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 .

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


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

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

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

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

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

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

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

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

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 of in - gel - digested HEK293 transfected with hNICD - GFP , 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 NICD - FBXW7 interaction . hNICD - GFP phospho - mutant peptides encoding non - phosphorylatable residues at S2513 / or 2516 ( serine to alanine ) were expressed in HEK293 . exogenously expressed was subsequently immunoprecipitated with anti - GFP antibody precipitated material was analysed by western blot using FBXW7 antibody . Wild - type hNICD - GFP GFP only vectors were included as positive negative controls , respectively . Western blot using GFP antibody served as immunoprecipitation efficiency control . β - Actin has been used as loading control for input lanes .

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 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 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

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 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 CDK8 . β - Actin served as loading control . of density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as fold changes compared to control ( scrambled siRNA transfected ) lysate . data represent mean ± from three independent experiments . Student ' s t - was performed , with * * p ≤ 0 . 01 .

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 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

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 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 CDK8 . β - Actin has been used as loading control . of density of western blot bands in ( C ) performed by ImageJ software . Data are expressed as fold changes compared to control ( scrambled siRNA transfected ) lysate . data represent mean ± from three independent experiments . Student ' s t - 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 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 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

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.


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

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 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 B-MEASURED_VAR 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

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 were treated with three highly selective CDK2 inhibitors ( 10 µM of Roscovitine , 0 . 1µM of Purvalanol B 10µM of GSK650394A ) for 3 hours . Endogenous of NICD were detected by western blot . β - Actin served as loading control . of density of western blot bands in ( F ) using ImageJ software . Data are expressed as fold changes compared to DMSO . 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_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 O 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 were treated with RO - 3306 , a specific CDK1 inhibitor , for 3 hours at 10 µM . of NICD , FBXW7 , phospho - Histone H3 Histone H3 were detected by western blot . β - Actin was used as loading control . of density of western blot bands in ( H ) using ImageJ software . Data are expressed as fold changes compared to DMSO . data represent mean ± from three independent experiments . Student ' s t - was performed , with * p ≤ 0 . 05 .

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


cycle profile for HEK293 released from synchronization after double thymidine block . were released harvested at indicated time points ( AS = asynchronous ) . of cycle arrest release was performed using propidium iodide ( PI ) 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 indicated in HEK293 was examined by western blotting , 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 of in given ‐ cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK2 . Graph represents mean of three independent experiments . data represent mean ± from three independent experiments . Student ' s t - 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 of in given ‐ cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK1 . Graph represents mean of three independent experiments . data represent mean ± from three independent experiments . Student ' s t - was performed , with * p ≤ 0 . 05 * * p ≤ 0 . 01 .

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

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

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 G2 phase have significantly higher NICD than S M stage ( p ≤ 0 . 001 ) . groups were compared against each other with a one - way ANOVA , followed by Turkey . M phase have lowest NICD . Each point in box - plot represents normalized of a single . Data from three different experiments ( n = 3 ) was randomised collated 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 NICD - FBXW7 interaction . 200 µg of HEK293 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 , precipitated material was analysed by western blot using NICD antibody . Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency . 10 % of lysate before immunoprecipitation was used as input control β - Actin has been used as loading control . of density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as changes compared to MLN4924 treated samples . data represent mean ± from three independent experiments . Student ' s t - was performed , with * * * p ≤ 0 . 001 .

B-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 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

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 midline . One half ( + ) was cultured for 4 hrs in presence of Purvalanol B ( 1 µM ) . half ( - ) was cultured for 4h in presence of DMSO . Control or treated explants were pooled NICD 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 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 B-MEASURED_VAR 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 midline . One half ( + ) was cultured for 4 hrs in presence of RO - 3306 ( 10 µM ) . half ( - ) was cultured for 4h in presence of DMSO . Control or treated explants were pooled NICD were detected by western blot . β - Actin has been used as loading control .

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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 absence ( - ) or presence ( + ) of 1µM of Purvalanol B for 4 hours then analysed by in situ hybridization for mLfng mRNA expression . Purvalanol B treated explant has one less somite than control explant treated explant is in same late phase 1 of oscillation cycle of dynamic mLfng mRNA expression indicating it is a whole cycle delayed compared to " - " 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 absence ( - ) or presence ( + ) of 10 µM of RO - 3306 for 4 hours then analysed by in situ hybridization for mLfng mRNA expression . RO - 3306 treated explant is two phases behind in oscillation cycle of dynamic mLfng mRNA expression indicating there is a delay in oscillation compared to " - " 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.


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

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

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

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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 terminal arbors by CAMDI deletion . Coronal sections through somatosensory cortex of P21 WT 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 anti - EGFP antibody . Scale bar , 1 mm . ( F ) of striatal . n = 3 mice / genotype . * * * , p < 0 . 001 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± .

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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.


( A , B ) Representative traces from adult control ( WT ) CAMDI - KO ( KO ) in open field . Locomotor activity ( A ) traveled ( B ) for 15 min . n = 8 for WT mice , n = 7 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 13 ) = 5 . 53 ) . Data are presented as mean ± .

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(A, B) Representative traces from adult control (WT) and CAMDI-KO (KO) in open field test. Locomotor activity (A) and distance traveled (B) for 15 min. n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.53). Data are presented as mean ± SEM.


( C ) Repetitive jumping behavior in open field . n = 8 for WT mice , n = 7 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 13 ) = 5 . 09 ) . Data are presented as mean ± .

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(C) Repetitive jumping behavior in open field test. n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.09). Data are presented as mean ± SEM.


( D ) Three - chamber social interaction . time spent sniffing with stranger mouse . n = 19 for WT mice , n = 15 for KO mice . * , p < 0 . 05 , * * * , p < 0 . 001 , Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 63 ) = 5 . 79 , main effect of chamber F ( 1 , 63 ) = 90 . 47 , interaction F ( 1 , 63 ) = 5 . 29 ) . Data are presented as mean ± .

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(D) Three-chamber social interaction test. The time spent sniffing with stranger mouse. n = 19 for WT mice, n = 15 for KO mice. *, p<0.05, ***, p<0.001,Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 63)=5.79, main effect of chamber F(1, 63)=90.47, interaction F(1, 63)=5.29). Data are presented as mean ± SEM.


( E ) Resident - intruder ( social sniffing ) . n = 7 for WT mice , n = 8 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 13 ) = 5 . 15 ) . Data are presented as mean ± .

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(E) Resident-intruder test (social sniffing). n = 7 for WT mice, n = 8 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 5.15). Data are presented as mean ± SEM.


( F ) Resident - intruder ( digging ) . n = 7 for WT mice , n = 8 for KO mice . * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 13 ) = 10 . 97 ) . Data are presented as mean ± .

(F) Resident-intruder test (digging). n = 7 for WT mice, n = 8 for KO mice. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test (F(1, 13) = 10.97). Data are presented as mean ± SEM.


( G ) Marble burying . n = 9 for WT mice , n = 25 for KO mice . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 32 ) = 5 . 39 ) . Data are presented as mean ± .

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(G) Marble burying test. n = 9 for WT mice, n = 25 for KO mice. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test (F(1, 32) = 5.39). Data are presented as mean ± SEM.


( H ) Forced swimming . n = 10 for WT mice , n = 13 for KO mice . * * , p < 0 . 01 , * * * , p < 0 . 001 Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 42 ) = 84 . 30 , main effect of session F ( 1 , 42 ) = 33 . 88 , interaction F ( 1 , 42 ) = 4 . 92 ) . Data are presented as mean ± .

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(H) Forced swimming test. n = 10 for WT mice, n = 13 for KO mice. **, p<0.01, ***, p<0.001 Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 42)=84.30, main effect of session F(1, 42)=33.88, interaction F(1, 42)=4.92). Data are presented as mean ± SEM.


( I , J ) - . Time in chamber ( I ) Latency to chamber ( J ) . n = 8 for WT mice , n = 7 for KO mice . * , p < 0 . 05 , * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc , ( ( I ) F ( 1 , 13 ) = 8 . 75 , ( J ) F ( 1 , 13 ) = 9 . 73 ) . Data are presented as mean ± .

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(I, J) Light-dark test. Time in light chamber (I) and Latency to light chamber (J). n = 8 for WT mice, n = 7 for KO mice. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni's post hoc test, ((I) F(1, 13) = 8.75, (J) F(1, 13)=9.73). Data are presented as mean ± SEM.


( K ) Hot plate . . n = 8 for WT mice , n = 12 for KO mice . * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc ( F ( 1 , 18 ) = 17 . 51 ) . Data are presented as mean ± .

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(K) Hot plate test.. n = 8 for WT mice, n = 12 for KO mice. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test (F(1, 18) = 17.51). Data are presented as mean ± SEM.


( A ) Co - expressed FLAG - CAMDI HDAC6 - HA interacted with each other in SH - SY5Y . n = 3 independent experiments .

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(A) Co-expressed FLAG-CAMDI and HDAC6-HA interacted with each other in SH-SY5Y cells. n=3 independent experiments.


( B ) Endogenous interaction of CAMDI with HDAC6 in mousebrain . Co - IP was performed with indicated antibodies using E16 mousebrain . n = 3 independent experiments .

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(B) Endogenous interaction of CAMDI with HDAC6 in the mousebrain. Co-IP assay was performed with the indicated antibodies using E16 mousebrain. n=3 independent experiments.


( C , D ) N - terminal region of CAMDI recognizes second HDAC domain of HDAC6 . SH - SY5Y were co - transfected with HDAC6 - HA each FLAG - CAMDI fragment ( C ) ( fragment 1 , 1 - 250 ; 2 , 251 - 500 ; 3 , 501 - 750 ; 4 , 751 - 1000 ; 5 , 1001 - 1451 ) or co - transfected with FLAG - CAMDI each HDAC6 - HA fragment ( D ) ( fragment 1 ( DD1 ) , 1 - 440 ; 2 ( DD2 ) , 441 - 840 ; 3 , 841 - 1215 ) . Co - IP was performed with indicated antibodies . n = 3 independent experiments . * * , p < 0 . 01 , * * * , p < 0 . 001 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± .

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(C, D) N-terminal region of CAMDI recognizes the second HDAC domain of HDAC6. SH-SY5Y cells were co-transfected with HDAC6-HA and each FLAG-CAMDI fragment (C) (fragment 1, 1-250; 2, 251-500; 3, 501-750; 4, 751-1000; 5, 1001-1451 amino acids) or co-transfected with FLAG-CAMDI and each HDAC6-HA fragment (D) (fragment 1 (DD1), 1-440; 2 (DD2), 441-840; 3, 841-1215 amino acids). Co-IP assay was performed with the indicated antibodies. n=3 independent experiments. **, p<0.01, ***, p<0.001, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( A ) CAMDI CAMDI Fragment 1 overexpression enhanced α - tubulin acetylation . HeLa overexpressing FLAG - CAMDI FLAG - CAMDI frag . 1 were immunostained with anti - FLAG or anti - Ac - tubulin antibodies . - scans along ( from x - to y - axis ) from . Scale bar , 10 μm .

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(A) CAMDI and CAMDI Fragment 1 overexpression enhanced α-tubulin acetylation. HeLa cells overexpressing FLAG-CAMDI and FLAG-CAMDI frag.1 were immunostained with anti-FLAG or anti-Ac-tubulin antibodies. Ratio line-scans along the line (from x- to y-axis) from images. Scale bar, 10 μm.


( B - C ) CAMDI blocked HDAC6 - mediated α - tubulin deacetylation . ( B ) Centrosomal fraction was isolated from lysates of SH - SY5Y expressing each construct , Ac - tubulin level was assessed by immunoblot . n = 3 independent experiments . * * , p < 0 . 01 , * * * , p < 0 . 001 , Two - way ANOVA followed by Scheffe ' s post - hoc . Data are presented as mean ± .

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(B-C) CAMDI blocked HDAC6-mediated α-tubulin deacetylation. (B) Centrosomal fraction was isolated from cell lysates of SH-SY5Y cells expressing each construct, and Ac-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, ***, p<0.001, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.


( B - C ) CAMDI blocked HDAC6 - mediated α - tubulin deacetylation . ( C ) CAMDI N - terminal domain alone increased Ac - tubulin level . n = 3 independent experiments . * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± .

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(B-C) CAMDI blocked HDAC6-mediated α-tubulin deacetylation. (C) CAMDI N-terminal domain alone increased Ac-tubulin level. n=3 independent experiments. **, p<0.01, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( D ) Deletion of CAMDI N - terminal domain ( 1 - 250 ) failed to inhibit HDAC6 . n = 3 independent experiments . N . S . , not significant . Two - way ANOVA followed by Scheffe ' s post - hoc . Data are presented as mean ± .

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(D) Deletion of CAMDI N-terminal domain (1-250 amino acids) failed to inhibit HDAC6. n = 3 independent experiments. N.S., not significant. Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.


( A ) Effect of HDAC6 over - expression or CAMDI co - expression on level of acetylated α - tubulin in centrosome . HeLa co - transfected with Centrin2 - EGFP with / without HDAC6 - HA FLAG - CAMDI were immunostained with anti - EGFP ( green ) anti - acetylated α - tubulin antibodies ( red ) . Scale bar , 1 μm . ( B ) of of acetylated α - tubulin at centrosome . n = 25 , 30 , 30 . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± .

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(A) Effect of HDAC6 over-expression or CAMDI co-expression on the level of acetylated α-tubulin in the centrosome. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-acetylated α-tubulin antibodies (red). Scale bar, 1 μm.(B) Quantification of intensity of acetylated α-tubulin at centrosome. n = 25, 30, 30 cells. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( C ) Effects of HDAC6 overexpression or CAMDI co - expression on centrosomal localization of γ - tubulin . HeLa co - transfected with Centrin2 - EGFP with / without HDAC6 - HA FLAG - CAMDI were immunostained with anti - EGFP ( green ) anti - γ - tubulin antibodies ( red ) . was stained with Hoechst 33258 . Scale bar , 1 μm . ( D ) of of γ - tubulin at centrosome . n = 39 , 16 , 17 . * , p < 0 . 05 , * * , p < 0 . 01 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± .

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(C) Effects of HDAC6 overexpression or CAMDI co-expression on the centrosomal localization of γ-tubulin. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). DNA was stained with Hoechst 33258. Scale bar, 1 μm.(D) Quantification of intensity of γ-tubulin at centrosome. n = 39, 16, 17 cells. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( E ) CAMDI stabilizes centrosomal γ - tubulin through HDAC6 inhibition . Centrosomal fraction was isolated from lysates of SH - SY5Y expressing each construct , γ - tubulin level was assessed by immunoblot . n = 3 independent experiments . * * , p < 0 . 01 , One - way ANOVA followed with Bonferroni ' s post hoc . Data are presented as mean ± .

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(E) CAMDI stabilizes centrosomal γ-tubulin through HDAC6 inhibition. Centrosomal fraction was isolated from cell lysates of SH-SY5Y cells expressing each construct, and γ-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, One-way ANOVA followed with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( F ) Effects of CAMDI knockdown on centrosomal localization of γ - tubulin in vivo . Coronal sections through somatosensory cortex of E17 . 5 were analyzed following in utero electroporation of EGFP CAMDI - sh plasmid at E14 . 5 . sections were immunostained with anti - EGFP ( green ) anti - γ - tubulin antibodies ( red ) . Representative of EGFP at Cortical Plateneuron expressing CAMDI - sh stained for endogenous γ - tubulin with Hoechst ( blue ) . Z - stack number 6 indicates centrosome of EGFP - positive CAMDI - sh - expressing neuron . Z - stack number 19 indicates centrosome of a nearby non - electroporated neuron . of γ - tubulin . EGFP ( - ) = 12 , EGFP ( + ) ( CAMDI - sh ) = 12 . * , p < 0 . 05 , One - way ANOVA with Bonferroni ' s post hoc . Data are presented as mean ± . Scale bar , 5 μm .

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(F) Effects of CAMDI knockdown on the centrosomal localization of γ-tubulin in vivo.Coronal sections through the somatosensory cortex of E17.5 were analyzed following in utero electroporation of EGFP and CAMDI-sh plasmid at E14.5. The sections were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). Representative images of EGFP at Cortical Plateneuron expressing CAMDI-sh stained for endogenous γ-tubulin and with Hoechst (blue). Z-stack number 6 indicates centrosome of EGFP-positive CAMDI-sh-expressing neuron. Z-stack number 19 indicates centrosome of a nearby non-electroporated neuron. Quantification of γ-tubulin intensity. EGFP (-) = 12 cells, EGFP (+) (CAMDI-sh) = 12 cells. *, p<0.05, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM. Scale bar, 5 μm.


( G ) Reduced γ - tubulin in centrosome fraction of CAMDI - KO brain . centrosomal fraction was isolated from brain lysates of WT CAMDI - KO mice γ - tubulin level was assessed by immunoblot . n = 3 independent experiments . * * , p < 0 . 01 , One - way ANOVA followed with Bonferroni ' s post hoc . Data are presented as mean ± .

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(G) Reduced γ-tubulin in the centrosome fraction of CAMDI-KO brain. The centrosomal fraction was isolated from brain lysates of WT and CAMDI-KO mice and γ-tubulin level was assessed by immunoblot analysis. n = 3 independent experiments. **, p<0.01, One-way ANOVA followed with Bonferroni's post hoc test. Data are presented as mean ± SEM.


( A ) Rescue of a delayed cortical migration in CAMDI - KO mice by Tubastatin A . Immunohistochemical of Cux1 expression in somatosensory cortex at P2 after intraperitoneal injections of Tubastatin A into pregnant CAMDI - KO mice from E12 . 5 - 17 . 5 . Scale bar , 100 μm . ( B ) of number of Cux1 - positive neurons . Note abnormal distribution of neurons in deep cortical layers of CAMDI - KO mice . n = 3 mice / genotype ( WT ( vehicle ) = 3 , 628 , KO ( vehicle ) = 2 , 816 , WT ( Tubastatin A ) = 1 , 847 , KO ( Tubastatin A ) = 2 , 364 ) . * , p < 0 . 05 , * * , p < 0 . 01 , Two - way ANOVA followed by Tukey HSD . Data are presented as mean ± .

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(A) Rescue of a delayed cortical migration in CAMDI-KO mice by Tubastatin A. Immunohistochemical analysis of Cux1 expression in the somatosensory cortex at P2 after intraperitoneal injections of Tubastatin A into pregnant CAMDI-KO mice from E12.5-17.5. 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 (vehicle) = 3,628 cells, KO (vehicle) = 2,816 cells, WT (Tubastatin A) = 1,847 cells, KO (Tubastatin A) = 2,364 cells). *, p<0.05, **, p<0.01, Two-way ANOVA followed by Tukey HSD test. Data are presented as mean ± SEM.


( C ) Enhanced α - tubulin acetylation at P0 by Tubastatin A . Ac - tubulin level was assessed by immunoblot . ( D ) of α - tubulin acetylation . n = 3 independent experiments . * , p < 0 . 05 , Two - way ANOVA followed by Scheffe ' s post - hoc . Data are presented as mean ± .

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(C) Enhanced α-tubulin acetylation at P0 by Tubastatin A. Ac-tubulin level was assessed by immunoblot analysis.(D) Quantification of the α-tubulin acetylation. n=3 independent experiments. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.


( A ) Representative traces from control ( WT ) CAMDI - KO ( KO ) mice in open field at P21 . Sample traces of locomotor activity were shown . ( B ) traveled for 10 min . n = 13 for WT ( vehicle ) mice , n = 13 for KO ( vehicle ) mice , n = 5 for WT ( Tubastatin A ) mice , n = 16 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 43 ) = 6 . 25 , main effect of drug F ( 1 , 43 ) = 2 . 14 , interaction F ( 1 , 43 ) = 12 . 68 ) . Data are presented as mean ± .

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(A) Representative traces from control (WT) and CAMDI-KO (KO) mice in the open field test at P21. Sample traces of locomotor activity were shown.(B) Distance traveled for 10 min. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 43)=6.25, main effect of drug F(1, 43)=2.14, interaction F(1, 43)=12.68). Data are presented as mean ± SEM.


( C ) Repetitive jumping behavior . n = 13 for WT ( vehicle ) mice , n = 27 for KO ( vehicle ) mice , n = 5 for WT ( Tubastatin A ) mice , n = 18 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 59 ) = 5 . 04 , main effect of drug F ( 1 , 59 ) = 10 . 13 , interaction F ( 1 , 59 ) = 4 . 98 ) . Data are presented as mean ± .

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(C) Repetitive jumping behavior. n = 13 for WT (vehicle) mice, n = 27 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 18 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 59)=5.04, main effect of drug F(1, 59)=10.13, interaction F(1, 59)=4.98). Data are presented as mean ± SEM.


( D ) Grooming time in small open field . n = 13 for WT ( vehicle ) mice , n = 13 for KO ( vehicle ) mice , n = 5 for WT ( Tubastatin A ) mice , n = 16 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , * * * , p < 0 . 001 . Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 43 ) = 11 . 18 , main effect of drug F ( 1 , 43 ) = 7 . 23 , interaction F ( 1 , 43 ) = 9 . 34 ) . Data are presented as mean ± .

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(D) Grooming time in small open field test. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 43)=11.18, main effect of drug F(1, 43)=7.23, interaction F(1, 43)=9.34). Data are presented as mean ± SEM.


( E ) traveled for 15 min . n = 10 for adult WT ( Vehicle ) mice , n = 5 for KO ( Vehicle ) mice , n = 6 for WT ( Tubastatin A ) mice , n = 11 for KO ( Tubastatin A ) mice . * * , p < 0 . 01 , Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 28 ) = 1 . 73 , main effect of drug F ( 1 , 28 ) = 3 . 96 , interaction F ( 1 , 28 ) = 11 . 80 ) . Data are presented as mean ± .

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(E) Distance traveled for 15 min. n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. **, p<0.01, Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 28)=1.73, main effect of drug F(1, 28)=3.96, interaction F(1, 28)=11.80). Data are presented as mean ± SEM.


( F ) - . Time in latency to chamber ( latency ) . n = 10 for adult WT ( Vehicle ) mice , n = 5 for KO ( Vehicle ) mice , n = 6 for WT ( Tubastatin A ) mice , n = 11 for KO ( Tubastatin A ) mice . * , p < 0 . 05 , Two - way ANOVA followed by Scheffe ' s post - hoc ( main effect of genotype F ( 1 , 28 ) = 2 . 34 , main effect of drug F ( 1 , 28 ) = 3 . 82 , interaction F ( 1 , 28 ) = 10 . 64 ) . Data are presented as mean ± .

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(F) Light-dark test. Time in latency to the light chamber (latency). n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 28)=2.34, main effect of drug F(1, 28)=3.82, interaction F(1, 28)=10.64). Data are presented as mean ± SEM.


Detection of native GFP - fused TgAtg8 . extracts corresponding to 107 tachyzoites from parental RHΔHX transgenic GFP - TgAtg8 lines were separated by SDS - PAGE analysed by Western blot using anti - TgAtg8 or anti - GFP antibodies . Overexpressed GFP - TgAtg8 native TgAtg8 are indicated by arrows .

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Detection of native and GFP-fused TgAtg8.Protein extracts corresponding to 107 tachyzoites from parental RHΔHX and transgenic GFP-TgAtg8 cell lines were separated by SDS-PAGE and analysed by Western blot using anti-TgAtg8 or anti-GFP antibodies. Overexpressed GFP-TgAtg8 and native TgAtg8 are indicated by arrows.


A . Autophagic vesicles marker GFP - TgAtg8 was found in cytosol of tachyzoites occasionally in punctate vesicles in recently egressed .

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A. Autophagic vesicles marker GFP-TgAtg8 was found in the cytosol of tachyzoites and occasionally in punctate vesicles in recently egressed parasites.


B . Extracellular tachyzoites were put to starve in HBSS medium for increased time periods proportions of displaying punctate or GFP - TgAtg8 signals were assessed . Data are mean from n = 4 independent experiments ± .

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B. Extracellular tachyzoites were put to starve in HBSS medium for increased time periods and the proportions of cells displaying punctate or cytosolic GFP-TgAtg8 signals were assessed. Data are mean from n = 4 independent experiments ±SEM.


A . Extracellular tachyzoites were starved for 8 hours in HBSS medium observed ( 2 , 3 ) . Unstarved control is shown for comparison ( 1 ) . Several vacuoles ( V ) can be seen in starved , with also cytosol - organelle - sequestrating membranous structures resembling autophagosomes ( Ap ) , as well as vacuoles containing partially digested material corresponding to autophagic degradative vacuoles ( Av ) . 2 ′ , 3 ′ 3 " pictures are a magnification of squared regions . Scale bar = 1 µm in initial ( 1 , 2 , 3 ) 0 . 2 µm in magnifications .

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A. Extracellular tachyzoites were starved for 8 hours in HBSS medium and observed (2, 3). Unstarved control is shown for comparison (1). Several vacuoles (V) can be seen in starved parasites, with also cytosol- and organelle-sequestrating membranous structures resembling autophagosomes (Ap), as well as vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). 2′, 3′ and 3" pictures are a magnification of squared regions. Scale bar = 1 µm in initial micrographs (1, 2, 3) and 0.2 µm in magnifications.


B . Partial co - localisation of GFP - TgAtg8 - labeled autophagosomes Toxoplasma mitochondrion . Extracellular tachyzoites expressing GFP - TgAtg8 were incubated in complete medium with Mitotracker Red to label mitochondrion ; partial co - localisation could be observed in some displaying autophagic vesicles .

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B. Partial co-localisation of GFP-TgAtg8-labeled autophagosomes and Toxoplasma mitochondrion. Extracellular tachyzoites expressing GFP-TgAtg8 were incubated in complete medium with Mitotracker Red to label the mitochondrion; partial co-localisation could be observed in some parasites displaying autophagic vesicles.


C . Identification of autophagic vesicles in extracellular tachyzoites by immuno - electron microscopy . Extracellular tachyzoites expressing GFP - TgAtg8 were starved for 8 hours in HBSS medium , fixed , cryosectioned stained with an anti - GFP antibody , then with gold - conjugated secondary antibody observed . Gold particles can be seen around cytosol - sequestrating membranous structures resembling autophagosomes ( Ap ) , as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles ( Av ) . Scale bar = 200 nm .

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C. Identification of autophagic vesicles in extracellular tachyzoites by immuno-electron microscopy. Extracellular tachyzoites expressing GFP-TgAtg8 were starved for 8 hours in HBSS medium, fixed, cryosectioned and stained with an anti-GFP antibody, then with gold-conjugated secondary antibody and observed. Gold particles can be seen around cytosol-sequestrating membranous structures resembling autophagosomes (Ap), as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). Scale bar = 200 nm.


A . extracts corresponding to tachyzoites incubated in same starvation conditions as in Figure 2B were separated by urea SDS - PAGE analysed with an anti - GFP antibody to detect TgAtg8 lipidated TgAtg8 - PE form . GFP - TgAtg8 extracts were analysed with anti GFP antibody ( left ) , while parental was analysed with anti - TgAtg8 antibody ( right ) . Anti - ROP5 was used as a loading control .

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A. Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect TgAtg8 and the lipidated TgAtg8-PE form. GFP-TgAtg8 parasites extracts were analysed with anti GFP antibody (left), while parental cell line was analysed with anti-TgAtg8 antibody (right). Anti-ROP5 was used as a loading control.


B . lysates from GFP - TgAtg8 were subjected to a centrifugation at 100 , 000 g to separate soluble fraction ( high speed supernatant , HSS ) from fraction ( high speed pellet , HSP ) . faster migrating form of GFP - TgAtg8 is exclusively present in fraction as revealed by Western blot after urea SDS - PAGE using anti -

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B. Cell lysates from GFP-TgAtg8 parasites were subjected to a centrifugation at 100,000 g to separate the soluble fraction (high speed supernatant, HSS) from the membrane fraction (high speed pellet, HSP). The faster migrating form of GFP-TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS-PAGE using anti-GFP


C . HSP fraction described in B . was extracted with various agents new HSS HSP fractions were separated analysed by Western blot after urea SDS - PAGE . Only a treatment with a detergent ( DOC ) resulted in GFP - TgAtg8 solubilisation from HSP fraction .

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C. The HSP fraction described in B. was extracted with various agents and new HSS and HSP fractions were separated and analysed by Western blot after urea SDS-PAGE. Only a treatment with a detergent (DOC) resulted in GFP-TgAtg8 solubilisation from the HSP fraction.


D . GFP - TgAtg8 - expressing were grown in host in presence of 3H - ethanolamine then starved in HBSS for 8 hours , still in presence of radioactive PE precursor . Tachyzoites were then lysed GFP - TgAtg8 was immunoprecipitated using anti - TgAtg8 antibody analysed by urea SDS - PAGE . immunoprecipitated forms of GFP - TgAtg8 were detected by Western blot using anti - GFP antibody radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP - TgAtg8 , as detected by fluorography ( 3H - etn ) .

D. GFP-TgAtg8-expressing parasites were grown in host cells in the presence of 3H-ethanolamine and then starved in HBSS for 8 hours, still in the presence of the radioactive PE precursor. Tachyzoites were then lysed and GFP-TgAtg8 was immunoprecipitated using anti-TgAtg8 antibody and analysed by urea SDS-PAGE. The immunoprecipitated forms of GFP-TgAtg8 were detected by Western blot using anti-GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP-TgAtg8, as detected by fluorography (3H-etn).


A . microscopy of extracellular tachyzoites expressing GFP - TgAtg8 or its glycine mutant version , before or after induction of autophagy for 8 hours in HBSS . Autophagosomes are labelled by GFP - TgAtg8 in control ( arrowheads ) , but not by glycine mutant version of . B . Extracellular tachyzoites expressing GFP - TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time proportions of displaying punctate or GFP signals were assessed . Data are mean from n = 4 independent experiments ± .

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A. Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version, before or after induction of autophagy for 8 hours in HBSS. Autophagosomes are labelled by GFP-TgAtg8 in control parasites (arrowheads), but not by the glycine mutant version of the protein. B. Extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed. Data are mean from n = 4 independent experiments ±SEM.


C . extracts corresponding to tachyzoites expressing GFP - TgAtg8 glycine mutant version incubated in HBSS for up to 8 hours , were separated by urea SDS - PAGE analysed with an anti - GFP antibody to detect GFP - TgAtg8 lipidated GFP - TgAtg8 - PE forms . Anti - ROP5 was used as a loading control .

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C. Protein extracts corresponding to tachyzoites expressing GFP-TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours, were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect GFP-TgAtg8 and lipidated GFP-TgAtg8-PE forms. Anti-ROP5 was used as a loading control.


A . Intracellular tachyzoites expressing GFP - TgAtg8 were used to invade host HFF proportions of displaying punctate or GFP signals were assessed along time during development . Data are mean from n = 5 independent experiments ± .

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A. Intracellular tachyzoites expressing GFP-TgAtg8 were used to invade host HFF and the proportions of cells displaying punctate or cytosolic GFP signals were assessed along time during development. Data are mean from n = 5 independent experiments ±SEM.


B . microscopy pictures of GFP - TgAtg8 - expressing tachyzoites during their intracellular development division . arrowhead indicates residual made of maternal material left after endodyogeny .

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B. Fluorescence microscopy pictures of GFP-TgAtg8-expressing tachyzoites during their intracellular development and division. The arrowhead indicates the residual body made of maternal material left after endodyogeny.


C . Detection of GFP - TgAtg8 - labelled autophagosomes in dividing . Progression of daughter division was followed by expression of an IMC1 - RFP construct .

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C. Detection of GFP-TgAtg8-labelled autophagosomes in dividing parasites. Progression of daughter cells division was followed by expression of an IMC1-RFP construct.