with shRNA targeting Chk1 (shChk1) or non-targeting shRNA (shScr) and Lenti-GFP-Polη or Lenti-GFP (-). Cell number was determined 7 days after transduction (5 days after maximal downregulation). Data represent the mean (+SD) of 3 independent experiments.


Western Blot of CDC45 Chk1 in U2OS after an extraction with CSK buffer to separate insoluble soluble fractions . Roscovitine ( 25μM ) was added 2 h before harvesting . H2B was used as a loading control .

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Western Blot of CDC45 and Chk1 in U2OS cells after an extraction with CSK buffer to separate the insoluble and soluble fractions. Roscovitine (25μM) was added 2 h before harvesting. H2B was used as a loading control.


IdU track lengths from U2OS . 200 obtained from 2 independent experiments were measured for each condition .

IdU track lengths from U2OS cells. 200 DNA fibers obtained from 2 independent experiments were measured for each condition.


IdU track lengths from U2OS . > 170 obtained from 2 independent experiments were measured for each condition . For comparison , data showing that Polη is required for roscovitine - dependent rescue of fork elongation in Chk1 - depleted ( Fig 3B ) is shown on right . lower panel shows corresponding Western Blot of Chk1 , Polη CDC45 . Actin was used as a loading control .

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IdU track lengths from U2OS cells. >170 DNA fibers obtained from 2 independent experiments were measured for each condition. For comparison, the data showing that Polη is required for roscovitine-dependent rescue of fork elongation in Chk1-depleted cells (Fig 3B) is shown on the right. The lower panel shows the corresponding Western Blot of Chk1, Polη and CDC45. Actin was used as a loading control.


of origin firing ( mean + SD ) from in C ( n = 2 ) . > 490 per condition were scored .

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Percentage of origin firing (mean+SD) from DNA fibers in C (n=2). >490 fibers per condition were scored.


Sensitivity of U2OS to Chk1 CDC45 depletion . number was determined 7 days after transfection ( 5 days after maximal downregulation ) . Data represent mean ( + SD ) of 2 independent experiments . Representative image of data shown in ( E ) . Scale bar : 100 μm .

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Sensitivity of U2OS cells to Chk1 and CDC45 depletion. Cell number was determined 7 days after transfection (5 days after maximal downregulation). Data represent the mean (+SD) of 2 independent experiments. Representative image of data shown in (E). Scale bar: 100 μm.


Sensitivity of U2OS to Chk1 inhibition roscovitine ( 5 μM ) . number was determined 4 days after a 24 h treatment ( 5 days in total ) with Chk1i ± Rosc . Data represent mean ( + SD ) of 2 independent experiments . Representative image of data shown in ( G ) . Scale bar : 100 μm .

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Sensitivity of U2OS cells to Chk1 inhibition and roscovitine (5 μM). Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 2 independent experiments. Representative image of data shown in (G). Scale bar: 100 μm.


Flow cytometry of PD - L1 expression in ( A ) KPC ( B ) Pan02 following treatment with gemcitabine RT . Gemcitabine was diluted in DMSO ( vehicle ) DMSO was used for ' untreated ' control group ( mean ± SD , n = 3 , One - way ANOVA , Bonferroni ) .

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Flow cytometry analysis of PD-L1 expression in (A) KPC and (B) Pan02 cells following treatment with gemcitabine and RT. Gemcitabine was diluted in DMSO (vehicle) and DMSO was used for the 'untreated' control group (mean ± SD, n=3, One-way ANOVA, Bonferroni test).


Western blot of indicated in both KPC ( C ) Pan02 ( D ) following radiation chemotherapy ± AG490 , a JAK / Stat kinase inhibitor . Actin represents loading control .

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Western blot analysis of indicated proteins in both KPC (C) and Pan02 (D) cells following radiation and chemotherapy ± AG490, a JAK/Stat kinase inhibitor. Actin represents loading control.


Flow cytometry of PD - L1 after gemcitabine RT ( as described above ) in KPC ( E ) Pan02 ( F ) following Stat1 downregulation by siRNA ( mean ± SD , n = 3 , Student ' s t - ) . MFI , mean index ;

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Flow cytometry analysis of PD-L1 after gemcitabine and RT (as described above) in KPC (E) and Pan02 (F) following Stat1 downregulation by siRNA (mean ± SD, n=3, Student's t-test). MFI, mean fluorescence index;


( A ) growth delay was measured in different groups , as indicated ( n = 6 mice per group ) . Anti - PD - L1 was given at days 0 3 , 6 9 ( black arrows ) . average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 is shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni ) . No loss was observed in in vivo

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(A) Tumor growth delay was measured in the different groups, as indicated (n=6 mice per group). Anti-PD-L1 was given at days 0 3, 6 and 9 (black arrows). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment


( B ) Quantitative data on of gated are shown ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni ) .

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(B) Quantitative data on the percentage of gated cells are shown (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


( A ) growth delay was measured by treating with RT ( 12 Gy or 5x3 Gy daily ; initiated at day 0 ) anti - PD - L1 alone or combination of RT ( n = 6 mice per group ) . Anti - PD - L1 was given at days 0 , 3 , 6 9 ( black arrows ) . radiosensitizing effect of anti - PD - L1 after 12 Gy 5x3 Gy was assessed as shown by capped lines ( means ± SD , n = 1 , One - way ANOVA , Bonferroni ) . No loss was observed in in vivo experiment .

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(A) Tumor growth delay was measured by treating tumor with RT (12 Gy or 5x3 Gy daily; initiated at day 0) and anti-PD-L1 alone or combination of RT (n=6 mice per group). Anti-PD-L1 was given at days 0, 3, 6 and 9 (black arrows). The radiosensitizing effect of anti-PD-L1 after 12 Gy and 5x3 Gy was assessed as shown by the capped lines (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment.


( A ) KPC growth delay after treatment with either 12 Gy ( day 0 ) , anti - PD - L1 ( days 0 , 3 , 6 9 ; black arrows ) , anti - CD8 alone ( days 0 3 , 6 9 ) or their combinations , as indicated .

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(A) KPC tumor growth delay after treatment with either 12 Gy (day 0), anti-PD-L1 (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0 3, 6 and 9) or their combinations, as indicated.


( B ) KPC growth delay after treatment with either 5x3 Gy ( day 0 - 4 ) , anti - PD - L1 alone ( days 0 , 3 , 6 9 ; black arrows ) , anti - CD8 alone ( days 0 , 3 , 6 9 ) or their combinations , as indicated .

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(B) KPC tumor growth delay after treatment with either 5x3 Gy (day 0-4), anti-PD-L1 alone (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0, 3, 6 and 9) or their combinations, as indicated.


( C ) KPC growth delay after either simultaneous ( day 0 , 3 , 6 9 ; black arrows - upper row ) or sequential ( day 6 , 9 , 13 16 ; black arrows - lower row ) addition of anti - PD - L1 to RT ( day 0 ) . average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 for Figure 4A - C is also shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni ) . No loss was observed in in vivo experiments .

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(C) KPC tumor growth delay after either simultaneous (day 0, 3, 6 and 9; black arrows-upper row) or sequential (day 6, 9, 13 and 16; black arrows-lower row) addition of anti-PD-L1 to RT (day 0). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 for Figure 4A-C is also shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiments.


( A ) Mice were treated with either gemcitabine ( day 0 , 3 ) , anti - PD - L1 ( day 4 , 7 , 10 13 ; black arrows ) , 12 Gy ( day 4 ) alone or in combinations , as indicated ( n = 8 mice per group ) . average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 is shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni ) No loss was observed in in vivo experiment .

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(A) Mice were treated with either gemcitabine (day 0, 3), anti-PD-L1 (day 4, 7, 10 and 13; black arrows), 12 Gy (day 4) alone or in combinations, as indicated (n=8 mice per group). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test) No weight loss was observed in the in vivo experiment.


RT plus anti - PD - L1 ( B ) reverses T - exhaustion . Bar graphs show quantitation of T activation markers in samples on flow cytometry , as indicated ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni ) .

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RT plus anti-PD-L1 (B) reverses T-cell exhaustion . Bar graphs show quantitation of T cell activation markers in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


RT plus anti - PD - L1 ( C ) increases CD8 : Treg . Bar graphs show of CD8 : Treg in samples on flow cytometry , as indicated ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni ) .

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RT plus anti-PD-L1 (C) increases CD8:Treg ratio. Bar graphs show ratio of CD8:Treg ratio in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


( A ) Representative of livers from different treatment groups , as indicated . corresponding liver weights are shown below ( n = 6 mice per group ) .

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(A) Representative images of livers from the different treatment groups, as indicated. The corresponding liver weights are shown below (n=6 mice per group).


( B ) Flow cytometry of CD45 + CD8 + , CD11b + Gr1 + CD4 + CD25 + FOXP3 + populations in livers of mice from different treatment groups , as indicated ( n = 5 per group ; ( means ± SD , n = 1 , One - way ANOVA , Bonferroni ) .

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(B) Flow cytometry analysis of CD45+CD8+, CD11b+Gr1+ and CD4+CD25+FOXP3+ cell populations in the livers of the mice from the different treatment groups, as indicated (n=5 per group; (means ± SD, n=1, One-way ANOVA, Bonferroni test).


( C ) Co - immunoprecipitation of Cdc4 using either unphosphorylated or Clb5 - Cdk1 - phosphorylated Far1 ( 85 - 150 ) as bait . experiment was repeated twice , a representative example is shown .

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(C) Co-immunoprecipitation of Cdc4 using either unphosphorylated or Clb5-Cdk1-phosphorylated Far1(85-150) as bait. The experiment was repeated twice, a representative example is shown.


( D ) Time - lapse showing expression degradation of wild - type S87A S91A mutant Far1 ( 1 - 150 ) - GFP - shuttling of Whi5 - mCherry . export of Whi5 - mCherry takes place at 0 min ( Start point ) was used to synchronize for quantitative .

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(D) Time-lapse fluorescence images showing the expression and degradation of wild-type and S87A S91A mutant Far1(1-150)-GFP and nuclear-cytoplasmic shuttling of Whi5-mCherry. Nuclear export of Whi5-mCherry takes place at 0 min (Start point) and was used to synchronize the cells for quantitative analysis.


( E , F ) Mean ± intensities of indicated Far1 ( 1 - 150 ) - GFP mutants over cycle .

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(E, F) Mean±SEM fluorescence intensities of the indicated Far1(1-150)-GFP mutants over the cell cycle.


( G ) Time from Start to degradation of 50 % of indicated Far1 - GFP sensor in single . bars show median ± 95 % confidence intervals . numbers above plot show median values . * * * * indicates p - value < 0 . 0001 , * * * < 0 . 001 ns > 0 . 05 for pair - wise comparisons with wild - type using Mann Whitney U .

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(G) Time from Start to degradation of 50% of the indicated Far1-GFP sensor in single cells. The bars show median ±95% confidence intervals. The numbers above the plot show median values. **** indicates p-value <0.0001, *** <0.001 and ns >0.05 for pair-wise comparisons with wild-type using Mann Whitney U test.


( H ) In vitro kinase showing effect of N130A , L131A , L135A L136A mutations on Far1 ( 1 - 150 ) - GFP phosphorylation by Clb5 - Cdk1 . WT / mut shows decrease in phosphorylation rate of mutant compared to wild - type . experiment was repeated twice , a representative example is shown .

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(H) In vitro kinase assay showing the effect of N130A, L131A, L135A and L136A mutations on Far1(1-150)-GFP phosphorylation by Clb5-Cdk1. WT/mut shows the decrease in phosphorylation rate of the mutant compared to wild-type. The experiment was repeated twice, a representative example is shown.


( A ) Autoradiograph of a cyclin specificity of indicated substrates with wild - type or docking site mutant cyclin - Cdk1 complexes . Far1 ( 1 - 150 mut ) is L131A L135A L136A mutant , Sic1ΔC is non - inhibitory mutant of Sic1 ( deletion of 216 - 284 ) . experiment was repeated twice , a representative example is shown .

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(A) Autoradiograph of a cyclin specificity analysis of the indicated substrates with wild-type or docking site mutant cyclin-Cdk1 complexes. Far1(1-150 mut) is L131A L135A L136A mutant, Sic1ΔC is the non-inhibitory mutant of Sic1 (deletion of 216-284). The experiment was repeated twice, a representative example is shown.


( B ) Mean ± intensities of wild - type NLxxxL motif mutant Far1 ( 1 - 150 ) - GFP in either wild - type or clb5 ( hpm ) clb6 ( hpm ) over cycle .

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(B) Mean±SEM fluorescence intensities of wild-type and NLxxxL motif mutant Far1(1-150)-GFP in either wild-type or clb5(hpm) clb6(hpm) strain over the cell cycle.


( C ) Plot showing mean ± intensities of Far1 ( 1 - 150 ) - GFP constructs with indicated docking site . In rxl mutant , NLxxxL motif is replaced with a mutated non - functional RxL motif ( VKATAF ) .

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(C) Plot showing the mean±SEM fluorescence intensities of Far1(1-150)-GFP constructs with the indicated docking site. In rxl mutant, the NLxxxL motif is replaced with a mutated non-functional RxL motif (VKATAF).


( D ) Kinetic characterization of Far1 ( 1 - 150 WT ) - GFP Far1 ( 1 - 150 RxL ) - GFP phosphorylation by Clb5 - Cdk1 . data is from two experimental replicates .

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(D) Kinetic characterization of Far1(1-150 WT)-GFP and Far1(1-150 RxL)-GFP phosphorylation by Clb5-Cdk1. The data is from two experimental replicates.


( E ) Mean ± intensities of wild - type or RxL - dependent Far1 ( 1 - 150 ) - GFP in wild - type or indicated cyclin hpm mutant strains .

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(E) Mean±SEM fluorescence intensities of wild-type or RxL-dependent Far1(1-150)-GFP in wild-type or the indicated cyclin hpm mutant strains.


( F ) Pheromone sensitivity halo with PGAL1 - 3HA - CLB5 sic1Δ strains carrying either wild - type FAR1 , far1Δ , far1 ( L135A ) or far1 ( RxL ) . Different concentrations of α - factor were pipetted on paper discs . On glucose plates , α - factor triggers cycle arrest . CLB5 overexpression from PGAL1 in sic1Δ causes lethality presumably by inhibition of replication licensing , which is rescued by presence of α - factor , which leads to Far1 - depedent inhibition of excess Clb5 - Cdk1 activity . experiment was repeated twice , a representative example is shown .

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(F) Pheromone sensitivity halo assay with PGAL1-3HA-CLB5 sic1Δ strains carrying either wild-type FAR1, far1Δ, far1(L135A) or far1(RxL). Different concentrations of α-factor were pipetted on the paper discs. On glucose plates, α-factor triggers cell cycle arrest. CLB5 overexpression from PGAL1 in sic1Δ strain causes lethality presumably by inhibition of replication licensing, which is rescued by the presence of α-factor, which leads to Far1-depedent inhibition of excess Clb5-Cdk1 activity. The experiment was repeated twice, a representative example is shown.


( B ) degradation of Far1 ( 1 - 150 ) - GFP with either wild - type or serine - phosphorylation sites indicated docking motifs was measured in time - lapse microscopy . Plot shows mean ± of GFP intensities from Start .

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(B) The degradation of Far1(1-150)-GFP with either wild-type or all serine-based phosphorylation sites and the indicated docking motifs was measured in time-lapse microscopy. Plot shows mean±SEM of GFP fluorescence intensities from Start.


( C ) Phos - tag SDS - PAGE autoradiographs showing Cks1 - dependency of Clb5 - Cdk1 - mediated multisite phosphorylation of Far1 ( 1 - 150 ) with either NLxxxL ( WT ) motif or RxL motif . experiment was performed twice , a representative example is shown .

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(C) Phos-tag SDS-PAGE autoradiographs showing the Cks1-dependency of Clb5-Cdk1-mediated multisite phosphorylation of Far1(1-150) with either NLxxxL (WT) motif or RxL motif. The experiment was performed twice, a representative example is shown.


( D ) Plot showing kinetics of Clb5 - Cdk1 - mediated phosphorylation of Far1 ( 85 - 150 ) with either wild - type ( NLxxxL ) or RxL docking motif . data is from two replicates .

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(D) Plot showing the kinetics of Clb5-Cdk1-mediated phosphorylation of Far1(85-150) with either wild-type (NLxxxL) or RxL docking motif. The data is from two replicates.


( F ) Mean ± GFP intensities of indicated Far1 ( 1 - 85 ) constructs , synchronized at time of Start .

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(F) Mean±SEM GFP fluorescence intensities of the indicated Far1(1-85) constructs, synchronized at the time of Start.


( G ) Plot showing timing of 50 % degradation of indicated Far1 ( 85 - 150 ) constructs in single . numbers above plot show median values . bars show median ± 95 % CI . * * * * ns indicate p - values < 0 . 0001 > 0 . 05 by Mann - Whitney for pair - wise comparisons with WT .

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(G) Plot showing the timing of 50% degradation of the indicated Far1(85-150) constructs in single cells. The numbers above the plot show median values. The bars show median ±95% CI. **** and ns indicate p-values <0.0001 and >0.05 by Mann-Whitney test for pair-wise comparisons with WT.


( C ) Plot showing mean ± intensities of Sic1 - threshold sensors shown in ' A ' containing indicated cyclin docking motif .

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(C) Plot showing the mean±SEM fluorescence intensities of the Sic1-based threshold sensors shown in 'A' containing the indicated cyclin docking motif.


( D ) time from Start to of threshold sensors with indicated docking motifs decreasing to 50 % of peak level was measured in single . black lines show median ± 95 % CI . numbers above plot show median degradation timings of indicated sensors .

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(D) The time from Start to the fluorescence levels of the threshold sensors with indicated docking motifs decreasing to 50% of peak level was measured in single cells. The black lines show median±95% CI. The numbers above the plot show median degradation timings of the indicated sensors.


( E ) In vitro phosphorylation kinetics of Sic1 ( T33 + 16T45 rxl llpp ΔC ) with indicated cyclin docking motif by Clb5 - Cdk1 . data is from two replicate experiments .

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(E) In vitro phosphorylation kinetics of Sic1(T33+16T45 rxl llpp ΔC) with the indicated cyclin docking motif by Clb5-Cdk1. The data is from two replicate experiments.


( F ) of multisite phosphorylation of Sic1 ( T33 + 16T45 rxl llpp ΔC ) with different docking motifs using Phos - tag SDS - PAGE autoradiography . experiment was performed twice , a representative example is shown .

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(F) Analysis of multisite phosphorylation of Sic1(T33+16T45 rxl llpp ΔC) with different docking motifs using Phos-tag SDS-PAGE autoradiography. The experiment was performed twice, a representative example is shown.


( G ) fraction of maximally phosphorylated form from total phosphorylation signal was quantified from Phos - tag gels as shown in panel ' F ' .

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(G) The fraction of maximally phosphorylated form from total phosphorylation signal was quantified from Phos-tag gels as shown in panel 'F'.


( B ) Autoradiograph showing cyclin specificity docking dependency of Lif1 Slx4 phosphorylation . experiments were repeated twice , representative examples are shown .

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(B) Autoradiograph showing the cyclin specificity and docking dependency of Lif1 and Slx4 phosphorylation. The experiments were repeated twice, representative examples are shown.


( C ) NLxxxL is necessary for efficient phosphorylation of Lif1 Slx4 by Clb5 - Cdk1 , as shown by in vitro kinase . experiments were repeated twice , representative examples are shown .

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(C) NLxxxL is necessary for efficient phosphorylation of Lif1 and Slx4 by Clb5-Cdk1, as shown by the in vitro kinase assay. The experiments were repeated twice, representative examples are shown.


( D ) Autoradiograph of Clb5 - Cdk1 - mediated phosphorylation of indicated Fin1 mutants . experiments were repeated twice , representative examples are shown .

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(D) Autoradiograph of Clb5-Cdk1-mediated phosphorylation of indicated Fin1 mutants. The experiments were repeated twice, representative examples are shown.


( G ) effect of different docking motifs on degradation of Far1 ( 1 - 150 SP ) - GFP , where TP sites are mutated to SP . Plot shows mean ± of intensities in cycle .

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(G) The effect of different docking motifs on the degradation of Far1(1-150 SP)-GFP, where all TP sites are mutated to SP. Plot shows mean±SEM of fluorescence intensities in the cell cycle.


( H ) time from Start to degradation of 50 % of Far1 ( 1 - 150 SP ) - GFP with indicated docking motif was measured in single . numbers above plot show median values for sensors , black lines show median ± 95 % CI . * * * * ns denote p - value < 0 . 0001 > 0 . 05 , respectively , for pair - wise comparisons with SP WT using Mann - Whitney U .

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(H) The time from Start to the degradation of 50% of Far1(1-150 SP)-GFP with the indicated docking motif was measured in single cells. The numbers above the plot show median values for the sensors, the black lines show median±95% CI. **** and ns denote p-value <0.0001 and >0.05, respectively, for pair-wise comparisons with SP WT using Mann-Whitney U test.


( A ) Time - lapse microscopy was used to measure cyclin - Citrine during mitotic exit in unperturbed cycles . were synchronized by import of 50 % of Whi5 - mCherry at mitotic exit . plot shows mean ± intensities averaged over a population of . vertical at - 14 minutes marks estimated time of anaphase entry .

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(A) Time-lapse microscopy was used to measure cyclin-Citrine levels during mitotic exit in unperturbed cell cycles. The cells were synchronized by the nuclear import of 50% of Whi5-mCherry at mitotic exit. The plot shows mean ±SEM fluorescence intensities averaged over a population of cells. The vertical line at -14 minutes marks the estimated time of anaphase entry.


( B ) Cyclin - Citrine degradation relative to import of 50 % Whi5 - mCherry was followed in single . Plot shows time from import of 50 % Whi5 - mCherry to degradation of 50 % of indicated cyclin in . error bars show 95 % CI of mean .

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(B) Cyclin-Citrine degradation relative to the nuclear import of 50% Whi5-mCherry was followed in single cells. Plot shows the time from nuclear import of 50% Whi5-mCherry to the degradation of 50% of the indicated cyclin in individual cells. The error bars show 95% CI of the mean.


( E ) effect of cyclin specificity on phosphorylation of Fin1 , Cnn1 Spc110 was studied in a kinase . plots show phosphorylation by Clb5 - , Clb3 - Clb2 - Cdk1 , error bars show standard deviation ( n = 2 ) .

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(E) The effect of cyclin specificity on the phosphorylation of Fin1, Cnn1 and Spc110 was studied in a kinase assay. The plots show the phosphorylation by Clb5-, Clb3- and Clb2-Cdk1, the error bars show standard deviation (n=2).


( F ) from a time - lapse microscopy experiment of an exemplar expressing Spc42 - mCherry for detection of SPBs EGFP - Fin1 . were imaged every 2 minutes , arrow indicates onset of anaphase .

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(F) Images from a time-lapse microscopy experiment of an exemplar cell expressing Spc42-mCherry for detection of SPBs and EGFP-Fin1. Cells were imaged every 2 minutes, the white arrow indicates onset of anaphase.


( G ) accumulation of EGFP - Fin1 to SPBs in anaphase was measured in experiments described in ' F ' . plot shows mean EGFP intensities at SPBs around time of anaphase onset . error bars are ± .

(G) The accumulation of EGFP-Fin1 to SPBs in anaphase was measured in experiments described in 'F'. The plot shows mean EGFP fluorescence intensities at SPBs around the time of anaphase onset. The error bars are ±SEM.


Expression of Kit ( magenta ) largely overlaps with expression of CD41 : YFP ( yellow ) in IAHC . Multistack reconstruction of confocal . Scale bars : 30μm overview , 10μm magnification . ( C ) Snapshots of Movie EV1 . Time - lapse of embryonic organotypic slice culture from CD41 : YFP ( yellow ) : H2B - GFP ( blue ) reporter mouse shows emergence of a single protruding from endothelial layer into aortic lumen . Scale bars : 50μm ( overview ) , 10μm ( magnification ) . Time expressed in hh : mm : ss . ( D ) Snapshots of Movie EV2 . Time - lapse of embryonic organotypic slice culture from CD41 : YFP ( yellow ) : H2B - GFP ( blue ) reporter mouse shows a single CD41 : YFP expressing that divides once on aortic endothelium . Scale bars : 100μm ( overview ) , 20μm ( magnification ) . Time expressed in hh : mm : ss .

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Expression of Kit (magenta) largely overlaps with the expression of CD41:YFP (yellow) in IAHC. Multistack reconstruction of confocal images. Scale bars: 30μm overview, 10μm magnification. (C) Snapshots of Movie EV1. Time-lapse of embryonic organotypic slice culture from CD41:YFP (yellow): H2B-GFP (blue) reporter mouse shows the emergence of a single cell protruding from the endothelial layer into the aortic lumen. Scale bars: 50μm (overview), 10μm (magnification). Time expressed in hh:mm:ss. (D) Snapshots of Movie EV2. Time-lapse of embryonic organotypic slice culture from CD41:YFP (yellow): H2B-GFP (blue) reporter mouse shows a single CD41:YFP expressing cell that divides once on the aortic endothelium. Scale bars: 100μm (overview), 20μm (magnification). Time expressed in hh:mm:ss.


BrdU incorporation in Kit + IAHC upon cumulative administration . Multistack reconstruction of confocal . Representative picture of a short BrdU ( 1h ) . Scale bars : 50μm ( overview ) , 10μm ( magnification ) . ( F ) of of Kit + within IAHCs incorporating BrdU using confocal . ( Labelling Index = 100 % ) can be labelled after 8h , indicating lack of quiescent inside IAHCs . Graph represents mean ± SE .

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BrdU incorporation in the Kit+ IAHC upon cumulative administration. Multistack reconstruction of confocal images. Representative picture of a short BrdU pulse (1h). Scale bars: 50μm (overview), 10μm (magnification). (F) Quantification of the percentage of Kit+ cells within IAHCs incorporating BrdU using confocal images. All cells (Labelling Index=100%) can be labelled after 8h, indicating the lack of quiescent cells inside the IAHCs. Graph represents mean±SE.


of recombination efficiency at E10 . 5 E11 . 5 using confocal . Bars represents mean ± SD of of expressing single colored cassettes in CD31 + endothelial ( n = 2 ) .

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Quantification of recombination efficiency at E10.5 and E11.5 using confocal images. Bars represents the mean±SD of the percentage of cells expressing single colored cassettes in CD31+ endothelial cells (n=2).


Transversal section of a recombined VeCadCreER : Confetti AGM from an E10 . 5 immunostained with CD31 ( C ) ( in image , black in label ) Multistack reconstruction of confocal . Scale bars

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Transversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with CD31 (C) (white in image, black in label) Multistack reconstruction of confocal images. Scale bars 30μm


Transversal section of a recombined VeCadCreER : Confetti AGM from an E10 . 5 immunostained with Kit ( D ) ( in image , black in label ) . Multistack reconstruction of confocal . Scale bars 20

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Transversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with Kit (D) (white in image, black in label). Multistack reconstruction of confocal images. Scale bars 20 μm


Analyses of recombination in 40 clusters . Bars representing single clusters are grouped on number of Kit + . Recombination induction follows schema in black square . ( n = 12 ) .

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Analyses of recombination in 40 clusters. Bars representing single clusters are grouped based on the number of Kit+ cells. Recombination induction follows the schema in the black square. (n=12 embryo).


Representative confetti recombination on clusters of different sizes . Small cluster are monoclonal ( left panel ) , while larger clusters are polyclonal ( right panel ) . Inlet shows relevant confetti channel . Kit is shown in in image , in black in label . Multistack reconstruction of confocal . Scale bars 5 μm .

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Representative confetti recombination on clusters of different sizes. Small cluster are monoclonal (left panel), while larger clusters are polyclonal (right panel). Inlet shows relevant confetti channel. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bars 5 μm.


Detail of Dll4 in Kit + of IAHC of different sizes . Dll4 expression inversely correlates with of cluster : 2 - 5 IAHCs are uniformly Dll4 - positive ( upper middle panel ) , > 5 IAHCs are partially Dll4 positive ( lower panel ) . Multistack reconstruction of confocal . Scale bars : 10 μm .

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Detail images of Dll4 staining in Kit+ cells of IAHC of different sizes. Dll4 expression inversely correlates with the size of the cluster: 2-5 cell IAHCs are uniformly Dll4-positive (upper and middle panel), >5 cell IAHCs are partially Dll4 positive (lower panel). Multistack reconstruction of confocal images. Scale bars: 10 μm.


of Kit + Dll4 + using confocal . Bars represent of Dll4 expressing within a cluster in each category of cluster . Mean ± SD ( total 17 clusters analysed , n = 2 ) .

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Quantification of Kit+ Dll4+ cells using confocal images. Bars represent the percentage of Dll4 expressing cells within a cluster in each category of cluster size. Mean±SD (total 17 clusters analysed, n=2).


Overview of Dll4 - ligand expression in embryonic aorta at E10 . 5 . Dll4 is expressed in majority of endothelial layer . Scale bar :

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Overview of Dll4-ligand expression in the embryonic aorta at E10.5. Dll4 is expressed in the majority of the endothelial layer. Scale bar: 30μm


( A ) Whole mount of aorta endothelium stained for CD31 ( in image , black in label ) Kit ( IAHC , cyan ) upon IgG ( left ) or αDll4 antibody treatment ( right ) . Multistack reconstruction of confocal . Red arrows indicate IAHCs on ventral part of aortic wall . Asterisks indicate aspecific autofluorescence . Scale bar : 50μm .

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(A)Whole mount images of the aorta endothelium stained for CD31 (white in image, black in label) and Kit (IAHC, cyan) upon IgG (left) or αDll4 antibody treatment (right). Multistack reconstruction of confocal images. Red arrows indicate IAHCs on the ventral part of the aortic wall. Asterisks indicate aspecific autofluorescence. Scale bar: 50μm.


Representative IAHCs of IgG αDll4 treated . Kit is shown in in image , in black in label . Multistack reconstruction of confocal . Note increased IAHC in right panel . Scale bars : 50μm ( overview ) , 10μm ( magnification ) .

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Representative IAHCs of IgG and αDll4 treated embryos. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Note the increased IAHC size in the right panel. Scale bars: 50μm (overview), and 10μm (magnification).


Frequency of IAHC categories on number of Kit + forming a cluster . of confocal . Bars represent mean ± SE ( n = 6 )

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Frequency of IAHC categories based on the number of Kit+ cells forming a cluster. Quantification of confocal images. Bars represent the mean±SE (n=6)


Number of Kit + per IAHC increases following αDll4 antibody treatment . Each dot represents one cluster . on confocal . Statistical : Mann - Whitney U . * * * * p < 0 . 0001 .

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Number of Kit+ cells per IAHC increases following αDll4 antibody treatment. Each dot represents one cluster. Quantification on confocal images. Statistical analysis: Mann-Whitney U test. ****p< 0.0001.


Total number of IAHCs per 100μm . of confocal . Mean ± SE . Statistical : t - . ns p > 0 . 05 ; ( n = 6 ) . ( F ) of number of Kit + per 100 μm using confocal . Bars represent mean ± SE . Statistical : t - . ns p > 0 . 05 ; * * * p < 0 . 001 ( n = 6 ) .

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Total number of IAHCs per 100μm. Quantification of confocal images. Mean±SE. Statistical analysis: t-test. ns p> 0.05; (n=6). (F) Quantification of the number of Kit+ cells per 100 μm using confocal images. Bars represent the mean±SE. Statistical analysis: t-test. ns p> 0.05; ***p< 0.001 (n=6).


Experimental design : E10 . 5 were injected with IgG or αDll4 , incubated for 5h then dissected . indicated IAHC subpopulations ( kit + CD45 + ; kit + CD45 - ; kit - CD45 - CD41 + were purified was sequenced .

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Experimental design: E10.5 embryos were injected with IgG or αDll4, incubated for 5h and then dissected. The indicated IAHC subpopulations (kit+CD45+; kit+CD45-;kit-CD45-CD41+ were purified and RNA was sequenced.


Experimental design sorting gate for Kit + CD45 - for single RNAseq ( n = 483 ) .

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Experimental design and sorting gate for Kit+CD45- cells for single cell RNAseq (n=483 cells).


Violin plots of Notch family elements expression in IgG αDll4 treated ( adj p val : * < 0 . 1 * * * p < 0 . 00001 ) .

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Violin plots of Notch family elements expression in IgG and αDll4 treated cells (adj p val: *<0.1 and *** p<0.00001).


of in each cycle phase extracted from single - seq . Bars represent average ± SE ( n = 483 ) .

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Percentage of cells in each cell cycle phase extracted from single cell RNA-seq analysis. Bars represent the average±SE (n=483).


Bar graph represents frequency of IAHCs containing recombined per under indicated conditions . of confocal . Mean ± SE ( n = 15 for untreated , n = 7 for IgG αDll4 treated ) .

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Bar graph represents the frequency of IAHCs containing recombined cells per embryo under the indicated conditions. Quantification of confocal images. Mean±SE (n=15 for untreated, n=7 for IgG and αDll4 treated).


Representative of recombined IAHCs following IgG or αDll4 conditions . Kit is shown in in image , in black in label . Multistack reconstruction of confocal . Scale bar : 5μm .

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Representative images of recombined IAHCs following IgG or αDll4 conditions. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 5μm.


classification of IAHC containing recombined in TAM - induced VeCadCreER : Confetti after IgG or αDll4 treatment . treated with αDll4 contain more IAHC with colored . of confocal ( n = 7 ) .

Quantification and classification of IAHC containing recombined cells in TAM-induced VeCadCreER:Confetti embryos after IgG or αDll4 treatment. Embryos treated with αDll4 contain more IAHC with colored cells. Quantification of confocal images (n=7 embryos).


Representative of recombined endothelial in transversal sections of aorta . distribution amount of recombined endothelial is similar in IgG αDll4 conditions . CD31 is shown in in image , in black in label . Multistack reconstruction of confocal . Scale bar : 50μm . of recombined in aortic endothelium using confocal . Mean ± SE ( n = 3 ) .

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Representative images of recombined endothelial cells in transversal sections of the aorta. The distribution and amount of recombined endothelial cells is similar in IgG and αDll4 conditions. CD31 is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 50μm. Quantification of recombined cells in the aortic endothelium using confocal images. Mean±SE (n=3).


Snapshots of Movie EV5 . Time Lapse of embryonic organotypic slice from a CD41 : YFP : H2B - GFP reporter mouse showing recruitment of CD41 : YFP negative ( magenta central point - tracking analyses ) migrating towards IAHC ( CD41 : YFP + ) . Green central point relative tracking identify with non - directional movement . Scale bar : 5μm . Time expressed in hh : mm : ss .

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Snapshots of Movie EV5. Time Lapse of embryonic organotypic slice from a CD41:YFP: H2B-GFP reporter mouse showing the recruitment of CD41:YFP negative cells (magenta central point-tracking analyses) migrating towards the IAHC (CD41:YFP+ cells). Green central point and relative tracking identify cells with non-directional movement. Scale bar: 5μm. Time expressed in hh:mm:ss.


Colony - forming assays using E10 . 5 AGMs derived from treated with IgG or αDll4 .

Colony-forming assays using E10.5 AGMs derived from embryos treated with IgG or αDll4.


FACS analyses of Gfi1 + derived from explants treated with IgG or αDll4 for 5 hours ( C ) . Analyses of hemogenic Gfi1 + Kit - ( D ) Gfi1 + Kit + ( E ) within CD31 + CD45 - population .

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FACS analyses of Gfi1+ cells derived from explants treated with IgG or αDll4 for 5 hours (C). Analyses of the hemogenic Gfi1+Kit-(D) and the Gfi1+Kit+ (E) within the CD31+CD45- population.


of reconstitution ( chimerism determined by CD45 . 1 CD45 . 2 expression ) in peripheral blood ( PB ) . treated with IgG or αDll4 were kept in explant culture for 48hours then transplanted into irradiated recipients . Peripheral blood was analyzed at different time points . Each dot / square represents a single transplanted mouse .

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Percentage of reconstitution (chimerism determined by CD45.1 and CD45.2 expression) in peripheral blood (PB). Embryos treated with IgG or αDll4 were kept in explant culture for 48hours and then transplanted into irradiated recipients. Peripheral blood was analyzed at different time points. Each dot/square represents a single transplanted mouse.


of progenitors ( lin - sca1 + kit + ) HSCs ( Lin - sca1 + kit + CD48 - CD150 + ) in donor population in bone marrow after 4 months of transplantation .

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Analysis of progenitors (lin-sca1+kit+) and HSCs (Lin-sca1+kit+CD48-CD150+) in the donor population in the bone marrow after 4 months of transplantation.


B . DIC microscopic image of WTSOD12SH diluted to 100 µM in HEPES buffer , pH 7 . 4 100 mM NaCl incubated 37 ° C , 180 RPM for 30 min shows no droplet formation ; scale bar : 100 µm ; inset shows Alexa Fluor 488 maleimide labelled WTSOD12SH ;

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B. DIC microscopic image of WTSOD12SH diluted to 100 µM in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated 37 °C, 180 RPM for 30 min shows no droplet formation; scale bar: 100 µm; inset shows Alexa Fluor 488 maleimide labelled WTSOD12SH;


C . Solution turbidity plot ( absorbance at 600 nm ) shows that WTSOD12SH ( at concentrations ranging from 25 to 200 µM does not undergo LLPS in presence or absence of heparin , LLPS inducer ; data is represented as mean ± SD ( from 3 biological replicates ) .

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C. Solution turbidity plot (absorbance at 600 nm) shows that WTSOD12SH (at concentrations ranging from 25 to 200 µM does not undergo LLPS in presence or absence of heparin, LLPS inducer; data is represented as mean ± SD (from 3 biological replicates).


D . DIC microscopic image of ApoSOD12SH under condensate inducing conditions ( 37  C , 180 RPM ) at a of 100 µM incubated 100 mM NaCl ; scale bar : 100 µm ( inset on bottom left shows Alexa Fluor 488 maleimide labeled condensates ;

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D. DIC microscopic image of ApoSOD12SH under condensate inducing conditions (37 C, 180 RPM) at a concentration of 100 µM incubated 100 mM NaCl; scale bar:100 µm (inset on bottom left shows Alexa Fluor 488 maleimide labeled protein condensates;


E . microscopic of Alexa Fluor 488 maleimide labeled ApoSOD12SH condensates diluted in HEPES buffer , pH 7 . 4 100 mM NaCl incubated ( 180 RPM , 37  C ) in absence ( left ) presence ( right ) of heparin at 30 min time -

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E. Fluorescent microscopic images of Alexa Fluor 488 maleimide labeled ApoSOD12SH condensates diluted in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated (180 RPM, 37 C) in absence (left) and presence (right) of heparin at 30 min time-point


F . Solution turbidity measurements with ApoSOD12SH ( absorbance at 600 nm ) shows that while in absence of heparin turbidity increases with time , presence of heparin results in faster LLPS ;

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F. Solution turbidity measurements with ApoSOD12SH (absorbance at 600 nm) shows that while in the absence of heparin the turbidity increases with time, the presence of heparin results in faster LLPS;


G . Comparison of ApoSOD12SH droplet numbers per microscopic field of view ( view 0 . 001963 mm2 ) calculated from 5 different of droplets incubated with without heparin at 2 h timepoint .

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G. Comparison of ApoSOD12SH droplet numbers per microscopic field of view (view area 0.001963 mm2) calculated from 5 different images of droplets incubated with and without heparin at 2 h timepoint.


H . Liquid nature of ApoSOD12SH showing droplet fusion surface

H. Liquid nature of ApoSOD12SH showing droplet fusion and surface wetting


I . Amplitude normalized FCS curves of ApoSOD12SH ( in blue ) of dilute phase ( DP ) condensed phase ( CP ) . Intense color indicates CP variant indicates DP . increase in τD ( as shown by arrow ) translates to a decrease in

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I. Amplitude normalized FCS curves of ApoSOD12SH (in blue) of dilute phase (DP) and condensed phase (CP). Intense color indicates CP and light variant indicates DP. The increase in τD (as shown by the arrow) translates to a decrease in D


J . Scheme depicting that presence of Zn not Cu in pre - incubation mixture inhibits LLPS . K . DIC microscopic of ApoSOD12SH in presence of Zn ( top ) showing no droplet formation Cu ( below ) showing presence of

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J. Scheme depicting that presence of Zn and not Cu in pre-incubation mixture inhibits LLPS. K. DIC microscopic images of ApoSOD12SH in presence of Zn (top) showing no droplet formation and Cu (below) showing the presence of droplets


L . Solution turbidity plot ( absorbance at 600 nm ) of ApoSOD12SH subjected to increasing concentrations of Cu Zn ; turbidity decreases in a dose dependent manner for Zn while no significant change is observed in presence of Cu ;

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L. Solution turbidity plot (absorbance at 600 nm) of ApoSOD12SH subjected to increasing concentrations of Cu and Zn; turbidity decreases in a dose dependent manner for Zn while no significant change is observed in presence of Cu;


A . DIC microscopic of I113T SOD12SH ( top ) G85R SOD12SH ( bottom ) droplets incubated in absence presence of Zn Cu respectively ( scale bar : 100 µm ) ; insets on bottom left show Alexa Fluor 488 maleimide labeled condensates for I113T SOD12SH G85R SOD12SH respectively ; scale bar : 5 µm . Droplet dissolution was observed for I113T SOD12SH on addition of Zn while droplets persisted on addition of Cu . Red arrowhead indicates fusion of liquid droplets .

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A. DIC microscopic images of I113T SOD12SH (top) and G85R SOD12SH (bottom) droplets incubated in the absence and presence of Zn and Cu respectively (scale bar: 100 µm); insets on bottom left show Alexa Fluor 488 maleimide labeled protein condensates for I113T SOD12SH and G85R SOD12SH respectively; scale bar: 5 µm. Droplet dissolution was observed for I113T SOD12SH on addition of Zn while droplets persisted on addition of Cu. Red arrowhead indicates fusion of liquid droplets.


B . Amplitude normalized FCS curves of I113T SOD12SH ( in green ) G85R SOD12SH ( in grey ) of dilute phase ( DP ) condensed phase ( CP ) showing an increase in diffusion time from DP to CP . intense color indicates CP variant indicates DP .

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B. Amplitude normalized FCS curves of I113T SOD12SH (in green) and G85R SOD12SH (in grey) of dilute phase (DP) and condensed phase (CP) showing an increase in diffusion time from DP to CP. The intense color indicates CP and light variant indicates DP.


C . On metal cofactor removal , ApoG37R SOD12SH formed condensates which ceased to exist when was incubated with Zn , while condensates persisted on Cu addition ( left to right ) ;

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C. On metal cofactor removal, ApoG37R SOD12SH formed condensates which ceased to exist when protein was incubated with Zn, while condensates persisted on Cu addition (left to right);


B . Plot of loge of hydrodynamic radius ( rH ) versus log e of number of residues in polypeptide chain . fitted to these data for native folded ( yellow dashed ) has a slope of 0 . 29 ± 0 . 02 a y - axis intercept of 1 . 56 ± 0 . 1 , while other fitted to chemically denatured ( grey dashed ) data has a slope of 0 . 57 ± 0 . 02 a y - axis intercept of 0 . 79 ± 0 . 07 . Literature data have been used for folded chemically denatured while we employed FCS to calculate rH of SOD12SH variants with ( solid shapes ) without Zn ( hollow shapes ) . hollow grey circle of G85R SOD12SH is directly under hollow green I113T SOD12SH in log - log plot .

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B. Plot of the loge of the hydrodynamic radius (rH) versus the log e of the number of residues in the polypeptide chain. The line fitted to these data for the native folded proteins (yellow dashed) has a slope of 0.29 ± 0.02 and a y-axis intercept of 1.56 ± 0.1, while the other fitted to the chemically denatured protein (grey dashed) data has a slope of 0.57 ± 0.02 and a y-axis intercept of 0.79 ± 0.07. Literature data have been used for folded and chemically denatured proteins while we employed FCS to calculate the rH of all SOD12SH variants with (solid shapes) and without Zn (hollow shapes). The hollow grey circle of G85R SOD12SH is directly under hollow green I113T SOD12SH in the log-log plot.


C . Variation of diffusion coefficients of de - metalated variants determined from FCS measurement with increasing Zn ( inset shows a comparison of binding constants between mutants Zn ; ApoSOD12SH ApoG37R SOD12SH has a higher binding affinity to Zn than ApoI113T SOD12SH ApoG85R SOD12SH ) .

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C. Variation of diffusion coefficients of de-metalated protein variants determined from FCS measurement with increasing Zn concentration (inset shows a comparison of binding constants between mutants and Zn; ApoSOD12SH and ApoG37R SOD12SH has a higher binding affinity to Zn than ApoI113T SOD12SH and ApoG85R SOD12SH).


D . Disordered / extended conformation content decreases in ApoSOD12SH ApoI113T SOD12SH with addition of Zn calculated from FTIR spectra ;

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D. Disordered/ extended conformation content decreases in ApoSOD12SH and ApoI113T SOD12SH with the addition of Zn calculated from FTIR spectra;


C . Salt - bridge of ApoG85R SOD12SH ( dist . cutoff < 0 . 5 nm = salt - bridge ) .

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C. Salt-bridge analysis of ApoG85R SOD12SH (dist. cutoff <0.5 nm = salt-bridge).


C . Upper panel shows of maturation of Alexa Fluor 488 labeled ApoSOD12SH droplets with time , samples were incubated at 37  C up to ~ 25 hours . are taken at indicated time point : 0 * hour ( time after incubation required for droplet formation ) , 12 hours > 24 hours ; below show magnified scale bar : 5 µm . middle panel shows magnified view of marked region from corresponding on upper panel . Red arrowhead indicates matured ( solid ) portion inside droplet . Lower panel shows coexistence of liquid solid phase inside droplet ; scale bar : 10 µm . plot ( bottom right ) shows distribution of high region within matured droplet .

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C. Upper panel shows fluorescence images of the maturation of Alexa Fluor 488 labeled ApoSOD12SH droplets with time, samples were incubated at 37 C up to ~25 hours. Images are taken at indicated time point: 0* hour (the time after incubation required for droplet formation), 12 hours and >24 hours; images below show magnified scale bar: 5 µm. The middle panel shows magnified view of the marked region from corresponding images on the upper panel. Red arrowhead indicates matured (solid) portion inside the droplet. Lower panel shows coexistence of liquid and solid phase inside droplet; scale bar: 10 µm. Intensity plot (bottom right) shows the distribution of high intensity region within matured droplet.


D . Diffusion coefficient obtained from point FCS measurement at different regions of droplet during maturation course ; 0 * ( x - axis label ) indicates time after incubation required for droplet formation .

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D. Diffusion coefficient obtained from point FCS measurement at different regions of the droplet during maturation course; 0* (x-axis label) indicates the time after incubation required for droplet formation.


E . ThT plot showing aggregation kinetics of SOD12SH variants . ApoSOD12SH , I113T SOD12SH G85R SOD12SH show high ThT .

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E. ThT fluorescence assay plot showing aggregation kinetics of all SOD12SH variants. ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show high ThT fluorescence.


F . AFM of ApoSOD12SH , I113T SOD12SH G85R SOD12SH show fibrillar after prolonged incubation at 37 ℃ , 180 RPM in absence of Zn ( left panel ) ; AFM of ApoSOD12SH , I113T SOD12SH G85R SOD12SH incubated with Zn ( at 1 : 5 : Zn molar ) show presence of oligomers for ApoSOD12SH , I113T SOD12SH short fibrils for G85R SOD12SH ( right panel ) ;

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F. AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show fibrillar aggregates after prolonged incubation at 37 ℃, 180 RPM in absence of Zn (left panel); AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH incubated with Zn (at 1:5 protein: Zn molar ratio) show presence of oligomers for ApoSOD12SH, I113T SOD12SH and short fibrils for G85R SOD12SH (right panel);


G . proliferation of SHSY5Y human neuroblastoma assessed by MTT post 12 h treatment with 5 µM ApoSOD12SH , I113T SOD12SH G85R SOD12SH condensates ( fibrils ) . culture medium ( DMEM ) was used as negative control untreated SHSY5Y were used as positive control .

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G. Cell proliferation of SHSY5Y human neuroblastoma cells assessed by MTT assay post 12 h treatment with 5 µM ApoSOD12SH, I113T SOD12SH and G85R SOD12SH condensates and aggregates (fibrils). Cell culture medium (DMEM) was used as negative control and untreated SHSY5Y cells were used as positive control.


H . Dot plots showing flow cytometric of annexin V propidium iodide ( PI ) of apoptotic SHSY5Y neuroblastoma following a 12 h treatment with 5 µM ApoSOD12SH condensates , I113T SOD12SH condensates , G85R SOD12SH condensates , ApoSOD12SH fibrils , I113T SOD12SH fibrils G85R SOD12SH fibrils respectively ( see Methods for details ) . High cytotoxicity was observed for SOD12SH condensates .

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H. Dot plots showing flow cytometric analysis of annexin V and propidium iodide (PI) staining of apoptotic SHSY5Y neuroblastoma cells following a 12 h treatment with 5 µM ApoSOD12SH condensates, I113T SOD12SH condensates, G85R SOD12SH condensates, ApoSOD12SH fibrils, I113T SOD12SH fibrils and G85R SOD12SH fibrils respectively (see Methods for details). High cytotoxicity was observed for SOD12SH condensates.


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

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


R - loop unwinding in presence of increasing DDX5 . top panel shows a typical image obtained after . bar graph ( bottom ) shows . average is expressed as unwinding , standard error of mean ( S . E . M . ) , n = 4 .

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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 nucleoplasm of U2OS . An average from 3 independent experiments performed in triplicate . In total , ~ 90 for each condition ( 10 / slide 9 slides for each condition ) were taken two per image were quantified . Statistical significance was assessed using one - way ANOVA t - . * : p < 0 . 05 ; * * * * : p < 0 . 0001 .

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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.