( H ) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population ; binary logistic regression analysis ; mean ± SEM . For all replications p - values for one - sided tests are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population; binary logistic regression analysis; mean±SEM. For all replications p-values for one-sided tests are shown.


( A ) FXR1 expression in PBMC of individuals carrying the rs2601 GG ( low risk ; N = 16 ) versus AA genotype ( high risk ; N = 27 ) .

O O O O O O O O O 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) FXR1 expression in PBMC of individuals carrying the rs2601 GG (low risk; N=16) versus AA genotype (high risk; N=27).


( B ) PBMC microRNA expression was normalized and data is plotted from all 4 miR - 181 - 5p members ( miR - 181a , b , c , d - 5p ) .

O O O O O O O O O O O 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) PBMC microRNA expression was normalized and data is plotted from all 4 miR-181-5p members (miR-181a, b, c, d-5p).


( C ) Sequence homology of all 4 human mature miR - 181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR - 181 family ( seed matches identified using Target Scan Human and SFOLD ) . The red letters specify seed sequences and seed matches , respectively . Chromosome positions for each seed sequence and seed match are denoted ( human genome assembly GRCh38 / hg38 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sequence homology of all 4 human mature miR-181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR-181 family (seed matches identified using Target Scan Human and SFOLD). The red letters specify seed sequences and seed matches, respectively. Chromosome positions for each seed sequence and seed match are denoted (human genome assembly GRCh38/hg38).


( D ) Denoted are ΔΔG values for binding of each of the miR - 181 family members to different 3 ' UTR positions . Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ΔΔG values . ΔΔG is an energetic score , and the more negative its value , the stronger is the expected binding of the microRNA to the given

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Denoted are ΔΔG values for binding of each of the miR-181 family members to different 3'UTR positions. Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ΔΔG values. ΔΔG is an energetic score, and the more negative its value, the stronger is the expected binding of the microRNA to the given site


( D ) The indicated amounts of recombinant wild - type and mutant PPM1H or PPM1J ( with a His - Sumo N - terminal tag , expressed in E . coli ) were incubated in vitro with 2 . 5 µg pThr72 phosphorylated Rab8a ( left ) or pThr73 phosphorylated Rab10 ( right ) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7 . 5 buffer . Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos - tag gel electrophoresis that separates phosphorylated ( slow migrating ) and dephosphorylated Rabs . The gel was stained with Instant Blue Coomassie . D288A is a substrate - trapping ( inactive ) variant of PPM1H and was used as a 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72 phosphorylated Rab8a (left) or pThr73 phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A is a substrate-trapping (inactive) variant of PPM1H and was used as a control.


( D ) Kinetics of phosphate hydrolysis . 25 nM recombinant wild - type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a ( GTPγS or GDP ) as described in Materials and Methods . Initial velocity ( V0 ) was calculated by dividing the concentration of released phosphatase ( μM ) by time ( min ) and plotted against substrate concentration for pThr72 phosphorylated Rab8a [ GTP bound conformation ] ( blue ) and pThr72 phosphorylated Rab8a [ GDP bound conformation ] ( red ) . The experiments were repeated twice , and both data points are shown in curves . Line fittings for the left and middle panel were performed using the mean of the two values . Kinetic constants ( Kcat , Vmax , Km ) were obtained using GraphPad software , and their uncertainties ( ± ) correspond to the SE of mean .

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

(D) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean.


( E ) Kinetic analysis of PPM1H as in ( D ) using 50 nM PPM1H and phosphopeptide substrates , as described in Materials and Methods . Initial velocity ( V0 ) was calculated by dividing the concentration of phosphatase ( μM ) by time ( min ) and plotted against substrate concentration for pThr72 Rab8a phospho - peptide ( blue ) and Rab10 pThr73 phospho - peptide ( red ) . Each experiment was performed twice ( individual data points shown ) .

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

(E) Kinetic analysis of PPM1H as in (D) using 50 nM PPM1H and phosphopeptide substrates, as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 Rab8a phospho-peptide (blue) and Rab10 pThr73 phospho-peptide (red). Each experiment was performed twice (individual data points shown).


( F ) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis . 50 nM recombinant wild - type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein ( blue ) or Rab8a pThr72 phosphorylated peptide ( red ) for indicated times and analysed as described in Materials and Methods . The experiments were repeated 3 times . The error bars represent SE of mean of the technical replicates .

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

(F) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis. 50 nM recombinant wild-type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein (blue) or Rab8a pThr72 phosphorylated peptide (red) for indicated times and analysed as described in Materials and Methods. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates.


( C ) His6 - SUMO - tagged full - length variants of PPM1H were used for catalytic assays ( left panel ) . 3 μg of recombinant wild - type or indicated mutant PPM1H proteins were resolved on 4 - 12 % Bis - Tris gradient gel and stained with Instant Blue Coomassie . Right , mass photometry histogram for 40nM His6 - SUMO - PPM1H WT ( blue ) and His6 - SUMO - PPM1H 2Glu ( brown ) , where WT is 130kDa ( ± 15 . 8 kDa , with 1004 single molecules counted ) and 2Glu is 83kDa ( ± 13 . 9 kDa , with 4117 single molecules counted ) . The calculated molecular weight of the fusion protein is approximately 68 . 5 kDa .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) His6-SUMO-tagged full-length variants of PPM1H were used for catalytic assays (left panel). 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie. Right, mass photometry histogram for 40nM His6-SUMO-PPM1H WT (blue) and His6-SUMO-PPM1H 2Glu (brown), where WT is 130kDa (±15.8 kDa, with 1004 single molecules counted) and 2Glu is 83kDa (±13.9 kDa, with 4117 single molecules counted). The calculated molecular weight of the fusion protein is approximately 68.5 kDa.


( D ) In vitro malachite green assay time course of recombinant His6 - SUMO - tagged PPM1H wild - type ( blue ) , 2Glu ( red ) , or D288A ( grey ) against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS , left ) , 32 μM pThr72 phosphorylated Rab8a peptide ( middle ) , or 32 μM pThr73 phosphorylated Rab10 peptide ( right ) . The experiments were repeated 4 times . The error bars represent SE of mean of the technical replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro malachite green assay time course of recombinant His6-SUMO-tagged PPM1H wild-type (blue), 2Glu (red), or D288A (grey) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.


( C ) In vitro malachite green assay time course to determine enzyme activity of recombinant PPM proteins ( 25 nM for the protein substrate , 50 nM for the peptide substrate ) against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS ) . The experiments were repeated 3 times . The error bars represent SE of mean of the technical replicates . ( D ) In vitro malachite green assay against 32 μM pThr72 - Rab8a peptide ( left ) and pThr73 - Rab10 peptide mimic ( right ) . The experiments were repeated 3 times . The error bars represent standard error ( SE ) of mean of the technical replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro malachite green assay time course to determine enzyme activity of recombinant PPM proteins (25 nM for the protein substrate, 50 nM for the peptide substrate) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS). The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. (D) In vitro malachite green assay against 32 μM pThr72-Rab8a peptide (left) and pThr73-Rab10 peptide mimic (right). The experiments were repeated 3 times. The error bars represent standard error (SE) of mean of the technical replicates.


( E , F ) ( E ) HEK293 cells overexpressing indicated constructs were treated with ± 200 nM MLi - 2 for 90 min and then lysed . 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg / ml final concentration and membranes were analysed using the OdysseyClx Western Blot imaging system . Left , each lane represents cell extract obtained from a different dish of cells ( two biological replicates per condition without MLi - 2 treatment , one biological replicate per condition with MLi - 2 treatment ) . Right , the ratio of phospho - Rab8a / total Rab8a was quantified using Image Studio software and data presented relative to the phosphorylation ratio observed in PPM1H wild - type expressing cells . ( F ) As in ( E ) assessing phospho - Rab10 levels . For quantitation the two replicates from the presented blots were used . The data points are shown on the graphs with the bar levels representing the mean value of the biological replicates .

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

(E, F) (E) HEK293 cells overexpressing indicated constructs were treated with ± 200 nM MLi-2 for 90 min and then lysed. 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg/ml final concentration and membranes were analysed using the OdysseyClx Western Blot imaging system. Left, each lane represents cell extract obtained from a different dish of cells (two biological replicates per condition without MLi-2 treatment, one biological replicate per condition with MLi-2 treatment). Right, the ratio of phospho-Rab8a/total Rab8a was quantified using Image Studio software and data presented relative to the phosphorylation ratio observed in PPM1H wild-type expressing cells. (F) As in (E) assessing phospho-Rab10 levels. For quantitation the two replicates from the presented blots were used. The data points are shown on the graphs with the bar levels representing the mean value of the biological replicates.


( B ) In vitro malachite green assay time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS , left ) , 32 μM pThr72 phosphorylated Rab8a peptide ( middle ) , or 32 μM pThr73 phosphorylated Rab10 peptide ( right ) . The experiments were repeated 4 times . The error bars represent SE of mean of the technical replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro malachite green assay time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.


( C ) 3 μg of recombinant wild - type or indicated mutant PPM1H proteins were resolved on 4 - 12 % Bis - Tris gradient gel and stained with Instant Blue Coomassie .

O O O O O O O O O O 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) 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie.


( E ) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS ) using the malachite green assay time course . The experiments were repeated 3 times . The error bars represent SE of mean of the technical replicates . Quality of purified proteins ( right panel ) is shown using the protocol in ( C ) .

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

(E) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS) using the malachite green assay time course. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. Quality of purified proteins (right panel) is shown using the protocol in (C).


( F ) HEK293 cells overexpressing indicated constructs were treated and analysed as described in ( D ) , with pRab10 analysed in the upper panel , and pRab8a in the lower panel .

(F) HEK293 cells overexpressing indicated constructs were treated and analysed as described in (D), with pRab10 analysed in the upper panel, and pRab8a in the lower panel.


( B ) Incremental deletions of the N - terminus from residues 1 - 37 to 1 - 44 , one residue at a time . Upon deletion of R43 ( 44 - end ) , a reduced level of soluble PPM1H expression has no significant catalytic activity .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Incremental deletions of the N-terminus from residues 1-37 to 1-44, one residue at a time. Upon deletion of R43 (44-end), a reduced level of soluble PPM1H expression has no significant catalytic activity.


( B ) HEK293T cells were transfected with SFB control , SFB - tagged PR130 and SFB - B56δ , and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot ( WB ) .

O O O O O O O O O O O O O O O O O O O O O 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) HEK293T cells were transfected with SFB control, SFB-tagged PR130 and SFB-B56δ, and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot (WB).


( C ) Glutathione Sepharose beads bound with bacterially expressed recombinant GST , GST - B56δ or GST - PPP2R1A proteins were incubated with bacterially purified recombinant MBP - PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody . Interaction of B56δ - PPP2R1A was shown similarly using MBP - B56δ and GST - PPP2R1A proteins . Recombinant protein expression is shown by Coomassie staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Glutathione Sepharose beads bound with bacterially expressed recombinant GST, GST-B56δ or GST-PPP2R1A proteins were incubated with bacterially purified recombinant MBP-PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody. Interaction of B56δ-PPP2R1A was shown similarly using MBP- B56δ and GST-PPP2R1A proteins. Recombinant protein expression is shown by Coomassie staining.


( D ) HEK293T cells were transfected with SFB control , SFB - PPP2CA , SFB - PPP2CB , SFB - PPP2R1A , SFB - PPP2R1B and SFB - B56δ , and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293T cells were transfected with SFB control, SFB- PPP2CA, SFB-PPP2CB, SFB-PPP2R1A, SFB-PPP2R1B and SFB- B56δ, and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody.


( E ) Cell lysates from HEK293T cells expressing SFB vector , SFB - B56α , SFB - B56β , SFB - B56γ , SFB - B56δ and SFB - B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody .

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

(E) Cell lysates from HEK293T cells expressing SFB vector, SFB-B56α, SFB-B56β, SFB-B56γ, SFB-B56δ and SFB-B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody.


( G ) Cells expressing B56δ full length ( FL ) , Δ91 - 102 , L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting .

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

(G) Cells expressing B56δ full length (FL), Δ91-102, L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting.


( H ) Various mutants of B56δ were transfected along with myc - PPP2R1A and pulled down using streptavidin beads and interaction with myc - PPP2R1A was detected by immunoblotting .

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(H) Various mutants of B56δ were transfected along with myc-PPP2R1A and pulled down using streptavidin beads and interaction with myc-PPP2R1A was detected by immunoblotting.


( I ) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio - layer interferometry . 6xHis - tagged PPM1G and PPP2R1A were immobilized on Ni - NTA biosensors and incubated with the GST - B56δ wild type ( WT ) or Δ91 - 102 mutant at various concentrations ( 100 - 400 nm ) . Curves represent experimental trace obtained from the BLI experiments . Binding affinities ( Kd ) ( + SD ; n = 3 independent experiments ) for B56δ - PPM1G and B56δ - PPP2R1A are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio-layer interferometry. 6xHis-tagged PPM1G and PPP2R1A were immobilized on Ni-NTA biosensors and incubated with the GST-B56δ wild type (WT) or Δ91-102 mutant at various concentrations (100-400 nm). Curves represent experimental trace obtained from the BLI experiments. Binding affinities (Kd) (+SD; n=3 independent experiments) for B56δ-PPM1G and B56δ-PPP2R1A are shown.


( A ) MCF7 and MDA - MB - 231 cells transfected with MYC - B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization . Representative images from three independent experiments are shown , Scale bars 5 µm . ( B ) Quantification of cytoplasmic to nuclear ( Cyto / Nuc ) ratio of PPM1G intensity per each cell was plotted ( n = 35 for MCF - 7 cells ; n = 25 for MDA - MB - 231 cells ) . Error bars indicate SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MCF7 and MDA-MB-231 cells transfected with MYC-B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization. Representative images from three independent experiments are shown, Scale bars 5 µm. (B) Quantification of cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell was plotted (n=35 for MCF-7 cells; n=25 for MDA-MB-231 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( C ) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies .

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

(C) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies.


( D ) Cells expressing either vector or SFB - B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting . Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively .

O O O O O O O O O O O O O O O O O O O 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) Cells expressing either vector or SFB-B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.


( E ) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting . Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively .

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

(E) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.


( F ) Localization of PPM1G in cells expressing either empty vector ( control ) , full length B56δ ( FL ) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies . Representative images from two independent experiments are shown , Scale bars 10 µm . ( G ) Cytoplasmic to nuclear ( Cyto / Nuc ) ratio of PPM1G intensity per each cell expressing either vector , B56δ full length ( FL ) or mutant versions was quantified and plotted ( n = 10 ) . Error bars represents SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

(F) Localization of PPM1G in cells expressing either empty vector (control), full length B56δ (FL) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies. Representative images from two independent experiments are shown, Scale bars 10 µm. (G) Cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell expressing either vector, B56δ full length (FL) or mutant versions was quantified and plotted (n=10). Error bars represents SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( A ) Endogenous association of PPM1G with α - Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α - Catenin antibody .

O O O O O O 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) Endogenous association of PPM1G with α-Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α-Catenin antibody.


( B ) SFB - PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads . Interaction with α - catenin was determined by western blotting .

(B) SFB-PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads. Interaction with α-catenin was determined by western blotting.


( C ) In vitro phosphorylated MBP - tagged α - Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant ( D496A ) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent ( A620 nm ) . Data represents mean absorbance from three independent experiments . Error bars indicate SD , * * p < 0 . 01 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

(C) In vitro phosphorylated MBP-tagged α-Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant (D496A) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represents mean absorbance from three independent experiments. Error bars indicate SD, **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( D ) In vitro phosphate release assay was performed using wild type α - Catenin and S641A mutant as substrates and PPM1G activity on these proteins is plotted , n = 3 , Error bars indicate SD ; * * p < 0 . 01 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro phosphate release assay was performed using wild type α-Catenin and S641A mutant as substrates and PPM1G activity on these proteins is plotted, n=3, Error bars indicate SD; **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( E ) MDA - MB - 231 breast cancer cells were transfected with vector , wild type PPM1G or catalytically inactive PPM1G D496A ( D / A ) mutant . Levels of phosphorylated α - catenin were detected by using phospho - specific antibodies .

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

(E) MDA-MB-231 breast cancer cells were transfected with vector, wild type PPM1G or catalytically inactive PPM1G D496A (D/A) mutant. Levels of phosphorylated α-catenin were detected by using phospho-specific antibodies.


( F ) Cells treated with control shRNA and PPM1G specific shRNAs were lysed and blotted against specific antibodies .

(F) Cells treated with control shRNA and PPM1G specific shRNAs were lysed and blotted against specific antibodies.


( G ) Cells transfected with vector , SFB - PR130 and SFB - B56δ and levels of pS641 α - catenin was detected using western blotting .

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

(G) Cells transfected with vector, SFB-PR130 and SFB-B56δ and levels of pS641 α-catenin was detected using western blotting.


( H ) 293T Cells transfected with either empty vector , full length B56δ or indicated mutants were lysed and the levels of phosphorylated α - catenin was determined using pS641 α - catenin specific antibody .

O O O O O O O O O O O O O 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) 293T Cells transfected with either empty vector, full length B56δ or indicated mutants were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody.


( I ) Cells treated with control shRNA and B56δ specific shRNAs were lysed and the levels of phosphorylated α - catenin was determined using pS641 α - catenin specific antibody .

O O O O O O O O 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) Cells treated with control shRNA and B56δ specific shRNAs were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody.


Cells were transduced with shRNAs as indicated and the effect of ( J ) B56δ expression on α - Catenin phosphorylation was assessed .

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

Cells were transduced with shRNAs as indicated and the effect of (J) B56δ expression on α-Catenin phosphorylation was assessed.


, K ) Cells were transduced with shRNAs as indicated and the effect of PPM1G expression on α - Catenin phosphorylation was assessed .

, K) Cells were transduced with shRNAs as indicated and the effect of PPM1G expression on α-Catenin phosphorylation was assessed.


( L ) SFB - PPM1G and SFB - PPM1G ΔNLS were transfected in both control shRNA and B56δ shRNA transduced cells . Lysates were pulled down using streptavidin beads and interaction with α - catenin was detected by western blotting using α - catenin antibody .

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

(L) SFB-PPM1G and SFB-PPM1G ΔNLS were transfected in both control shRNA and B56δ shRNA transduced cells. Lysates were pulled down using streptavidin beads and interaction with α-catenin was detected by western blotting using α-catenin antibody.


( M ) Control shRNA and B56δ shRNA cells were transfected with vector , MYC - PPM1G and MYC - PPM1G ΔNLS in indicated combination and pS641 α - catenin levels were determined using western blotting .

(M) Control shRNA and B56δ shRNA cells were transfected with vector, MYC-PPM1G and MYC-PPM1G ΔNLS in indicated combination and pS641 α-catenin levels were determined using western blotting.


( A ) Cells transduced with the indicated shRNAs were fixed and localization of α - catenin and β - catenin was determined by imaging with a confocal microscope after staining with antibodies against α - catenin and β - catenin . Representative images from three independent experiments are shown . Scale bars , 10 μm . ( B ) Quantification of plasma membrane ( PM ) to cytoplasmic ratio of α - catenin intensity per each cell from figure 4A was plotted ( n = 40 cells ) . Error bars indicate SD . * * * p < 0 . 001 , ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells transduced with the indicated shRNAs were fixed and localization of α-catenin and β-catenin was determined by imaging with a confocal microscope after staining with antibodies against α-catenin and β-catenin. Representative images from three independent experiments are shown. Scale bars, 10 μm. (B) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4A was plotted (n=40 cells). Error bars indicate SD. ***p < 0.001, (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( C ) Control , PPM1G and B56δ shRNA transduced cells were transfected with SFB - α - catenin . Cell lysates were pulled down using streptavidin beads and interaction with β - catenin was determined by western blotting .

O O O O O O O O O O O O O O O O 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) Control, PPM1G and B56δ shRNA transduced cells were transfected with SFB-α-catenin. Cell lysates were pulled down using streptavidin beads and interaction with β-catenin was determined by western blotting.


( D ) F - Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope . Scale bars , 20 μm .

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

(D) F-Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope. Scale bars, 20 μm.


E ) Localization of α - catenin and β - catenin in control shRNA cells or B56δ depleted cells with or without expression of SFB - PPM1G and SFB - PPM1G ΔNLS was detected by imaging with confocal microscope after staining with specific antibodies . Representative images are shown , Scale bars , 10 μm . ( F ) Quantification of plasma membrane ( PM ) to cytoplasmic ratio of α - catenin intensity per each cell from figure 4F was plotted ( n = 30 cells ) . Error bars indicate SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

E) Localization of α-catenin and β-catenin in control shRNA cells or B56δ depleted cells with or without expression of SFB-PPM1G and SFB-PPM1G ΔNLS was detected by imaging with confocal microscope after staining with specific antibodies. Representative images are shown, Scale bars, 10 μm. (F) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4F was plotted (n=30 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( A , B ) MDA - MB - 231 cells were transfected with vector control , PPM1G wild type , catalytically inactive PPM1G D496A mutant or α - Catenin and transwell migration assay was performed . Phase contrast microscopic images ( A ) were taken after staining the migrated cells with crystal violet and ( B ) Quantification data for migrated cells per field ( derived from average of 3 different fields ) from three independent experiments is shown . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

(A, B) MDA-MB-231 cells were transfected with vector control, PPM1G wild type, catalytically inactive PPM1G D496A mutant or α-Catenin and transwell migration assay was performed. Phase contrast microscopic images (A) were taken after staining the migrated cells with crystal violet and (B) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( C , D ) MCF - 7 cells were transfected with SFB - vector , SFB - B56δ or indicated mutants and transwell migration assay was performed . Phase contrast microscopic images ( C ) were taken after staining the migrated cells with crystal violet and ( D ) Quantification data for migrated cells per field ( derived from average of 3 different fields ) from three independent experiments is shown . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) MCF-7 cells were transfected with SFB-vector, SFB-B56δ or indicated mutants and transwell migration assay was performed. Phase contrast microscopic images (C) were taken after staining the migrated cells with crystal violet and (D) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( E , F ) SFB - B56δ was expressed in control shRNA and PPM1G shRNA transduced MCF7 cells . Representative images from transwell migration assay ( E ) are shown and ( F ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

(E, F) SFB-B56δ was expressed in control shRNA and PPM1G shRNA transduced MCF7 cells. Representative images from transwell migration assay (E) are shown and (F) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( G , H ) SFB - PPM1G was expressed in control shRNA and B56δ shRNA transduced MCF7 cells . Representative images from transwell migration assay ( G ) are shown and ( H ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

(G, H) SFB-PPM1G was expressed in control shRNA and B56δ shRNA transduced MCF7 cells. Representative images from transwell migration assay (G) are shown and (H) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( I , J ) Control shRNA and B56δ shRNA transduced MCF7 cells were transfected with MYC - PPM1G and MYC - PPM1G ΔNLS . Representative images from transwell migration assay ( I ) performed with these cells are shown and ( J ) quantified data for migrated cells from three independent experiments per field was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, J) Control shRNA and B56δ shRNA transduced MCF7 cells were transfected with MYC-PPM1G and MYC-PPM1G ΔNLS. Representative images from transwell migration assay (I) performed with these cells are shown and (J) quantified data for migrated cells from three independent experiments per field was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( K , L ) SFB - tagged α - catenin S641A and S641D mutants were expressed in PPM1G shRNA and B56δ shRNA transduced MCF7 cells as shown . Representative images from transwell migration assay ( K ) performed with these cells are shown and ( L ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

(K, L) SFB-tagged α-catenin S641A and S641D mutants were expressed in PPM1G shRNA and B56δ shRNA transduced MCF7 cells as shown. Representative images from transwell migration assay (K) performed with these cells are shown and (L) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


( B ) Hoechst ( DNA ) staining and IF for Tbr2 to calculate the thickness ( white bars ) of the VZ , SVZ and intermediate zone + cortical plate ( IZ + CP ) in control and DKO E13 . 5 neocortex . Scale bar 20 μm . ( C ) Quantification of VZ , SVZ and IZ + CP thickness in control and DKO E13 . 5 neocortex . Each zone is represented as percentage of total thickness ( measured in µm ) of the neocortex . Columns are means ± SEM ( n = 6 embryos , 4 litters ) . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hoechst (DNA) staining and IF for Tbr2 to calculate the thickness (white bars) of the VZ, SVZ and intermediate zone + cortical plate (IZ+CP) in control and DKO E13.5 neocortex. Scale bar 20 μm. (C) Quantification of VZ, SVZ and IZ+CP thickness in control and DKO E13.5 neocortex. Each zone is represented as percentage of total thickness (measured in µm) of the neocortex. Columns are means ± SEM (n=6 embryos, 4 litters). (


( D ) IF for Pax6 and Tbr2 ( not shown ) in control and DKO E13 . 5 neocortex . White bars indicate thickness of Pax6 + zone ( i . e . VZ ) . Scale bar 20 μm . ( E ) Quantification of the number of APs ( Pax6 + Tbr2 - ) from ( D ) . Pax6 + Tbr2 + cells were excluded from final counts . AP numbers are expressed as percentage of total cell number ( sum of D , F and H ) . Columns are means ± SEM ( n = 4 embryos , 3 litters ) . ( F ) IF for Tbr2 in control and DKO E13 . 5 neocortex . White bars indicate thickness of Tbr2 + zone ( i . e . SVZ ) . Scale bar 20 μm . ( G ) Quantification of the number of BPs from ( F ) . Columns are means ± SEM ( n = 6 embryos , 3 litters ) . ( H ) IF for Tbr1 in control and DKO E13 . 5 neocortex . White bars indicate thickness of Tbr1 + zone ( i . e . mostly CP ) . Scale bar 15 μm . ( I ) Quantification the number of deep - layer post - mitotic neurons from ( H ) . Columns are means ± SEM ( n = 3 embryos , 3 litters ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IF for Pax6 and Tbr2 (not shown) in control and DKO E13.5 neocortex. White bars indicate thickness of Pax6+ zone (i.e. VZ). Scale bar 20 μm. (E) Quantification of the number of APs (Pax6+Tbr2-) from (D). Pax6+Tbr2+ cells were excluded from final counts. AP numbers are expressed as percentage of total cell number (sum of D, F and H). Columns are means ± SEM (n=4 embryos, 3 litters). (F) IF for Tbr2 in control and DKO E13.5 neocortex. White bars indicate thickness of Tbr2+ zone (i.e. SVZ). Scale bar 20 μm. (G) Quantification of the number of BPs from (F). Columns are means ± SEM (n=6 embryos, 3 litters). (H) IF for Tbr1 in control and DKO E13.5 neocortex. White bars indicate thickness of Tbr1+ zone (i.e. mostly CP). Scale bar 15 μm. (I) Quantification the number of deep-layer post-mitotic neurons from (H). Columns are means ± SEM (n=3 embryos, 3 litters).


( J ) IF for Ccny ( a ) , Ccnyl1 ( b ) and LRP6 ( c ) ( low - density lipoprotein receptor - related protein 6 ) phosphorylated at T1479 ( pLRP6 ) in the neocortex of E13 . 5 control embryos . White arrowheads in ( a , b ) indicate apical membrane / cortex , shown at higher magnification in the insets . High levels of LRP6 phosphorylation are detected in mitotic APs ( c , white arrowheads ) . Scale bar 50 μm . ( K ) IF for Ccny ( a ) , Ccnyl1 ( b ) and pLRP6 ( c ) , each together with IF for Tbr2 , in the neocortex of E12 . 5 control embryos . White arrowheads in the insets of ( a , b ) depict Ccny / l1 puncta in Tbr2 + BPs . High levels of LRP6 phosphorylation are detected in mitotic BPs ( c , inset , white arrowhead ) . DNA staining for mitotic pLRP6 + BP shown in bottom right inset ( c , green arrowhead ) . Scale bar 20 μm .

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

(J) IF for Ccny (a), Ccnyl1 (b) and LRP6 (c) (low-density lipoprotein receptor-related protein 6) phosphorylated at T1479 (pLRP6) in the neocortex of E13.5 control embryos. White arrowheads in (a,b) indicate apical membrane / cortex, shown at higher magnification in the insets. High levels of LRP6 phosphorylation are detected in mitotic APs (c, white arrowheads). Scale bar 50 μm. (K) IF for Ccny(a), Ccnyl1(b) and pLRP6(c), each together with IF for Tbr2, in the neocortex of E12.5 control embryos. White arrowheads in the insets of (a, b) depict Ccny/l1 puncta in Tbr2+ BPs. High levels of LRP6 phosphorylation are detected in mitotic BPs (c, inset, white arrowhead). DNA staining for mitotic pLRP6+ BP shown in bottom right inset (c, green arrowhead). Scale bar 20 μm.


( L ) IF for pLRP6 ( T1479 ) in the neocortex of E13 . 5 control and DKO embryos . White arrowheads depict mitotic APs and purple arrowheads depict mitotic BPs . Scale bar 20 μm . ( M ) Quantification of relative pLRP6 staining intensity in mitotic APs and BPs from ( L ) . Data are expressed as fold change vs . controls and are means ± SEM ( n = 3 embryos , 3 litters ) .

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

(L) IF for pLRP6 (T1479) in the neocortex of E13.5 control and DKO embryos. White arrowheads depict mitotic APs and purple arrowheads depict mitotic BPs. Scale bar 20 μm. (M) Quantification of relative pLRP6 staining intensity in mitotic APs and BPs from (L). Data are expressed as fold change vs. controls and are means ± SEM (n=3 embryos, 3 litters).


( O ) Expression analysis of the β - catenin target gene Axin2 by RNAScope in situ hybridization on neocortex sections from E13 . 5 control and DKO embryos . Scale bar 20 μm . ( P ) Quantification of ( O ) . Axin2 + area was calculated and normalized to total neocortex area . Data are expressed as fold change vs . controls and are means ± SEM ( n = 4 embryos , 3 litters ) .

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

(O) Expression analysis of the β-catenin target gene Axin2 by RNAScope in situ hybridization on neocortex sections from E13.5 control and DKO embryos. Scale bar 20 μm. (P) Quantification of (O). Axin2+ area was calculated and normalized to total neocortex area. Data are expressed as fold change vs. controls and are means ± SEM (n=4 embryos, 3 litters).


( A ) Triple - IF for BrdU , Pax6 and Tbr2 ( not shown ) in E12 . 5 control and DKO neocortices . BrdU was injected into pregnant dams one hour before sacrifice . ( B ) Quantification of the proportion of Pax6 + Tbr2 - cells that are BrdU + , in control and DKO neocortices at E11 . 5 , E12 . 5 and E13 . 5 . Data are expressed as fold change vs . controls , with the control values set to 1 . 0 , and are means ± SEM ( E11 . 5 n = 4 embryos , 3 litters ; E12 . 5 n = 5 embryos , 5 litters and E13 . 5 n = 5 embryos , 4 litters ) . ( C ) Average percentage of APs ( Pax6 + Tbr2 - ) ( from B ) that are BrdU + , in E13 . 5 control and DKO neocortices . Data are means ± SEM . ( D ) Co - IF for Tbr2 and BrdU ( double positive cells depicted by white arrowheads ) in E12 . 5 control and DKO neocortices . BrdU was injected into pregnant dams one hour before sacrifice . ( E ) Quantification of the proportion of Tbr2 + cells that are BrdU + , in control and DKO neocortices at E11 . 5 , E12 . 5 and E13 . 5 . Data are expressed as fold change vs controls , with the control values set to 1 . 0 , and are means ± SEM ( E11 . 5 n = 4 embryos , 3 litters ; E12 . 5 n = 5 embryos , 5 litters and E13 . 5 n = 5 embryos , 4 litters ) . ( F ) Average percentage of BPs ( Tbr2 + ) ( from E ) that are BrdU + , in E13 . 5 control and DKO neocortices . Data are means ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Triple-IF for BrdU, Pax6 and Tbr2 (not shown) in E12.5 control and DKO neocortices. BrdU was injected into pregnant dams one hour before sacrifice. (B) Quantification of the proportion of Pax6+Tbr2- cells that are BrdU+, in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are expressed as fold change vs. controls, with the control values set to 1.0, and are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=5 embryos, 5 litters and E13.5 n=5 embryos, 4 litters). (C) Average percentage of APs (Pax6+Tbr2-) (from B) that are BrdU+, in E13.5 control and DKO neocortices. Data are means ± SEM. (D) Co-IF for Tbr2 and BrdU (double positive cells depicted by white arrowheads) in E12.5 control and DKO neocortices. BrdU was injected into pregnant dams one hour before sacrifice. (E) Quantification of the proportion of Tbr2+ cells that are BrdU+, in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are expressed as fold change vs controls, with the control values set to 1.0, and are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=5 embryos, 5 litters and E13.5 n=5 embryos, 4 litters). (F) Average percentage of BPs (Tbr2+) (from E) that are BrdU+, in E13.5 control and DKO neocortices. Data are means ± SEM.


( K ) Co - IF for the proliferation marker Ki67 , the mitotic marker phospho - histone H3 ( pHH3 ) , and Tbr2 to identify mitotic APs ( division at apical surface ) and BPs ( division in basal VZ or SVZ ) in control and DKO E13 . 5 neocortices . Arrowheads depict pHH3 + Tbr2 + cells . ( L ) Average percentage of Ki67 + APs and BPs that are pHH3 + in control and DKO E13 . 5 neocortices . Data are means ± SEM ( n = 4 embryos , 3 litters ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-IF for the proliferation marker Ki67, the mitotic marker phospho-histone H3 (pHH3), and Tbr2 to identify mitotic APs (division at apical surface) and BPs (division in basal VZ or SVZ) in control and DKO E13.5 neocortices. Arrowheads depict pHH3+Tbr2+ cells. (L) Average percentage of Ki67+ APs and BPs that are pHH3+ in control and DKO E13.5 neocortices. Data are means ± SEM (n=4 embryos, 3 litters).


( N ) IF for cleaved caspase 3 ( Casp3 ) to identify apoptotic cells in E13 . 5 control and DKO neocortices . ( O ) Quantification of apoptotic cells in control and DKO neocortices at E11 . 5 , E12 . 5 and E13 . 5 . Data are represented as number of Casp3 + cells per 5000 cells . Data are means ± SEM ( E11 . 5 n = 4 embryos , 3 litters ; E12 . 5 n = 3 embryos , 3 litters , and E13 . 5 n = 4 embryos , 3 litters ) .

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

(N) IF for cleaved caspase 3 (Casp3) to identify apoptotic cells in E13.5 control and DKO neocortices. (O) Quantification of apoptotic cells in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are represented as number of Casp3+ cells per 5000 cells. Data are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=3 embryos, 3 litters, and E13.5 n=4 embryos, 3 litters).


( C ) IF for alpha - tubulin to visualize mitotic spindle and astral microtubules in E13 . 5 control and DKO neocortices . Central and apical - basal astral microtubules denoted by white and purple arrows , respectively . Scale bar 2 μm . ( D ) Quantification of apical - basal , central and total ( sum ) number of astral microtubules in mitotic APs of control and DKO E13 . 5 neocortices . Data are means of microtubules per cell ± SEM ( at least 10 cells counted per embryo ) ( n = 3 embryos , 3 litters ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IF for alpha-tubulin to visualize mitotic spindle and astral microtubules in E13.5 control and DKO neocortices. Central and apical-basal astral microtubules denoted by white and purple arrows, respectively. Scale bar 2 μm. (D) Quantification of apical-basal, central and total (sum) number of astral microtubules in mitotic APs of control and DKO E13.5 neocortices. Data are means of microtubules per cell ± SEM (at least 10 cells counted per embryo)(n=3 embryos, 3 litters).


( E , F ) In utero electroporation ( IUE ) of plasmids expressing GFP plus shControl ( Co ) or shCcny / l1 into the lateral ventricles of E13 . 5 wildtype embryos , followed by analysis at E15 . 5 or E17 . 5 . ( E ) IF for GFP and Tbr2 in the E15 . 5 neocortex . ( F ) IF for GFP and the deep - layer neuron marker CTIP2 in the E17 . 5 neocortex . ( E , F ) White arrowheads denote double - positive cells . Insets of magnified cells ( indicated by blue arrowheads ) depict representative GFP + Tbr2 ( E ) or GFP + Ctip2 ( F ) co - staining . Scale bars 20 μm . ( G ) Quantification of the percentage of GFP + cells that are Tbr2 + , in control and shCcny / l1 neocortices at E15 . 5 upon IUE at E13 . 5 . Data are means ± SEM ( n = 3 embryos ) . ( H ) Quantification of the percentage of GFP + cells that are Ctip2 + , in control and shCcny / l1 neocortices at E17 . 5 upon IUE at E13 . 5 . Data are means ± SEM ( n = 4 embryos ) .

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

(E, F) In utero electroporation (IUE) of plasmids expressing GFP plus shControl (Co) or shCcny/l1 into the lateral ventricles of E13.5 wildtype embryos, followed by analysis at E15.5 or E17.5. (E) IF for GFP and Tbr2 in the E15.5 neocortex. (F) IF for GFP and the deep- layer neuron marker CTIP2 in the E17.5 neocortex. (E, F) White arrowheads denote double-positive cells. Insets of magnified cells (indicated by blue arrowheads) depict representative GFP + Tbr2 (E) or GFP + Ctip2 (F) co-staining. Scale bars 20 μm. (G) Quantification of the percentage of GFP+ cells that are Tbr2+, in control and shCcny/l1 neocortices at E15.5 upon IUE at E13.5. Data are means ± SEM (n=3 embryos). (H) Quantification of the percentage of GFP+ cells that are Ctip2+, in control and shCcny/l1 neocortices at E17.5 upon IUE at E13.5. Data are means ± SEM (n=4 embryos).


( I ) IF for βIII - tubulin ( Tuj1 ) of control ( Ccny + / - ; Ccnyl1 - / - ; shRNA Co ) and mutant ( Ccny + / - ; Ccnyl1 - / - ; shRNA Ccny ) NPC monolayer cultures grown in differentiation medium for seven days . Insets depict neurons with representative neurite morphology for controls and mutants . Scale bar 20 μm . ( J ) Quantification of the percentage of Tuj1 + cells from ( I ) . Data are means ± SEM ( experiment performed twice in triplicates , representative experiment shown ) .

(I) IF for βIII-tubulin (Tuj1) of control (Ccny+/-; Ccnyl1-/-; shRNA Co) and mutant (Ccny+/-; Ccnyl1-/-; shRNA Ccny) NPC monolayer cultures grown in differentiation medium for seven days. Insets depict neurons with representative neurite morphology for controls and mutants. Scale bar 20 μm. (J) Quantification of the percentage of Tuj1+ cells from (I). Data are means ± SEM (experiment performed twice in triplicates, representative experiment shown).


( C ) Analysis of Flag - tagged Sox4 wildtype ( WT ) protein overexpressed in HEK293T ( 293T ) cells and detected with FLAG antibody . Lysates were treated with or without lambda - phosphatase ( λ - Phos ) . Antibody against phosphorylated β - catenin ( pβCAT ) was used to validate λ - Phos treatment . Purple arrowheads indicate Sox4 phosho - shift from 70kDa to 65kDa . ERK blot shows similar loading of samples . ( D ) Analysis of Flag - tagged Sox11 WT protein overexpressed in 293T cells and detected with FLAG antibody . Lysates treated with or without λ - Phos . Upper smear ( red arrowhead ) represents phosphorylated Sox11 . Antibody for pβCAT was used to validate λ - Phos treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of Flag-tagged Sox4 wildtype (WT) protein overexpressed in HEK293T (293T) cells and detected with FLAG antibody. Lysates were treated with or without lambda-phosphatase (λ-Phos). Antibody against phosphorylated β-catenin (pβCAT) was used to validate λ-Phos treatment. Purple arrowheads indicate Sox4 phosho-shift from 70kDa to 65kDa. ERK blot shows similar loading of samples. (D) Analysis of Flag-tagged Sox11 WT protein overexpressed in 293T cells and detected with FLAG antibody. Lysates treated with or without λ-Phos. Upper smear (red arrowhead) represents phosphorylated Sox11. Antibody for pβCAT was used to validate λ-Phos treatment.


( G ) Co - transfection of myc - tagged GSK3β and Flag - Sox4 in 293T cells . BIO treatment ( 0 . 5 µM ) performed for 1 hour prior to harvest . Lower band ( 65kDa ) represents non - phosphorylated Sox4 . High and low exposure ( exp ) blots shown to better visualize non - phosphorylated Sox4 . Numbers above blot are quantification of Sox4 protein levels normalized to GFP and are expressed as fold change vs . controls . The sum of upper and lower bands was quantified ( n = 8 biological replicates ) .

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

(G) Co-transfection of myc-tagged GSK3β and Flag-Sox4 in 293T cells. BIO treatment (0.5 µM) performed for 1 hour prior to harvest. Lower band (65kDa) represents non-phosphorylated Sox4. High and low exposure (exp) blots shown to better visualize non-phosphorylated Sox4. Numbers above blot are quantification of Sox4 protein levels normalized to GFP and are expressed as fold change vs. controls. The sum of upper and lower bands was quantified (n=8 biological replicates).


( B ) IF for Sox4 and pHH3 on neocortex sections of E13 . 5 control and DKO embryos . The level of Sox4 immunoreactivity is reduced in pHH3 + cells ( white arrowheads ) . ( C ) Quantification of Sox4 staining in SVZ mitotic cells from E13 . 5 control and DKO embryos . Mitotic Sox4 staining was defined as high if the pixel intensity was similar to , or higher than , non - mitotic cells , or low if the levels were below those observed in non - mitotic cells . Data are means ± SEM for ratios of cells with high vs . low Sox4 , and are displayed as fold change vs . controls ( n = 4 embryos , 3 litters ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IF for Sox4 and pHH3 on neocortex sections of E13.5 control and DKO embryos. The level of Sox4 immunoreactivity is reduced in pHH3+ cells (white arrowheads). (C) Quantification of Sox4 staining in SVZ mitotic cells from E13.5 control and DKO embryos. Mitotic Sox4 staining was defined as high if the pixel intensity was similar to, or higher than, non-mitotic cells, or low if the levels were below those observed in non-mitotic cells. Data are means ± SEM for ratios of cells with high vs. low Sox4, and are displayed as fold change vs. controls (n=4 embryos, 3 litters).


( H ) IF for Sox11 and pHH3 in E13 . 5 control and DKO neocortices . Sox11 staining intensity is decreased in mitotic cells ( white arrowheads , insets ) . ( I ) Quantification of Sox11 protein levels in SVZ mitotic cells from E13 . 5 control and DKO embryos . Quantification performed as in ( C ) . Data are means ± SEM for ratios of high vs . low Sox11 and are displayed as fold change vs . controls ( n = 5 embryos , 3 litters ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IF for Sox11 and pHH3 in E13.5 control and DKO neocortices. Sox11 staining intensity is decreased in mitotic cells (white arrowheads, insets). (I) Quantification of Sox11 protein levels in SVZ mitotic cells from E13.5 control and DKO embryos. Quantification performed as in (C). Data are means ± SEM for ratios of high vs. low Sox11 and are displayed as fold change vs. controls (n=5 embryos, 3 litters).


( K ) Co - IF for Sox11 and Tbr2 in sections of neocortex of E13 . 5 control and DKO embryos . Sox11 immunoreactivity is reduced in some Tbr2 + cells ( asterisks , insets ) . ( L ) Quantification of percentage of Tbr2 + cells that show with high Sox11 staining intensity , in control and DKO E13 . 5 neocortices . High or low staining intensity was judged based on visual inspection , taking background staining levels into account . Data are means ± SEM and are shown as fold change vs . controls ( n = 5 embryos , 3 litters ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-IF for Sox11 and Tbr2 in sections of neocortex of E13.5 control and DKO embryos. Sox11 immunoreactivity is reduced in some Tbr2+ cells (asterisks, insets). (L) Quantification of percentage of Tbr2+ cells that show with high Sox11 staining intensity, in control and DKO E13.5 neocortices. High or low staining intensity was judged based on visual inspection, taking background staining levels into account. Data are means ± SEM and are shown as fold change vs. controls (n=5 embryos, 3 litters).


( B ) IF for Tuj1 and Map2 ( not shown ) to identify newly formed neurons in NPCs after seven - day culture in differentiation medium . Empty CAG - IRES - EGFP vector was used as control in cells not transduced with Sox4 / 11 . Transduced cells were identified by IF for GFP ( Co and DKO ) or FLAG ( Co + Sox4 / 11 and DKO + Sox4 / 11 ) . Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension . Scale bar 20 μm . ( C ) Quantification of transduced Tuj1 + cells from ( B ) . Note the decrease in neuron number in DKO cultures compared to control , and the rescue in DKO cultures upon Sox4 / 11 expression ( experiment performed twice in triplicates , representative experiment shown ) . Data are means ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IF for Tuj1 and Map2 (not shown) to identify newly formed neurons in NPCs after seven-day culture in differentiation medium. Empty CAG-IRES-EGFP vector was used as control in cells not transduced with Sox4/11. Transduced cells were identified by IF for GFP (Co and DKO) or FLAG (Co + Sox4/11 and DKO + Sox4/11). Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension. Scale bar 20 μm. (C) Quantification of transduced Tuj1+ cells from (B). Note the decrease in neuron number in DKO cultures compared to control, and the rescue in DKO cultures upon Sox4/11 expression (experiment performed twice in triplicates, representative experiment shown). Data are means ± SEM.


( E ) IF for Tuj1 to identify newly formed neurons in NPCs after seven - day culture in differentiation medium . Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension . Scale bar 20 μm . ( F ) Quantification of Tuj1 + cells in control , control + CHIR ( 1 µM , 3 µM ) , DKO , and DKO + CHIR NPC cultures . Data are means ± SEM ( experiment performed twice in triplicates , representative experiment is shown ) . Numbers below asterisks show fold change .

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

(E) IF for Tuj1 to identify newly formed neurons in NPCs after seven-day culture in differentiation medium. Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension. Scale bar 20 μm. (F) Quantification of Tuj1+ cells in control, control + CHIR (1 µM, 3 µM), DKO, and DKO + CHIR NPC cultures. Data are means ± SEM (experiment performed twice in triplicates, representative experiment is shown). Numbers below asterisks show fold change.


( G ) U2OS ( left panel , n = 6 biological replicates ) or HeLa cells ( right panel , n = 3 biological replicates ) were transfected with the indicated siRNA ; 48 hours post - transfection , they were treated with the radiomimetic antibiotic , phleomycin ( 50 μg / mL ) , and collected at the indicated time points . Flow cytometry analysis of phosphorylated - H2AX signal was used to measure γ - H2AX endogenous signal . Data are represented as a bar graph showing the mean ± SEM , each replicate being represented by a round symbol . Significance was determined by two - way ANOVA followed by a Holm - Sidak ' s test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 0005 .

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

(G) U2OS (left panel, n=6 biological replicates) or HeLa cells (right panel, n = 3 biological replicates) were transfected with the indicated siRNA; 48 hours post-transfection, they were treated with the radiomimetic antibiotic, phleomycin (50 μg/mL), and collected at the indicated time points. Flow cytometry analysis of phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal. Data are represented as a bar graph showing the mean ± SEM, each replicate being represented by a round symbol. Significance was determined by two-way ANOVA followed by a Holm-Sidak's test. *P<0.05, **P<0.01, ***P<0.0005.


( H ) U2OS ( n = 8 biological replicates ) were transfected with the indicated siRNA ; 48 hours post - transfection , they were treated with the radiomimetic antibiotic , phleomycin ( 50 μg / mL ) , and collected at the indicated time points . Data are represented as a bar graph showing the mean ± SEM , each replicate being representing as a round symbol . Significance was determined by two - way ANOVA followed by a Sidak ' s test . * P < 0 . 05 .

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

(H) U2OS (n=8 biological replicates) were transfected with the indicated siRNA; 48 hours post-transfection, they were treated with the radiomimetic antibiotic, phleomycin (50 μg/mL), and collected at the indicated time points. Data are represented as a bar graph showing the mean ± SEM, each replicate being representing as a round symbol. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05.


( A ) U2OS ( left panel ) , RPE1 - hTERT ( middle panel ) , and HeLa cells ( right panel ) were monitored for their sensitivity to the radiomimetic drug NCS using the SRB assay . For each cell line , the following conditions were used : U2OS cells were transfected with a nontargeting siRNA ( siCtrl ) or an siRNA targeting human POGZ ( siPOGZ - 1 or - 2 ) ; RPE1 - hTERT cells were transduced a control shRNA ( shCtrl ) or a shRNA directed against human POGZ ( shPOGZ - 1 or - 2 ) ; HeLa cells expressing a non - targeting sgRNA ( sgCtrl ) or a sgRNA targeting human POGZ and sub - cloned ( POGZ ∆ - 1 or - 2 ) . Cells were pulsed with NCS at the indicated concentrations for 1 hour , replenished with fresh medium and incubated for 4 days before being processed for SRB assays . Data are represented as a bar graph showing the relative mean ± SEM , each replicate being representing as a round symbol ( 3 biological replicates ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s test . * P < 0 . 01 .

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

(A) U2OS (left panel), RPE1-hTERT (middle panel), and HeLa cells (right panel) were monitored for their sensitivity to the radiomimetic drug NCS using the SRB assay. For each cell line, the following conditions were used: U2OS cells were transfected with a nontargeting siRNA (siCtrl) or an siRNA targeting human POGZ (siPOGZ-1 or -2); RPE1-hTERT cells were transduced a control shRNA (shCtrl) or a shRNA directed against human POGZ (shPOGZ-1 or -2); HeLa cells expressing a non-targeting sgRNA (sgCtrl) or a sgRNA targeting human POGZ and sub-cloned (POGZ∆-1 or -2). Cells were pulsed with NCS at the indicated concentrations for 1 hour, replenished with fresh medium and incubated for 4 days before being processed for SRB assays. Data are represented as a bar graph showing the relative mean ± SEM, each replicate being representing as a round symbol (3 biological replicates). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.01.


( D ) U2OS ( left panel ) and HeLa ( right panel ) cells containing the DR - GFP reporter construct were transfected with the indicated siRNA . 24h post - transfection . Cells were transfected with the I - SceI expression plasmid or an empty vector ( EV ) , and the GFP + population was analyzed 48h post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition provided with I - SceI ( siCtrl , I - SceI ) . Data are represented as the mean ± SEM , each replicate being representing as a round symbol ( n = 3 biological replicates ) . Significance was determined by one - way ANOVA followed by a Dunnett ' s test . * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (left panel) and HeLa (right panel) cells containing the DR-GFP reporter construct were transfected with the indicated siRNA. 24h post-transfection. Cells were transfected with the I-SceI expression plasmid or an empty vector (EV), and the GFP+ population was analyzed 48h post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition provided with I-SceI (siCtrl, I-SceI). Data are represented as the mean ± SEM, each replicate being representing as a round symbol (n=3 biological replicates). Significance was determined by one-way ANOVA followed by a Dunnett's test. *P≤0.0001.


( F ) Quantification of γ - H2AX foci in HeLa cells where POGZ has been targeted by CRISPR technology ( POGZ ∆ - 1 or - 2 ) and in control HeLa cells ( sgCtrl ) . Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered at the indicated time points . Cells were fixed , stained , and imaged via confocal microscopy . Data are the total number of γ - H2AX foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 , * * P < 0 . 0005 . ( G ) Representative images used for quantification in ( F ) . Scale bar = 5 μm .

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

(F) Quantification of γ-H2AX foci in HeLa cells where POGZ has been targeted by CRISPR technology (POGZ∆-1 or -2) and in control HeLa cells (sgCtrl). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered at the indicated time points. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0005. (G) Representative images used for quantification in (F). Scale bar = 5 μm.


( A ) U2OS stably expressing mCherry - LacR - FokI were transfected with the indicated siRNA . 24h post - transfection , DNA damage was induced using Shield1 and 4 - OHT . Immunofluorescence against the indicated DNA repair proteins was subsequently performed to monitor their accumulation at sites of DNA damage by confocal microscopy . Data are represented as a box - and - whisker ( 10 - 90 percentile ) where the fluorescent signal at the mCherry focus was normalized to nuclear background . At least 100 cells per condition were counted ( n = 3 biological replicates ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 , * * P < 0 . 005 , * * P ≤ 0 . 0005 .

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

(A) U2OS stably expressing mCherry-LacR-FokI were transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield1 and 4-OHT. Immunofluorescence against the indicated DNA repair proteins was subsequently performed to monitor their accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 100 cells per condition were counted (n=3 biological replicates). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.005, **P≤0.0005.


( B ) U2OS ( left panel ) , RPE1 - hTERT ( middle panel ) , and HeLa cells ( right panel ) were monitored for their capacity to form IR - induced BRCA1 foci . For each cell line , the following conditions were used : U2OS cells were transfected with a nontargeting siRNA ( siCtrl ) or an siRNA targeting human POGZ ( siPOGZ - 1 or - 2 ) ; RPE1 - hTERT cells were transduced a control shRNA ( shCtrl ) or a shRNA directed against human POGZ ( shPOGZ - 1 or - 2 ) ; HeLa cells were expressing a non - targeting sgRNA ( sgCtrl ) or a sgRNA targeting human POGZ and sub - cloned ( POGZ ∆ - 1 or - 2 ) . Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post - exposure to IR . Cells were fixed , stained , and imaged via confocal microscopy . Data are the total number of ΒRCA1 foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 005 , * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (left panel), RPE1-hTERT (middle panel), and HeLa cells (right panel) were monitored for their capacity to form IR-induced BRCA1 foci. For each cell line, the following conditions were used: U2OS cells were transfected with a nontargeting siRNA (siCtrl) or an siRNA targeting human POGZ (siPOGZ-1 or -2); RPE1-hTERT cells were transduced a control shRNA (shCtrl) or a shRNA directed against human POGZ (shPOGZ-1 or -2); HeLa cells were expressing a non-targeting sgRNA (sgCtrl) or a sgRNA targeting human POGZ and sub-cloned (POGZ∆-1 or -2). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.001.


( E ) U2OS stably expressing mCherry - LacR - FokI were transduced with the indicated shRNA and subsequently transfected with the indicated siRNA . 24h post - transfection , DNA damage was induced using Shield - 1 and 4 - OHT . Immunofluorescence against BRCA1 was subsequently performed to monitor its accumulation at sites of DNA damage by confocal microscopy . Data are represented as a box - and - whisker ( 10 - 90 percentile ) where the fluorescent signal at the mCherry focus was normalized to nuclear background . At least 50 cells per condition were counted . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 0001 .

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

(E) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated shRNA and subsequently transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against BRCA1 was subsequently performed to monitor its accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 50 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.0001.


( G ) U2OS stably expressing mCherry - LacR - FokI were transduced with the indicated siRNA . 24h post - transfection , DNA damage was induced using Shield - 1 and 4 - OHT . Immunofluorescence against the indicated HP1 isoform was subsequently performed to monitor its respective accumulation at sites of DNA damage by confocal microscopy . Data are represented as a box - and - whisker ( 10 - 90 percentile ) where the fluorescent signal at the mCherry focus was normalized to nuclear background . At least 75 cells per condition were counted . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 005 , * * P < 0 . 0001 .

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

(G) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against the indicated HP1 isoform was subsequently performed to monitor its respective accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 75 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.0001.


( C ) HeLa cells transfected with the indicated construct were monitored for their capacity to form IR - induced BRCA1 foci . HeLa cells where POGZ has been targeted by CRISPR technology ( POGZ ∆ - 1 ) and in control HeLa cells ( sgCtrl ) were transfected by an empty Flag vector ( EV ) or a sgRNA - resistant FLAG - tagged POGZ cDNA construct corresponding to indicated rescue mutant ( full - length , FL ; POGZ801 - 848 , HPZ ) . Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post - exposure to IR . Cells were fixed , stained , and imaged via confocal microscopy . Data are the total number of ΒRCA1 foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 , * * P < 0 . 0001 . ( D ) Representative images used for quantification in ( C ) . Scale bar = 5 μm .

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

(C) HeLa cells transfected with the indicated construct were monitored for their capacity to form IR-induced BRCA1 foci. HeLa cells where POGZ has been targeted by CRISPR technology (POGZ∆-1) and in control HeLa cells (sgCtrl) were transfected by an empty Flag vector (EV) or a sgRNA-resistant FLAG-tagged POGZ cDNA construct corresponding to indicated rescue mutant (full-length, FL; POGZ801-848, HPZ). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0001. (D) Representative images used for quantification in (C). Scale bar = 5 μm.


( E ) Similar as in ( C ) except that γ - H2AX foci were monitored at the indicated time points by confocal microscopy . Data are the total number of γ - H2AX foci in EdU + cells and represented as a bar graph showing the mean ± SD ( n = 3 biological replicates , with at least 100 cells analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s test . * P < 0 . 05 . ( F ) Representative images used for quantification in ( E ) . Scale bar = 5 μm .

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

(E) Similar as in (C) except that γ-H2AX foci were monitored at the indicated time points by confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05. (F) Representative images used for quantification in (E). Scale bar = 5 μm.


Observed genotypic distribution of offspring of heterozygous Pogz + / ∆ crosses at specified embryonic day . Each litter is considered a biological replicate . Data are represented as a bar graph showing the mean ± SEM ( n for each embryonic day is specified ) . ( D )

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

Observed genotypic distribution of offspring of heterozygous Pogz+/∆ crosses at specified embryonic day. Each litter is considered a biological replicate. Data are represented as a bar graph showing the mean ± SEM (n for each embryonic day is specified). (D)


( E ) The body mass of male wild - type ( WT ) or Pogz + / ∆ mice was monitored weekly for 4 weeks post - weaning . Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a round dot ( WT ) or a square ( Pogz + / ∆ ) . At least 4 mice per genotype was monitored . Significance was determined by two - way ANOVA followed by a Sidak ' s test . * P < 0 . 05 .

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

(E) The body mass of male wild-type (WT) or Pogz+/∆ mice was monitored weekly for 4 weeks post-weaning. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ). At least 4 mice per genotype was monitored. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05.


( H ) Quantification of the distance travelled ( left panel ) and the average speed ( right panel ) of each mouse ( n = 6 mice per genotype ) in the open field . Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a a round dot ( WT ) or a square ( Pogz + / ∆ ) . Significance was determined by unpaired two - tailed t - test . * P < 0 . 005 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the distance travelled (left panel) and the average speed (right panel) of each mouse (n=6 mice per genotype) in the open field. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a a round dot (WT) or a square (Pogz+/∆ ). Significance was determined by unpaired two-tailed t-test. *P<0.005.


( L ) Plasma was isolated from cardiac punctures of wild - type ( WT ) or Pogz + / ∆ mice ( 8 weeks ) and assessed for circulating levels of specified immunoglobulin isotypes . Each mouse is represented by a round dot ( WT ) or a square ( Pogz + / ∆ ) ( n = 6 mice per genotype ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s test . * P < 0 . 005 .

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

(L) Plasma was isolated from cardiac punctures of wild-type (WT) or Pogz+/∆ mice (8 weeks) and assessed for circulating levels of specified immunoglobulin isotypes. Each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ) (n=6 mice per genotype). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.005.


( A ) Wild - type ( WT ) and Pogz + / ∆ mice were subjected to a lethal dose of ionizing radiation ( 8 . 5 Gy ) before recovering in sterile conditions and being assessed for their sensitivity to IR . Data are represented as a Kaplan - Meier survival curve of each genotype ( ( n = 6 mice per genotype ) . Significance was determined by log - rank ( Mantel - Cox ) test . * P < 0 . 005 .

(A) Wild-type (WT) and Pogz+/∆ mice were subjected to a lethal dose of ionizing radiation (8.5 Gy) before recovering in sterile conditions and being assessed for their sensitivity to IR. Data are represented as a Kaplan-Meier survival curve of each genotype ((n=6 mice per genotype). Significance was determined by log-rank (Mantel-Cox) test. *P<0.005.


( D ) MEFs were monitored for their sensitivity to the radiomimetic drug phleomycin using the SRB assay . Immortalized MEFS obtained from the indicated genotype were treated with increasing concentrations of phleomycin for 1hr , replenished with fresh medium and incubated for 4 days before being processed for SRB assays . Data are represented as a bar graph showing the relative mean ± SEM , each replicate being representing as a round dot ( WT ) or a square ( Pogz + / ∆ ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s test . * P < 0 . 005 , * * P < 0 . 0005 .

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

(D) MEFs were monitored for their sensitivity to the radiomimetic drug phleomycin using the SRB assay. Immortalized MEFS obtained from the indicated genotype were treated with increasing concentrations of phleomycin for 1hr, replenished with fresh medium and incubated for 4 days before being processed for SRB assays. Data are represented as a bar graph showing the relative mean ± SEM, each replicate being representing as a round dot (WT) or a square (Pogz+/∆ ). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.005, **P<0.0005.


( H ) Stimulated CD43 - negative splenocytes were loaded on slides via Cytospin and processed for γ - H2AX immunofluorescence . Cells were fixed , stained , and imaged via confocal microscopy . Data are the percentage of cells in a field of view with indicated γ - H2AX foci and are represented as a bar graph showing the mean ± SEM . At least 100 cells per genotype were counted . Significance was determined by two - way ANOVA followed by a Sidak ' s test . * P < 0 . 05 , * * P < 0 . 005 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stimulated CD43-negative splenocytes were loaded on slides via Cytospin and processed for γ-H2AX immunofluorescence. Cells were fixed, stained, and imaged via confocal microscopy. Data are the percentage of cells in a field of view with indicated γ-H2AX foci and are represented as a bar graph showing the mean ± SEM. At least 100 cells per genotype were counted. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05, **P<0.005.


A Volcano plot representing the statistical analysis of the fold enrichment of proteins after affinity purification in HEPES epes buffer of ABCE1 - TAP followed by label - free quantification ( LFQ ) using liquid chromatography - tandem mass spectrometry ( LC - MS / MS ) . Proteins above the curved lines show a statistically significant enrichment according to the t test value .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Volcano plot representing the statistical analysis of the fold enrichment of proteins after affinity purification in HEPES epes buffer of ABCE1-TAP followed by label-free quantification (LFQ) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins above the curved lines show a statistically significant enrichment according to the t test value.


B , C Sucrose density gradient UV - profile after in vitro splitting assays ( B ) and relative abundance of 80S and subunits as calculated from triplicates and displayed as mean ± SD . ( C ) ; SF = splitting factors including Dom34 , Hbs1 , ABCE1 and eIF6 ; ( + ) = 4 - fold molar excess of eIF3j ; ( + + ) = 20 - fold molar excess of eIF3j .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C Sucrose density gradient UV-profile after in vitro splitting assays (B) and relative abundance of 80S and subunits as calculated from triplicates and displayed as mean ± SD. (C); SF = splitting factors including Dom34, Hbs1, ABCE1 and eIF6; (+) = 4-fold molar excess of eIF3j; (++) = 20-fold molar excess of eIF3j.


( A ) Top 2 significantly overrepresented expression patterns for ERS UP and ERS DOWN genes following PHx . Data show changes in expression at 4 , 10 , 48h and 1 week after PHx ( 0h ) for genes comprised within each model profile of dynamic expression identified by STEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top 2 significantly overrepresented expression patterns for ERS UP and ERS DOWN genes following PHx. Data show changes in expression at 4, 10, 48h and 1 week after PHx (0h) for genes comprised within each model profile of dynamic expression identified by STEM.