( 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 ) .

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(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 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 .

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(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 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 ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O 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 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

(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 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 .

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

(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 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 .

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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 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 .

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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 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 ) .

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

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


( 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 ) .

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

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

(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 ) 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 .

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 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 B-MEASURED_VAR O 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 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 ) .

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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 ) 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 ) .

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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 ) 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 .

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

(K) 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 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 and green arrowheads in ( N ) point to a SOX2 + ( red ) progenitor and 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 ) , 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 .

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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 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 .

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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 assay on UD and Tg templates . S ; scrambled oligonucleotide .

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


( 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 .

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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 the absence and presence of RFC / PCNA on the Tg template .

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


( B ) Pulse chase reactions on the Tg template in the absence and 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 ) Pulse chase reaction on the Tg template in the absence and 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 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 ) .

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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 pulse chase reaction on the Tg template . Pol δ was added after the 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 assay on a Tg template in the presence of PCNA and RFC . The primer / template sequence and location of the 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 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 .

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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 the Tg template with 5 nM Pol δ or Pol ε and 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 ) 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 .

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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 assay on undamaged ( UD ) , 8 - oxoguanine ( 8oxoG ) , and thymine glycol ( Tg ) templates in the 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 ) Pulse chase reaction on UD and 8oxoG templates . Where present , Pol δcat ( 10 nM ) was added directly after addition of the 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 ) Pulse chase reactions on the 8oxoG template in the absence and 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 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 .

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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 RNA 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 RNA 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 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 .

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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 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 .

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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 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 .

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

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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 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 .

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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 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 .

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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 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 .

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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 cells were treated with ( + ) or without ( - ) 2 μM doxycycline ( Dox ) to induce Cas9 as indicated . Western blot shows FLAG - Cas9 and 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 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 .

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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 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 .

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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 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 .

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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 and Pim2 mRNA expression . Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and 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 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 .

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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 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 .

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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 analysis of FLAG - Cas9 , p - KAP1 , NF - κB2 , and 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 and Pim2 mRNA expression . Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and 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 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 .

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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 analysis of FLAG - RAG1 , p - KAP1 , NF - κB2 , and 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.


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E. Cd40 and Pim2 mRNA expression.


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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 ) .

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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 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 .

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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.


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 ) .

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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 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 .

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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 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 ) .

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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 , 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 .

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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 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 .

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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.


iPS 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 has been used as vehicle 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 indicative that this top band is a phosphorylated isoform . β - Actin served as loading control . Quantification of the density of western blot bands in ( C ) 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 , * * * p ≤ 0 . 001 and ns = not significant .

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iPS 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 has been used as vehicle 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 indicative that this top band is a phosphorylated isoform. β-Actin served as loading control.   Quantification of the density of western blot bands in (C) 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, ***p≤0.001 and ns= not significant.


HEK293 cells were treated with the inhibitors NICD phosphorylation status was analysed by a Phos - tag assay . DMSO served as vehicle control . NICD phosphorylation profile varies following Roscovitine , DRB , XAV939 or MLN4924 treatment . Following λ phosphatase treatment none of the high molecular weight bands are visible indicating they are all phosphorylated isoforms .

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HEK293 cells were treated with the inhibitors NICD phosphorylation status was analysed by a Phos-tag assay. DMSO served as vehicle control. NICD phosphorylation profile varies following Roscovitine, DRB, XAV939 or MLN4924 treatment. Following λ phosphatase treatment none of the high molecular weight bands are visible indicating they are all phosphorylated isoforms.


NICD interaction with FBXW7 at endogenous levels in HEK293 cells . 500 µg of HEK293 cell lysates treated with DMSO or MLN4924 were subjected to immunoprecipitation using FBXW7 antibody , or IgG antibody as negative control , and precipitated material was analysed by western blot using NICD antibody . Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency . 10 % of cell lysate before immunoprecipitation was used as input control and β - Actin served as loading control . Quantification of the density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as percentage changes compared to MLN4924 . All data represent the mean ± SEM from three independent experiments . Student t - test was used to determine p values , with * * p ≤ 0 . 01 .

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NICD interaction with FBXW7 at endogenous levels in HEK293 cells. 500 µg of HEK293 cell lysates treated with DMSO or MLN4924 were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924. All data represent the mean ± SEM from three independent experiments. Student t-test was used to determine p values, with **p≤0.01.


Roscovitine treatment reduced the NICD - FBXW7 interaction . 500 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 together with Roscovitine were subjected to immunoprecipitation using FBXW7 antibody , or IgG antibody as negative control , and precipitated material was analysed by western blot using NICD antibody . Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency . 10 % of cell lysate before immunoprecipitation was used as input control and β - Actin served as loading control . Quantification of the density of western blot bands in ( C ) performed by ImageJ software . Data are expressed as percentage changes compared to MLN4924 treated samples . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * * p ≤ 0 . 001 .

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Roscovitine treatment reduced the NICD-FBXW7 interaction. 500 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 together with Roscovitine were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.


LC - MS - MS analysis of in - gel - digested HEK293 cells transfected with hNICD - GFP , and subjected to immunoprecipitation using GFP antibody , identified multiple phosphorylation sites in NICD , highlighted in green .

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LC-MS-MS analysis of in-gel-digested HEK293 cells transfected with hNICD-GFP, and subjected to immunoprecipitation using GFP antibody, identified multiple phosphorylation sites in NICD, highlighted in green.


Phosphorylation of serine 2513 , but not serine 2516 , appears to be essential for the NICD - FBXW7 interaction . hNICD - GFP phospho - mutant peptides encoding non - phosphorylatable residues at S2513 and / or 2516 ( serine to alanine ) were expressed in HEK293 cells . The exogenously expressed protein was subsequently immunoprecipitated with anti - GFP antibody and precipitated material was analysed by western blot using FBXW7 antibody . Wild - type hNICD - GFP and GFP only vectors were included as positive and negative controls , respectively . Western blot using GFP antibody served as immunoprecipitation efficiency control . β - Actin has been used as loading control for the input lanes .

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Phosphorylation of serine 2513, but not serine 2516, appears to be essential for the NICD-FBXW7 interaction. hNICD-GFP phospho-mutant peptides encoding non-phosphorylatable residues at S2513 and/or 2516 (serine to alanine) were expressed in HEK293 cells. The exogenously expressed protein was subsequently immunoprecipitated with anti-GFP antibody and precipitated material was analysed by western blot using FBXW7 antibody. Wild-type hNICD-GFP and GFP only vectors were included as positive and negative controls, respectively. Western blot using GFP antibody served as immunoprecipitation efficiency control. β-Actin has been used as loading control for the input lanes.


HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA ( - ) or siRNA specific for CDK2 ( + ) followed by western blot for NICD , CDK2 , CDK1 , CDK4 and CDK8 . β - Actin served as loading control . Quantification of the density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as fold changes compared to control ( scrambled siRNA transfected ) cell lysate . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * p ≤ 0 . 01 .

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HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK2 (+) followed by western blot for NICD, CDK2, CDK1, CDK4 and CDK8. β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01.


HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA ( - ) or siRNA specific for CDK1 ( + ) followed by western blot for NICD , CDK1 , CDK2 , CDK4 and CDK8 . β - Actin has been used as loading control . Quantification of the density of western blot bands in ( C ) performed by ImageJ software . Data are expressed as fold changes compared to control ( scrambled siRNA transfected ) cell lysate . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * p ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK1 (+) followed by western blot for NICD, CDK1, CDK2, CDK4 and CDK8. β-Actin has been used as loading control.   Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.


Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases - a selection of those tested is shown here . At both 1 and 10µM , Roscovitine is able to inhibit more than 95 % of CDK2 activity , but is far less effective against other kinases . Purvalanol B ( 0 . 1 and 1µM ) inhibits more than 94 % of CDK2 activity . At both 1 and 10 µM , GSK650394A is able to inhibit more than 98 % of CDK2 activity . Source : The Kinase Profiling Inhibitor

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Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases - a selection of those tested is shown here. At both 1 and 10µM, Roscovitine is able to inhibit more than 95% of CDK2 activity, but is far less effective against other kinases. Purvalanol B (0.1 and 1µM) inhibits more than 94% of CDK2 activity. At both 1 and 10 µM, GSK650394A is able to inhibit more than 98% of CDK2 activity. Source: The Kinase Profiling Inhibitor Database


HEK293 cells were treated with three highly selective CDK2 inhibitors ( 10 µM of Roscovitine , 0 . 1µM of Purvalanol B and 10µM of GSK650394A ) for 3 hours . Endogenous levels of NICD were detected by western blot . β - Actin served as loading control . Quantification of the density of western blot bands in ( F ) using ImageJ software . Data are expressed as fold changes compared to DMSO . 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 .

O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 with three highly selective CDK2 inhibitors (10 µM of Roscovitine, 0.1µM of Purvalanol B and 10µM of GSK650394A) for 3 hours. Endogenous levels of NICD were detected by western blot. β-Actin served as loading control.   Quantification of the density of western blot bands in (F) using ImageJ software. Data are expressed as fold changes compared to DMSO. 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.


HEK293 cells were treated with RO - 3306 , a specific CDK1 inhibitor , for 3 hours at 10 µM . Levels of NICD , FBXW7 , phospho - Histone H3 and Histone H3 were detected by western blot . β - Actin was used as loading control . Quantification of the density of western blot bands in ( H ) using ImageJ software . Data are expressed as fold changes compared to DMSO . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * p ≤ 0 . 05 .

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

HEK293 cells were treated with RO-3306, a specific CDK1 inhibitor, for 3 hours at 10 µM. Levels of NICD, FBXW7, phospho-Histone H3 and Histone H3 were detected by western blot. β-Actin was used as loading control.   Quantification of the density of western blot bands in (H) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.


Cell cycle profile for HEK293 cells released from synchronization after double thymidine block . Cells were released and harvested at the indicated time points ( AS = asynchronous ) . Analysis of cell cycle arrest and release was performed using propidium iodide ( PI ) staining and flow cytometry . A representative experiment of three performed is shown .

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Cell cycle profile for HEK293 cells released from synchronization after double thymidine block. Cells were released and harvested at the indicated time points (AS= asynchronous). Analysis of cell cycle arrest and release was performed using propidium iodide (PI) staining and flow cytometry. A representative experiment of three performed is shown.


Expression of the indicated proteins in HEK293 cells was examined by western blotting , and β - Actin was used as loading control . This summary is a representation of three independent experiments .

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Expression of the indicated proteins in HEK293 cells was examined by western blotting, and β-Actin was used as loading control. This summary is a representation of three independent experiments.


Graph of flow cytometry data shows the percentage of cells in given cell ‐ cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK2 . Graph represents the mean of three independent experiments . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * p ≤ 0 . 01 ( ns = not significant ) .

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Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK2. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01 (ns=not significant).


Graph of flow cytometry data shows the percentage of cells in given cell ‐ cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK1 . Graph represents the mean of three independent experiments . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * p ≤ 0 . 05 and * * p ≤ 0 . 01 .

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Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK1. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05 and **p≤0.01.


iPS cells in G2 express high levels of Cyclin A and do not incorporate EdU ( marked in red within the NICD panel ) . EdU incorporation occurs during S phase ( marked by yellow in the NICD panel ) . Cells with low Cyclin A intensity and no EdU incorporation are either in G1 or M phase . M phase cells are distinguishable by their chromatin condensation , observable in the DAPI panel ( dividing cells are indicated with white in the NICD panel ) . G1 cells do not have any of the features described above ( blue in NICD panel ) . Scale bars are 20 µm .

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iPS cells in G2 express high levels of Cyclin A and do not incorporate EdU (marked in red within the NICD panel). EdU incorporation occurs during S phase (marked by yellow in the NICD panel). Cells with low Cyclin A intensity and no EdU incorporation are either in G1 or M phase. M phase cells are distinguishable by their chromatin condensation, observable in the DAPI panel (dividing cells are indicated with white in the NICD panel). G1 cells do not have any of the features described above (blue in NICD panel). Scale bars are 20 µm.


G1 and G2 phase cells have significantly higher NICD protein levels than S and M stage cells ( p ≤ 0 . 001 ) . All groups were compared against each other with a one - way ANOVA analysis , followed by Turkey test . M phase cells have the lowest NICD . Each point in the box - plot represents the normalized intensity of a single cell . Data from three different experiments ( n = 3 ) was randomised collated and plotted .

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G1 and G2 phase cells have significantly higher NICD protein levels than S and M stage cells (p≤0.001). All groups were compared against each other with a one-way ANOVA analysis, followed by Turkey test. M phase cells have the lowest NICD. Each point in the box-plot represents the normalized intensity of a single cell. Data from three different experiments (n=3) was randomised collated and plotted.


Purvalanol B treatment reduced the NICD - FBXW7 interaction . 200 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 in combination with 0 . 1 µM of Purvalanol B were subjected to immunoprecipitation using FBXW7 antibody , or IgG antibody as negative control , and precipitated material was analysed by western blot using NICD antibody . Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency . 10 % of cell lysate before immunoprecipitation was used as input control and β - Actin has been used as loading control . Quantification of the density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as percentage changes compared to MLN4924 treated samples . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * * p ≤ 0 . 001 .

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

Purvalanol B treatment reduced the NICD-FBXW7 interaction. 200 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 in combination with 0.1 µM of Purvalanol B were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin has been used as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.


E10 . 5 mouse tails were bissected down the midline . One half ( + ) was cultured for 4 hrs in the presence of Purvalanol B ( 1 µM ) . The contralateral half ( - ) was cultured for 4h in the presence of DMSO . Control or treated explants were pooled and NICD levels were detected by western blot . β - Actin was used as loading control .

O 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 O O O O 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

E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of Purvalanol B (1 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin was used as loading control.


E10 . 5 mouse tails were bissected down the midline . One half ( + ) was cultured for 4 hrs in the presence of RO - 3306 ( 10 µM ) . The contralateral half ( - ) was cultured for 4h in the presence of DMSO . Control or treated explants were pooled and NICD levels were detected by western blot . β - Actin has been used as loading control .

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

E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of RO-3306 (10 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin has been used as loading control.


Bissected E10 . 5 mouse PSM explants were cultured in the absence ( - ) or presence ( + ) of 1µM of Purvalanol B for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression . Purvalanol B treated explant has one less somite than the control explant and the treated explant is in the same late phase 1 of the oscillation cycle of dynamic mLfng mRNA expression indicating it is a whole cycle delayed compared to the " - " explant . n = 18 . Scale bar is 100 µm .

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Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 1µM of Purvalanol B for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. Purvalanol B treated explant has one less somite than the control explant and the treated explant is in the same late phase 1 of the oscillation cycle of dynamic mLfng mRNA expression indicating it is a whole cycle delayed compared to the "-" explant. n=18. Scale bar is 100 µm.


Bissected E10 . 5 mouse PSM explants were cultured in the absence ( - ) or presence ( + ) of 10 µM of RO - 3306 for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression . RO - 3306 treated explant is two phases behind in the oscillation cycle of dynamic mLfng mRNA expression indicating there is a delay in the oscillation compared to the " - " explant . n = 10 . Scale bar is 100 µm .

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Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 10 µM of RO-3306 for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. RO-3306 treated explant is two phases behind in the oscillation cycle of dynamic mLfng mRNA expression indicating there is a delay in the oscillation compared to the "-" explant. n=10. Scale bar is 100 µm.


The mean log2 fold change in successive somite length at position + 1 ( last formed somite ) is plotted for different treatment conditions . Error bars denote standard error of the mean . Paired t - test analysis was performed at 5 % significance level , with p = 0 . 0437 for RO - 3006 and p = 0 . 0524 for PurB , compared to DMSO . n = 14 . The mean log2 fold change in successive somite length at position + 2 ( penultimate formed somite ) is plotted for different treatment conditions . Error bars denote standard error of the mean . Paired t - test analysis was performed at 5 % significance level , with p = 0 . 6409 for RO - 3006 and p = 0 . 4065 for PurB , compared to DMSO . n = 14 .

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

The mean log2 fold change in successive somite length at position +1 (last formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.0437 for RO-3006 and p= 0.0524 for PurB, compared to DMSO. n=14.   The mean log2 fold change in successive somite length at position +2 (penultimate formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.6409 for RO-3006 and p= 0.4065 for PurB, compared to DMSO. n=14.


( A ) Abnormal distribution of Cux1 - positive neurons in CAMDI - KO mice . Expression of Cux1 in the somatosensory cortex was compared between P2 wild - type ( WT ) and CAMDI - KO ( KO ) mice . Scale bar , 100 μm . ( B ) Quantification of the number of Cux1 - positive neurons . Note the abnormal distribution of neurons in deep cortical layers of CAMDI - KO mice . n = 3 mice / genotype ( WT = 788 cells , KO = 2 , 139 cells ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , Two - way ANOVA followed by Scheffe ' s post - hoc test . Data are presented as mean ± SEM .

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(A) Abnormal distribution of Cux1-positive neurons in CAMDI-KO mice. Expression of Cux1 in the somatosensory cortex was compared between P2 wild-type (WT) and CAMDI-KO (KO) mice. Scale bar, 100 μm.(B) Quantification of the number of Cux1-positive neurons. Note the abnormal distribution of neurons in deep cortical layers of CAMDI-KO mice. n = 3 mice/genotype (WT = 788 cells, KO = 2,139 cells). *, p<0.05, **, p<0.01, ***, p<0.001, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.


( C ) Delay in neuronal migration by CAMDI deletion . Coronal sections through the somatosensory cortex of P21 WT and CAMDI - KO mice were analyzed following in utero electroporation of EGFP plasmid at E14 . 5 . Scale bar , 100 μm . ( D ) Quantification of the number of EGFP - positive neurons . Note the abnormal distribution of neurons in lower cortical layers of CAMDI - KO mice . n = 3 for WT mice ( 546 cells ) , n = 4 for KO mice ( 680 cells ) . * , p < 0 . 05 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc test . Data are presented as mean ± SEM .

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(C) Delay in neuronal migration by CAMDI deletion. Coronal sections through the somatosensory cortex of P21 WT and CAMDI-KO mice were analyzed following in utero electroporation of EGFP plasmid at E14.5. Scale bar, 100 μm.(D) Quantification of the number of EGFP-positive neurons. Note the abnormal distribution of neurons in lower cortical layers of CAMDI-KO mice. n = 3 for WT mice (546 cells), n = 4 for KO mice (680 cells). *, p<0.05, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.


( E ) Aberrant axonal projection and terminal arbors by CAMDI deletion . Coronal sections through the somatosensory cortex of P21 WT and CAMDI - KO mice were analyzed following in utero electroporation of EGFP plasmid at E14 . 5 . Representative pictures show an aberrant axonal projection of corticostriatal neurons in CAMDI - KO mice . Sections were stained with Hoechst 33258 and anti - EGFP antibody . Scale bar , 1 mm . ( F ) Quantification of the striatal intensity . n = 3 mice / genotype . * * * , p < 0 . 001 , One - way ANOVA with Bonferroni ' s post hoc test . Data are presented as mean ± SEM .

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(E) Aberrant axonal projection and terminal arbors by CAMDI deletion. Coronal sections through the somatosensory cortex of P21 WT and CAMDI-KO mice were analyzed following in utero electroporation of EGFP plasmid at E14.5. Representative pictures show an aberrant axonal projection of corticostriatal neurons in CAMDI-KO mice. Sections were stained with Hoechst 33258 and anti-EGFP antibody. Scale bar, 1 mm.(F) Quantification of the striatal intensity. n = 3 mice/genotype. ***, p<0.001, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.