( A ) CSCs stimulated with or without CpG ODN ( 10 μg / ml ) were analyzed for expression of phosphor - AMPKα . Representative mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(A) CSCs stimulated with or without CpG ODN (10 μg/ml) were analyzed for expression of phosphor-AMPKα. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( B ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) in presence or absence of DAPT ( 10 μM ) , analyzed for phosphor - AMPKα . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(B) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( C ) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN ( 10 μg / ml ) analyzed for phosphor - AMPKα . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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 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) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN (10 μg/ml) and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( D ) CSCs were treated with DAPT ( 10 μM ) in presence or absence of A769662 ( 10 μM ) , analysed for mitochondrial OCR . Ctrl represents CSCs without any treatment . Mean ± from 6 . Data information : ANOVA with Turkey method ( D ) , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(D) CSCs were treated with DAPT (10 μM) in the presence or absence of A769662 (10 μM), and analysed for mitochondrial OCR. Ctrl represents CSCs without any treatment. Mean±SEM from 6 patients. Data information: ANOVA with Turkey method (D), **p < 0.01, ***p < 0.001, ****p < 0.0001.


( E ) CSCs were stimulated with or without CpG ODN ( 10 μg / ml ) analyzed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(E) CSCs were stimulated with or without CpG ODN (10 μg/ml) and analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( F ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) in presence or absence of Compound C ( 10 μM ) analysed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(F) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of Compound C (10 μM) and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( G ) CSCs were transfected with 2 different AMPKα shRNA or control , stimulated with CpG ODN ( 10 μg / ml ) , analysed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(G) CSCs were transfected with 2 different AMPKα shRNA or the control, stimulated with CpG ODN (10 μg/ml), and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( A ) CSCs transfected with or without Notch1 ICD were analyzed for AMP / ATP plus LKB1 phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O 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 O O O O O 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) CSCs transfected with or without Notch1 ICD were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( B ) CSCs treated with or without DAPT ( 10 μM ) were analyzed for AMP / ATP plus LKB1 phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(B) CSCs treated with or without DAPT (10 μM) were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( C ) CSCs treated with or without CpG ODN ( 10 μg / ml ) were analysed for interactions between Notch1 AMPK . Representative mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C) CSCs treated with or without CpG ODN (10 μg/ml) were analysed for interactions between Notch1 and AMPK. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs transfected with LKB1 shRNA （ right ） or control ( left ) were treated with CpG ODN ( 10 μg / ml ) analysed for AMPK phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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 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

(D) CSCs transfected with LKB1 shRNA（right）or the control (left) were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E ) CSCs transfected with TAK1 shRNA or control were treated with CpG ODN ( 10 μg / ml ) analysed for AMPK phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O 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) CSCs transfected with TAK1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( F ) CSCs transfected with LAMTOR1 shRNA or control were treated with CpG ODN ( 10 μg / ml ) analysed for AMPK phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

(F) CSCs transfected with LAMTOR1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( G ) CSCs transfected with AXIN1 shRNA or control were treated with CpG ODN ( 10 μg / ml ) analysed for AMPK phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

(G) CSCs transfected with AXIN1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) NSCLC PDX models treated with or without chemotherapy for first 2 were analyzed for growth at indicated time . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(A) NSCLC PDX models treated with or without chemotherapy for the first 2 weeks were analyzed for tumor growth at the indicated time. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( B ) Frequencies of CD133 - expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(B) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 weeks. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( C - E ) Flow cytometric analyses of expressions of TLR9 , activated Notch1 , phosphor - AMPKα in cancer from NSCLC xenografts treated with or without chemotherapy after 4 . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O O O B-MEASURED_VAR 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 O O O O O O 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-E) Flow cytometric analyses of protein expressions of TLR9, activated Notch1, and phosphor-AMPKα in cancer cells from NSCLC xenografts treated with or without chemotherapy after 4 weeks. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( F ) NSCLC PDX models were treated with chemotherapy alone or together with indicated inhibitors , respectively , for first 2 . volume was determined at indicated time . Mean ± from 6 experiments . ( G ) Frequencies of CD133 - expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with indicated inhibitors after 4 . Mean ± from 6 experiments . Data information : ANOVA with Turkey ' s method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(F) NSCLC PDX models were treated with chemotherapy alone or together with the indicated inhibitors, respectively, for the first 2 weeks. Tumor volume was determined at the indicated time. Mean±SEM from 6 experiments. (G) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with the indicated inhibitors after 4 weeks. Mean±SEM from 6 experiments. Data information: ANOVA test with Turkey's method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


Cortical malformations observed in six novel BBSOAS ( P ) 1 shows abnormally elongated occipital convolutions ( arrowheads in A ) , whereas abnormal gyrification of supramarginal angular gyri is present in five out of six individuals ( P2 - P6 B ' , C ' C ' ' highlight abnormally convoluted regions boxed in B C .

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

Cortical malformations observed in six novel BBSOAS patients Patient (P)1 shows abnormally elongated occipital convolutions (arrowheads in A), whereas abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6 B', C' and C'' highlight the abnormally convoluted regions boxed in B and C.


Cortical malformations observed in six novel BBSOAS ( P ) 1 shows abnormal gyrification of supramarginal angular gyri is present in five out of six individuals ( P2 - P6 ;

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

Cortical malformations observed in six novel BBSOAS patients Patient (P)1 shows abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6;


( H , H ' ) TBR2 ( green in H ) , SOX2 ( red in H ) NR2F1 ( yellow in H ' ) immunofluorescence ( IF ) of a GW11 section of human neocortex Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; oSVZ : outer subventricular zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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

(H,H') TBR2 (green in H), SOX2 (red in H) and NR2F1 (yellow in H') immunofluorescence (IF) of a GW11 section of human neocortex Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.


( I ) NR2F1 expression ( quantified by pixel ) at different L - M in different regions along A - P extent of cortex , as indicated . n ≥ 3 sections from n = 1 brain . Data information number of positive was quantified in 100µm - width boxes , randomly placed across cortex . In graphs , data are represented as means ± . 2 - way ANOVA ( I ) ( * P < 0 . 05 , * * * P < 0 . 001 ) .

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

(I) NR2F1 expression (quantified by pixel intensity) at different L-M levels and in different regions along the A-P extent of the cortex, as indicated. n ≥ 3 sections from n = 1 fetal brain. Data information the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. 2-way ANOVA (I) (*P<0.05, ***P<0.001).


( J - L ' ) NR2F1 ( red ) IF of human GW14 neocortex , showing expression in CP ( K , L ) VZ ( K ' , L ' ) around convolutions in posterior - most cortex High NR2F1 is detected in neurogenic of a gyrus ( L ' ) . Data information : Nuclei ( blue ) were stained with DAPI . In Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; oSVZ : outer subventricular zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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 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

(J-L') NR2F1 (red) IF of human GW14 neocortex, showing expression levels in the CP (K,L) and VZ (K',L') around primary convolutions in the posterior-most cortex High NR2F1 is detected in the neurogenic area of a gyrus (L'). Data information: Nuclei (blue) were stained with DAPI. In Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.


( M , M ' ) NR2F1 level as measured on single in VZ ( M ) CP ( M ' ) of sulci gyri n ≥ 4 sections from n = 1 brain . ( N - O ) NR2F1 ( red ) TBR2 ( green ) immunostaining NR2F1 level as measured in single TBR2 + IPs by pixel is shown in graph ( O ) . n ≥ 4 convolutions from n = 1 brain ( n = 2 sections ) . Data information In graphs , data are represented as means ± . Student t - ( M , M ' , O ( * P < 0 . 05 , * * * P < 0 . 001 ) .

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

(M,M') NR2F1 level as measured on single cells in the VZ (M) and CP (M') of sulci and gyri n ≥ 4 sections from n = 1 fetal brain. (N-O) NR2F1 (red) and TBR2 (green) immunostaining NR2F1 level as measured in single TBR2+ IPs by pixel intensity is shown in graph (O). n ≥ 4 convolutions from n = 1 fetal brain (n = 2 sections). Data information In graphs, data are represented as means ± SEM. Student t-test (M,M',O (*P<0.05, ***P<0.001).


( P - R ) NR2F1 ( red ) HOPX ( green ) immunostaining Virtually HOPX + bRGs are also NR2F1 + ( magnification in P ' ' ) their number is greatly increased in gyri ( P ' ' ) compared to sulci ( P ' ) , as quantified in ( R ) . At a single level , NR2F1 pixel is higher in oSVZ HOPX + in gyri compared to sulci ( Q ) . n ≥ 4 sections from n = 1 brain . Data information : Nuclei ( blue ) were stained with DAPI . number of positive was quantified in 100µm - width boxes , randomly placed across cortex . In graphs , data are represented as means ± . Student t - Q , R ) ( * P < 0 . 05 , * * * P < 0 . 001 ) . oSVZ : outer subventricular

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

(P-R) NR2F1 (red) and HOPX (green) immunostaining Virtually all HOPX+ bRGs are also NR2F1+ (magnification in P'') and their number is greatly increased in gyri (P'') compared to sulci (P'), as quantified in (R). At a single cell level, NR2F1 pixel intensity is higher in oSVZ HOPX+ cells in gyri compared to sulci (Q). n ≥ 4 sections from n = 1 fetal brain. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. Student t-test Q,R) (*P<0.05, ***P<0.001). oSVZ: outer subventricular zone


( A ) Representative of wild - type ( WT ) mutant ( KO ) neurospheres obtained from E15 . 5 neocortices cultured in vitro . culture passage ( " step " ) corresponds to 3 days of culture after dissociation re - plating . KO are more numerous larger in . Data information : Scale bars : 50µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of wild-type (WT) and mutant (KO) neurospheres obtained from E15.5 neocortices and cultured in vitro. The culture passage ("step") corresponds to 3 days of culture after dissociation and re-plating. KO aggregates are more numerous and larger in size. Data information: Scale bars: 50µm.


( B ) Graph showing number of neurospheres per P100 plate at different steps , as indicated . Nr2f1KO neurospheres proliferate for a longer time ( up to 30 passages tested ) , whereas WT exhaust around step 9 . n ≥ 3 culture wells from n = 2 batches . Data information : data are represented as means ± . 2 - way ANOVA ( B ) ( * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(B) Graph showing the number of neurospheres per P100 cell plate at different steps, as indicated. Nr2f1KO neurospheres proliferate for a longer time (up to 30 passages tested), whereas WT cells exhaust around step 9. n ≥ 3 culture wells from n = 2 batches. Data information: data are represented as means ± SEM. 2-way ANOVA (B) (**P<0.01, ***P<0.001).


( C ) Panel of WT KO isolated neurospheres grown from day 1 to day 10 . Blue red lines represent diameter of WT KO neurospheres , respectively . Data information Scale bars : 50µm .

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(C) Panel of WT and KO isolated neurospheres grown from day 1 to day 10. Blue and red lines represent the diameter of WT and KO neurospheres, respectively. Data information Scale bars: 50µm.


( D , E ) PH3 ( green ; dividing ) IF on cross - sections of day 4 neurospheres . arrowheads point at mitotic figures , which are quantified in ( E ) as of dividing over DAPI - stained nuclei ( blue ) . n ≥ 3 neurospheres from n = 2 batches . Data information : data are represented as means ± . Student t - ( E ) ( * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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

(D,E) PH3 (green; dividing cells) IF on cross-sections of day 4 neurospheres. White arrowheads point at mitotic figures, which are quantified in (E) as percentage of dividing cells over DAPI-stained nuclei (blue). n ≥ 3 neurospheres from n = 2 batches. Data information: data are represented as means ± SEM. Student t-test (E) (**P<0.01, ***P<0.001). Scale bars: 50µm.


( A - D ) Ki67 ( green ; mitotically active progenitors ) Tuj1 ( red ; post - mitotic neurons ) IF at E14 . 5 ( A , A ' ) , E16 . 5 ( B , B ' ) E18 . 5 ( C , C ' ) of lateral pallia of wild - type ( WT ) versus mutant ( KO ) brains . Ki67 + proliferating ( quantified in D ) accumulate in mutant cortices . n ≥ 3 brains . Data information : Nuclei ( blue ) were stained with DAPI . number of positive was quantified in 100µm - width boxes , randomly placed across LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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

(A-D) Ki67 (green; mitotically active progenitors) and Tuj1 (red; post-mitotic neurons) IF at E14.5 (A,A'), E16.5 (B,B') and E18.5 (C,C') of lateral pallia of wild-type (WT) versus mutant (KO) brains. Ki67+ proliferating cells (quantified in D) accumulate in mutant cortices. n ≥ 3 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.


( E - F ' ) Tbr2 ( green ; IPs ) Pax6 ( red ; radial glia ) IF of E13 . 5 ( E , E ' ) E17 . 5 ( F , F ' ) lateral pallia of WT KO brains . Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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') Tbr2 (green; IPs) and Pax6 (red; radial glia cells) IF of E13.5 (E,E') and E17.5 (F,F') lateral pallia of WT and KO brains. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.


of different classes of cortical progenitors : apical radial glia ( aRGs ) as Pax6 + NPs in VZ ( G ) , basal intermediate progenitors ( IPs ) as Tbr2 + in SVZ ( H ) n ≥ 3 brains . Data information : number of positive was quantified in 100µm - width boxes , randomly placed across LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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 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

Quantification of different classes of cortical progenitors: apical radial glia cells (aRGs) as Pax6+ NPs in VZ (G), basal intermediate progenitors (IPs) as Tbr2+ cells in SVZ (H) n ≥ 3 brains. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


of different classes of cortical progenitors : , basal RGs as Pax6 + in outer SVZ ( I ) . n ≥ 3 brains . Data information : number of positive was quantified in 100µm - width boxes , randomly placed across LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

Quantification of different classes of cortical progenitors: , basal RGs as Pax6+ cells in outer SVZ (I). n ≥ 3 brains. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


( J - L ) E12 . 5 dorsal brain views ( J , J ' ) representative sections of posterior hemispheres ( K , K ' ) , showing a vesicle enlargement in mutant brains ( K ' ) . extension of ventricular surface was quantified along A - P axis ( L ) ; red color code represents Nr2f1 gradient in WT brains . 12µm - thick sections were collected on series of 10 slides ; consecutive measurement are 120µm apart from each other . n ≥ 3 brains . Data information : In graphs , data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L) E12.5 dorsal brain views (J,J') and representative sections of posterior hemispheres (K,K'), showing a vesicle enlargement in mutant brains (K'). The extension of the ventricular surface was quantified along the A-P axis (L); red color code represents Nr2f1 gradient in WT brains. 12µm-thick sections were collected on series of 10 slides; consecutive measurement levels are 120µm apart from each other. n ≥ 3 brains. Data information: In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


( N - P ) GFP ( green ; expressed under Sox2 promoter ) RFP ( red ; expressed under Tis21 promoter ) IF of E13 . 5 lateral pallia , 18 - hours after IUE . proportion of single or double positive NPs is shown in pie charts in ( P ) . n = 3 electroporated brains . Data information : Nuclei ( blue ) were stained with DAPI . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(N-P) GFP (green; expressed under Sox2 promoter) and RFP (red; expressed under Tis21 promoter) IF of E13.5 lateral pallia, 18-hours after IUE. The proportion of single or double positive NPs is shown in pie charts in (P). n = 3 electroporated brains. Data information: Nuclei (blue) were stained with DAPI. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


( Q - R ' ) Ki67 ( green ; progenitors ) EdU ( red ) IF of E13 . 5 WT ( Q , Q ' ) KO ( R , R ' ) , injected with EdU at E12 . 5 . Differentiating ( EdU + Ki67 - ) are located in IZ / CP ; percentages shown in ( Q ' , R ' ) were obtained from n ≥ 4 sections of n ≥ 2 brains . Data information : Nuclei ( blue ) were stained with DAPI . Student t - ( Q ' , R ' ; E13 . 5 , EdU injected at E12 . 5 ; WT / KO : * = 0 . 03967 ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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

(Q-R') Ki67 (green; progenitors) and EdU (red) IF of E13.5 WT (Q,Q') and KO (R,R') embryos, injected with EdU at E12.5. Differentiating cells (EdU+Ki67-) are located in the IZ/CP; percentages shown in (Q',R') were obtained from n ≥ 4 sections of n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. Student t-test (Q',R'; E13.5, EdU injected at E12.5; WT/KO: *=0.03967 (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.


( A - D ) Graphs showing average number of PH3 + mitotic figures in posterior lateral pallium of WT mutant cortices . total number of mitoses ( A ) as well as number of dividing in VZ ( Pax6 + Sox2 + aRGs ; B ) , SVZ ( Tbr2 + IPs ; C ) outer ( basal - most ) region of SVZ ( oSVZ / IZ ; Pax6 + oRGs ; D ) are shown . For graphs ( B - D ) , statistical by 2 - way ANOVA n ≥ 3 brains per / genotype . Data information : number of positive was quantified in 100µm - width boxes , randomly placed across lateral pallium . In graphs , data are represented as means ± . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . SVZ : subventricular zone ; VZ : ventricular zone .

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

(A-D) Graphs showing the average number of PH3+ mitotic figures in the posterior lateral pallium of WT and mutant cortices. The total number of mitoses (A) as well as the number of dividing cells in VZ (Pax6+Sox2+ aRGs; B), SVZ (Tbr2+ IPs; C) and outer (basal-most) region of SVZ (oSVZ/IZ; Pax6+ oRGs; D) are shown. For graphs (B-D), statistical analysis by 2-way ANOVA n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001). SVZ: subventricular zone; VZ: ventricular zone.


( E ) cycle duration at different embryonic ages ( from E10 . 5 to E16 . 5 ) in WT ( blue bars ) or KO ( orange ) , as quantified upon a double EdU / BrdU injection protocol in posterior - most LP n ≥ 3 brains per / genotype . Data information : number of positive was quantified in 100µm - width boxes , randomly placed across lateral pallium . In graphs , data are represented as means ± . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell cycle duration at different embryonic ages (from E10.5 to E16.5) in WT (blue bars) or KO embryos (orange line), as quantified upon a double EdU/BrdU injection protocol in posterior-most LP n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).


( F - G ' ) Ki67 ( green ; NPs ) EdU ( red ) IF in WT ( F , F ' ) KO ( G , G ' ) after 6 hours consecutive EdU incorporation . Note higher number of labelled NPs in KO cortices ( G ' ) suggesting faster cycle progression / or longer S - phase . Data information : Nuclei ( blue ) were stained with DAPI .

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

(F-G') Ki67 (green; NPs) and EdU (red) IF in WT (F,F') and KO (G,G') after 6 hours consecutive EdU incorporation. Note the higher number of labelled NPs in KO cortices (G') suggesting faster cell cycle progression and/or longer S-phase. Data information: Nuclei (blue) were stained with DAPI.


( H ) Best linear fit of EdU + Ki67 + NP after 8 hours EdU cumulative labelling in WT ( blue ) KO ( orange ) brains . x - y - intercept are proportional to S - phase to number of cycling , respectively , while steepness of is proportional to cycle duration . WT : y = 11 . 66x + 24 . 62 ; KO : y = 13 . 54x + 32 . 96 ) . n ≥ 2 brains per time point / genotype . Data information : number of positive was quantified in 100µm - width boxes , randomly placed across lateral pallium . In graphs , data are represented as means ± . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(H) Best linear fit of EdU+Ki67+ NP percentage after 8 hours EdU cumulative labelling in WT (blue line) and KO (orange line) brains. The x- and y-intercept are proportional to the S-phase length and to the number of cycling cells, respectively, while the steepness of the line is proportional to cell cycle duration. WT: y = 11.66x +24.62; KO: y = 13.54x +32.96). n ≥ 2 brains per time point/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).


( I - K ) PH3 ( green ; dividing NPs ) Pax6 ( red ; aRGs ) IF of E13 . 5 WT ( I ) KO ( J ) lateral pallia . Strong weak / punctate PH3 patterns show M - phase G2 - phase , respectively . G2 - phase NP is quantified in ( K ) . As a result of shorter G1 - phase globally shorter cycle time , of G2 - phase is increased in KO NP pool . n ≥ 2 brains . Data information : Nuclei ( blue ) were stained with DAPI . number of positive was quantified in 100µm - width boxes , randomly placed across lateral pallium . In graphs , data are represented as means ± . Student t - ( K ; * P < 0 . 05 ) Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone .

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

(I-K) PH3 (green; dividing NPs) and Pax6 (red; aRGs) IF of E13.5 WT (I) and KO (J) lateral pallia. Strong and weak/punctate PH3 patterns show M-phase and G2-phase cells, respectively. G2-phase NP percentage is quantified in (K). As a result of shorter G1-phase and globally shorter cell cycle time, the percentage of G2-phase cells is increased in the KO NP pool. n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. Student t-test (K; *P<0.05) Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.


( L ) cycle time of E13 . 5 NPs in lateral pallia of WT ( upper column ) KO ( lower column ) , as calculated by EdU cumulative labelling PH3 KO NPs have a 16 . 1 % faster cycle time , due to a 30 . 9 % shorter G1 - phase compensating a 14 . 3 % longer S - phase . n ≥ 3 brains . In graphs , data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(L) Cell cycle time of E13.5 NPs in the lateral pallia of WT (upper column) and KO cells (lower column), as calculated by EdU cumulative labelling and PH3 staining KO NPs have a 16.1% faster cell cycle time, due to a 30.9% shorter G1-phase compensating a 14.3% longer S-phase. n ≥ 3 brains. In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


( A - B ' ) Pax6 ( green ; aRGs ) Nr2f1 ( red ) IF of E12 . 5 WT ( A , A ' ) KO ( B , B ' ) lateral pallia . Data information : nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone .

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 O O O O O O O O O O O O O O O O O O O O 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') Pax6 (green; aRGs) and Nr2f1 (red) IF of E12.5 WT (A,A') and KO (B,B') lateral pallia. Data information: nuclei (blue) were stained with DAPI. Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.


( C ) Pax6 Nr2f1 pixel at E12 . 5 E14 . 5 indicating increased Pax6 upon Nr2f1 removal . n ≥ 3 brains . Data information : pixel was quantified in 100µm - width boxes , randomly placed across lateral pallium . Data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(C) Pax6 and Nr2f1 pixel intensity quantification at E12.5 and E14.5 indicating increased Pax6 levels upon Nr2f1 removal. n ≥ 3 brains. Data information: the pixel intensity was quantified in 100µm-width boxes, randomly placed across the lateral pallium. Data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


( D ) Real time RT - PCR of Nr2f1 Pax6 expression in E12 . 5 cortices . n ≥ 3 cortices . Data information : Data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Real time RT-PCR quantification of Nr2f1 and Pax6 expression in E12.5 cortices. n ≥ 3 cortices. Data information: Data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


( E - G ' ' ) GFP ( green ; electroporated ) , Pax6 ( red ) Nr2f1 ( blue ) IF of pCIG2 - Nr2f1 - IRES - GFP electroporated brains at E13 . 5 ( 24 - hours after IUE ) . VZ ( F - F ' ' ) CP ( G - G ' ' ) regions are shown at high magnification . Arrowheads point to GFP + electroporated over - expressing Nr2f1 ( blue in F ' , G ' ) , down - regulating Pax6 ( F ' ' ) rapidly migrating out of VZ ( G - G ' ' ) . Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone .

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 O O O O O O O O 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 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

(E-G'') GFP (green; electroporated cells), Pax6 (red) and Nr2f1 (blue) IF of pCIG2-Nr2f1-IRES-GFP electroporated brains at E13.5 (24-hours after IUE). VZ (F-F'') and CP (G-G'') regions are shown at high magnification. Arrowheads point to GFP+ electroporated cells over-expressing Nr2f1 (blue in F',G'), down-regulating Pax6 (F'') and rapidly migrating out of the VZ (G-G''). Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.


( H ) Scatter plot showing Pax6 Nr2f1 pixel of VZ electroporated progenitors , comparing Nr2f1 overexpressing ( orange dots ) with control pCIG2 - GFP electroporated ( blue dots ) . Average Nr2f1 Pax6 pixel intensities of 2 populations were compared by 2 - way ANOVA resulted significantly different ( * * * = < 0 . 0001 ) . n = 2 electroporated brains . Data information Data are represented as means ± . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(H) Scatter plot showing Pax6 and Nr2f1 pixel intensity of VZ electroporated progenitors, comparing Nr2f1 overexpressing cells (orange dots) with control pCIG2-GFP electroporated cells (blue dots). Average Nr2f1 and Pax6 pixel intensities of the 2 populations were compared by 2-way ANOVA and resulted significantly different (***= <0.0001). n = 2 electroporated brains. Data information Data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).


( I ) Real time RT - PCR of cycle genes comparing WT ( blue ) KO ( orange ) cortices . CyclinD1 P21 transcripts are down - regulated whereas Dct is up - regulated in mutants . n = 3 cortices . Data information : Data are represented as means ± . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(I) Real time RT-PCR of cell cycle genes comparing WT (blue line) and KO (orange line) cortices. CyclinD1 and P21 transcripts are down-regulated whereas Dct is up-regulated in mutants. n = 3 cortices. Data information: Data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).


( J - L ) GFP ( green ; electroporated ) P21 ( red ) IF of E14 . 5 cortex electroporated 48 hours earlier with control PX458 plasmid ( J ) or CRISPR / Cas9 - expressing plasmid directed against Nr2f1 ( PX458 - αNr2f1 ; K ) . of P21 / GFP double positive ( L ) . Arrowheads in ( J , K ) point to P21 + . n = 2 electroporated brains . Data information , nuclei ( blue ) were stained with DAPI . Student t - ( L ; * P < 0 .

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 B-CONTROLLED_VAR 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 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

(J-L) GFP (green; electroporated cells) and P21 (red) IF of E14.5 cortex electroporated 48 hours earlier with control PX458 plasmid (J) or CRISPR/Cas9-expressing plasmid directed against Nr2f1 sequence (PX458-αNr2f1; K). Percentage of P21/GFP double positive cells (L). Arrowheads in (J,K) point to P21+ cells. n = 2 electroporated brains. Data information , nuclei (blue) were stained with DAPI. Student t-test (L; *P<0.05


( N ) Graph showing number of differentially expressed genes ( DEG up - regulated ( UP ) or down - regulated ( DOWN ) in KO compared to WT , as detected by - Seq of E15 . 5 neocortices . Data information : For statistical of - Seq data , see Methods .

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(N) Graph showing the number of differentially expressed genes (DEG up-regulated (UP) or down-regulated (DOWN) in KO compared to WT, as detected by RNA-Seq of E15.5 neocortices. Data information: For statistical analysis of RNA-Seq data , see Methods.


( P ) Hierarchical clustering of expression level of DEGs belonging to " neuron differentiation " Heat map color scale indicates normalized gene expression from high ( red ) to low ( ) level . Data information : For statistical of - Seq data see Methods .

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(P) Hierarchical clustering and heatmap of the expression level of DEGs belonging to the "neuron differentiation" Heat map color scale indicates normalized gene expression from high (red) to low (white) level. Data information: For statistical analysis of RNA-Seq data see Methods.


( A ) Real time RT - PCR of Pax6 expression in neurospheres with different genotypes , as indicated . n = 3 cortices . N KO P HET : Nr2f1 KO , Pax6 HET . Data information : Data are represented as means ± . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(A) Real time RT-PCR analysis of Pax6 expression in neurospheres with different genotypes, as indicated. n = 3 cortices. N KO P HET: Nr2f1 KO, Pax6 HET. Data information: Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).


( B ) Representative of Nr2f1 KO Pax6 HET neurospheres obtained from E15 . 5 neocortices cultured in vitro . Scale bars : 50µm .

O 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) Representative images of Nr2f1 KO Pax6 HET neurospheres obtained from E15.5 neocortices and cultured in vitro. Scale bars: 50µm.


( C ) Graph showing number of neurospheres per P100 plate , at different steps , as indicated . While Nr2f1 KO neurospheres ( orange ) proliferate for a longer time compared to WT ones ( blue ) , loss of one Pax6 allele ( gray ) almost restores normal proliferation rate exhaustion time . Complete Pax6 loss is not compatible with renewal ( yellow ) . n ≥ 3 culture wells from n = 2 independent batches . Data information : Data are represented as means ± . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(C) Graph showing the number of neurospheres per P100 cell plate, at different steps, as indicated. While Nr2f1 KO neurospheres (orange line) proliferate for a longer time compared to WT ones (blue line), the loss of one Pax6 allele (gray line) almost restores normal proliferation rate and exhaustion time. Complete Pax6 loss is not compatible with stem cell renewal (yellow line). n ≥ 3 culture wells from n = 2 independent batches. Data information: Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).


( D - E ) Paired after neurosphere dissociation 24 - hour culture at clonal density . Couples of dividing were identified by Map2 ( red ; N , neurons ) BLBP ( green ; P , progenitors ) IF . Pie charts in ( E ) show proportion of P - P ( proliferative ; D ) , P - N ( asymmetric differentiative ; D ' ) or N - N ( symmetric differentiative ; D ' ' ) couples in WT , Nr2f1KO Nr2f1KO Pax6HET . n ≥ 3 samples from n = 2 culture batches . Data information : Nuclei ( blue ) were stained with DAPI . Data are represented as means ± . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 Scale bars : 50µm .

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

(D-E) Paired cell analysis after neurosphere dissociation and 24-hour culture at clonal density. Couples of dividing cells were identified by Map2 (red; N, neurons) and BLBP (green; P, progenitors) IF. Pie charts in (E) show the proportion of P-P (proliferative; D), P-N (asymmetric differentiative; D') or N-N (symmetric differentiative; D'') couples in WT, Nr2f1KO and Nr2f1KO Pax6HET animals. n ≥ 3 samples from n = 2 culture batches. Data information: Nuclei (blue) were stained with DAPI. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001 Scale bars: 50µm.


( F - I ) Pax6 ( green ) Nr2f1 ( red ) IF in posterior - most region of E12 . 5 cortex , showing Pax6 upregulation in Nr2f1 KO ( G , G ' ) compared to WT ( F ' , F ' ' ) . Normal Pax6 can be restored by loss of one Pax6 allele ( Nr2f1 KO , Pax6 HET ; H , H ' ) . n ≥ 3 brains . N KO : Nr2f1 KO ; P HET : Pax6 HET . Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm .

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

(F-I) Pax6 (green) and Nr2f1 (red) IF in the posterior-most region of E12.5 cortex, showing Pax6 upregulation in Nr2f1 KO animals (G,G') compared to WT (F',F''). Normal Pax6 levels can be restored by loss of one Pax6 allele (Nr2f1 KO, Pax6 HET; H,H'). n ≥ 3 brains. N KO: Nr2f1 KO; P HET: Pax6 HET. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm.


Pixel of Nr2f1 ( blue ) Pax6 ( orange ) is shown in ( I ) . n ≥ 3 brains . N KO : Nr2f1 KO ; P HET : Pax6 HET . Data information : pixel or number of positive was quantified in 100µm - width boxes , randomly placed across LP . Data are represented as means ± . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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Pixel intensity quantification of Nr2f1 (blue) and Pax6 (orange) is shown in (I). n ≥ 3 brains. N KO: Nr2f1 KO; P HET: Pax6 HET. Data information: the pixel intensity or the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).


( J ) cycle duration at E12 . 5 in different mutants as indicated , quantified in posterior regions with double EdU / BrdU injection protocol n ≥ 6 sections from n = 2 brains . Data information : pixel or number of positive was quantified in 100µm - width boxes , randomly placed across LP . Data are represented as means ± . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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(J) Cell cycle duration at E12.5 in different mutants as indicated, quantified in posterior regions with double EdU/BrdU injection protocol n ≥ 6 sections from n = 2 brains. Data information: the pixel intensity or the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).


( K ) Insets showing representative γ - Tubulin ( green ) Phospho - Vimentin ( red ) IF of cortical VZ surface to evaluate orientation of cleavage plane of dividing aRGs . Pie charts show of vertical ( green ) , oblique ( yellow ) horizontal ( red ) division planes of mitotic figures in E12 . 5 lateral pallium of with different genotypes , as indicated . n ≥ 2 brains per genotype . Data information : Nuclei ( blue ) were stained with DAPI . Data are represented as means ± . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . cale bars : 50µm .

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(K) Insets showing representative γ-Tubulin (green) and Phospho-Vimentin (red) IF of the cortical VZ surface to evaluate the orientation of the cleavage plane of dividing aRGs. Pie charts show the percentage of vertical (green), oblique (yellow) and horizontal (red) division planes of mitotic figures in the E12.5 lateral pallium of animals with different genotypes, as indicated. n ≥ 2 brains per genotype. Data information: Nuclei (blue) were stained with DAPI. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001). cale bars: 50µm.


( A - C ) NR2F1 expression in cerebral organoids during neural induction differentiation in vitro ( day 40 day 70 , respectively ) , together with NP markers PAX6 ( green in A ) , SOX2 ( green in B , B ' ) DCX as a neural marker ( blue in A , B , B ' ' ) . of NR2F1 in NPs is shown in ( C ) . n ≥ 4 organoids from n = 2 batches . Data information : pixel or number of positive were quantified in 100µm - width boxes , randomly placed across cortex / organoid neuroepithelia In graphs , data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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(A-C) NR2F1 expression in cerebral organoids during neural induction and differentiation in vitro (day 40 and day 70, respectively), together with the NP markers PAX6 (green in A), SOX2 (green in B,B') and DCX as a neural marker (blue in A,B,B''). Quantification of NR2F1 levels in NPs is shown in (C). n ≥ 4 organoids from n = 2 batches. Data information: the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( D - G ) NR2F1 ( red ) PAX6 ( green ) IF in day 40 organoids . Neuroepithelia with high NR2F1 show weak PAX6 expression ( E , E ' ) , vice versa ( F , F ' ) . by pixel ( G ) . n ≥ 4 organoids from n = 2 batches . Data information : Nuclei ( blue stained with DAPI . pixel or number of positive were quantified in 100µm - width boxes , randomly placed across cortex / organoid neuroepithelia In graphs , data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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(D-G) NR2F1 (red) and PAX6 (green) IF in day 40 organoids. Neuroepithelia with high NR2F1 levels show weak PAX6 expression (E,E'), and vice versa (F,F'). Quantification by pixel intensity analysis (G). n ≥ 4 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( I - J ) NR2F1 ( red ) GFP ( green ) IF in day 40 human brain organoids upon electroporation of PX458 - αNR2F1 plasmid . In ( I , I ' ) , GFP + NR2F1 - ( empty arrowheads ) a GFP + NR2F1 + ( arrowhead ) are shown ; in ( J ) . n ≥ 6 organoids from n = 2 batches . Data information : Nuclei ( blue stained with DAPI . pixel or number of positive were quantified in 100µm - width boxes , randomly placed across cortex / organoid neuroepithelia In graphs , data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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(I-J) NR2F1 (red) and GFP (green) IF in day 40 human brain organoids upon electroporation of the PX458-αNR2F1 plasmid. In (I,I'), GFP+NR2F1- cells (empty arrowheads) and a GFP+NR2F1+ cell (white arrowhead) are shown; quantification in (J). n ≥ 6 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( K - M ) TUJ1 ( red ; differentiating neurons ) GFP ( green ) IF 7 days after electroporation of control ( K ) PX458 - αNr2f1 plasmids ( L ) . Co - expression of GFP with SOX2 or TUJ1 distinguishes neural progenitors ( NPs ) from neurons ( Ns ) , as quantified in ( M ) . Arrowheads in ( K , L ) point to TUJ1 + GFP + . n ≥ 6 organoids from n = 2 batches . Data information : Nuclei ( blue stained with DAPI . number of positive was normalized over total number of GFP + . In graphs , data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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(K-M) TUJ1 (red; differentiating neurons) and GFP (green) IF 7 days after electroporation of control (K) and PX458-αNr2f1 plasmids (L). Co-expression of GFP with SOX2 or TUJ1 distinguishes neural progenitors (NPs) from neurons (Ns), as quantified in (M). Arrowheads in (K,L) point to TUJ1+ GFP+ cells. n ≥ 6 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the number of positive cells was normalized over the total number of GFP+ cells. In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( N ) Red green arrowheads in ( N ) point to a SOX2 + ( red ) progenitor a TUJ1 + ( blue ) neuron , respectively . Scale bars : 50µm .

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(N) Red and green arrowheads in (N) point to a SOX2+ (red) progenitor and a TUJ1+ (blue) neuron, respectively. Scale bars: 50µm.


( C , D ) Standard replication assays on undamaged ( UD ) , thymine glycol ( Tg ) , cyclobutane pyrimidine dimer ( CPD ) templates in presence ( C ) absence ( D ) of Pol η , Pol ζ , Rev1 , Rad6 - Rad18 , Uba1 , ubiquitin ( TLS machinery ) . Reactions contained 2 . 5 nM Pol δ .

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(C, D) Standard replication assays on undamaged (UD), thymine glycol (Tg), and cyclobutane pyrimidine dimer (CPD) templates in the presence (C) and absence (D) of Pol η, Pol ζ, Rev1, Rad6-Rad18, Uba1, and ubiquitin (TLS machinery). Reactions contained 2.5 nM Pol δ.


( B ) Oligonucleotide competition on UD Tg templates . S ; scrambled oligonucleotide . ( C ) Lane profiles of lanes 4 - 6 in ( B ) normalised to run off product . FL ; FL - lead , OR ; oligonucleotide restart , S ; Stall , RO ; Run off , OF ; Okazaki fragments .

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(B) Oligonucleotide competition assay on UD and Tg templates. S; scrambled oligonucleotide. (C) Lane profiles of lanes 4-6 in (B) normalised to the run off product. FL; FL-lead, OR; oligonucleotide restart, S; Stall, RO; Run off, OF; Okazaki fragments.


( B ) Oligonucleotide competition on UD Tg templates . S ; scrambled oligonucleotide .

(B) Oligonucleotide competition assay on UD and Tg templates. S; scrambled oligonucleotide.


( D ) chase reaction on UD Tg templates performed as shown . Where present , Pol δcat ( 10 nM ) was added directly after addition of chase .

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(D) Pulse chase reaction on UD and Tg templates performed as shown. Where present, Pol δcat (10 nM) was added directly after addition of the chase.


( E ) Standard replication reaction performed in absence presence of RFC / PCNA on Tg template .

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(E) Standard replication reaction performed in the absence and presence of RFC/PCNA on the Tg template.


( B ) chase reactions on Tg template in absence presence of 10 nM Pol δ analysed on a denaturing gel .

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(B) Pulse chase reactions on the Tg template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel.


( D ) chase reaction on Tg template in absence presence of 10 nM Pol δ analysed on a urea polyacrylamide gel .

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(D) Pulse chase reaction on the Tg template in the absence and presence of 10 nM Pol δ analysed on a urea polyacrylamide gel.


( E ) Titration of Pol δ in a chase reaction on Tg template . Pol δ was added after 0 min time point was taken . ( F ) of bypass 5 min after addition of chase for data in ( E ) .

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(E) Titration of Pol δ in a pulse chase reaction on the Tg template. Pol δ was added after the 0 min time point was taken. (F) Quantification of bypass 5 min after addition of the chase for the data in (E).


( E ) Titration of Pol δ in a chase reaction on Tg template . Pol δ was added after 0 min time point was taken .

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(E) Titration of Pol δ in a pulse chase reaction on the Tg template. Pol δ was added after the 0 min time point was taken.


( G ) Titration of Pol δ or Pol ε ( 1 , 2 . 5 , 5 , 7 . 5 , 10 nM ) into a primer extension on a Tg template in presence of PCNA RFC . primer / template location of lesion are shown above .

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(G) Titration of Pol δ or Pol ε (1, 2.5, 5, 7.5, 10 nM) into a primer extension assay on a Tg template in the presence of PCNA and RFC. The primer/template sequence and location of the lesion are shown above.


( B ) Mapping 3ʹ - end of stalled nascent leading strand . chase reaction on Tg CPD templates . Where present , Pol δ ( 10 nM ) was added immediately after 0 min time point . Products were with SwaI BamHI resolved alongside a sequencing ladder corresponding to nascent strand . dd ; dideoxy .

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(B) Mapping the 3ʹ-end of the stalled nascent leading strand. Pulse chase reaction on Tg and CPD templates. Where present, Pol δ (10 nM) was added immediately after the 0 min time point. Products were cleaved with SwaI and BamHI and resolved alongside a sequencing ladder corresponding to the nascent strand. dd; dideoxy.


( A ) Primer extension reactions on Tg template with 5 nM Pol δ or Pol ε either dATP , dCTP , dGTP , or dTTP .

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(A) Primer extension reactions on the Tg template with 5 nM Pol δ or Pol ε and either dATP, dCTP, dGTP, or dTTP.


( C ) of nucleotide incorporation opposite Tg from replication reactions processed as shown in ( B ) . Three experiments were performed , two in presence one in absence of Pol δ ( 10 nM ) , giving 99 , 97 , 95 successful sequencing reads respectively .

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(C) Analysis of nucleotide incorporation opposite Tg from replication reactions processed as shown in (B). Three individual experiments were performed, two in the presence and one in the absence of Pol δ (10 nM), giving 99, 97, and 95 successful sequencing reads respectively.


( A ) Standard replication on undamaged ( UD ) , 8 - oxoguanine ( 8oxoG ) , thymine glycol ( Tg ) templates in absence of TLS components .

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(A) Standard replication assay on undamaged (UD), 8-oxoguanine (8oxoG), and thymine glycol (Tg) templates in the absence of TLS components.


( B ) chase reaction on UD 8oxoG templates . Where present , Pol δcat ( 10 nM ) was added directly after addition of chase .

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(B) Pulse chase reaction on UD and 8oxoG templates. Where present, Pol δcat (10 nM) was added directly after addition of the chase.


( C ) chase reactions on 8oxoG template in absence presence of 10 nM Pol δ analysed on a denaturing gel .

(C) Pulse chase reactions on the 8oxoG template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel.


A ) Assessment of detection limit of synthetic SARS - CoV - 2 by CRISPR - , inferred from values after 30 min . Background subtracted represents sample minus control without target .

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A) Assessment of the detection limit of synthetic SARS-CoV-2 by the CRISPR-based assay, inferred from fluorescence values after 30 min. Background subtracted fluorescence represents sample minus control fluorescence without target.


Detection of synthetic SARS - CoV - 2 using commercially available paper strips ( lateral - flow ) in buffer ( B )

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Detection of synthetic SARS-CoV-2 RNA using commercially available paper strips (lateral-flow) in buffer (B)


Detection of synthetic SARS - CoV - 2 using commercially available paper strips ( lateral - flow ) in saliva ( C ) .

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Detection of synthetic SARS-CoV-2 RNA using commercially available paper strips (lateral-flow) in saliva (C).


Rag1 - / - : Bcl2 Art - / - : Bcl2 abl pre - B were treated with imatinib for 48 h to induce G1 arrest . ( A ) DSBs quantified by qPCR of Igk ( Jκ1 ) genomic . Schematic shows germline ( GL ) Igk locus , unrepaired Jκ1 coding end ( Cut ) primer location . Results are normalized to Rag1 - / - : Bcl2 abl pre - B , which do not generate RAG DSBs have only germline Igk . Loss of Igk germline product is representative of DSB generation .

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Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (A) DSBs quantified by qPCR analysis of Igk (Jκ1) genomic DNA. Schematic shows germline (GL) Igk locus, unrepaired Jκ1 coding end (Cut) and primer location. Results are normalized to Rag1-/-:Bcl2 abl pre-B cells, which do not generate RAG DSBs and have only germline Igk DNA. Loss of Igk germline product is representative of DSB generation.


Rag1 - / - : Bcl2 Art - / - : Bcl2 abl pre - B were treated with imatinib for 48 h to induce G1 arrest . ( B ) Representative of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows of with indicated number of foci from three biological replicates . In each replicate , 100 per condition were quantified .

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Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (B) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.


Rag1 - / - : Bcl2 Art - / - : Bcl2 abl pre - B were treated with imatinib for 48 h to induce G1 arrest . ( C ) Western blot of KAP1 phosphorylation ( p - KAP1 ) , NF - κB2 ( p100 p52 ) PIM2 . GAPDH is shown as loading control .

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Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (C) Western blot analysis of KAP1 phosphorylation (p-KAP1), NF-κB2 (p100 and p52) and PIM2. GAPDH is shown as loading control.


Rag1 - / - : Bcl2 Art - / - : Bcl2 abl pre - B were treated with imatinib for 48 h to induce G1 arrest . ( D ) Cd40 Pim2 mRNA expression .

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Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (D) Cd40 and Pim2 mRNA expression.


Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( E ) DSBs quantified by qPCR of Igh ( JH1 ) genomic analyzed as in A with results normalized to empty vector control , which has only germline Igh .

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Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (E) DSBs quantified by qPCR analysis of Igh (JH1) genomic DNA and analyzed as in A with results normalized to empty vector control, which has only germline Igh DNA.


Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( F ) Representative of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows of with indicated number of foci from three biological replicates . In each replicate , 100 per condition were quantified .

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Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (F) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.


E - H . Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( G ) Western blot of FLAG - RAG1 , p - KAP1 , NF - κB2 , PIM2 . GAPDH is shown as loading control .

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E-H. Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (G) Western blot analysis of FLAG-RAG1, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control.


Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B were transduced with an empty vector ( control ) or vector expressing RAG1 then treated with imatinib for 48 h . ( H ) Cd40 Pim2 mRNA expression .

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Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (H) Cd40 and Pim2 mRNA expression.


A . Rag1 - / - : Lig4 - / - : Bcl2 : iCas9 abl pre - B were treated with ( + ) or without ( - ) 2 μM doxycycline ( Dox ) to induce Cas9 as indicated . Western blot shows FLAG - Cas9 GAPDH ( loading control ) .

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A. Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with (+) or without (-) 2 μM doxycycline (Dox) to induce Cas9 as indicated. Western blot shows FLAG-Cas9 and GAPDH (loading control).


Rag1 - / - : Lig4 - / - : Bcl2 : iCas9 abl pre - B were treated with doxycycline as in A imatinib to trigger cycle arrest for 24 h . were then transfected with indicated gRNA maintained in doxycycline imatinib . analyses were completed 48 h after gRNA transfection . DSBs quantified by qPCR of Eb ( B ) Gapdh ( C ) genomic . Results are normalized to without gRNA ( - ) . Schematic shows germline ( GL ) locus , unrepaired cut end ( Cut ) location of gRNA primers . Loss of germline product is representative of DSB generation .

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Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with doxycycline as in A and imatinib to trigger cell cycle arrest for 24 h. Cells were then transfected with indicated gRNA and maintained in doxycycline and imatinib. All analyses were completed 48 h after gRNA transfection. DSBs quantified by qPCR analysis of Eb (B) and Gapdh (C) genomic DNA. Results are normalized to cells without gRNA (-). Schematic shows germline (GL) locus, unrepaired cut end (Cut) and location of gRNA and primers. Loss of germline product is representative of DSB generation.


( D ) Representative of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows of with indicated number of foci from three biological replicates . In each replicate , 100 per condition were quantified .

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(D) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.


( E ) Western blot of FLAG - tagged Cas9 or RAG1 ( as indicated ) , p - KAP1 , NF - κB2 , PIM2 . GAPDH is shown as loading control . Responses to RAG DSBs in Rag1 - / - : Lig4 - / - : Bcl2 abl pre - B transduced with empty vector or vector expressing RAG1 RAG DSBs from Fig 1G are included for direct comparison . # denotes band for FLAG - Cas9 . * denotes band for FLAG - RAG1 .

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(E) Western blot analysis of FLAG-tagged Cas9 or RAG1 (as indicated), p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control. Responses to RAG DSBs in Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells transduced with empty vector or vector expressing RAG1 RAG DSBs from Fig 1G are included for direct comparison. # denotes band for FLAG-Cas9. * denotes band for FLAG-RAG1.


( F ) Cd40 Pim2 mRNA expression . Blue dashed indicates mRNA expression in response to RAG DSBs from Fig 1H is included for comparison .

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(F) Cd40 and Pim2 mRNA expression. Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison.


A - B . DSBs quantified by qPCR of Jκ1 ( A ) JH1 JHD1 - 1 ( B ) genomic . Schematics show germline ( GL ) locus location of respective gRNAs primers . PCR is normalized to without gRNA ( - ) . Loss of germline product is representative of DSB generation .

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A-B. DSBs quantified by qPCR analysis of Jκ1 (A) and JH1 and JHD1-1 (B) genomic DNA. Schematics show germline (GL) locus and location of respective gRNAs and primers. PCR is normalized to cells without gRNA (-). Loss of germline product is representative of DSB generation.


C . Representative of 53BP1 foci . Scale bar denotes 8 μm . Bar graph shows of with indicated number of foci from three biological replicates . In each replicate , 100 per condition were quantified .

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C. Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.


D . Western blot of FLAG - Cas9 , p - KAP1 , NF - κB2 , PIM2 . GAPDH is shown as loading control .

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D. Western blot analysis of FLAG-Cas9, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control.


E . CD40 Pim2 mRNA expression . Blue dashed indicates mRNA expression in response to RAG DSBs from Fig 1H is included for comparison .

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E. CD40 and Pim2 mRNA expression. Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison.


B . DSBs quantified by qPCR of Igh ( JH1 ) genomic analyzed as in Fig 1E with results normalized to empty vector control , which has only germline Igh . There is no statistically significant difference in DSBs generated by cRAG1 or RAG1P326G compared to full - RAG1 .

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B. DSBs quantified by qPCR analysis of Igh (JH1) genomic DNA and analyzed as in Fig 1E with results normalized to empty vector control, which has only germline Igh DNA. There is no statistically significant difference in DSBs generated by cRAG1 or RAG1P326G compared to full-length RAG1.


D . Western blot of FLAG - RAG1 , p - KAP1 , NF - κB2 , PIM2 . GAPDH is shown as loading control .

D. Western blot analysis of FLAG-RAG1, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control.


E. Cd40 and Pim2 mRNA expression.


Photograph of with FMR1G - ins . allele . Typical physical behavioral features of FXS were noted in , who shows moderate to severe intellectual disability .

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Photograph of male patient with FMR1G-ins. allele. Typical physical and behavioral features of FXS were noted in the patient, who shows moderate to severe intellectual disability.


FMR1 mRNA were analyzed via RT - qPCR in EBV - transformed lymphocyte derived from ' s blood samples . showed a ˜ 60 % decrease in FMR1 mRNA compared to control ( * P = 0 . 010 , 0 . 383 ± 0 . 110 SD , n = 3 ) . Treatment with translational blocker puromycin restored FMR1 mRNA in ( * P = 0 . 048 , 0 . 383 ± 0 . 110 SD versus 1 . 25 ± 0 . 166 SD , n = 3 ) , suggesting that reduction of FMR1 mRNA in these is primarily due to nonsense - mediated decay . RT - qPCR reactions were run in triplicate in three independent experiments . Fold changes in FMR1 expression , normalized to HRPT expression , were calculated using ΔΔCT method , analyzed statistically with a two - t - ( GraphPad ) . Error bars represent mean values with SD .

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FMR1 mRNA levels were analyzed via RT-qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔCT method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD.


Western blot shows a significant decrease ( > 90 % ) in FMRP in - derived ( * * * P = 0 . 008 , 0 . 13 ± 0 . 031 SD , n = 2 ) , FMRP migrates slightly lower than wild - type . Band intensities for different lines were quantified , normalized for actin analyzed statistically with a twot - t - ( GraphPad ) . Error bars represent mean values with SD .

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Western blot analysis shows a significant decrease (> 90%) in FMRP protein levels in patient-derived cells (***P = 0.008, 0.13 ± 0.031 SD, n = 2), and the patient FMRP migrates slightly lower than the wild-type protein. Band intensities for the different cell lines were quantified, normalized for actin and analyzed statistically with a twot-tailed t-test (GraphPad). Error bars represent mean values with SD.


A - C HEK293 ( A ) SH - Sy5y ( B ) , as well as neurons from FMR1 KO mice ( C ) , were transfected with vectors encoding GFP - tagged wild - type FMRP ( GFP - FMR1WT ) or GFP - tagged FMRP ( GFP - FMR1G - insertion ) under control of a β - actin promoter . Wild - type FMRP localizes predominantly in cytoplasm , whereas FMRP forms nucleolar that colocalize with nucleophosmin . Scale bars in ( A , B ) represent 10 μm . Scale bar in ( C ) represents 5 μm .

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A-C HEK293 (A) and SH-Sy5y (B) cells, as well as primary neurons from FMR1 KO mice (C), were transfected with vectors encoding the GFP-tagged wild-type FMRP (GFP-FMR1WT) or the GFP-tagged patient FMRP (GFP-FMR1G-insertion) under the control of a β-actin promoter. Wild-type FMRP localizes predominantly in the cytoplasm, whereas the patient FMRP forms nuclear and nucleolar aggregates that colocalize with nucleophosmin. Scale bars in (A, B) represent 10 μm. Scale bar in (C) represents 5 μm.


Transfected HEK293 expressing a GFP - tagged FMRP in which C - terminus is truncated following G - ins . mutation site ( GFP - FMR1ΔCt ) . GFP - FMR1ΔCt is exclusively , suggesting that truncation of C - terminus alone is not sufficient to explain localization change observed for patientFMRP . Transfected HEK293 expressing a modified version of GFP - tagged patientFMRP ( GFP - FMR1G - ins . [ NLSmutated ] ) . novel acid in C - terminus of patientFMRP [ RKRTRRKRTWRRLQRKRRSLPNR ] contains stretches of arginines lysines predicted to function as a localization signal ( on Expasy motif scanner , ScanProsite ) . Mutating adjacent R K residues into alanines [ RARTRAARTWARLQRAARSLPNR ] abolishes nucleolar localization of patientFMRP , strongly suggesting that novel acid present in patientFMRP C - terminus is a functional localization signal ( NLS ) .

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Transfected HEK293 cells expressing a GFP-tagged FMRP in which the C-terminus is truncated following the G-ins. mutation site (GFP-FMR1ΔCt). GFP-FMR1ΔCt protein is exclusively cytoplasmic, suggesting that the truncation of the C-terminus alone is not sufficient to explain the localization change observed for the patientFMRP.Transfected HEK293 cells expressing a modified version of GFP-tagged patientFMRP (GFP-FMR1G-ins. [NLSmutated]). The novel amino acid sequence in the C-terminus of patientFMRP [RKRTRRKRTWRRLQRKRRSLPNR] contains stretches of arginines and lysines predicted to function as a nuclear localization signal (based on Expasy protein motif scanner, ScanProsite). Mutating adjacent R and K residues into alanines [RARTRAARTWARLQRAARSLPNR] abolishes the nucleolar localization of the patientFMRP, strongly suggesting that the novel amino acid sequence present in the patientFMRP C-terminus is a functional nuclear localization signal (NLS).


Transfected HEK293 expressing GFP - tagged wild - type FMRP fused to NLS motif ( GFP - FMR1wt + NLS ) . Unlike FMRP , GFP - FMR1wt + NLS is predominantly does not aggregate in nucleus . However , treatment of HEK293 expressing GFP - FMR1wt + NLS with leptomycin B - an inhibitor of export - resulted in appearance of nucleolar inclusions . This suggests that presence of a full - C - terminus facilitates export of FMRP in this context . Transfected HEK293 expressing a modified version of GFP - tagged FMRP , in which truncation of C - terminus is reverted by restoring open reading frame ( GFP - FMR1G - ins . [ Ct restored ] ) . is not detected in nucleus when C - terminus is intact ; however , nucleolar retention of is observed upon leptomycin B treatment of transfected . In with results from ( A ) , this suggests that an intact C - terminus enables export of FMR1 . Data information : Scale bar represents 10 μm .

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Transfected HEK293 cells expressing GFP-tagged wild-type FMRP fused to the patient NLS motif (GFP-FMR1wt+NLS). Unlike the patient FMRP, GFP-FMR1wt+NLS protein is predominantly cytoplasmic and does not aggregate in the nucleus. However, treatment of HEK293 cells expressing GFP-FMR1wt+NLS with leptomycin B-an inhibitor of nuclear export-resulted in the appearance of nucleolar inclusions. This suggests that the presence of a full-length C-terminus facilitates the nuclear export of FMRP in this context.Transfected HEK293 cells expressing a modified version of GFP-tagged patient FMRP, in which the truncation of the C-terminus is reverted by restoring the open reading frame (GFP-FMR1G-ins.[Ct restored]). The patient protein is not detected in the nucleus when the C-terminus is intact; however, nucleolar retention of the protein is observed upon leptomycin B treatment of the transfected cells. In line with results from (A), this suggests that an intact C-terminus enables nuclear export of the FMR1 protein.Data information: Scale bar represents 10 μm.


of small lateral ventral neurons ( sLNvs ) is sensitive to dfmr1 activity . Overexpression of wild - type dfmr1 in sLNv neurons results in a consistent " axonal collapse " phenotype , where branching of axonal termini of sLNvs is reduced . Scale bars represent 50 10 μm ( magnified ) .

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The morphology of small lateral ventral neurons (sLNvs) is sensitive to dfmr1 activity. Overexpression of wild-type dfmr1 in the sLNv neurons results in a consistent "axonal collapse" phenotype, where the branching of axonal termini of sLNvs is reduced. Scale bars represent 50 and 10 μm (magnified images).


Overexpression of wild - type dfmr1 ( UAS - dfmr1wt ) using a LNv neuron - specific driver ( Pdf - Gal4 ) causes reduction of branching - " axonal collapse " - in sLNv axonal termini . dfmr1ΔCt dfmr1wt + NLS overexpression phenocopies overexpression of dfmr1wt , suggesting that truncation of C - terminus or presence of NLS alone does not impair or alter functional efficacy of dfmr1 in this context . However , overexpression of dfmr1ΔCt + NLS fails to cause collapse of sLNv termini , indicating a significant loss or change of Dfmrp function . Axonal is visualized by expressing CD8 - GFP under control of same neuronal driver . Scale bar represents 10 μm . of axonal termini branching in LNv neurons overexpressing various dfmr1 alleles . Overexpression of dfmr1wt , dfmr1ΔCt , or dfmr1wt + NLS significantly reduced axonal branching [ * * * * P < 0 . 0001 ; control 101 . 3 ± 29 . 76 SD , n = 30 ; UAS - dfmrwt 45 . 41 ± 21 . 69 SD , n = 29 ; UAS - dfmrΔCt 52 . 31 ± 14 . 67 SD , n = 32 ; UAS - dfmrwt + NLS 72 . 45 ± 21 . 83 SD , n = 29 ; UAS - dfmrΔCt + NLS 117 . 8 ± 36 . 96 SD , n = 28 ] . dfmr1ΔCt + NLS overexpression does not result in a significant change of axonal branching compared to background control [ Pdf - Gal4 , UAS - GFP ] ( P = 0 . 672 ) . For purposes , each branching ( n ) was manually outlined , starting from first point of defasculation of axonal bundle . These measurements were normalized for variability in brain across samples using LNv commissure measurements for each brain . Two - t - using Welch ' s correction were then used to compare controls with mutant ( GraphPad ) . Error bars represent mean values with SD . Dots visible for box plots of UAS - dfmrwt UAS - dfmrΔCt represent samples that outlier whiskers .

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Overexpression of wild-type dfmr1 (UAS-dfmr1wt) using a LNv neuron-specific driver line (Pdf-Gal4) causes reduction of branching area-"axonal collapse"-in the sLNv axonal termini. dfmr1ΔCt and dfmr1wt+NLS overexpression phenocopies overexpression of dfmr1wt, suggesting that the truncation of the C-terminus or the presence of the patient NLS alone does not impair or alter the functional efficacy of dfmr1 in this context. However, overexpression of dfmr1ΔCt+NLS fails to cause collapse of sLNv termini, indicating a significant loss or change of Dfmrp protein function. Axonal morphology is visualized by expressing CD8-GFP under the control of the same neuronal driver. Scale bar represents 10 μm.Quantification of axonal termini branching in LNv neurons overexpressing various dfmr1 alleles. Overexpression of dfmr1wt, dfmr1ΔCt, or dfmr1wt+NLS significantly reduced axonal branching area [****P < 0.0001; control 101.3 ± 29.76 SD, n = 30; UAS-dfmrwt 45.41 ± 21.69 SD, n = 29; UAS-dfmrΔCt 52.31 ± 14.67 SD, n = 32; UAS-dfmrwt+NLS 72.45 ± 21.83 SD, n = 29; UAS-dfmrΔCt+NLS 117.8 ± 36.96 SD, n = 28]. dfmr1ΔCt+NLS overexpression does not result in a significant change of axonal branching area compared to the background control [Pdf-Gal4, UAS-GFP] (P = 0.672). For quantification purposes, each branching area (n) was manually outlined, starting from the first point of defasculation of the axonal bundle. These measurements were normalized for variability in brain size across samples using LNv commissure length measurements for each brain. Two-tailed t-tests using Welch's correction were then used to compare controls with mutant phenotypes (GraphPad). Error bars represent mean values with SD. Dots visible for box plots of UAS-dfmrwt and UAS-dfmrΔCt represent samples that outlier the whiskers.


Overexpression of dfmr1ΔCt + NLS results in aberrant axonal termini . Axonal guidance defects were frequently observed ( arrowheads ) scored manually for bifurcations of axon bundle ( 9 / 48 ) , tangling of axons in termini ( 13 / 48 ) , apparent looping of axons ( 7 / 48 ) .

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Overexpression of dfmr1ΔCt+NLS results in aberrant axonal termini morphology. Axonal guidance defects were frequently observed (arrowheads) and scored manually for bifurcations of the axon bundle (9/48), tangling of axons in the termini (13/48), and apparent looping of axons (7/48).


A - D Subcellular localization of transgenically expressed dfmr1wt , dfmr1ΔCt , dfmr1wt + NLS , dfmr1ΔCt + NLS in LNv neurons . DfmrpΔCt + NLS shows localization ( A ) , colocalizing with genetically encoded marker RedStinger , while Dfmrpwt , DfmrpΔCt , Dfmrpwt + NLS are predominantly ( B - D ) . These results suggest that an intact C - terminus can mediate export of Dfmrp . Scale bar represents 5 μm .

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

A-D Subcellular localization of transgenically expressed dfmr1wt, dfmr1ΔCt, dfmr1wt+NLS, and dfmr1ΔCt+NLS proteins in LNv neurons. DfmrpΔCt+NLS shows nuclear localization (A), colocalizing with the genetically encoded nuclear marker RedStinger, while Dfmrpwt, DfmrpΔCt, and Dfmrpwt+NLS are predominantly cytoplasmic (B-D). These results suggest that an intact C-terminus can mediate the nuclear export of the Dfmrp protein. Scale bar represents 5 μm.


HEK293 were treated for 3 hours with 150 nM of LY411575 , 1 µM of MLN4924 , 10 µM of Roscovitine , 10 µM DRB or 10 µM XAV939 . DMSO served as vehicle control . Transfection with pCS2 - NICD vector served as positive control . Western blot reveals that NICD were increased upon treatment with Roscovitine , DRB , XAV939 , or MLN4924 . NICD is undetectable following LY411575 treatment . NICD antibody detects a doublet following inhibitor treatment top band disappears following λ phosphatase treatment indicating that this top band is a phosphorylated isoform . β - Actin served as loading control . of density of western blot bands in ( A ) using ImageJ software . Data are expressed as fold changes compared to DMSO treatment . data represent mean ± from three independent experiments . One - way ANOVA , followed by Dunnett , was performed with * p ≤ 0 . 05 , * * p ≤ 0 . 01 ns = not significant .

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

HEK293 cells were treated for 3 hours with 150 nM of LY411575, 1 µM of MLN4924, 10 µM of Roscovitine, 10 µM DRB or 10 µM XAV939. DMSO served as vehicle control. Transfection with pCS2-NICD vector served as positive control. Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine, DRB, XAV939, or MLN4924. NICD is undetectable following LY411575 treatment. NICD antibody detects a doublet following inhibitor treatment and the top band disappears following λ phosphatase treatment indicating that this top band is a phosphorylated isoform. β-Actin served as loading control.   Quantification of the density of western blot bands in (A) using ImageJ software. Data are expressed as fold changes compared to DMSO treatment. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed with *p≤0.05, **p≤0.01 and ns=not significant.