( 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 .

O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O B-TISSUE O B-TISSUE O O O O O O B-EXP_ASSAY O B-TISSUE O B-TISSUE O O O B-TISSUE O B-TISSUE O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O B-TISSUE O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-TISSUE O B-TISSUE O O B-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O B-TISSUE O O O O B-TISSUE O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O O B-TISSUE I-TISSUE O O 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-TISSUE O B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O B-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O B-TISSUE I-TISSUE O B-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O B-TISSUE O B-TISSUE O O 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 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

O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O 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-TISSUE I-TISSUE O O O O O O O O O O O B-TISSUE O O B-TISSUE I-TISSUE 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O B-TISSUE 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 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

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O O O B-TISSUE I-TISSUE 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 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 ) .

O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O O O O O B-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE 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 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 .

O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O 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 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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-GENEPROD O O B-ORGANISM 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 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 , 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

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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 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

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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 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

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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 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 .

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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 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 .

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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 . 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 .

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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 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 ) .

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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).


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


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

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


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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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.


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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 .

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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 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 .

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

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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 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 .

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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 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 .

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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 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-DISEASE B-CELL_TYPE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY 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 protein abundance and synergy delta scores for ara - C and HU or dF - dC in primary patient - derived AML blasts ( n = 23 ) .

O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-DISEASE B-CELL_TYPE 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 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 .

B-EXP_ASSAY O O O O O B-DISEASE B-CELL_TYPE O O O O O O O B-GENEPROD O O O 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-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O 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 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 .

O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-DISEASE B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

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 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 .

O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) 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 .

O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

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 . 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O

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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE O O O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O

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 and 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 the gs2 background by western blotting using an anti - GLN1 / GLN2 antibody . Ponceau S staining was used as the 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 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 .

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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 the F test on differential expression between WT and mdx + - mice .

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


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 .

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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 the F tests on differential expression between mdx + + and mdx + - mice .

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


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

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


Scatter plots comparing the 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 the likelihood ratio p - values for the test on differences between WT and mdx at any time point .

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


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 .

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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 the trajectories of Chordc1 and Psat1 in WT and 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 the trajectories of Cd59a , Chordc1 , Hpgd , Ldhb , Lincred1 , Myo6 , Slc39a11 and Tlr1 in WT , mdx + + and 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 the estimated logFC of mdx + + vs WT ( x axis ) and of mdx + - vs WT ( y axis ) at weeks 6 , 12 , 18 , 24 and 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 analysis ( H ) performed by Ingenuity Pathway Analysis of the comparison between mdx and wt mice .

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


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 .

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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 and 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 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 .

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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 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 ) .

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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 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 ) .

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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 the F test on differential expression between healthy individuals and DMD patients .

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


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 .

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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 the F test on differential expression between treated and untreated DMD patients .

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


F ) Barplot comparing the logFC of the genes associated with steroid treatment estimated in this study to the 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 . The group effects at baseline are plotted on the x - axis , while the effects of steroids are plotted on the 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 images of time - lapse imaging of H3 . 3 - mCherry and H3 . 2 - EGFP proteins 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 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 .

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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 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-SUBCELLULAR O B-ORGANISM B-TISSUE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O B-GENEPROD O O 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-SUBCELLULAR O O O O O O O O B-TISSUE O O O O O O O O O B-SUBCELLULAR O O O O O O O

(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.