C ) Left panel : representative of Western Blot of CD36 , PPARγ GAPDH expression of ACM HC C - MSC extracts in AM supplemented or not with 20μg / ml 13HODE , 5mmol / L NAC or both ( n = 8 biological replicates ) . Right panels : d . a . normalized on house - keeping GAPDH ( Two - Way Anova ) . Data information : mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

C) Left panel: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both (n=8 biological replicates). Right panels: d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.


D ) Left panel : representative of internalization of oxLDL ( red ) in ACM C - MSC treated with scramble siRNA or CD36 siRNA , cultured in AM subjected to 10μg / ml DiI oxLDL treatment . Right panel : of DiI normalized on nuclei number ( n = 3 biological replicates , arbitrary units ; Two - Student ' s t - ) . Data information : mean ± . * p < 0 . 05 ; * * p < 0 . 01 .

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 B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O

D) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC treated with scramble siRNA or CD36 siRNA, cultured in AM and subjected to 10μg/ml DiI oxLDL treatment. Right panel: quantification of the DiI fluorescence normalized on nuclei number (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.


E ) Left panel : representative of internalization of oxLDL ( red ) in ACM C - MSC cultured in AM or AM + 5μM GW9662 subjected to 10μg / ml DiI oxLDL treatment . Nuclei are counterstained with Hoechst33342 ( blue ) . Right panel : of DiI normalized on nuclei number for each sample ( n = 3 biological replicates , arbitrary units ; Two - Student ' s t - ) . Data information : mean ± . * p < 0 . 05 ; * * p < 0 . 01 .

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 B-CONTROLLED_VAR 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

E) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC cultured in AM or AM+5μM GW9662 and subjected to 10μg/ml DiI oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Right panel: quantification of the DiI fluorescence normalized on nuclei number for each sample (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.


F ) Left panel : representative of Western Blot of CD36 GAPDH expression of extracts of ACM C - MSC cultured in AM or AM + 5μM GW9662 . Right panels : d . a . normalized on house - keeping GAPDH ( n = 7 biological replicates ; Two - Student ' s t - ) . Data information : mean ± . * p < 0 . 05 ; * * p < 0 . 01 .

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 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

F) Left panel: representative images of Western Blot of CD36 and GAPDH expression of protein extracts of ACM C-MSC cultured in AM or AM+5μM GW9662. Right panels: d.a. normalized on the house-keeping GAPDH (n=7 biological replicates; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.


C ) Left panels : representative of ORO of HFD - fed WT Pkp2 + / - cardiac sections . Right panel : of ORO positive ( n = 10 ) . For comparison , of ORO positive of cardiac sections of CD - fed WT Pkp2 + / - mice ( n = 9 ; Two - Way Anova ) is shown ( Appendix Figure S3 ) . D ) Representative of PPARγ ( green ) immunostaining on HFD - fed WT Pkp2 + / - mice cardiac sections ( n = 10 ; Two - Way Anova ) . of PPARγ in HFD is shown relative to values of CD ( n = 9 ) . E ) Representative of MDA ( green ) immunostaining on HFD - fed WT Pkp2 + / - mice cardiac sections ( n = 10 ; Two - Way Anova ) . of MDA in HFD is shown relative to values of CD ( n = 9 ) . F ) Representative of CD36 immunostaining ( green ) on HFD - fed WT Pkp2 + / - mice cardiac sections ( n = 10 ; Two - Way Anova ) . Nuclei are counterstained with Hoechst33342 ( blue ) . of in HFD is shown relative to values of CD ( n = 9 ) . Data information : mean ± . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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 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-MEASURED_VAR 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 B-MEASURED_VAR 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 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 B-MEASURED_VAR 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

C) Left panels: representative images of ORO staining of HFD-fed WT and Pkp2+/- cardiac sections. Right panel: quantification of ORO positive area percentage (n=10). For comparison, quantification of ORO positive area of cardiac sections of CD-fed WT and Pkp2+/- mice (n=9; Two-Way Anova) is shown (Appendix Figure S3). D) Representative images of PPARγ (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the PPARγ staining in HFD is shown relative to the values of CD (n=9). E) Representative images of MDA (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the MDA staining in HFD is shown relative to the values of CD (n=9). F) Representative images of CD36 immunostaining (green) on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Nuclei are counterstained with Hoechst33342 (blue). Quantification of the staining in HFD is shown relative to the values of CD (n=9). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


C ) Left panel : representative of ORO of cardiac sections of Pkp2 + / - mice , fed a 3 - month HFD plus atorvastatin . Right panel : of of ORO positive ( n = 9 ) is compared to that in HFD ( as in Figure 6C ; Two - Student ' s t - ) . D ) Representative of PPARγ immunostaining ( green ) on cardiac sections of Pkp2 + / - mice fed a 3 - month HFD plus atorvastatin ( n = 9 ) . is compared to values of Pkp2 + / - in HFD relative to WT in CD ( as in Figure 6D ; Two - Student ' s t - ) . E ) Representative of MDA immunostaining ( green ) on cardiac sections of Pkp2 + / - mice fed a 3 - month HFD plus atorvastatin ( n = 9 ) . is compared to values of Pkp2 + / - in HFD relative to WT in CD ( as in Figure 6E ; Two - Student ' s t - ) . F ) Representative of CD36 immunostaining ( green ) on cardiac sections of Pkp2 + / - mice fed a 3 - month HFD plus atorvastatin ( n = 9 ) . is compared to values of Pkp2 + / - HFD relative to WT in CD ( as in Figure 6F ; Two - Student ' s t - ) . Nuclei are counterstained with Hoechst33342 ( blue ) . Data information : mean ± . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

C) Left panel: representative images of ORO staining of cardiac sections of Pkp2+/- mice, fed a 3-month HFD plus atorvastatin. Right panel: quantification of the percentage of ORO positive area (n=9) is compared to that in HFD (as in Figure 6C; Two-tailed Student's t-test). D) Representative images of PPARγ immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6D; Two-tailed Student's t-test). E) Representative images of MDA immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6E; Two-tailed Student's t-test). F) Representative images of CD36 immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- HFD and relative to WT in CD (as in Figure 6F; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


( A ) E7 . 0 whole transverse section ( TSs ) ( dashed lines ) through ( a1 ) extra - embryonic tissue showing expression in a ring of extra - embryonic ectoderm ( ee ) extraembryonic mesoderm ( em ) absence of transcripts in allantois ( al ) ; ( a2 ) revealing expression in epiblast ( ep ) in mesendoderm ( me ) emerging from primitive streak ( ps ) , dashed box ( a2 ' ) defines higher magnification region shown in a2 . ( B ) E8 . 5 whole dorsal view TSs ( dashed lines ) showing expression in : ( b1 , b2 , b3 ) open anterior neural plate ( future fore , mid hindbrain ) including ( b3 ) neural crest ( nc ) emerging from hindbrain ; ( b4 ) in dorsal neural tube ( prospective neural crest ) dorsal somites ( s ) ; ( b5 ) in closing preneural tube neural ( pnt ) epithelising somites ; ( b6 ) in caudal lateral epiblast ( cle ) adjacent to node ( n ) at anterior tip of primitive streak , but not in presomitic mesoderm ( psm ) . arrows indicate open anterior posterior neural plate . ( C ) E9 . 5 whole side view TSs ( dashed lines ) showing expression in : ( c1 ) forebrain ( fb ) hindbrain , including optic vesicles ( opv ) , ( c1 ' ) seen at high magnification ( indicated by box grey dashed ) otic vesicles ( ov ) ( c1 ' ' ) seen at high magnification ( box grey dashed ) ; ( c2 ) high expression in dorsal hindbrain ( hb ) ( c3 ) spinal cord ( sc ) dermamyotome ( dm ) of differentiating somite ; ( c5 ) throughout open posterior neural plate . ( D ) High magnification of E9 . 5 whole showing expression in first branchial arch ( ba ) forming cranial ganglia , which are just becoming apparent , ( V ) , ( VII ) , ( VIII ) ( IX ) ( X ) . OV , otic vesicle . ( E ) Expression in mesenchyme underlying apical ectodermal ridge ( aer ) in progress zone ( pz ) in zone of polarising activity ( zpa ) of forelimb ( fl ) . Data information : representative of n ≥ 4 for each stage . Scale bars 200 µm , except sections 100 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) E7.0 whole embryo and transverse section (TSs) (dashed lines) through (a1) extra-embryonic tissue showing expression in a ring of extra-embryonic ectoderm (ee) and extraembryonic mesoderm (em) and absence of transcripts in the allantois (al); (a2) revealing expression in epiblast (ep) and in mesendoderm (me) emerging from the primitive streak (ps), dashed box (a2') defines higher magnification region shown in a2. (B) E8.5 whole embryo dorsal view and TSs (dashed lines) showing expression in: (b1,b2,b3) the open anterior neural plate (future fore, mid and hindbrain) including (b3) neural crest (nc) emerging from hindbrain; (b4) in the dorsal neural tube (prospective neural crest) and dorsal somites (s); (b5) in closing preneural tube neural (pnt) and epithelising somites; (b6) in caudal lateral epiblast (cle) adjacent to the node (n) at the anterior tip of the primitive streak, but not in presomitic mesoderm (psm). White arrows indicate open anterior and posterior neural plate . (C) E9.5 whole embryo side view and TSs (white dashed lines) showing expression in: (c1) forebrain (fb) and hindbrain , including optic vesicles (opv), (c1') seen at high magnification (and indicated by box grey dashed line) and otic vesicles (ov) (c1'') seen at high magnification (box grey dashed line); (c2) high expression in dorsal hindbrain (hb) and (c3) spinal cord (sc) and dermamyotome (dm) of the differentiating somite; (c5) throughout open posterior neural plate. (D) High magnification of E9.5 whole embryo showing expression in first branchial arch (ba) and forming cranial ganglia, which are just becoming apparent, (V), (VII), (VIII) (IX) and (X). OV, otic vesicle. (E) Expression in mesenchyme cells underlying the apical ectodermal ridge (aer) in the progress zone (pz) and in the zone of polarising activity (zpa) of the forelimb (fl). Data information: Images representative of n≥ 4 embryos for each stage. Scale bars 200 µm, except sections 100 µm.


( A - H ) Live wildtype littermate Slc7a5 - null imaged shortly after dissection . ( A - D ) Wildtype ( E - H ) Slc7a5 - null E9 . 75 from lateral ( A , D , E , H ) , frontal ( B , F ) or dorsal ( C , G ) views ; ( e1 - h1 ) higher magnification of Slc7a5 - null . arrowheads indicate smaller limb bud ( compare B F ) , open / reduced forebrain ( compare D H ) , open neural tube at posterior ( compare C E , e1 ) anterior ( compare B F , f1 ) regions . Black arrowheads indicate kinked neural tube ( compare C G , g1 ) apparently missing optic smaller otic vesicles ( ov ) ( compare D h1 ) . Data information : Scale bars 200

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 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 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

(A-H) Live wildtype littermate and Slc7a5-null embryos imaged shortly after dissection. (A-D) Wildtype and (E-H) Slc7a5-null E9.75 embryos from lateral (A, D, E, H), frontal (B, F) or dorsal (C, G) views; (e1-h1) higher magnification images of the Slc7a5-null embryo. White arrowheads indicate the smaller limb bud (compare B and F), open/reduced forebrain (compare D and H), open neural tube at posterior (compare C and E, e1) and anterior (compare B and F, f1) regions. Black arrowheads indicate kinked neural tube (compare C and G, g1) and apparently missing optic and smaller otic vesicles (ov) (compare D and h1). Data information: Scale bars 200 µm


mRNA in situ hybridization immunofluorescence in E9 . 5 or E10 . 5 wildtype Slc7a5 - null for key marker genes . ( I - L ) Fgf8 mRNA transcripts were detected in wildtype ( I - j1 ) in Slc7a5 - null ( K - l1 ) E10 . 5 ( n = 4 each ) ( arrowheads indicate limb buds in K isthmus in L ) with ( j1 , l1 ) sections through isthmus at midbrain / hindbrain border ( i1 , k1 ) lateral views of forelimb buds . Data information : Scale bars 200 µm , except sections 100 µm .

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 B-MEASURED_VAR 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

mRNA in situ hybridization and immunofluorescence in E9.5 or E10.5 wildtype and Slc7a5-null embryos for key marker genes. (I-L) Fgf8 mRNA transcripts were detected in wildtype (I-j1) and in Slc7a5-null (K-l1) E10.5 embryos (n=4 each) (white arrowheads indicate limb buds in K and the isthmus in L) with (j1, l1) sections through isthmus at midbrain/hindbrain border and (i1, k1) lateral views of forelimb buds. Data information: Scale bars 200 µm, except sections 100 µm.


mRNA in situ hybridization immunofluorescence in E9 . 5 or E10 . 5 wildtype Slc7a5 - null for key marker genes . ( M - N ) Wholemount Tubulin - β - III ( Tuj1 ) immunofluorescence performed on ( M , m1 ) wildtype ( N , n1 ) Slc7a5 - null E9 . 5 - E10 . 5 ( n = 5 each condition ) . Dorsal root ganglia ( DRG ) Sympathetic Chain Ganglia ( SCG ) are indicated with open arrowheads together with cranial ganglia IX , X XI . in ( m1 , n1 ) show higher magnification of cranial ganglia V , VII / VIII ( open arrowheads ) . Data information : Scale bars 200

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 B-MEASURED_VAR I-MEASURED_VAR I-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 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

mRNA in situ hybridization and immunofluorescence in E9.5 or E10.5 wildtype and Slc7a5-null embryos for key marker genes. (M-N) Wholemount Tubulin-β-III (Tuj1) immunofluorescence performed on (M, m1) wildtype and (N, n1) Slc7a5-null E9.5-E10.5 embryos (n=5 each condition). Dorsal root ganglia (DRG) and Sympathetic Chain Ganglia (SCG) are indicated with open arrowheads together with cranial ganglia IX, X and XI. Images in (m1, n1) show higher magnification of the cranial ganglia V, VII/VIII (open arrowheads). Data information: Scale bars 200 µm


mRNA in situ hybridization immunofluorescence in E9 . 5 or E10 . 5 wildtype Slc7a5 - null for key marker genes . ( O - P ) Neurog2 expression in wildtype ( O - o4 ) Slc7a5 - null ( P - p4 ) ( n = 5 n = 6 each ) , showing reduced expression in forebrain ( o2 , p2 ) , cranial ganglia ( o1 , o3 , p1 , p3 , arrowheads ) in spinal cord at level of forelimb ( o4 , p4 , arrowheads indicate position of neural crest ) . Data information : Scale bars 200 µm , except sections 100 µm .

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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA in situ hybridization and immunofluorescence in E9.5 or E10.5 wildtype and Slc7a5-null embryos for key marker genes. (O-P) Neurog2 expression in wildtype (O-o4) and Slc7a5-null embryos (P-p4) (n= 5 and n=6 each), showing reduced expression in forebrain (o2, p2), cranial ganglia (o1, o3, p1, p3, arrowheads) and in spinal cord at level of forelimb (o4, p4, arrowheads indicate position of neural crest). Data information: Scale bars 200 µm, except sections 100 µm.


( A - B ) Sox10 in situ hybridization in ( A ) wildtype ( B ) Slc7a5 - null E9 . 5 ( n = 4 each ) with TSs of ( a1 , b1 ) hindbrain at level of trigeminal ganglion V , ( a2 , b2 ) otic vesicles neural tube at more posterior ( a3 , a4 , b3 , b4 ) . Scale bars 200 µm , except sections a1 - b4 , 100 µm . Closed arrowheads in ( a3 , a4 ) indicate neural crest , open arrowheads indicate depleted neural crest in ( b3 ) position where neural crest should be in ( b4 ) .

O O O O O B-MEASURED_VAR 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

(A-B) Sox10 in situ hybridization in (A) wildtype and (B) Slc7a5-null E9.5 embryos (n=4 each) with TSs of (a1, b1) the hindbrain at level of trigeminal ganglion V, (a2, b2) otic vesicles and the neural tube at more posterior levels (a3, a4, b3, b4). Scale bars 200 µm, except sections a1 - b4, 100 µm. Closed white arrowheads in (a3, a4) indicate neural crest, open white arrowheads indicate depleted neural crest in (b3) and position where neural crest should be in (b4).


( C - G ) Immunofluorescence on TSs caudal spinal cord of E9 . 5 wildtype Slc7a5 - null ; Pax3 FoxA2 were used as indicators for dorso - ventral organisation in ( C , F ) wildtype , n = 2 ( for FoxA2 ) n = 4 ( For Pax3 ) ( D , G ) Slc7a5 - null , n = 3 ( for FoxA2 ) n = 3 for Pax3 ) neural tube . Arrowheads indicate border of Pax3 expression domain . Scale bars 50 µm . ( E ) of Pax3 expressing was determined by counting these DAPI labelled nuclei in neural tube comparison made between wildtype ( 4 , 6 sections each ) Slc7a5 - null ( 3 , 6 sections each ) , each dot represents average for one , unpaired t - , * p = 0 . 018 ( see original source data ) . Error bars indicate .

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

(C-G) Immunofluorescence on TSs caudal spinal cord of E9.5 wildtype littermates and Slc7a5-null embryos; Pax3 and FoxA2 were used as indicators for dorso-ventral organisation in (C, F) wildtype, n=2 (for FoxA2) and n= 4 (For Pax3) and (D, G) Slc7a5-null, n=3 (for FoxA2 ) and n = 3 for Pax3) neural tube. Arrowheads indicate border of Pax3 expression domain. Scale bars 50 µm. (E) Percentage of Pax3 expressing cells was determined by counting these cells and all DAPI labelled nuclei in the neural tube and comparison made between wildtype (4 embryos, 6 sections each) and Slc7a5-null (3 embryos, 6 sections each), each dot represents the average for one embryo, unpaired t-test, *p = 0.018 (see original source data). Error bars indicate SEM.


( A - D ) Proliferation was assessed in spinal cord ( forelimb level ) ( A , B ) using a phospho - Histone3 ( phospho - H3 ) antibody ( green ) to identify mitotic DAPI ( blue ) to label nuclei ; were counted in 3 wildtype ( 18 sections ) 3 Slc7a5 - null ( 18 sections ) mitotic index calculated ; ( C , D ) Proliferation was also assessed in forebrain in 3 wildtype ( 17 sections ) 3 Slc7a5 - null ( 16 sections ) . dashed lines indicate midline . Data information scale 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 B-MEASURED_VAR 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-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 O O O O O O O O O O O O O O O O O O

(A-D) Proliferation was assessed in the spinal cord (forelimb level) (A, B) using a phospho-Histone3 (phospho-H3) antibody (green) to identify mitotic cells and DAPI (blue) to label nuclei; cells were counted in 3 wildtype (18 sections) and 3 Slc7a5-null embryos (18 sections) and mitotic index calculated; (C, D) Proliferation was also assessed in the forebrain in 3 wildtype embryos (17 sections) and 3 Slc7a5-null embryos (16 sections). White dashed lines indicate midline. Data information All scale bars 50 µm.


( E , F ) Comparison of mitotic index in wildtype Slc7a5 - null ( E ) spinal cord , p = 0 . 872 , ( F ) forebrain * p = 0 . 049 , each dot represents average for one , unpaired t - Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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(E, F) Comparison of mitotic index in wildtype and Slc7a5-null (E) spinal cord, p = 0.872 ,and (F) forebrain *p = 0.049, each dot represents the average for one embryo, unpaired t-test Data information: *p<0.05, **p<0.01, ***p<0.001


( G , H ) mTORC1 activity in spinal cord ( hindlimb level ) was assessed by immunofluorescence using an antibody against phospho - ribosomal S6 ( phosho - S6 Ser 235 / 236 ) . Data information scale bars 50 µm .

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(G, H) mTORC1 activity in the spinal cord (hindlimb level) was assessed by immunofluorescence using an antibody against the phospho-ribosomal protein S6 (phosho-S6 Ser 235/236). Data information All scale bars 50 µm.


( I ) Labelling was measured plotted relative to DAPI ; 6 wildtype ( 18 sections ) 5 Slc7a5 - null ( 19 sections ) . Each dot represents average for one , ( * p = 0 . 0457 ) unpaired t - with Welch correction Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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(I) Labelling intensity was measured and plotted relative to DAPI intensity; 6 wildtype embryos (18 sections) and 5 Slc7a5-null embryos (19 sections). Each dot represents the average for one embryo, (*p = 0.0457) unpaired t-test with Welch correction Data information: *p<0.05, **p<0.01, ***p<0.001


( J ) Western blot of E9 . 5 wildtype ( n = 3 ) Slc7a5 - null ( n = 4 ) lysates immunoblotted using an antibody against phospho - S6K ( Thr389 ) total P70S6K for loading control . Band intensities were measured with FIJI an unpaired t - was performed for statistical Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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(J) Western blot of individual E9.5 wildtype (n=3) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-S6K (Thr389) and total P70S6K for loading control. Band intensities were measured with FIJI and an unpaired t-test was performed for statistical analysis Data information: *p<0.05, **p<0.01, ***p<0.001


( K - L ) ( K ) Validation by qPCR of targets identified by RNAseq ; qPCR was performed on E8 . 5 of each genotype ( wildtype n = 5 , heterozygous n = 9 Slc7a5 - null n = 7 ) . ( L ) qPCR was performed on E9 . 5 ( wildtype n = 5 , heterozygous n = 3 Slc7a5 - null n = 4 ) using primers specific for genes associated with integrated stress response . A one - way Anova with a Turkey post - was performed . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 Error bars indicate .

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(K-L) (K) Validation by qPCR of the targets identified by RNAseq; qPCR was performed on individual E8.5 embryos of each genotype (wildtype n=5, heterozygous n=9 and Slc7a5-null n=7). (L) qPCR was performed on individual E9.5 embryos (wildtype n=5, heterozygous n=3 and Slc7a5-null n=4) using primers specific for genes associated with the integrated stress response. A one-way Anova test with a Turkey post-test was performed. Data information: *p<0.05, **p<0.01, ***p<0.001 Error bars indicate SEM.


mRNA in situ hybridization was performed at E9 . 5 to detect Chac1 in ( A - a6 ) wildtype ( B - b6 ) Slc7a5 - null littermate ( n = 3 for each condition ) ; Chac1 expression at E10 in emerging limb buds assessed in ( C ) wildtype ( D ) Slc7a5 - null littermate ( n = 2 for each condition ) Scale bars 200 µm for whole 100 µm for sections .

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mRNA in situ hybridization was performed at E9.5 to detect Chac1 in (A-a6) wildtype and (B-b6) Slc7a5-null littermate embryos (n=3 for each condition); Chac1 expression at E10 in emerging limb buds assessed in (C) wildtype and (D) Slc7a5-null littermate embryos (n=2 for each condition) Scale bars 200 µm for whole embryo images and 100 µm for sections.


mRNA in situ hybridization was performed at E9 . 5 to detect Trib3 mRNA detected in ( E - e6 ) wildtype ( F - f6 ) Slc7a5 - null littermate ( n = 3 for each condition ) in emerging limb buds at E10 . 5 assessed in ( G ) wildtype ( H ) Slc7a5 - null littermate ( n = 2 for each condition ) . Scale bars 200 µm for whole 100 µm for sections .

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mRNA in situ hybridization was performed at E9.5 to detect Trib3 mRNA detected in (E-e6) wildtype and (F-f6) Slc7a5-null littermate embryos (n=3 for each condition) and in emerging limb buds at E10.5 assessed in (G) wildtype and (H) Slc7a5-null littermate embryos (n=2 for each condition). Scale bars 200 µm for whole embryo images and 100 µm for sections.


mRNA in situ hybridization was performed at E9 . 5 Chac1 Trib3 transcripts were also detected in wildtype E9 . 5 after > 3 days in situ hybridisation reaction ( n = 3 n = 6 respectively ) . Lateral view ( I K ) , optic vesicles ( arrowheads ) , branchial arches ( ba ) otic vesicles ( ov ) . Dorsal view ( J L ) , neural tube ( nt ) limb buds ( arrowheads ) . Scale bars 200 µm for whole 100 µm for sections .

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mRNA in situ hybridization was performed at E9.5 Chac1 and Trib3 transcripts were also detected in wildtype E9.5 embryos after >3 days in situ hybridisation reaction (n=3 and n=6 respectively). Lateral view (I and K), optic vesicles (white arrowheads), branchial arches (ba) and otic vesicles (ov). Dorsal view (J and L), neural tube (nt) and limb buds (white arrowheads). Scale bars 200 µm for whole embryo images and 100 µm for sections.


Western blot of E9 . 5 wildtype ( n = 4 ) Slc7a5 - null ( n = 4 ) lysates immunoblotted using an antibody against phospho - GCN2 ( Thr899 ) total GCN2 ( loading control ) , * * * p = 0 . 0001 ; band intensities measured with FIJI analysed with unpaired t - Data information : error bars indicate .

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Western blot of individual E9.5 wildtype (n=4) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-GCN2 (Thr899) and total GCN2 (loading control), ***p = 0.0001 ; band intensities measured with FIJI and analysed with unpaired t-test Data information: All error bars indicate SEM.


Western blot of E9 . 5 wildtype ( n = 4 ) Slc7a5 - null ( n = 4 ) lysates immunoblotted using an antibody against phospho - eIF2α ( Ser51 ) total eIFα ( loading control ) , * p = 0 . 0482 , band intensities measured with FIJI analysed with unpaired t - Data information : error bars indicate .

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Western blot of individual E9.5 wildtype (n=4) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-eIF2α (Ser51) and total eIFα (loading control), *p = 0.0482, band intensities measured with FIJI and analysed with unpaired t-test Data information: All error bars indicate SEM.


( C - F ) phosphorylated eIFα was assessed by immunofluorescence in E9 . 5 in forebrain , wildtype ( C , c1 ) ( n = 2 , 0 / 14 sections ) Slc7a5 - null ( D , d1 ) ( n = 2 16 / 16 sections ) , in hindbrain / anterior spinal cord , wildtype ( E - e2 ) ( n = 4 , 0 / 62 sections ) Slc7a5 - null ( F - f2 ) ( n = 4 , 54 / 54 sections ) including in otic vesicle ( compare e2 , f2 ) . Scale bars in ( C - e1 , D - f1 ) 100 µm in ( e2 f2 ) 25µm . Arrowheads indicate ventral midline .

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(C-F) phosphorylated eIFα was assessed by immunofluorescence in E9.5 embryos in the forebrain, wildtype (C,c1) (n=2 embryos, 0/14 sections) and Slc7a5-null (D, d1) (n=2 embryos 16/16 sections), and in hindbrain/anterior spinal cord, wildtype (E-e2) (n= 4 embryos, 0/62 sections) and Slc7a5-null (F-f2) (n= 4 embryos, 54/54 sections) including in the otic vesicle (compare e2, f2). Scale bars in (C-e1, D-f1) 100 µm and in (e2 and f2) 25µm. Arrowheads indicate ventral midline.


( G ) An increase in Trib3 was detected by Western blot in Slc7a5 - null , each lane represents an E9 . 5 lysate ( wildtype n = 3 , Slc7a5 - null , n = 4 ) with α - Tubulin loading control , unpaired t - * p = 0 . 0153 Data information : error bars indicate .

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(G) An increase in Trib3 protein was detected by Western blot in Slc7a5-null embryos, each lane represents an E9.5 embryo lysate (wildtype n=3, Slc7a5-null, n=4) with α-Tubulin loading control, unpaired t-test *p = 0.0153 Data information: All error bars indicate SEM.


( H - L ) TUNEL to detect apoptotic in wildtype ( n = 3 , 32 sections ) or Slc7a5 - null ( n = 5 , 47 sections ) ; ( H , I ) transverse sections through head at level of forebrain ( J , K ) spinal cord , ( H - K ) scale bars 50 µm , arrowheads indicate ventral midline . ( L ) of TUNEL positive within spinal cord sections was performed Each dot represents average apoptosis for a single , Welch ' s correction unpaired t - , p = 0 . 250 , F - to compare variances , p = 0 . 0003 Data information : error bars indicate .

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(H-L) TUNEL assay to detect apoptotic cells in wildtype (n=3 embryos, 32 sections) or Slc7a5-null (n=5 embryos, 47 sections); (H, I) transverse sections through the head at level of the forebrain and (J, K) the spinal cord, (H-K) scale bars 50 µm, arrowheads indicate ventral midline. (L) Quantification of TUNEL positive cells within spinal cord sections was performed Each dot represents the average apoptosis cell count for a single embryo, Welch's correction unpaired t-test, p = 0.250, F-test to compare variances, p = 0.0003 Data information: All error bars indicate SEM.


( A - B ) ( A , a1 ) Wnt β - catenin target Axin2 strongly expressed in littermate , ( a2 , a3 ) in TS , ( B , b1 ) in Scl7a5 - null ( b2 , b3 ) in TS ( n = 2 , n = 6 / 6 Slc7a5 - null ) . Data information : dashed lines indicate position of sections . Scale bars 200

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(A-B) (A,a1) Wnt β-catenin target Axin2 strongly expressed in littermate, (a2, a3) in TS, and (B, b1) in Scl7a5-null embryos (b2, b3) in TS (n=2 littermates, n=6/6 Slc7a5-null embryos). Data information: White dashed lines indicate position of sections. Scale bars 200 µm


( C , d1 ) Axin2 transcripts in trunk explant exposed to DMSO ( C , c1 , n = 12 / 12 ) were reduced in Wnt - c59 exposed explants ( D , d1 , n = 14 / 14 ) . in ( c1 , d1 ) show TS through explants ( data from 3 independent explant experiments ) . ( E , F ) Slc7a5 transcripts in trunk explant exposed to DMSO ( E , e1 , n = 9 / 10 ) or Wnt - c59 ( F , f1 , n = 15 / 15 ) . in ( e1 , f1 ) show TS through explants ( data from 3 independent explant experiments ) . Data information : Scale bars = 100

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(C, d1) Axin2 transcripts in embryo trunk explant exposed to DMSO (C, c1, n=12/12) were reduced in Wnt-c59 exposed explants (D, d1, n=14/14). Images in (c1, d1) show TS through explants (data from 3 independent embryo explant experiments). (E, F) Slc7a5 transcripts in embryo trunk explant exposed to DMSO (E, e1, n=9/10) or Wnt-c59 (F, f1, n=15/15). Images in (e1, f1) show TS through explants (data from 3 independent embryo explant experiments). Data information: Scale bars = 100 µm


( G , H ) Slc7a5 expression in control T - Cre ; Ctnnb1flLOF / Δ heterozygous littermate ( G , g1 ) in TS ( g2 , g3 ) , in T - Cre ; Ctnnb1flLOF / Δ mutant where Slc7a5 transcripts are absent in posterior neural tube ( region of T - cre recombination , indicated with arrowhead in H ) shown in TS in ( h2 , h3 ) ( n = 0 / 6 littermate n = 6 / 6 mutant ) . Data information : dashed lines indicate position of sections . Scale bars , 200

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(G, H) Slc7a5 expression in control T-Cre;Ctnnb1flLOF/Δ heterozygous littermate embryo (G, g1) and in TS (g2, g3), and in T-Cre;Ctnnb1flLOF/Δ mutant embryo where Slc7a5 transcripts are absent in posterior neural tube (the region of T-cre recombination, indicated with white arrowhead in H) and shown in TS in (h2, h3) (n= 0/6 littermate and n=6/6 mutant embryos). Data information: White dashed lines indicate position of sections. Scale bars, 200 µm


( I , J ) Trib3 is lacking in T - Cre ; Ctnnb1flLOF / Δ heterozygous control at E7 . 75 ( I ) , caudal region dissected from same ( dashed box in I ) ( i1 ) viewed in TS ( i2 ) ; Trib3 detected in homozygous T - Cre ; Ctnnb1flLOF / Δ ( J ) , in caudal region dissected from same ( dashed box in J ) ( j1 ) shown in TS in caudal epiblast remnant primitive streak ( ps ) ( j2 ) ( n = 3 / 3 T - Cre ; Ctnnb1flLOF / Δ 14 littermate controls ) . Trib3 was also detected in more rostral forming neural tube at this early stage ( seen in J ) so in not directly experiencing β - catenin loss ; this may reflect failure to form paraxial mesoderm , which provides signals that promote support neurogenesis . Data information : dashed lines indicate position of sections . Scale bars sections 50 µm .

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(I, J) Trib3 is lacking in T-Cre;Ctnnb1flLOF/Δ heterozygous control embryos at E7.75 (I), caudal region dissected from the same embryo (white dashed box in I) (i1) and viewed in TS (i2); Trib3 detected in homozygous T-Cre;Ctnnb1flLOF/Δ embryo (J), in caudal region dissected from the same embryo (white dashed box in J) (j1) and shown in TS in caudal epiblast and remnant primitive streak (ps) (j2) (n= 3/3 T-Cre;Ctnnb1flLOF/Δ embryos and 14 littermate controls). Trib3 was also detected in the more rostral forming neural tube at this early stage (seen in J) and so in cells not directly experiencing β-catenin loss; this may reflect the failure to form paraxial mesoderm, which provides signals that promote and support neurogenesis. Data information: White dashed lines indicate position of sections. Scale bars all sections 50 µm.


( K - M ) At E9 . 5 Trib3 was not detected in T - Cre ; Ctnnb1flLOF / Δ heterozygous littermate ( K , k1 ) in TS ( k2 , k3 ) , but was detected in homozygous T - Cre ; Ctnnb1flLOF / Δ mutant ( L - m3 ) in patches of in neural tube ( nt ) ( L - l2 ) , gut ( g ) ( l3 ) somites ( s ) ( m2 , arrowheads ) ( n = 9 / 9 T - Cre ; Ctnnb1flLOF / Δ 12 littermate controls ) . Data information : dashed lines indicate position of sections . Scale bars , sections 50 µm .

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(K-M) At E9.5 Trib3 was not detected in T-Cre;Ctnnb1flLOF/Δ heterozygous littermate embryos (K, k1) and in TS ( k2, k3), but was detected in homozygous T-Cre;Ctnnb1flLOF/Δ mutant embryos (L-m3) in patches of cells in neural tube (nt) (L - l2), gut (g) (l3) and somites (s) (m2, arrowheads) (n=9/9 T-Cre;Ctnnb1flLOF/Δ embryos and 12 littermate controls). Data information: White dashed lines indicate position of sections. Scale bars, all sections 50 µm.


Selected BioID REV7 results , shown as dot plots . spectral for each indicated prey are shown as AvgSpec . were selected on iProphet probability of > 0 . 95 , BFDR of < 0 . 05 ≥ 10 peptide . circle represents relative abundance of preys over baits .

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Selected BioID REV7 results, shown as dot plots. The spectral counts for each indicated prey protein are shown as AvgSpec. Proteins were selected based on and iProphet probability of >0.95, BFDR of <0.05 and ≥10 peptide count. The circle size represents the relative abundance of preys over baits.


U2OS EJ5 - GFP were transfected with indicated siRNAs . At 24hr post - transfection , were transfected with I - SceI expression plasmid , GFP + population was analyzed 48 hr post - plasmid transfection . of GFP + was determined for each condition subsequently normalized to non - targeting condition ( siCTRL ) .

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U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).


of Neutral Comet . U2OS stably expressing shCtrl , shREV7 or shSHLD2 were exposed to IR ( 10 Gy ) run in low melting agarose under neutral conditions . Immunofluorescence against stained with SYBR Gold was performed to measure tail moment .

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Quantification of the Neutral Comet assay. U2OS cells stably expressing shCtrl, shREV7 or shSHLD2 were exposed to IR (10 Gy) and run in low melting agarose under neutral conditions. Immunofluorescence against DNA stained with SYBR Gold was performed to measure the tail moment.


U2OS were transfected with indicated siRNA . 48h post - transfection were treated with NCS to induce damage were harvested at 0 , 1 , 2 , 4 24 hr post NCS treatment . Flow Cytometry of phosphorylated - H2AX signal was used to measure γ - H2AX endogenous signal .

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U2OS cells were transfected with the indicated siRNA. 48h post-transfection the cells were treated with NCS to induce DNA damage and the cells were harvested at 0, 1, 2, 4 and 24 hr post NCS treatment. Flow Cytometry analysis of phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal.


Sensitivity to IR monitored by colony formation . MCF - 7 stably expressing indicated shRNAs were exposed to increasing doses of IR . 24 h post - irradiation , were re - seeded to allow colony formation , fixed stained with 0 . 4 % crystal violet . Shown is of colonies per condition which possessed more than 50 colonies .

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Sensitivity to IR monitored by colony formation assay. MCF-7 cells stably expressing the the indicated shRNAs were exposed to increasing doses of IR. 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies.


MCF - 7 stably expressing indicated shRNAs 24 h post - irradiation , were re - seeded to allow colony formation , fixed stained with 0 . 4 % crystal violet . Shown is of colonies per condition which possessed more than 50 colonies . were exposed to increasing doses of UV radiation .

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MCF-7 cells stably expressing the the indicated shRNAs 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies. cells were exposed to increasing doses of UV radiation.


U2OS stably expressing HA - REV7 ( Top ) or HA - SHLD2 ( Bottom ) were pre - sensitized with 10ug / mL Hoescht 33342 before exposed to UV micro - irradiation . Immunofluorescence against endogenous HA γ - H2AX epitope was subsequently performed to monitor REV7 SHLD2 accumulation at sites of damage . Shown are representative . Scale bar = 5µm .

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

U2OS cells stably expressing HA-REV7 (Top) or HA-SHLD2 (Bottom) were pre-sensitized with 10ug/mL Hoescht 33342 before exposed to UV micro-irradiation. Immunofluorescence against endogenous HA and γ-H2AX epitope was subsequently performed to monitor REV7 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.


U2OS LacR - Fok1 were transfected with GFP or GFP - SHLD2 24 h later damage was induced using Shield - 1 4 - OHT . were then processed for GFP mCherry immunofluorescence . Shown are representative . Scale bar = 5µm . of experiments shown in ( C ) . Data are represented as mean ± SD ( n = 3 ) . At least 100 per condition were counted .

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U2OS LacR-Fok1 cells were transfected with GFP or GFP-SHLD2 and 24 h later DNA damage was induced using Shield-1 and 4-OHT. The cells were then processed for GFP and mCherry immunofluorescence. Shown are representative micrographs. Scale bar = 5µm. Quantification of the experiments shown in (C). Data are represented as the mean ± SD (n=3). At least 100 cells per condition were counted.


of experiments shown in ( C ) . Shown is of GFP signal at mCherry - LacR - Fok1 focus .

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

Quantification of the experiments shown in (C). Shown is the quantification of the GFP signal at the mCherry-LacR-Fok1 focus.


293T lines expressing ER - AsiSI with Flag - SHLD2 treated with 1 µM of 4 - OHT . 6h later were processed immunoprecipitated with Anti - FLAG Magnetic Beads anti - γ - H2AX . x / A / G magnetic beads . was purified subjected to qPCR detection . Shown is of IP efficiency as of precipitated from input ( Bottom ) .

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

293T cell lines expressing ER-AsiSI with Flag-SHLD2 and treated with 1 µM of 4-OHT. 6h later the cells were processed and immunoprecipitated with Anti-FLAG Magnetic Beads and anti-γ-H2AX.x/Protein A/G magnetic beads. DNA was purified and subjected to qPCR detection. Shown is the quantification of IP efficiency as the percentage of DNA precipitated from input (Bottom).


U2OS stably expressing HA - SHLD2 ∆ 720 - 904 ( Left ) or HA - SHLD2 ∆ 1 - 680 ( Right ) were processed as in ( B ) . Immunofluorescence against endogenous HA RPA32 epitope was subsequently performed to monitor RPA32 SHLD2 accumulation at sites of damage . Shown are representative . Scale bar = 5µm .

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U2OS cells stably expressing HA-SHLD2∆720-904 (Left) or HA-SHLD2∆1-680 (Right) were processed as in (B). Immunofluorescence against endogenous HA and RPA32 epitope was subsequently performed to monitor RPA32 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.


U2OS mCherry - LacR - Fok1 were treated with indicated siRNA subsequently transfected with a GFP - SHLD2 construct . 24 h post - transfection damage was induced using Shield - 1 4 - OHT . were then fixed analyzed for of GFP - SHLD2 signal at mCherry - LacR - Fok1 focus . Shown is of GFP - SHLD2 signal at Fok1 focus . Data are represented as a box - - whisker plot where whiskers represent 10 - 90 percentile . At least 75 were counted per condition .

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

U2OS mCherry-LacR-Fok1 cells were treated with the indicated siRNA and subsequently transfected with a GFP-SHLD2 construct. 24 h post-transfection DNA damage was induced using Shield-1 and 4-OHT. The cells were then fixed and analyzed for the intensity of the GFP-SHLD2 signal at mCherry-LacR-Fok1 focus. Shown is the quantification of the GFP-SHLD2 signal at the Fok1 focus. Data are represented as a box-and-whisker plot where the whiskers represent the 10-90 percentile. At least 75 cells were counted per condition.


293T were transfected with Flag - REV7 GFP - SHLD2 expression vectors as indicated . 24h post - transfection were treated with DMSO or with 10uM of ATM inhibitor KU - 60019 for 1 h prior to irradiation . 1h post - irradiation ( 10 Gy ) extracts were prepared REV7 complexes were immunoprecipitated using anti - Flag ( M2 ) Resin then analyzed by immunoblotting using GFP , REV7 p - Chk1 antibodies .

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293T cells were transfected with Flag-REV7 and GFP-SHLD2 expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP, REV7 and p-Chk1 antibodies.


CH12F3 - 2 stably expressing indicated shRNAs were stimulated with a cocktail of cytokines ( CIT ) to induce class switching to IgA . of IgA + was monitored 24 48h post - stimulation by with an anti - IgA antibody followed by flow cytometry .

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

CH12F3-2 cells stably expressing the indicated shRNAs were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis.


U2OS DR - GFP were transfected with indicated siRNAs . At 24hr post - transfection , were transfected with I - SceI expression plasmid , GFP + population was analyzed 48 hr post - plasmid transfection . of GFP + was determined for each condition subsequently normalized to non - targeting condition ( siCTRL ) .

U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).


U2OS were transfected with indicated siRNAs then treated with CldU , IdU NCS 48h post - transfection as indicated . slides were stained , dehydrated , mounted visualized shown is of CldU / IdU tract in order to visualize end - resection ( Right ) . At least 500 tracks were measured per condition .

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

U2OS cells were transfected with the indicated siRNAs and then treated with CldU, IdU and NCS 48h post-transfection as indicated. The slides were stained, dehydrated, mounted and visualized and shown is the quantification of CldU/IdU tract length in order to visualize DNA end-resection (Right). At least 500 DNA tracks were measured per condition.


Genes significantly enriched or dropped out after a 14 - day treatment with Doxorubicin were identified by plotting as a Log2 fold change compared to untreated . Ranking was determined on Log2 fold score ( Left ) . top ten Doxorubicin sensitizers are indicated on right with their respective fold change ( Fc ) in Log2 .

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

Genes significantly enriched or dropped out after a 14-day treatment with Doxorubicin were identified by plotting as a Log2 fold change compared to untreated. Ranking was determined based on the Log2 fold score (Left). The top ten Doxorubicin sensitizers are indicated on the right with their respective fold change (Fc) in Log2.


293T were transfected with Flag - REV7 GFP - SHLD1 ( Left ) or Flag - SHLD2 GFP - SHLD1 ( Right ) expression vectors as indicated . 24h post - transfection were treated with DMSO or with 10uM of ATM inhibitor KU - 60019 for 1 h prior to irradiation . 1h post - irradiation ( 10 Gy ) extracts were prepared REV7 or SHLD2 complexes were immunoprecipitated using anti - Flag ( M2 ) Resin then analyzed by immunoblotting using GFP REV7 antibodies .

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

293T cells were transfected with Flag-REV7 and GFP-SHLD1 (Left) or Flag-SHLD2 and GFP-SHLD1 (Right) expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 or SHLD2 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP and REV7 antibodies.


U2OS EJ5 - GFP were transfected with either siCTRL , siSHLD1 # 1 or siSHLD1 # 2 . At 24hr post - transfection , were transfected with I - SceI expression plasmid , GFP + population was analyzed 48 hr post - plasmid transfection . of GFP + was determined for each condition subsequently normalized to non - targeting condition ( siCTRL ) .

O O O 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 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

U2OS EJ5-GFP cells were transfected with either siCTRL, siSHLD1 #1 or siSHLD1 #2. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).


CH12F3 - 2 stably expressing either shCTRL , shSHLD1 # 1 or shSHLD1 # 2 were stimulated with a cocktail of cytokines ( CIT ) to induce class switching to IgA . of IgA + was monitored 24 48h post - stimulation by with an anti - IgA antibody followed by flow cytometry .

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

CH12F3-2 cells stably expressing either shCTRL, shSHLD1#1 or shSHLD1#2 were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis.


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


Survival of low high expressers of SHLD2 in a cohort of 24 affected by Triple Negative Breast cancer ( TNBC ) . Data are represented as Kaplan - Meyer curves with expression classified as low high .

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

Survival analysis of low and high expressers of SHLD2 in a cohort of 24 patients affected by Triple Negative Breast cancer (TNBC). Data are represented as Kaplan-Meyer curves with expression classified as low and high.


Relapse - free Survival ( Basal Subtype ) of low high expressers of SHLD2 obtained from KM - plotter database . Data are represented as Kaplan - Meyer curves with expression classified as low high .

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

Relapse-free Survival (Basal Subtype) of low and high expressers of SHLD2 obtained from the KM-plotter database. Data are represented as Kaplan-Meyer curves with expression classified as low and high.


Immunoblot of lysates prepared from indicated SAMHD1 - proficient ( + / + ) , deficient ( - / - ) rescue ( WT , H233A ) pairs with indicated antibodies . Representative of 2 independent experiments .

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

Immunoblot of lysates prepared from the indicated SAMHD1-proficient (+/+), deficient (-/-) and rescue (WT, H233A) cell line pairs with the indicated antibodies. Representative of 2 independent experiments.


Proliferation inhibition of ara - C RNRi combination treatment in SAMHD1 + / + or - / - THP - 1 . Error bars indicate s . e . m . of two ( HU dF - dC ) or three ( 3 - AP ) independent experiments , each performed in duplicate .

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

Proliferation inhibition analysis of ara-C and RNRi combination treatment in SAMHD1+/+ or -/- THP-1 cells. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate.


Ara - C EC50 values plotted as a function of RNRi in SAMHD1 + / + , - / - rescue ( WT , H233A ) THP - 1 pairs . EC50 values in absence of RNRi are indicated by black red dotted . Error bars indicate s . e . m . of two ( HU dF - dC ) or three ( 3 - AP ) independent experiments , each performed in duplicate .

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

Ara-C EC50 values plotted as a function of RNRi concentration in SAMHD1+/+, -/- and rescue (WT, H233A) THP-1 cell line pairs. EC50 values in the absence of RNRi are indicated by the black and red dotted line. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate.


Drug synergy plots for ara - C indicated RNRi in SAMHD1 + / + , - / - rescue ( WT , H233A ) pairs . Each data point indicates an average delta score from a single dose - response matrix experiment performed in duplicate . Zero , > 0 , or < 0 corresponds to additive , synergy , or antagonism , respectively , whilst > 5 indicates strong synergy . horizontal error bars indicate mean s . d . , respectively , statistical significance was determined using a two - unpaired t - : ns , not significant , P ≥ 0 . 05 ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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

Drug synergy plots for ara-C and the indicated RNRi in SAMHD1+/+, -/- and rescue (WT, H233A) cell line pairs. Each data point indicates an average delta score from a single dose-response matrix experiment performed in duplicate. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. The horizontal line and the error bars indicate the mean and s.d., respectively, statistical significance was determined using a two-tailed unpaired t-test: ns, not significant, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.


Spearman correlation of relative SAMHD1 abundance synergy delta scores for ara - C versus HU or dF - dC in a panel ( n = 9 ) of haematological cancer lines . Error bars indicate s . e . m . Each data point corresponds to SAMHD1 determined by immunoblot ( n = 4 for each , representative blot shown in Appendix Fig S2 ) an average delta score from repeated dose - response matrix experiments each performed in triplicate : THP - 1 , n = 4 ; HuT - 78 , n = 2 ; HL - 60 / iva , n = 1 ; KBM - 7 , n = 2 ( HU ) 3 ( dF - dC ) ; K562 , n = 3 ( HU ) 4 ( dF - dC ) ; CCRF - CEM , n = 3 ( HU ) 4 ( dF - dC ) ; MV - 4 - 11 , n = 2 ( HU ) 3 ( dF - dC ) ; Jurkat , n = 2 ( HU ) 3 ( dF - dC ) ; MOLT - 4 , n = 2 ( HU ) an 3 ( dF - dC ) .

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

Spearman correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C versus HU or dF-dC in a panel (n = 9) of haematological cancer cell lines. Error bars indicate s.e.m. Each data point corresponds to SAMHD1 protein levels determined by immunoblot analysis (n = 4 for each cell line, representative blot shown in Appendix Fig S2) and an average delta score from repeated dose-response matrix experiments each performed in triplicate: THP-1, n =4; HuT-78, n = 2; HL-60/iva, n = 1; KBM-7, n = 2 (HU) and 3 (dF-dC); K562, n = 3 (HU) and 4 (dF-dC); CCRF-CEM, n =3 (HU) and 4 (dF-dC); MV-4-11, n = 2 (HU) and 3 (dF-dC); Jurkat, n = 2 (HU) and 3 (dF-dC); MOLT-4, n = 2 (HU) an 3 (dF-dC).


Immunoblot of lysates prepared from SAMHD1 + / + or - / - THP - 1 treated for 24 h with ara - C HU , as indicated . Representative of 3 independent experiments .

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

Immunoblot analysis of lysates prepared from SAMHD1+/+ or -/- THP-1 cells treated for 24 h with ara-C and HU, as indicated. Representative of 3 independent experiments.


Kaplan - Meier of NOD / SCID mice injected i . v . with luciferase - expressing SAMHD1 + / + or - / - THP - 1 clones ( day 0 ) treated with ara - C / or HU as indicated ( day 6 ) . n = 6 per treatment group .

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Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ or -/- THP-1 cell clones (day 0) and treated with ara-C and/or HU as indicated (day 6). n = 6 per treatment group.


Kaplan - Meier of NOD / SCID mice injected i . v . with luciferase - expressing SAMHD1 + / + or - / - HL - 60 / iva clones ( day 0 ) treated with ara - C / or HU as indicated ( day 6 ) . n = 6 per treatment group .

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

Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ or -/- HL-60/iva cell clones (day 0) and treated with ara-C and/or HU as indicated (day 6). n = 6 per treatment group.


Kaplan - Meier of NOD / SCID mice injected i . v . with luciferase - expressing SAMHD1 + / + THP - 1 clone ( day 0 ) treated with ara - C / or dF - dC as indicated ( day 6 ) . n = 7 per treatment group .

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

Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ THP-1 cell clone (day 0) and treated with ara-C and/or dF-dC as indicated (day 6). n = 7 per treatment group.


Kaplan - Meier of CD45 . 2 C57BL / 6J mice injected i . v . with murine MLL - AF9 - transformed AML blasts ( day 0 ) treated with ara - C / or HU days 20 - 24 . n = 5 per treatment group , except for vehicle ( n = 4 ) .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O

Kaplan-Meier analysis of CD45.2 C57BL/6J mice injected i.v. with murine MLL-AF9-transformed AML blasts (day 0) and treated with ara-C and/or HU days 20-24. n = 5 per treatment group, except for vehicle (n = 4).


Drug synergy plots for ara - C HU or dF - dC in - derived AML blasts . Each data point indicates an average delta score from a single sample subjected to a dose - response matrix experiment performed in triplicate , n = 16 for HU n = 9 for dF - dC . Zero , > 0 , or < 0 corresponds to additive , synergy , or antagonism , respectively , whilst > 5 indicates strong synergy . Median , upper lower quartiles , range of delta scores are indicated by box - - whisker plots .

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

Drug synergy plots for ara-C and HU or dF-dC in primary patient-derived AML blasts. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate, n = 16 for HU and n = 9 for dF-dC. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. Median, upper and lower quartiles, and range of delta scores are indicated by box-and-whisker plots.


Pearson correlation of relative SAMHD1 abundance synergy delta scores for ara - C HU or dF - dC in - derived AML blasts ( n = 23 ) .

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

Pearson correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C and HU or dF-dC in primary patient-derived AML blasts (n = 23).


Immunoblot of - derived AML blasts treated with control ( dX ) or Vpx - containing ( X ) - like particles ( VLPs ) : A2953 ( C ) , ALG17 _ 001 ( E ) . Accompanying proliferation inhibition of ara - C indicated RNRi combination in these samples : A2953 ( D ) , ALG17 _ 001 ( D ) . Error bars indicate s . d . of single experiment performed in triplicate .

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 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 O O O O O O O O O

Immunoblot of primary patient-derived AML blasts treated with control (dX) or Vpx-containing (X) virus-like particles (VLPs): patient A2953 (C), ALG17_001 (E). Accompanying proliferation inhibition analysis of ara-C and indicated RNRi combination in these samples: patient A2953 (D), ALG17_001 (D). Error bars indicate s.d. of single experiment performed in triplicate.


Paired drug synergy plot for ara - C RNRi ( HU , n = 7 ; dF - dC , n = 5 ) in - derived AML blasts pre - treated with control ( dX ) or Vpx - containing ( X ) VLPs . Zero , > 0 , or < 0 corresponds to additive , synergy , or antagonism , respectively , whilst > 5 indicates strong synergy < 5 indicates strong antagonism . Each data point indicates an average delta score from a single sample subjected to a dose - response matrix experiment performed in triplicate . Statistical testing was performed using two - way ANOVA .

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Paired drug synergy plot for ara-C and RNRi (HU, n = 7; dF-dC, n = 5) in primary patient-derived AML blasts pre-treated with control (dX) or Vpx-containing (X) VLPs. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy and <5 indicates strong antagonism. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate. Statistical testing was performed using two-way ANOVA.


Intracellular dNTP measurements using a primer - extension in SAMHD1 + / + THP - 1 treated for 4 or 24 h with either 50 µM HU ( middle panel ) or 2 . 5 nM 3 - AP ( right panel ) , ratios of dCTP - to - dATP were calculated . Bars indicate mean values of three independent experiments , error bars indicate s . e . m . Statistical analyses were done using unpaired two - t - : * * , P < 0 . 01 .

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Intracellular dNTP measurements using a primer-extension assay in SAMHD1+/+ THP-1 cells treated for 4 or 24 h with either 50 µM HU (middle panel) or 2.5 nM 3-AP (right panel), ratios of dCTP-to-dATP were calculated. Bars indicate mean values of three independent experiments, error bars indicate s.e.m. Statistical analyses were done using unpaired two-tailed t-tests: **, P < 0.01.


Intracellular ara - CTP ( upper panel ) dCTP : dATP ( lower panel ) in indicated lines following indicated treatment determined using HPLC - MS / MS . SAMHD1 - / - THP - 1 were treated with 500 nM ara - C SAMHD1 + / + THP - 1 were treated with either solvent , 500 nM ara - C or a combination of 500 nM ara - C an RNRi ( HU , 50 µM ; dF - dC , 10 nM ; 3 - AP , 150 nM ) for 24 h prior . Values relative to mean ara - CTP amounts in ara - C treated SAMHD1 + / + THP - 1 shown ( indicated by dashed ) . Circles , columns error bars correspond to values , means s . e . m . of at least three experiments performed independently .

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Intracellular ara-CTP (upper panel) and dCTP:dATP ratio (lower panel) in the indicated cell lines following the indicated treatment determined using HPLC-MS/MS. SAMHD1-/- THP-1 cells were treated with 500 nM ara-C and SAMHD1+/+ THP-1 cells were treated with either solvent, 500 nM ara-C or a combination of 500 nM ara-C and an RNRi (HU, 50 µM; dF-dC, 10 nM; 3-AP, 150 nM) for 24 h prior. Values relative to mean ara-CTP amounts in ara-C treated SAMHD1+/+ THP-1 cells shown (indicated by dashed line). Circles, columns and error bars correspond to individual values, means and s.e.m. of at least three experiments performed independently.


Quantificaiton of dCK phosphorylation at serine - 74 ( S74 ) with respect to total dCK in SAMHD1 + / + THP - 1 treated with either solvent , 500 nM ara - C or a combination of 500 nM ara - C an RNRi ( HU , 50 µM ; dF - dC , 10 nM ; 3 - AP , 150 nM ) for 24 h . Circles squares , columns error bars correspond to measurements , means s . e . m . of one representative out of two independent experiments performed in triplicates .

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Quantificaiton of dCK phosphorylation at serine-74 (S74) with respect to total dCK in SAMHD1+/+ THP-1 cells treated with either solvent, 500 nM ara-C or a combination of 500 nM ara-C and an RNRi (HU, 50 µM; dF-dC, 10 nM; 3-AP, 150 nM) for 24 h. Circles and squares, columns and error bars correspond to individual measurements, means and s.e.m. of one representative out of two independent experiments performed in triplicates.


Measurement of released inorganic triphosphate ( PPPi ) from hydrolysis of ara - CTP ( 200 µM ) by recombinant SAMHD1 ( 0 . 35 µM ) in presence of GTP ( 200 µM ) a titration of different non - hydrolysable dNTP analogues ( dNTPαS ) in enzyme - coupled malachite green . Error bars indicate s . e . m . of two independent experiments performed in triplicate quadruplet .

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Measurement of released inorganic triphosphate (PPPi) from hydrolysis of ara-CTP (200 µM) by recombinant SAMHD1 (0.35 µM) in the presence of GTP (200 µM) and a titration of different non-hydrolysable dNTP analogues (dNTPαS) in the enzyme-coupled malachite green assay. Error bars indicate s.e.m. of two independent experiments performed in triplicate and quadruplet.


In vitro pull - down assays . GST or GST - PSKR1JK fusion coupled with glutathione beads were used to pull down His - CPK28 . amount of fusion loaded on gel was indicated by Coomassie brilliant blue ( CBB ) . Asterisks indicate specific bands of fusion .

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In vitro pull-down assays. GST or GST-PSKR1JK fusion proteins coupled with glutathione beads were used to pull down His-CPK28. The amount of fusion proteins loaded on the gel was indicated by Coomassie brilliant blue (CBB) staining. Asterisks indicate the specific bands of fusion proteins.


Co - IP assays . PSKR1 - HA was co - expressed with CPK28 - GFP or GFP in N . benthamiana leaves . PSK ( 10 μM ) or H2O was injected 2 hours before leaves sampling . was precipitated using GFP - Trap . Both immunoprecipitated input samples were subjected to western blotting using an anti - HA or anti - GFP antibody .

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Co-IP assays. PSKR1-HA was co-expressed with CPK28-GFP or GFP in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The protein was precipitated using GFP-Trap. Both immunoprecipitated and input samples were subjected to western blotting using an anti-HA or anti-GFP antibody.


A . Co - IP showing that PSK promotes CPK28 - GS2 interaction . GS2 - HA was co - expressed with CPK28 - GFP or GFP in N . benthamiana leaves . PSK ( 10 μM ) or H2O was injected 2 hours before leaves sampling . was precipitated by GFP - Trap . Immunoprecipitated input samples were subjected to western blotting using an anti - HA anti - GFP antibody . Data information : Experiments were repeated twice with similar results .

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A. Co-IP assay showing that PSK promotes CPK28-GS2 interaction. GS2-HA was co-expressed with CPK28-GFP or GFP in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The protein was precipitated by GFP-Trap. Immunoprecipitated and input samples were subjected to western blotting using an anti-HA and anti-GFP antibody. Data information: Experiments were repeated twice with similar results.


B . Co - IP showing that CPK28 - GS2 interaction was enhanced in presence of PSKR1 . GS2 - HA CPK28 - GFP were co - expressed with or without PSKR1 - FLAG in N . benthamiana leaves . was precipitated by GFP - Trap . Immunoprecipitated input samples were subjected to western blotting using an anti - HA , anti - GFP or anti - FLAG antibody . Data information : Experiments were repeated twice with similar results .

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B. Co-IP assay showing that CPK28-GS2 interaction was enhanced in the presence of PSKR1. GS2-HA and CPK28-GFP were co-expressed with or without PSKR1-FLAG in N. benthamiana leaves. The protein was precipitated by GFP-Trap. Immunoprecipitated and input samples were subjected to western blotting using an anti-HA, anti-GFP or anti-FLAG antibody. Data information: Experiments were repeated twice with similar results.


C . In vitro pull - down assays showing that CPK28 - GS2 interaction was enhanced in presence of PSKR1 . Fixed amounts of His - CPK28 GST - GS2 fusion were incubated with an increasing amount of MBP - PSKR1 fusion MBP . Asterisks indicate specific bands of fusion . Data information : Experiments were repeated twice , with similar results .

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C. In vitro pull-down assays showing that CPK28-GS2 interaction was enhanced in the presence of PSKR1. Fixed amounts of His-CPK28 and GST-GS2 fusion proteins were incubated with an increasing amount of MBP-PSKR1 fusion protein and MBP protein. Asterisks indicate the specific bands of fusion proteins. Data information: Experiments were repeated twice, with similar results.


A . PSK induces phosphorylation of GS2 by CPK28 in vivo . GS2 - GFP was co - expressed with CPK28 - HA ( empty vector as a negative control ) in N . benthamiana leaves . PSK ( 10 μM ) or H2O was injected 2 hours before leaves sampling . phosphorylated GS2 was precipitated by GFP - Trap , then immunoblotted with anti - pSer / pThr anti - pSer antibodies .

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A. PSK induces the phosphorylation of GS2 by CPK28 in vivo. GS2-GFP was co-expressed with CPK28-HA (empty vector as a negative control) in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The phosphorylated GS2 protein was precipitated by GFP-Trap, and then immunoblotted with anti-pSer/pThr and anti-pSer antibodies.


C . Site - directed mutation of S334 S360 residues in GS2 inhibited its phosphorylation by CPK28 .

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C. Site-directed mutation of S334 and S360 residues in GS2 inhibited its phosphorylation by CPK28.


D . Identification of OE - GS2 transgenic lines expressing phospho - mutated versions of GS2 in gs2 background by western blotting using an anti - GLN1 / GLN2 antibody . Ponceau S was used as loading control .

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D. Identification of OE-GS2 transgenic lines expressing phospho-mutated versions of GS2 in the gs2 background by western blotting using an anti-GLN1/GLN2 antibody. Ponceau S staining was used as the loading control.


B ) Volcano plot for F on differential expression between WT mdx mice . C ) Volcano plot for F on differential expression between WT mdx + + mice .

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B) Volcano plot for the F test on differential expression between WT and mdx mice. C) Volcano plot for the F test on differential expression between WT and mdx++ mice.


D ) Volcano plot for F on differential expression between WT mdx + - mice .

D) Volcano plot for the F test on differential expression between WT and mdx+- mice.


E , F ) Boxplot comparing distribution of Dmd Prune2 genes in WT , mdx , mdx + + mdx + - mice groups . central band of boxplot denotes median ; box denotes first third quartile ; whiskers are computed as min ( max ( x ) , Q3 + 1 . 5 IQR ) max ( min ( x ) , Q1 - 1 . 5 * IQR ) , where min ( x ) max ( x ) denote minimum maximum of distribution , Q1 Q3 first third quartile , IQR interquartile range .

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E, F) Boxplot comparing the distribution of the Dmd and Prune2 genes in the WT, mdx, mdx++ and mdx+- mice groups. The central band of the boxplot denotes the median; the box denotes the first and third quartile; the whiskers are computed as min(max(x), Q3 + 1.5 IQR) and max(min(x), Q1 - 1.5 * IQR), where min(x) and max(x) denote the minimum and maximum of the distribution, Q1 and Q3 the first and third quartile, and IQR the interquartile range.


G ) Volcano plot for F on differential expression between mdx + + mdx + - mice .

G) Volcano plot for the F tests on differential expression between mdx++ and mdx+- mice.


Scatter plots comparing estimated logFCs across mice groups . Panel H : mdx vs mdx + + .

Scatter plots comparing the estimated logFCs across mice groups. Panel H: mdx vs mdx++.


Scatter plots comparing estimated logFCs across mice groups . + + . Panel I : mdx vs mdx + - . Panel J : mdx + + vs mdx + - .

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Scatter plots comparing the estimated logFCs across mice groups. ++. Panel I: mdx vs mdx+-. Panel J: mdx++ vs mdx+-.


A ) Histogram of likelihood p - values for on differences between WT mdx at any time point .

A) Histogram of the likelihood ratio p-values for the test on differences between WT and mdx at any time point.


B ) Distribution of estimated logFCs for mdx versus WT mice at 6 , 12 , 18 , 24 30 for 1532 genes with significant differences between WT mdx .

B) Distribution of the estimated logFCs for mdx versus WT mice at weeks 6, 12, 18, 24 and 30 for the 1532 genes with significant differences between WT and mdx.


D ) Trajectory plots comparing trajectories of Chordc1 Psat1 in WT mdx mice .

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D) Trajectory plots comparing the trajectories of Chordc1 and Psat1 in WT and mdx mice.


E ) Trajectory plots comparing trajectories of Cd59a , Chordc1 , Hpgd , Ldhb , Lincred1 , Myo6 , Slc39a11 Tlr1 in WT , mdx + + mdx + - mice .

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E) Trajectory plots comparing the trajectories of Cd59a, Chordc1, Hpgd, Ldhb, Lincred1, Myo6, Slc39a11 and Tlr1 in WT, mdx++ and mdx+- mice.


F ) Scatter plots comparing estimated logFC of mdx + + vs WT ( x axis ) of mdx + - vs WT ( y axis ) at 6 , 12 , 18 , 24 30 .

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F) Scatter plots comparing the estimated logFC of mdx++ vs WT (x axis) and of mdx+- vs WT (y axis) at weeks 6, 12, 18, 24 and 30.


H ) upstream regulator ( H ) performed by Ingenuity Pathway of comparison between mdx wt mice .

H) upstream regulator analysis (H) performed by Ingenuity Pathway Analysis of the comparison between mdx and wt mice.


A ) Barplot showing of significant genes in muscle modules . Next to each module name we report total number of genes that it comprises . B ) Barplot showing of significant genes in blood modules . Next to each module name we report total number of genes that it comprises . C ) Matrix with values of cross - correlation between blood muscle module eigengenes . To enhance visibility , only correlations with absolute value above 0 . 5 are shown .

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A) Barplot showing the percentage of significant genes in the muscle modules. Next to each module name we report the total number of genes that it comprises. B) Barplot showing the percentage of significant genes in the blood modules. Next to each module name we report the total number of genes that it comprises. C) Matrix with the values of the cross-correlation between blood and muscle module eigengenes. To enhance visibility, only correlations with absolute value above 0.5 are shown.


A ) RT - PCR ( 30 cycles ) showing skipping of exon 23 for PMO PS49 oligos . No skipping was visible in saline treated mice .

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A) RT-PCR (30 cycles) showing skipping of exon 23 for PMO and PS49 oligos. No skipping was visible in saline treated mice.


B ) Western blot showing of restoration of dystrophin in mdx mice following antisense oligonucleotides administration . M is molecular ladder . WT gastrocnemius represents standard curve using extracts from gastrocnemius muscle . NT is saline treated mdx mice .

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B) Western blot showing the percentage of restoration of dystrophin protein in mdx mice following antisense oligonucleotides administration. M is molecular ladder. WT gastrocnemius represents the standard curve using protein extracts from gastrocnemius muscle. NT is saline treated mdx mice.


C ) Barplot comparing estimated logFC for PS49 PMO treatments in MoTreat dataset to desired drug effect obtained from MoLong dataset ( logFC WT vs mdx at week 12 ) .

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C) Barplot comparing the estimated logFC for the PS49 and PMO treatments in the MoTreat dataset to the desired drug effect obtained from the MoLong dataset (logFC WT vs mdx at week 12).


D , E ) Histograms comparing distribution of selected genes across different mice groups ( PMO , PS49 saline in MoTreat dataset , WT mdx at week 12 in MoLong dataset ) .

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D, E) Histograms comparing the distribution of selected genes across different mice groups (PMO, PS49 and saline in the MoTreat dataset, WT and mdx at week 12 in the MoLong dataset).


B ) Volcano plot for F on differential expression between healthy individuals DMD .

B) Volcano plot for the F test on differential expression between healthy individuals and DMD patients.


C ) Scatter plot showing group effect at baseline ( x - axis ) effect of interaction between group ( y - axis ) . Black dots represent non differentially expressed genes , while blue dots represent differentially expressed genes .

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C) Scatter plot showing the group effect at baseline (x-axis) and the effect of the interaction between age and group (y-axis). Black dots represent non differentially expressed genes, while blue dots represent differentially expressed genes.


E ) Volcano plot for F on differential expression between treated untreated DMD .

E) Volcano plot for the F test on differential expression between treated and untreated DMD patients.


F ) Barplot comparing logFC of genes associated with steroid treatment estimated in this study to estimates of Liu et al . ( 2012 ) .

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F) Barplot comparing the logFC of the genes associated with steroid treatment estimated in this study to the estimates of Liu et al. (2012).


G ) Scatter plot showing genes for which a significant effect of treatment with steroids . group effects at baseline are plotted on x - axis , while effects of steroids are plotted on y - axis .

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G) Scatter plot showing genes for which a significant effect of treatment with steroids. The group effects at baseline are plotted on the x-axis, while the effects of steroids are plotted on the y-axis.


( A ) Still of time - lapse imaging of H3 . 3 - mCherry H3 . 2 - EGFP in wild - type zygotes ( n = 17 ) .

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(A) Still images of time-lapse imaging of H3.3-mCherry and H3.2-EGFP proteins in wild-type zygotes (n=17).


( B ) IF of DAXX ATRX in zygotes at different pronuclear ( PN ) stages ( PN0 , n = 8 ; PN2 , n = 6 ; PN5 , n = 33 ) . Data information : panels : of paternal maternal pronuclei at given stage / condition are from same zygote .

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(B) IF of DAXX and ATRX in zygotes at different pronuclear (PN) stages (PN0, n=8; PN2, n=6; PN5, n= 33). Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote.


( C ) Boxplot displaying signal of DAXX at pat - mat - PCH in mouse zygotes at PN5 stage , relative to mean signal at pat - PCH . In boxplots , enter lines show medians ; box limits indicate 25th 75th percentiles as determined by R software ; whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 3 times . * * * P < 0 . 001 , n = 28 , paired t - .

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(C) Boxplot displaying signal intensity of DAXX at pat- and mat-PCH in mouse zygotes at PN5 stage, relative to mean signal at pat-PCH. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001, n= 28, paired t-test.


( D ) IF of DAXX ATRX in PN5 zygotes upon siRNA - mediated knockdown of Daxx ( n = 14 ) or with control siRNAs ( n = 8 ) . Data information : panels : of paternal maternal pronuclei at given stage / condition are from same zygote . Yellow dashed circles represent contours of pronuclei . scale bars , 10µm .

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(D) IF of DAXX and ATRX in PN5 zygotes upon siRNA-mediated knockdown of Daxx (n=14) or with control siRNAs (n=8). Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote. Yellow dashed circles represent the contours of the pronuclei. All scale bars, 10µm.


( E ) IF of DAXX ATRX in control ( n = 15 ) Atrxm - z + ( n = 14 ) PN5 zygotes . Data information : panels : of paternal maternal pronuclei at given stage / condition are from same zygote . Yellow dashed circles represent contours of pronuclei . scale bars , 10µm .

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(E) IF of DAXX and ATRX in control (n=15) and Atrxm-z+ (n=14) PN5 zygotes. Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote. Yellow dashed circles represent the contours of the pronuclei. All scale bars, 10µm.


( A ) IF of DAXX EZH2 in paternal pronuclei of Ezh1m - z + ( n = 5 ) , Ezh2m - z + ( n = 3 ) Ezh1m - z + ; Ezh2m - z + ( n = 4 ) late zygotes . Yellow dashed circles represent contours of pronuclei . Data information : scale bars , 10µm .

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(A) IF of DAXX and EZH2 in paternal pronuclei of Ezh1m-z+ (n=5), Ezh2m-z+ (n=3) and Ezh1m-z+; Ezh2m-z+ (n=4) late zygotes. Yellow dashed circles represent the contours of the pronuclei. Data information: All scale bars, 10µm.


( B ) IF of DAXX RNF2 in PN0 zygotes ( n = 10 ) . Data information : scale bars , 10µm .

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(B) IF of DAXX and RNF2 in PN0 zygotes (n=10). Data information: All scale bars, 10µm.


( C ) IF of DAXX in wild type ( n = 30 ) , Ring1m - z + ( n = 12 ) , Rnf2m - z + ( n = 11 ) Ring1m - z + ; Rnf2m - z + ( n = 12 ) paternal pronuclei of PN5 zygotes . Data information : scale bars , 10µm .

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

(C) IF of DAXX in wild type (n=30), Ring1m-z+ (n=12), Rnf2m-z+ (n=11) and Ring1m-z+; Rnf2m-z+ (n=12) paternal pronuclei of PN5 zygotes. Data information: All scale bars, 10µm.


( D ) Anti - Flag co - IP of DAXXMycHis , RNF2 H3K27me3 with CBX23 × Flag from extracts of HEK293 in presence or absence of benzonase , followed by immuno - blot analyses for indicated antigens .

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

(D) Anti-Flag co-IP of DAXXMycHis, RNF2 and H3K27me3 with CBX23×Flag from extracts of HEK293 cells in presence or absence of benzonase, followed by immuno-blot analyses for indicated antigens.


( C ) Occupancy ( % of IP - ed / input ) of GAL4DBDCBX2 , RNF2 , DAXXMycHis , H3 . 3 , H3 at 5 × UAS site as measured by ChIP - qPCR of from HEK2935xUAS . Data are means ± ( n = 3 ) .

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 I-MEASURED_VAR I-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

(C) Occupancy (% of IP-ed DNA / input DNA) of GAL4DBDCBX2, RNF2, DAXXMycHis, H3.3, and H3 proteins at 5×UAS site as measured by ChIP-qPCR of chromatin from HEK2935xUAS cells. Data are means ± SEM (n = 3).


( D ) Immunoblots for indicated in HEK2935xUAS

(D) Immunoblots for the indicated proteins in HEK2935xUAS cells