( B ) Immunoblots indicate suppression of serum - induced AKT phosphorylation at S473 and T308 in response to miR - 155 and suppression of T308 phosphorylation in response to miR - 181b .

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(B) Immunoblots indicate suppression of serum-induced AKT phosphorylation at S473 and T308 in response to miR-155 and suppression of T308 phosphorylation in response to miR-181b.


( C ) miR - 155 and miR - 181b reduced cell viability in GDC0941 - sensitive breast cancer cell lines . Error bars indicate mean ± SD ( n = 3 ) .

(C) miR-155 and miR-181b reduced cell viability in GDC0941-sensitive breast cancer cell lines. Error bars indicate mean ± SD (n=3).


( D ) Differential localization of E - cadherin in PEO1 and PEO4 cells 48 hours post transfection with the indicated oligos . Cells were counterstained with phalloidin and DAPI .

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(D) Differential localization of E-cadherin in PEO1 and PEO4 cells 48 hours post transfection with the indicated oligos. Cells were counterstained with phalloidin and DAPI.


( E ) Stimulation of PEO4 cells with 10 ng / mL TGFβ1 significantly sensitized the cells to AKT inhibition with AKT Inhibitor X . Each point represents the mean of 3 experiments ± SD and * denotes p - value < 0 . 05 by Student ' s T - test .

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(E) Stimulation of PEO4 cells with 10 ng/mL TGFβ1 significantly sensitized the cells to AKT inhibition with AKT Inhibitor X. Each point represents the mean of 3 experiments ± SD and * denotes p-value < 0.05 by Student's T-test.


( F ) Cell viability upon expression of miR - 155 or miR - 181a mimics in 41 NSCLC cell lines and 5 human bronchial epithelial cell lines . Each data point is the mean of n = 3 / cell line . R2 from Pearson correlation .

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(F) Cell viability upon expression of miR-155 or miR-181a mimics in 41 NSCLC cell lines and 5 human bronchial epithelial cell lines. Each data point is the mean of n=3/cell line. R2 from Pearson correlation.


( A ) Consequence of miR - 517a on EOC cell viability and 41 NSCLC cell lines ( as in 2A ) . N , normal cell lines ( IHH , HOSE , HBEC3 , HBEC13 , HBEC30 , and HBEC34 ) . miR - 517a was found to significantly reduce cell viability ( Z - score < - 2 ) in 5 cell lines in the screen , and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors .

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(A) Consequence of miR-517a on EOC cell viability and 41 NSCLC cell lines (as in 2A). N, normal cell lines (IHH, HOSE, HBEC3, HBEC13, HBEC30, and HBEC34). miR-517a was found to significantly reduce cell viability (Z-score < -2) in 5 cell lines in the screen, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors.


( B , C ) In vivo delivery of neutral liposome incorporated miR - 517a mimic reduced tumor burden by weight ( B ) and nodule number ( C ) in an orthotopic xenograft model using SKOV3 cells . Box - and - whisker plot from n = 8 . p - value from Student ' s T - Test . LD , low - dose ( 200 μg / kg ) ; HD , high - dose ( 400 μg / kg ) .

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(B, C) In vivo delivery of neutral liposome incorporated miR-517a mimic reduced tumor burden by weight (B) and nodule number (C) in an orthotopic xenograft model using SKOV3 cells. Box-and-whisker plot from n=8. p-value from Student's T-Test. LD, low-dose (200 μg/kg); HD, high-dose (400 μg/kg).


( D ) Correlation of the consequence of ARCN1 depletion and miR - 517a sensitivity in 12 NSCLC cell lines ( plot as in 2D ) .

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(D) Correlation of the consequence of ARCN1 depletion and miR-517a sensitivity in 12 NSCLC cell lines (plot as in 2D).


( E ) Immunoblots indicate miR - 517a - induced depletion of ARCN1 and USP1 in the indicated cell lines .

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(E) Immunoblots indicate miR-517a-induced depletion of ARCN1 and USP1 in the indicated cell lines.


( F ) Consequence of the indicated siRNAs and miR - 517a on the viability of the indicated cell lines .

(F) Consequence of the indicated siRNAs and miR-517a on the viability of the indicated cell lines.


( G ) Immunoblots indicate that miR - 517a suppresses expression of ARCN1 in multiple miR - 517a sensitive cell lines .

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(G) Immunoblots indicate that miR-517a suppresses expression of ARCN1 in multiple miR-517a sensitive cell lines.


( A ) Consequence of miR - 124 on EOC cell viability ( as in 2A ) . N , normal cell lines ( IHH and HOSE ) . In the screen , miR - 124 was found to significantly reduce cell viability ( Z - score < - 2 ) in 6 cell lines , and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors .

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(A) Consequence of miR-124 on EOC cell viability (as in 2A). N, normal cell lines (IHH and HOSE). In the screen, miR-124 was found to significantly reduce cell viability (Z-score < -2) in 6 cell lines, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors.


( B ) miR - 124 induced expression of neuronal marker proteins Tuj1 ( TUBB3 ) and MAP2 in ES2 cells 48 hours post transfection . Cells were counterstained with DAPI and phalloidin .

(B) miR-124 induced expression of neuronal marker proteins Tuj1 (TUBB3) and MAP2 in ES2 cells 48 hours post transfection. Cells were counterstained with DAPI and phalloidin.


( C ) miR - 124 responsive genes in PEO1 cells were enriched for TargetScan predicted targets . p - value from hypergeometric distribution .

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(C) miR-124 responsive genes in PEO1 cells were enriched for TargetScan predicted targets. p-value from hypergeometric distribution.


( A ) Immunoblots indicate miR - 124 - induced SIX4 depletion . siSIX4 is shown as an antibody control .

(A) Immunoblots indicate miR-124-induced SIX4 depletion. siSIX4 is shown as an antibody control.


( B ) Consequence of SIX4 depletion on EOCcell viability ( as in 2A , N , normal cell lines : HOSE , HBEC30 ) .

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(B) Consequence of SIX4 depletion on EOCcell viability (as in 2A, N, normal cell lines: HOSE, HBEC30).


( C ) in vivo knockdown of SIX4 using neutral liposome incorporated siRNA reduced tumor burden and ascites volume in an orthotopic xenograft mouse model . Box - and - whisker plot from n = 8 . p - value from Student ' s T - Test .

(C) in vivo knockdown of SIX4 using neutral liposome incorporated siRNA reduced tumor burden and ascites volume in an orthotopic xenograft mouse model. Box-and-whisker plot from n=8. p-value from Student's T-Test.


( D ) Consequence of SIX4 depletion in PEO1 cells on E - cadherinplasma membrane accumulation 48 hours after transfection . Cells were counterstained with phalloidin and DAPI .

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(D) Consequence of SIX4 depletion in PEO1 cells on E-cadherinplasma membrane accumulation 48 hours after transfection. Cells were counterstained with phalloidin and DAPI.


( E ) Immunoblots indicate consequence of SIX4 depletion on LKB1 accumulation and AMPK pathway activation .

(E) Immunoblots indicate consequence of SIX4 depletion on LKB1 accumulation and AMPK pathway activation.


A . The unfertilized oocytes were injected with spermatozoa with a variant in IQCN at 15 - 18 h post - injection . The white arrowheads represent the polar body ( PB ) . PN represents pronucleus .

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A. The unfertilized oocytes were injected with spermatozoa with a variant in IQCN at 15-18 h post-injection. The white arrowheads represent the polar body (PB). PN represents pronucleus.


B . The expression and localization of PLCζ in sperm from the affected individuals .

B. The expression and localization of PLCζ in sperm from the affected individuals.


C . H & E staining of spermatozoa from affected individuals . The black arrow represents normal spermatozoa . The black arrowheads represent tapered - head spermatozoa with increased major axis length and normal minor axis length . The white arrowheads represent thin spermatozoa with decreased major axis length and minor axis length .

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C. H&E staining of spermatozoa from affected individuals. The black arrow represents normal spermatozoa. The black arrowheads represent tapered-head spermatozoa with increased major axis length and normal minor axis length. The white arrowheads represent thin spermatozoa with decreased major axis length and minor axis length.


D . The ultrastructure of spermatozoa from normal and affected individual . The white squares represent the perinuclear ring . Ac represents acrosome . N represents nucleus .

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D. The ultrastructure of spermatozoa from normal and affected individual. The white squares represent the perinuclear ring. Ac represents acrosome. N represents nucleus.


E . The range of acrosomes in sperm heads from normal and affected individual ( n = 7 ) .

E. The range of acrosomes in sperm heads from normal and affected individual (n=7).


A . Fertility assessment experiments in WT and Iqcn - / - male mice after mating with WT female mice ( n = 18 ) .

A. Fertility assessment experiments in WT and Iqcn-/- male mice after mating with WT female mice (n = 18).


B . Size of the testes and epididymides in WT and Iqcn - / - male mice .

B. Size of the testes and epididymides in WT and Iqcn-/- male mice.


C . Quantification ratio of the testis weight / body weight in WT and Iqcn - / - male mice ( n = 3 ) .

C. Quantification ratio of the testis weight/body weight in WT and Iqcn-/- male mice (n = 3).


D . Sperm counts in the caudal epididymis from WT and Iqcn - / - male mice ( n = 3 ) .

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D. Sperm counts in the caudal epididymis from WT and Iqcn-/- male mice (n = 3).


E . H & E staining of sperm from WT and Iqcn - / - male mice . Red arrowheads represent sperm with no sickle shape heads .

E. H&E staining of sperm from WT and Iqcn-/- male mice. Red arrowheads represent sperm with no sickle shape heads.


F . Percentage of sperm without sickle shape heads from WT and Iqcn - / - male mice ( n = 3 ) .

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F. Percentage of sperm without sickle shape heads from WT and Iqcn-/- male mice (n = 3).


G . Ultrastructure of spermatozoa from WT and Iqcn - / - male mice . Ac represents the acrosome .

G. Ultrastructure of spermatozoa from WT and Iqcn-/- male mice. Ac represents the acrosome.


H . Immunostaining of lectin ( green ) in spermatozoa from WT and Iqcn - / - male mice .

H. Immunostaining of lectin (green) in spermatozoa from WT and Iqcn-/- male mice.


I . Percentage of abnormal acrosomes in spermatozoa from WT and Iqcn - / - male mice ( n = 3 ) .

I. Percentage of abnormal acrosomes in spermatozoa from WT and Iqcn-/- male mice (n = 3).


J . Percentage of spontaneous and induced acrosome reactions in spermatozoa from WT and Iqcn - / - male mice ( n = 3 ) .

J. Percentage of spontaneous and induced acrosome reactions in spermatozoa from WT and Iqcn-/- male mice (n = 3).


A . Representative two pronucleus ( 2PN ) zygotes , two - cell embryos , and blastocysts after ICSI in WT and Iqcn - / - male mice .

A. Representative two pronucleus (2PN) zygotes, two-cell embryos, and blastocysts after ICSI in WT and Iqcn-/- male mice.


B . Percentage of 2PN zygotes , two - cell embryos and blastocysts after ICSI in WT and Iqcn - / - male mice ( n = 3 ) .

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B. Percentage of 2PN zygotes, two-cell embryos and blastocysts after ICSI in WT and Iqcn-/- male mice (n = 3).


C . Profile of Ca2 + responses induced by oocyte injection with spermatozoa from WT and Iqcn - / - male mice ( n = 5 ) . Spermatozoa from WT male mice induced 8 - 9 Ca2 + spikes over a 3 - h period ( blue line ) , while spermatozoa from Iqcn - / - male mice failed to induce spikes ( red line ) .

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C. Profile of Ca2+ responses induced by oocyte injection with spermatozoa from WT and Iqcn-/- male mice (n = 5). Spermatozoa from WT male mice induced 8-9 Ca2+ spikes over a 3-h period (blue line), while spermatozoa from Iqcn-/- male mice failed to induce spikes (red line).


D . Immunostaining of oocytes injected with spermatozoa from WT and Iqcn - / - male mice . The expression of PLCζ ( red ) and α - tubulin ( green ) was detected in oocytes . M represents male nucleus and F represents the female nucleus .

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D. Immunostaining of oocytes injected with spermatozoa from WT and Iqcn-/- male mice. The expression of PLCζ (red) and α-tubulin (green) was detected in oocytes. M represents male nucleus and F represents the female nucleus.


E . Expression and localization of PLCζ in sperm from WT and Iqcn - / - male mice .

E. Expression and localization of PLCζ in sperm from WT and Iqcn-/- male mice.


F . Expression and localization of PLCζ during acrosomal biogenesis from WT and Iqcn - / - male mice . PLCζ was localized in the acroplaxome in WT spermatids . PLCζ was only localized in the tip of the head in the cap and acrosome phases ( white arrowheads ) and was lost during the maturation phase in Iqcn - / - mice .

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F. Expression and localization of PLCζ during acrosomal biogenesis from WT and Iqcn-/- male mice. PLCζ was localized in the acroplaxome in WT spermatids. PLCζ was only localized in the tip of the head in the cap and acrosome phases (white arrowheads) and was lost during the maturation phase in Iqcn-/- mice.


A . Expression and localization of IQCN during spermiogenesis in the testes of WT male mice . IQCN ( red ) was co - localized with α - tubulin ( green ) from steps 8 - 14 . Sb represents late spermatids . Scale bars , 20 μm .

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A. Expression and localization of IQCN during spermiogenesis in the testes of WT male mice. IQCN (red) was co-localized with α-tubulin (green) from steps 8-14. Sb represents late spermatids. Scale bars, 20 μm.


B . Ultrastructure of the testes from WT and Iqcn - / - male mice from steps 8 - 16 . MA represents manchette . PR represents perinuclear ring . CD represents cytoplasmic droplets . Scale bars , 2 μm ( steps 8 - 12 ; 15 - 16 ) and 5 μm ( steps 13 - 14 ) .

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B. Ultrastructure of the testes from WT and Iqcn-/- male mice from steps 8-16. MA represents manchette. PR represents perinuclear ring. CD represents cytoplasmic droplets. Scale bars, 2 μm (steps 8-12; 15-16) and 5 μm (steps 13-14).


C . Immunostaining of α - tubulin ( green ) in spermatids at steps 13 - 14 . MA represents manchette . PR represents perinuclear ring .

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C. Immunostaining of α-tubulin (green) in spermatids at steps 13-14. MA represents manchette. PR represents perinuclear ring.


D . Length of the manchette structure in spermatids at steps 13 - 14 from WT and Iqcn - / - male mice ( n = 10 ) .

D. Length of the manchette structure in spermatids at steps 13-14 from WT and Iqcn-/- male mice (n = 10).


A . Expression and localization of calmodulin during spermiogenesis in the testes of WT and Iqcn - / - male mice . Calmodulin ( red ) was co - localized with α - tubulin ( green ) from steps 8 - 14 in WT mice , but failed to co - localize in steps 13 - 14 in Iqcn - / - male mice .

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A. Expression and localization of calmodulin during spermiogenesis in the testes of WT and Iqcn-/- male mice. Calmodulin (red) was co-localized with α-tubulin (green) from steps 8-14 in WT mice, but failed to co-localize in steps 13-14 in Iqcn-/- male mice.


B . Interaction between IQCN and calmodulin ( CaM ) following co - IP by IQCN antibody in testes from WT and Iqcn - / - male mice ( n = 3 ) . IQCN interacts with calmodulin in WT mice , the interaction of which is completely lost in Iqcn - / - male mice . GAPDH was used as the internal control .

B. Interaction between IQCN and calmodulin (CaM) following co-IP by IQCN antibody in testes from WT and Iqcn-/- male mice (n = 3). IQCN interacts with calmodulin in WT mice, the interaction of which is completely lost in Iqcn-/- male mice. GAPDH was used as the internal control.


C . Immunostaining of calmodulin ( red ) and α - tubulin ( green ) in HeLa cells . The normal ( WT ) and mutant ( c . 910C > T and c . 2453 _ 2454del ) plasmids of IQCN were transfected into HeLa cells . HeLa cells were treated with an antagonist of calmodulin W - 7 . Multipolar spindles were observed in HeLa cells transfected with mutant plasmids and W - 7 treatment .

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C. Immunostaining of calmodulin (red) and α-tubulin (green) in HeLa cells. The normal (WT) and mutant (c.910C>T and c.2453_2454del) plasmids of IQCN were transfected into HeLa cells. HeLa cells were treated with an antagonist of calmodulin W-7. Multipolar spindles were observed in HeLa cells transfected with mutant plasmids and W-7 treatment.


D . Percentage of multipolar spindles in WT and mutant IQCN transfected HeLa cells . ( n = 3 biological replicates )

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D. Percentage of multipolar spindles in WT and mutant IQCN transfected HeLa cells. (n = 3 biological replicates)


B . Quantification of PNRC1 expression levels in primary normal ( blue circles ) and cancer ( red circles ) samples by Real Time PCR .

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B. Quantification of PNRC1 expression levels in primary normal (blue circles) and cancer (red circles) samples by Real Time PCR.


Immunofluorescence stainings of PNRC1 ( red ) and Ki - 67 ( green ) performed on a primary healthy colon ( C ) DAPI was used to stain cell nuclei . Scale bars : 50µm .

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Immunofluorescence stainings of PNRC1 (red) and Ki-67 (green) performed on a primary healthy colon (C) DAPI was used to stain cell nuclei. Scale bars:50µm.


Immunofluorescence stainings of PNRC1 ( red ) and Ki - 67 ( green ) performed on a malignant lymphoma ( D ) . DAPI was used to stain cell nuclei . Scale bars : 50µm .

Immunofluorescence stainings of PNRC1 (red) and Ki-67 (green) performed on a malignant lymphoma (D). DAPI was used to stain cell nuclei. Scale bars:50µm.


E . Proliferation curves of HeLa cells transfected with HRASG12V ( R ) , PNRC1 ( P ) , their combination ( RP ) or a LacZ control ( L ) . The results shown are the average ± SD of 2 biological replicates with 3 technical replicates each . A one - way ANOVA with Tukey multiple comparison test was performed for 72h dataset ; the statistically significant comparisons are as follows : L vs R ( p < 0 . 002 ) , L vs P ( p < 0 . 02 ) , R vs P ( p < 0 . 0002 ) and R vs RP ( p < 0 . 0004 )

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E. Proliferation curves of HeLa cells transfected with HRASG12V (R), PNRC1 (P), their combination (RP) or a LacZ control (L). The results shown are the average ± SD of 2 biological replicates with 3 technical replicates each. A one-way ANOVA with Tukey multiple comparison test was performed for 72h dataset; the statistically significant comparisons are as follows: L vs R (p<0.002), L vs P (p<0.02), R vs P (p<0.0002) and R vs RP (p<0.0004)


F . Proliferation curves of MCF7 cells transfected with HRASG12V ( R ) , PNRC1 ( P ) , their combination ( RP ) or a LacZ control ( L ) . The results shown are the average ± SD of 2 biological replicates with 3 technical replicates each .

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F. Proliferation curves of MCF7 cells transfected with HRASG12V (R), PNRC1 (P), their combination (RP) or a LacZ control (L). The results shown are the average ± SD of 2 biological replicates with 3 technical replicates each.


G . Proliferation curves of KRASG12S - mutated A549 cells transfected with PNRC1 or LacZ control . A representative experiment with the average ± SD of 3 technical replicates is shown . An unpaired t - test was performed for 144h dataset ( p < 0 . 0003 ) .

G. Proliferation curves of KRASG12S-mutated A549 cells transfected with PNRC1 or LacZ control. A representative experiment with the average ± SD of 3 technical replicates is shown. An unpaired t-test was performed for 144h dataset (p<0.0003).


H . Proliferation curves of BJ - T cells transduced with a non - targeting or a PNRC1 - specific shRNA construct . Results are shown as the average ± SD of 3 biological replicates with 3 technical replicates each . An unpaired t - test was performed for each time point ( * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 ) .

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H. Proliferation curves of BJ-T cells transduced with a non-targeting or a PNRC1-specific shRNA construct. Results are shown as the average ± SD of 3 biological replicates with 3 technical replicates each. An unpaired t-test was performed for each time point (** p<0.01, *** p<0.005, **** p<0.001).


A . Click - iT RNA imaging assay performed on HeLa cells expressing RFP - PNRC1 or RFP control . Confocal images of transfected cells pulsed with EU for 16 hours were collected at the indicated time points after EU removal ( green : Alexa 488 - EU , red : RFP , blue : DAPI , scale bar : 5µm ) . RFP - PNRC1 - transfected cells are indicated with arrowheads .

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A. Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1 or RFP control. Confocal images of transfected cells pulsed with EU for 16 hours were collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1-transfected cells are indicated with arrowheads.


Confocal microscopy images of HeLa cells expressing GFP - PNRC1 and stained with antibodies against NPM1 ( B ) nucleolar proteins ( nuclei are stained in blue with DAPI , scale bar : 5µm ) .

Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against NPM1 (B) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm).


Confocal microscopy images of HeLa cells expressing GFP - PNRC1 and stained with antibodies against UBF1 ( C ) nucleolar proteins ( nuclei are stained in blue with DAPI , scale bar : 5µm ) . A magnification of the merged channel images is provided for UBF1 staining .

Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against UBF1 (C) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm). A magnification of the merged channel images is provided for UBF1 staining.


D . Agarose / formaldehyde gel separation of neotranscribed rRNAs collected from LacZ and PNRC1 - expressing cells pulsed for 15 minutes with 3H - uridine and chased for the reported time points ( minutes ) . Samples were loaded according to the amount of incorporated 3H , quantified by liquid scintillation . FU was included as a control of a blocked rRNA processing .

D. Agarose/formaldehyde gel separation of neotranscribed rRNAs collected from LacZ and PNRC1-expressing cells pulsed for 15 minutes with 3H-uridine and chased for the reported time points (minutes). Samples were loaded according to the amount of incorporated 3H, quantified by liquid scintillation. FU was included as a control of a blocked rRNA processing.


E . Ratios between the intensity of 47S pre - rRNA band and bands belonging to 32S or 28S rRNA species relative to panel ( D ) and measured at 60 minutes after the chase for both LacZ or PNRC1 - expressing cells .

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E. Ratios between the intensity of 47S pre-rRNA band and bands belonging to 32S or 28S rRNA species relative to panel (D) and measured at 60 minutes after the chase for both LacZ or PNRC1-expressing cells.


F . Real Time PCR quantification of 5 ' - ETS steady - state levels in HeLa cells expressing PNRC1 or GFP control . The average ± SD of 3 biological replicates is shown .

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F. Real Time PCR quantification of 5'-ETS steady-state levels in HeLa cells expressing PNRC1 or GFP control. The average ± SD of 3 biological replicates is shown.


A - B . Coomassie blue staining and western blot analysis of the SDS - PAGE gel corresponding to PNRC1 IP . Red arrows represent the gel areas excised both from the IgG and IP lanes for mass spectrometry analysis . Input , unbound and IP fractions were loaded as reported . The protein band corresponding to PNRC1 is indicated .

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A-B. Coomassie blue staining and western blot analysis of the SDS-PAGE gel corresponding to PNRC1 IP. Red arrows represent the gel areas excised both from the IgG and IP lanes for mass spectrometry analysis. Input, unbound and IP fractions were loaded as reported. The protein band corresponding to PNRC1 is indicated.


C . Co - immunoprecipitation performed on HeLa cells expressing HA - PNRC1 with a specific anti - HA antibody in absence or presence of 1mg / ml RNaseA .

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C. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1 with a specific anti-HA antibody in absence or presence of 1mg/ml RNaseA.


D . Co - immunoprecipitation of DCP1α - interacting proteins performed on HA - PNRC1 - expressing HeLa cells with a specific anti - DCP1α antibody .

D. Co-immunoprecipitation of DCP1α-interacting proteins performed on HA-PNRC1-expressing HeLa cells with a specific anti-DCP1α antibody.


E . Co - immunoprecipitation performed on wild - type HeLa cells with an anti - PNRC1 antibody and probed for DCP1α .

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E. Co-immunoprecipitation performed on wild-type HeLa cells with an anti-PNRC1 antibody and probed for DCP1α.


F - G . Co - immunoprecipitation performed on HeLa cells expressing HA - PNRC1 with a specific anti - HA antibody and probed with antibodies specific for proteins involved in RNA decapping . The white arrow indicates the specific DCP2 band , as reported in the datasheet of the antibody employed .

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F-G. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1 with a specific anti-HA antibody and probed with antibodies specific for proteins involved in RNA decapping. The white arrow indicates the specific DCP2 band, as reported in the datasheet of the antibody employed.


A . Confocal microscopy images of RFP or RFP - PNRC1 - expressing cells stained for DCP1α , DDX6 and LSM1 . RFP - PNRC1 - transfected cells are indicated with arrowheads . Scale bar : 10µm . B . Quantification of cells classified according to the P - body localization of the three stained proteins . The average ± SD of 3 independent experiments is reported .

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A. Confocal microscopy images of RFP or RFP-PNRC1-expressing cells stained for DCP1α, DDX6 and LSM1. RFP-PNRC1-transfected cells are indicated with arrowheads. Scale bar: 10µm. B. Quantification of cells classified according to the P-body localization of the three stained proteins. The average ± SD of 3 independent experiments is reported.


C . Nucleolar fractionation performed on LacZ or PNRC1 - expressing HeLa cells . Total lysates , cytoplasmic ( Cyto ) , nucleoplasmic ( Nucl ) and nucleolar ( No ) fractions were collected . UBF1 , RNA PolII and MEK2 were used as markers for the nucleolar , non - nucleolar and cytoplasmic fractions , respectively . The electrophoretic mobility of proteins in the nucleoplasmic lanes is slightly diminished due to nuclear lysis buffer composition . D . Densitometric quantification of DCP1α nucleolar pool obtained from the western blot shown in panel ( C ) and normalized upon the amount of nucleolar UBF1 and the levels of DCP1α in the input fraction .

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C. Nucleolar fractionation performed on LacZ or PNRC1-expressing HeLa cells. Total lysates, cytoplasmic (Cyto), nucleoplasmic (Nucl) and nucleolar (No) fractions were collected. UBF1, RNA PolII and MEK2 were used as markers for the nucleolar, non-nucleolar and cytoplasmic fractions, respectively. The electrophoretic mobility of proteins in the nucleoplasmic lanes is slightly diminished due to nuclear lysis buffer composition. D. Densitometric quantification of DCP1α nucleolar pool obtained from the western blot shown in panel (C) and normalized upon the amount of nucleolar UBF1 and the levels of DCP1α in the input fraction.


E . Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing a shRNA - resistant GFP - DCP1α in combination with RFP or RFP - PNRC1 transgenes ( green : GFP , red : RFP , blue : DAPI , scale bar : 5µm ) .

E. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing a shRNA-resistant GFP-DCP1α in combination with RFP or RFP-PNRC1 transgenes (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm).


A . Co - immunoprecipitation performed on HeLa cells expressing HA - PNRC1WT , HA - PNRC1W300A or HA - PNRC1ΔNLS with a specific anti - HA antibody and probed with anti - NPM1 and anti DCP1α antibodies .

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A. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1WT, HA-PNRC1W300A or HA-PNRC1ΔNLS with a specific anti-HA antibody and probed with anti-NPM1 and anti DCP1α antibodies.


B - C . Co - immunoprecipitation performed on HeLa cells expressing HA - PNRC1W300A mutant with a specific anti - HA antibody and probed with antibodies specific for PNRC1 - interacting proteins . The white arrow indicates the specific DCP2 band .

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B-C. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1W300A mutant with a specific anti-HA antibody and probed with antibodies specific for PNRC1-interacting proteins. The white arrow indicates the specific DCP2 band.


D . Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing an shRNA - resistant GFP - DCP1α in combination with RFP , RFP - PNRC1WT or RFP - PNRC1W300A mutant ( green : GFP , red : RFP , blue : DAPI , scale bar : 5µm ) .

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D. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing an shRNA-resistant GFP-DCP1α in combination with RFP, RFP-PNRC1WT or RFP-PNRC1W300A mutant (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm).


E . Confocal microscopy images of RFP , RFP - PNRC1 or RFP - PNRC1W300A - expressing cells stained for DCP1α , DDX6 and LSM1 . RFP - PNRC1WT or RFP - PNRC1W300A - transfected cells are indicated with arrowheads . Scale bar :

E. Confocal microscopy images of RFP, RFP-PNRC1 or RFP-PNRC1W300A-expressing cells stained for DCP1α, DDX6 and LSM1. RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads. Scale bar: 10µm


F . Quantification of HeLa cells expressing RFP - PNRC1W300A classified according to the P - body localization of the three stained proteins . The average of 3 independent experiments ± SD is reported .

F. Quantification of HeLa cells expressing RFP-PNRC1W300A classified according to the P-body localization of the three stained proteins. The average of 3 independent experiments ± SD is reported.


G . Co - immunoprecipitation performed on HeLa cells expressing LacZ control , HA - PNRC1WT , HA - PNRC1W300A or HA - PNRC1ΔNLS constructs with a specific anti - DCP1α antibody and probed with anti - PNRC1 and anti - NPM1 antibodies .

G. Co-immunoprecipitation performed on HeLa cells expressing LacZ control, HA-PNRC1WT, HA-PNRC1W300A or HA-PNRC1ΔNLS constructs with a specific anti-DCP1α antibody and probed with anti-PNRC1 and anti-NPM1 antibodies.


A . Confocal images of a Click - iT RNA imaging assay performed on HeLa cells expressing RFP - PNRC1W300A pulsed with EU for 16 hours and collected at the indicated time points after EU removal ( green : Alexa 488 - EU , red : RFP , blue : DAPI , scale bar : 5µm ) . RFP - PNRC1WT or RFP - PNRC1W300A - transfected cells are indicated with arrowheads .

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A. Confocal images of a Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1W300A pulsed with EU for 16 hours and collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads.


B . Real Time PCR quantification of 5 ' - ETS steady - state levels in HeLa cells expressing PNRC1W300A or GFP control . The average of 3 independent experiments ± SD is reported .

B. Real Time PCR quantification of 5'-ETS steady-state levels in HeLa cells expressing PNRC1W300A or GFP control. The average of 3 independent experiments ± SD is reported.


C . Quantification of 5 ' - ETS RNA levels in HeLa cells infected with an anti - DCP2 or a non - targeting shRNA and expressing GFP , PNRC1WT or PNRC1W300A transgenes . The average of 2 independent experiments ± SD is reported .

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C. Quantification of 5'-ETS RNA levels in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average of 2 independent experiments ± SD is reported.


Levels of U3 ( D ) snoRNAs immunoprecipitated by the anti - m3G cap antibody measured by Real Time PCR . Values represents the average of two biological replicates with three technical replicates and

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Levels of U3 (D) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR. Values represents the average of two biological replicates with three technical replicates and are


Levels of U8 ( E ) snoRNAs immunoprecipitated by the anti - m3G cap antibody measured by Real Time PCR .

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Levels of U8 (E) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR.


F . Levels of U3 snoRNA immunoprecipitated by the anti - m3G cap antibody in HeLa cells infected with an anti - DCP2 or a non - targeting shRNA and expressing GFP , PNRC1WT or PNRC1W300A transgenes . The average ± SD of 2 independent experiments with three technical replicates is reported .

F. Levels of U3 snoRNA immunoprecipitated by the anti-m3G cap antibody in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average ± SD of 2 independent experiments with three technical replicates is reported.


G . Western blot analysis of input and Flag - IP fractions of RIP experiment . H . RNA levels of U3 , U8 and U1 co - immunoprecipitating with GFP , PNRC1WT or PNRC1W300A . Results are shown as the average ± SD of two biological replicates .

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G. Western blot analysis of input and Flag-IP fractions of RIP experiment. H. RNA levels of U3, U8 and U1 co-immunoprecipitating with GFP, PNRC1WT or PNRC1W300A. Results are shown as the average ± SD of two biological replicates.


( B ) CRMP2 expression in adult cortex ( layers VI - IV ) , corpus callosum ( CC ) and hippocampal area CA1 . CRMP2 ( green ) is expressed throughout the cortex and hippocampus , CRMP2A ( red ) is particularly strongly expressed in a subset of callosal axons . Nuclei are counterstained with Hoechst 33342 . Scale bars : 50 μm .

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(B) CRMP2 expression in adult cortex (layers VI - IV), corpus callosum (CC) and hippocampal area CA1. CRMP2 (green) is expressed throughout the cortex and hippocampus, CRMP2A (red) is particularly strongly expressed in a subset of callosal axons. Nuclei are counterstained with Hoechst 33342. Scale bars: 50 μm.


( C ) Whole - mount immunohistochemistry with anti - neurofilaments antibody for visualization of peripheral nerves . In crmp2 - / - embryos , the growth of ophthalmic branch of the trigeminal nerve is increased and axons are defasciculated ( first and second row , arrowheads ) . Similarly , we detected increased growth and branching of lateral branches of spinal nerves in crmp2 - / - embryos ( third row , arrowheads ) . Trigeminal nerve ( V ) and its branches ( Op - ophthalmic , Mx - maxillar , Md - mandibular ) are indicated . Scale bars : 500 μm .

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(C) Whole-mount immunohistochemistry with anti-neurofilaments antibody for visualization of peripheral nerves. In crmp2-/- embryos, the growth of ophthalmic branch of the trigeminal nerve is increased and axons are defasciculated (first and second row, arrowheads). Similarly, we detected increased growth and branching of lateral branches of spinal nerves in crmp2-/- embryos (third row, arrowheads). Trigeminal nerve (V) and its branches (Op - ophthalmic, Mx - maxillar, Md - mandibular) are indicated. Scale bars: 500 μm.


( D , E ) Quantification of areas innervated by ophthalmic branches of trigeminal ganglion and spinal nerve , normalized to WT littermates ( each dot depicts one embryo ) . Area of ophthalmic branch in crmp2 - / - was increased by 66 % ( p < 0 . 05 , n = 10 , 3 litters ) . For spinal nerves , total area in knockouts was increased by 45 % ( p < 0 . 05 , WT n = 8 , crmp2 - / - n = 7 , 3 litters ) . Mean ± SD , * p < 0 . 05 , t - test .

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(D, E) Quantification of areas innervated by ophthalmic branches of trigeminal ganglion and spinal nerve, normalized to WT littermates (each dot depicts one embryo). Area of ophthalmic branch in crmp2-/- was increased by 66% (p<0.05, n=10, 3 litters). For spinal nerves, total area in knockouts was increased by 45% (p<0.05, WT n=8, crmp2-/- n=7, 3 litters). Mean ± SD, * p<0.05, t-test.


( F ) Growth of motor neurons from E11 . 5 - E12 . 5 WT and crmp2 - / - spinal cord explants in microfluidic chambers . Excerpts from 14 hour time - lapse imaging are shown . Upon application of Sema3A into distal compartment , WT axons tend to stop or slow down their growth unlike crmp2 - / - axons . Purple lines highlight the growth path of individual axons in a distal chamber . Scale bars : 100 μm .

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(F) Growth of motor neurons from E11.5 - E12.5 WT and crmp2-/- spinal cord explants in microfluidic chambers. Excerpts from 14 hour time-lapse imaging are shown. Upon application of Sema3A into distal compartment, WT axons tend to stop or slow down their growth unlike crmp2-/- axons. Purple lines highlight the growth path of individual axons in a distal chamber. Scale bars: 100 μm.


( G ) Quantification of the fraction of growing ( > 50 μm ) axons in one imaging field ( WT : control 76 . 8 ± 5 . 7 % , Sema3A 57 . 7 ± 10 . 6 % , p < 0 . 001 , crmp2 - / - : control 75 . 4 ± 10 . 4 % , Sema3A 75 . 4 ± 6 . 7 % , p = 0 . 99 ) , n = 3 experiments per genotype , 7 - 8 explants per condition . Mean ± SD . * * * p < 0 . 001 , 2 - way ANOVA with Bonferroni ' s multiple comparisons test .

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(G) Quantification of the fraction of growing (>50 μm) axons in one imaging field (WT: control 76.8±5.7%, Sema3A 57.7±10.6%, p<0.001, crmp2-/-: control 75.4±10.4%, Sema3A 75.4±6.7%, p=0.99), n=3 experiments per genotype, 7-8 explants per condition. Mean ± SD. *** p<0.001, 2-way ANOVA with Bonferroni's multiple comparisons test.


( A ) CRMP2 deficiency leads to callosal hypoplasia . Shortening of corpus callosum ( arrowheads ) is apparent in both sagittal ( first row ) and coronal sections ( second row ) of adult brains . Scale bars : 1 mm .

(A) CRMP2 deficiency leads to callosal hypoplasia. Shortening of corpus callosum (arrowheads) is apparent in both sagittal (first row) and coronal sections (second row) of adult brains. Scale bars: 1 mm.


( B ) Quantification of callosal length in 30 - days old mice ( P30 , n = 3 , crmp2 - / - is 80 . 8 ± 5 % of WT , p = 0 . 003 ) and adult mice ( n = 3 , crmp2 - / - is 80 . 5 ± 3 % of WT , p = 0 . 002 ) , mean ± SD , * p < 0 . 05 , t - test .

(B) Quantification of callosal length in 30-days old mice (P30, n=3, crmp2-/- is 80.8±5% of WT, p=0.003) and adult mice (n=3, crmp2-/- is 80.5±3% of WT, p=0.002), mean ± SD, * p<0.05, t-test.


( C ) Labeling of adult corpus callosum with anti - neurofilaments antibody in sagittal sections . Outline depicts missing posterior part of the tract in crmp2 - / - mice . Caudal part of the corpus callosum in WTs is located dorsally above the habenular commissure ( hbc ) , while in crmp2 - / - mice callosum terminates rostrally before reaching the hbc ( arrows ) . PC indicates posterior commissure . Scale bars : 500 µm .

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(C) Labeling of adult corpus callosum with anti-neurofilaments antibody in sagittal sections. Outline depicts missing posterior part of the tract in crmp2-/- mice. Caudal part of the corpus callosum in WTs is located dorsally above the habenular commissure (hbc), while in crmp2-/- mice callosum terminates rostrally before reaching the hbc (arrows). PC indicates posterior commissure. Scale bars: 500 µm.


( D ) The growth of GFP - labeled callosal axons in the contralateral cortex at P6 ( embryos were electroporated at E15 . 5 ) . Note the disorganized paths of crmp2 - / - axons . Scale bars : 200 μm .

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(D) The growth of GFP-labeled callosal axons in the contralateral cortex at P6 (embryos were electroporated at E15.5). Note the disorganized paths of crmp2-/- axons. Scale bars: 200 μm.


( F ) Quantification of tortuosity of axons ( WT n = 6 pups , crmp2 - / - n = 3 pups ) upon their exit from the callosal tract ( WT 1 . 016 ± 0 . 003 vs . crmp2 - / - 1 . 044 ± 0 . 012 , p < 0 . 001 ) , mean ± SD , * * * p < 0 . 001 , t - test .

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(F) Quantification of tortuosity of axons (WT n=6 pups, crmp2-/- n=3 pups) upon their exit from the callosal tract (WT 1.016±0.003 vs. crmp2-/- 1.044±0.012, p<0.001), mean ± SD, ***p<0.001, t-test.


( G ) DiI - labeled callosal axons from P9 oblique brain sections and their reconstruction in Neurolucida 360 ( WT n = 6 , crmp2 - / - n = 9 ) . Scale bars : 200 μm .

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(G) DiI-labeled callosal axons from P9 oblique brain sections and their reconstruction in Neurolucida 360 (WT n=6, crmp2-/- n=9). Scale bars: 200 μm.


( H ) Polar histograms of callosal axons reconstructed Note the broader range of axon growth angles in crmp2 - / - mice .

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(H) Polar histograms of callosal axons reconstructed Note the broader range of axon growth angles in crmp2-/- mice.


( I ) Left : schematic representation of polar histogram analysis by clustering the traced axons ( WT n = 6 pups , crmp2 - / - n = 9 pups ) into 3 groups based on the growth angles . Right : proportion of axons growing in selected clusters ( WT , 0 ° - ± 20 ° : 67 . 3 ± 10 . 3 % ; ± 20 ° - ± 40 ° : 21 . 2 ± 5 . 3 % ; ± 40 ° - ± 60 ° : 6 . 22 ± 3 . 2 % ; crmp2 - / - , 58 . 5 ± 5 % , p = 0 . 047 , 27 . 7 ± 4 . 3 % , p = 0 . 022 , 8 . 64 ± 2 . 25 % , p = 0 . 11 ) , mean ± SD , * p < 0 . 05 , t - test .

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(I) Left: schematic representation of polar histogram analysis by clustering the traced axons (WT n=6 pups, crmp2-/- n=9 pups) into 3 groups based on the growth angles. Right: proportion of axons growing in selected clusters (WT, 0°-±20°: 67.3±10.3%; ±20°-±40°: 21.2±5.3%; ±40°-±60°: 6.22±3.2%; crmp2-/-, 58.5±5%, p=0.047, 27.7±4.3%, p=0.022, 8.64±2.25%, p=0.11), mean ± SD, *p<0.05, t-test.


( A , B ) ( A ) Coronal sections of P14 brains stained for calbindin and VgluT1 . Infrapyramidal bundle ( IPB ) progresses into hippocampal CA3 region in both WT and crmp2 - / - mice . Nuclei are counterstained with Hoechst 33342 . ( B ) Details from depicted regions in ( A ) , arrowheads show the course of IPB . Scale bars : 100 μm .

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(A, B) (A) Coronal sections of P14 brains stained for calbindin and VgluT1. Infrapyramidal bundle (IPB) progresses into hippocampal CA3 region in both WT and crmp2-/- mice. Nuclei are counterstained with Hoechst 33342. (B) Details from depicted regions in (A), arrowheads show the course of IPB. Scale bars: 100 μm.


( C , D ) ( C ) Staining for calbindin shows unpruned infrapyramidal bundle in adult crmp2 - / - mice . No VGluT2 signal in hippocampal CA3 region is present in WT and crmp2 - / - suggesting mature synapses . Scale bars : 500 μm . ( D ) Details from depicted regions in ( C ) , arrowheads show the course of IPB . Scale bars : 100 μm .

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(C, D) (C) Staining for calbindin shows unpruned infrapyramidal bundle in adult crmp2-/- mice. No VGluT2 signal in hippocampal CA3 region is present in WT and crmp2-/- suggesting mature synapses. Scale bars: 500 μm. (D) Details from depicted regions in (C), arrowheads show the course of IPB. Scale bars: 100 μm.


( E , F ) Staining for calbindin and VgluT1 shows mature synapses in the main bundle in both genotypes and in the IPB in crmp2 - / - mice ( arrowheads ) . Scale bars : 100 μm .

(E, F) Staining for calbindin and VgluT1 shows mature synapses in the main bundle in both genotypes and in the IPB in crmp2-/- mice (arrowheads). Scale bars: 100 μm.


( G ) Quantification of IPB index ( length of the IPB vs . main bundle , WT 0 . 43 ± 0 . 05 , crmp2 - / - 0 . 78 ± 0 . 05 , p < 0 . 001 ) counted from adult coronal sections ( WT n = 4 mice , crmp2 - / - n = 5 mice ) . Mean ± SD , * * * p < 0 . 001 , t - test .

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(G) Quantification of IPB index (length of the IPB vs. main bundle, WT 0.43±0.05, crmp2-/- 0.78±0.05, p<0.001) counted from adult coronal sections (WT n=4 mice, crmp2-/- n=5 mice). Mean ± SD, ***p<0.001, t-test.


( A ) Upper row : DiI tracing of the visual cortex axons at P9 ( before pruning ) , sagittal sections . Branching point of the tract is shown . Lower row : visual cortex axons in adult mice after pruning period . Note significantly reduced number of axons continuing into pyramidal tract in WT ( dashed arrow ) . ( arrows - 2 branches of corticospinal visual axons ) . Maximum projections are shown . Scale bars : 200 μm .

(A) Upper row: DiI tracing of the visual cortex axons at P9 (before pruning), sagittal sections. Branching point of the tract is shown. Lower row: visual cortex axons in adult mice after pruning period. Note significantly reduced number of axons continuing into pyramidal tract in WT (dashed arrow). (arrows - 2 branches of corticospinal visual axons). Maximum projections are shown. Scale bars: 200 μm.


( C ) Quantification of VP ( visual pruning ) index ( fluorescence intensity of pyramidal axons after vs . before the branch point ) . In WT animals after the pruning period , only a minor part of axons descends towards the pyramidal tract ( WT : P9 0 . 65 ± 0 . 09 , adults 0 . 17 ± 0 . 04 , p = 0 . 006 ) . However , in crmp2 - / - mice , corticospinal axons are still largely present , and their VP indexes are not significantly different between adult and P9 stages ( crmp2 - / - : P9 0 . 54 ± 0 . 18 , adults 0 . 62 ± 0 . 27 , p = 0 . 24 ) . Mean ± SD , * * p < 0 . 01 , 2 - way ANOVA with Bonferroni ' s multiple comparisons test . P9 : n = 3 pups / genotype , adults : n = 5 mice / genotype )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of VP (visual pruning) index (fluorescence intensity of pyramidal axons after vs. before the branch point). In WT animals after the pruning period, only a minor part of axons descends towards the pyramidal tract (WT: P9 0.65±0.09, adults 0.17±0.04, p=0.006). However, in crmp2-/- mice, corticospinal axons are still largely present, and their VP indexes are not significantly different between adult and P9 stages (crmp2-/-: P9 0.54±0.18, adults 0.62±0.27, p=0.24). Mean ± SD, **p<0.01, 2-way ANOVA with Bonferroni's multiple comparisons test. P9: n=3 pups/genotype, adults: n=5 mice/genotype)


( A ) Time - lapse imaging of DIV7 cultured hippocampal neurons before and after Semaphorin stimulation . Upper panel : Axon growth without Semaphorin stimulation . Middle panel : stimulation with Sema3A ( 0 hours ) ( 1nM , n = 669 axons for WT , 761 axons for knockout ) causes retraction of both WT and crmp2 - / - neurons . Lower panel : stimulation with Sema3F ( 5 nM , n = 602 axons for WT , 955 axons for knockout ) causes axon retraction in WT , but not in crmp2 - / - neurons . Red triangles depict growing axons , yellow retracting axons and blue asterisks indicate steady non - growing axons . Scale bars : 100 μm ( whole image field ) and 50 μm ( magnified ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time-lapse imaging of DIV7 cultured hippocampal neurons before and after Semaphorin stimulation. Upper panel: Axon growth without Semaphorin stimulation. Middle panel: stimulation with Sema3A (0 hours) (1nM, n=669 axons for WT, 761 axons for knockout) causes retraction of both WT and crmp2-/- neurons. Lower panel: stimulation with Sema3F (5 nM, n=602 axons for WT, 955 axons for knockout) causes axon retraction in WT, but not in crmp2-/- neurons. Red triangles depict growing axons, yellow retracting axons and blue asterisks indicate steady non-growing axons. Scale bars: 100 μm (whole image field) and 50 μm (magnified).


( C ) Quantification of retracting axons ( number of retracting vs . steady axons , 3 experiments ) . WT : control 13 . 4 ± 5 % , Sema3A 31 . 2 ± 6 % ( p < 0 . 001 ) , Sema3F 36 . 9 ± 8 . 7 % ( p < 0 . 001 ) ; crmp2 - / - : control 19 . 8 ± 1 . 8 % , Sema3A 28 . 5 ± 4 . 4 % ( p < 0 . 05 ) , Sema3F 22 . 4 ± 4 . 1 % ( p > 0 . 99 ) , mean ± SD are shown . * * * p < 0 . 001 , * p < 0 . 05 , 2 - way ANOVA with Bonferroni ' s multiple comparisons 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 O O O O O O O O O O O O O O O O O O

(C) Quantification of retracting axons (number of retracting vs. steady axons, 3 experiments). WT: control 13.4±5%, Sema3A 31.2±6% (p<0.001), Sema3F 36.9±8.7% (p<0.001); crmp2-/-: control 19.8±1.8%, Sema3A 28.5±4.4% (p<0.05), Sema3F 22.4±4.1% (p>0.99), mean ± SD are shown. ***p<0.001, *p<0.05, 2-way ANOVA with Bonferroni's multiple comparisons test.


( A , B ) Dendritic spine density in DiOlistically labeled DG granule cells is similar in WT and crmp2 - / - mice at P30 . In adults , however , spine density in crmp2 - / - granule cells is increased comparing to WT . Scale bars : 5 μm . ( C ) Quantification of dendritic spine density in DG granule cells in the inner molecular layer ( 50 - 100 μm away from the soma ) . WT , P30 : 12 . 5 ± 2 . 2 spines / 10 μm , adults : 9 . 95 ± 2 . 3 ; crmp2 - / - , P30 : 12 . 86 ± 2 . 1 ( p > 0 . 99 ) , adults : 12 . 88 ± 2 . 9 ( p < 0 . 001 ) , mean ± SD , * * * p < 0 . 001 , 2 - way ANOVA with Bonferroni correction . P30 : 3 animals / genotype , WT = 61 dendrites , knockout = 64 dendrites ; adults : 3 animals / genotype , WT = 41 dendrites , knockout = 37 dendrites .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dendritic spine density in DiOlistically labeled DG granule cells is similar in WT and crmp2-/- mice at P30. In adults, however, spine density in crmp2-/- granule cells is increased comparing to WT. Scale bars: 5 μm. (C) Quantification of dendritic spine density in DG granule cells in the inner molecular layer (50 - 100 μm away from the soma). WT, P30: 12.5±2.2 spines/10 μm, adults: 9.95±2.3; crmp2-/-, P30: 12.86±2.1 (p>0.99), adults: 12.88±2.9 (p<0.001), mean ± SD, ***p<0.001, 2-way ANOVA with Bonferroni correction. P30: 3 animals/genotype, WT=61 dendrites, knockout=64 dendrites; adults: 3 animals/genotype, WT=41 dendrites, knockout=37 dendrites.


( D , E ) Analysis of branching of DiOlistically labeled DG granule cell dendrites in adult WT and crmp2 - / - mice ( n = 3 animals , ≥ 25 dendrites ) . Quantification of granule cell branching by Sholl analysis showed no significant differences ( p > 0 . 99 ) . Scale bars : 50 μm , mean ± SEM , 2 - way ANOVA with Bonferroni correction .

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

(D, E) Analysis of branching of DiOlistically labeled DG granule cell dendrites in adult WT and crmp2-/- mice (n=3 animals, ≥25 dendrites). Quantification of granule cell branching by Sholl analysis showed no significant differences (p>0.99). Scale bars: 50 μm, mean ± SEM, 2-way ANOVA with Bonferroni correction.


( F ) Defects in synapse elimination in the inner molecular layer revealed by double immunostaining with PSD95 and VGluT2 antibodies ( n = 5 mice / genotype ) . GL indicates granule cell layer , IML / OML indicate inner / outer molecular layer , respectively . Density of colocalized PSD95 / VGLuT2 puncta was counted . IML : WT 109 ± 21 , crmp2 - / - 156 ± 15 ( p = 0 . 004 ) . OML : WT 281 ± 38 , crmp2 - / - 297 ± 80 ( p = 0 . 7 ) . Scale bars : 10 μm , mean ± SD , * p < 0 . 05 , t - 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

(F) Defects in synapse elimination in the inner molecular layer revealed by double immunostaining with PSD95 and VGluT2 antibodies (n=5 mice/genotype). GL indicates granule cell layer, IML/OML indicate inner/outer molecular layer, respectively. Density of colocalized PSD95/VGLuT2 puncta was counted. IML: WT 109±21, crmp2-/- 156±15 (p=0.004). OML: WT 281±38, crmp2-/- 297±80 (p=0.7). Scale bars: 10 μm, mean ± SD, *p<0.05, t-test.


( G ) Spine density in DiOlistically labeled prefrontal cortex ( PFC ) is similar in WT and crmp2 - / - mice in both juvenile and adult mice . Scale bars : 5 μm . ( H ) Quantification of dendritic spine density of PFC pyramidal neurons , basal dendrites ( WT , juvenile : 8 . 63 ± 1 . 44 spines / 10 μm , adults : 7 . 3 ± 1 . 4 ; crmp2 - / - , juvenile : 9 . 05 ± 1 . 73 , p = 0 . 97 , adults : 6 . 98 ± 1 . 53 , p > 0 . 99 ) , mean ± SD , 2 - way ANOVA with Bonferroni ' s multiple comparisons test . P25 : n = 3 , ≥ 50 dendrites ; adults : n = 3 , ≥ 50 dendrites .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Spine density in DiOlistically labeled prefrontal cortex (PFC) is similar in WT and crmp2-/- mice in both juvenile and adult mice. Scale bars: 5 μm. (H) Quantification of dendritic spine density of PFC pyramidal neurons, basal dendrites (WT, juvenile: 8.63±1.44 spines/10 μm, adults: 7.3±1.4; crmp2-/-, juvenile: 9.05±1.73, p=0.97, adults: 6.98±1.53, p>0.99), mean ± SD, 2-way ANOVA with Bonferroni's multiple comparisons test. P25: n=3, ≥50 dendrites; adults: n=3, ≥50 dendrites.


( A - B ) Ultrasonic vocalization was measured at P6 , P8 and P12 ( WT n = 14 pups , crmp2 - / - n = 13 pups ) . In crmp2 - / - mice , there is a significant decrease in the rate and duration of calls at P8 ( call rate , WT 352 ± 58 / 5 min , crmp2 - / - 164 ± 41 / 5 min , p = 0 . 015 ; call duration , WT 9 . 52 ± 2 ms , crmp2 - / - 3 . 36 ± 1 ms , p = 0 . 011 ) and P12 ( call rate , WT 46 ± 16 / 5 min crmp2 - / - 1 . 54 ± 1 . 24 / 5 min , p = 0 . 002 ; call duration , WT 0 . 68 ± 0 . 35 ms , crmp2 - / - 0 . 007 ± 0 . 006 ms , p = 0 . 002 ) . Mean ± SEM , * p < 0 . 05 , t - test ( P6 , P8 ) , Mann - Whitney test ( P12 ) . ( C ) Representative sonograms of the P8 mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ultrasonic vocalization was measured at P6, P8 and P12 (WT n=14 pups, crmp2-/- n=13 pups). In crmp2-/- mice, there is a significant decrease in the rate and duration of calls at P8 (call rate, WT 352±58/5 min, crmp2-/- 164±41/5 min, p=0.015; call duration, WT 9.52±2 ms, crmp2-/- 3.36±1 ms, p=0.011) and P12 (call rate, WT 46±16/5 min crmp2-/- 1.54±1.24/5 min, p=0.002; call duration, WT 0.68±0.35 ms, crmp2-/- 0.007±0.006 ms, p=0.002). Mean ± SEM, *p<0.05, t-test (P6, P8), Mann-Whitney test (P12). (C) Representative sonograms of the P8 mice.


( D ) 3 - chamber test ( WT n = 11 mice , crmp2 - / - n = 13 mice ) . In sociability phase , WT mice spent significantly more time with a social partner ( stranger 1 ) , unlike knockouts . ( WT - stranger 1 : 0 . 14 ± 0 . 01 , object : 0 . 07 ± 0 . 007 ( p = 0 . 0001 ) ; crmp2 - / - - stranger 1 : 0 . 07 ± 0 . 01 , object : 0 . 04 ± 0 . 006 ( p = 0 . 07 ) ) . In social novelty phase , when an object was substituted with a second social partner ( stranger 2 ) , both WTs and knockouts preferred novel mice to known mice . ( WT - stranger 1 : 0 . 03 ± 0 . 006 , stranger 2 : 0 . 09 ± 0 . 008 ( p < 0 . 0001 ) ; crmp2 - / - - stranger 1 : 0 . 02 ± 0 . 004 , stranger 2 : 0 . 05 ± 0 . 01 ( p = 0 . 02 ) ) . Mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 3-chamber test (WT n=11 mice, crmp2-/- n=13 mice). In sociability phase, WT mice spent significantly more time with a social partner (stranger 1), unlike knockouts. (WT - stranger 1: 0.14±0.01, object: 0.07±0.007 (p=0.0001); crmp2-/- - stranger 1: 0.07±0.01, object: 0.04±0.006 (p=0.07)). In social novelty phase, when an object was substituted with a second social partner (stranger 2), both WTs and knockouts preferred novel mice to known mice. (WT - stranger 1: 0.03±0.006, stranger 2: 0.09±0.008 (p<0.0001); crmp2-/- - stranger 1: 0.02±0.004, stranger 2: 0.05±0.01 (p=0.02)). Mean ± SEM, *p<0.05, ***p<0.001, t-test.


Elevated plus maze test ( n = 10 mice / genotype ) . ( E ) Total distance walked is similar in WT and crmp2 - / - ( WT 7 . 9 ± 0 . 45 m , crmp2 - / - 8 . 6 ± 0 . 5 m , p = 0 . 3 ) . ( F ) Frequency of open arm ( OA ) visits is increased in crmp2 - / - mice suggesting decreased anxiety . CA denotes closed arms , MID denotes the transition zone between arms , Total arm visits represents a sum of visits in all four arms . ( CA frequency : WT 13 . 7 ± 0 . 9 / 5 min , crmp2 - / - 10 . 5 ± 1 . 1 / 5 min , p = 0 . 04 ; OA frequency : WT 7 . 7 ± 0 . 8 / 5 min , crmp2 - / - 14 . 6 ± 1 . 7 / 5 min , p = 0 . 002 ; MID frequency : WT 20 . 6 ± 1 / 5 min , crmp2 - / - 24 . 6 ± 1 . 6 / 5 min , p = 0 . 06 mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Elevated plus maze test (n=10 mice/genotype). (E) Total distance walked is similar in WT and crmp2-/- (WT 7.9±0.45 m, crmp2-/- 8.6±0.5 m, p=0.3). (F) Frequency of open arm (OA) visits is increased in crmp2-/- mice suggesting decreased anxiety. CA denotes closed arms, MID denotes the transition zone between arms, Total arm visits represents a sum of visits in all four arms. (CA frequency: WT 13.7±0.9/5 min, crmp2-/- 10.5±1.1/5 min, p=0.04; OA frequency: WT 7.7±0.8/5 min, crmp2-/- 14.6±1.7/5 min, p=0.002; MID frequency: WT 20.6±1/5 min, crmp2-/- 24.6±1.6/5 min, p=0.06 mean ± SEM, *p<0.05, ***p<0.001, t-test.


Elevated plus maze test ( n = 10 mice / genotype ) . ( G ) duration of open arm ( OA ) visits is increased in crmp2 - / - mice suggesting decreased anxiety . CA denotes closed arms , MID denotes the transition zone between arms , Total arm visits represents a sum of visits in all four arms . Total arm visits : WT 21 . 4 ± 1 / 5 min , crmp2 - / - 25 . 1 ± 1 . 5 / 5 min , p = 0 . 07 ; CA duration : WT 177 ± 17 s . , crmp2 - / - 75 . 3 ± 12 . 6 s , p < 0 . 001 ; OA duration : WT 42 ± 12 . 6 s , crmp2 - / - 144 . 4 ± 21 s , p < 0 . 001 ; MID duration : WT 80 . 26 ± 11 . 5 s . , crmp2 - / - 80 . 17 ± 12 . 6 s . , p = 0 . 99 ) , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Elevated plus maze test (n=10 mice/genotype). (G) duration of open arm (OA) visits is increased in crmp2-/- mice suggesting decreased anxiety. CA denotes closed arms, MID denotes the transition zone between arms, Total arm visits represents a sum of visits in all four arms. Total arm visits: WT 21.4±1/5 min, crmp2-/- 25.1±1.5/5 min, p=0.07; CA duration: WT 177±17 s., crmp2-/- 75.3±12.6 s, p<0.001; OA duration: WT 42±12.6 s, crmp2-/- 144.4±21 s, p<0.001; MID duration: WT 80.26±11.5 s., crmp2-/- 80.17±12.6 s., p=0.99), mean ± SEM, *p<0.05, ***p<0.001, t-test.


( H , I ) Y maze test ( WT n = 9 mice , crmp2 - / - n = 8 mice ) . Decreased alternations between arms of the maze indicates impaired working memory ( number of visits , WT : 23 . 1 ± 1 . 9 , crmp2 - / - 30 . 4 ± 3 . 8 , p = 0 . 12 ; alternations , WT 68 . 8 ± 2 . 5 % , crmp2 - / - 57 . 3 ± 2 . 5 % , p = 0 . 006 ) , mean ± SEM , * p < 0 . 05 , t - 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

(H, I) Y maze test (WT n=9 mice, crmp2-/- n=8 mice). Decreased alternations between arms of the maze indicates impaired working memory (number of visits, WT: 23.1±1.9, crmp2-/- 30.4±3.8, p=0.12; alternations, WT 68.8±2.5%, crmp2-/- 57.3±2.5%, p=0.006), mean ± SEM, *p<0.05, t-test.


A , B Core histones were prepared from NSPCs treated with various concentrations of crotonate ( A ) and 1 μM MS - 275 ( B ) for 24 h and subjected to western blotting analysis using anti - Kcr antibody , and the experiments were repeated three times .

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

A, B Core histones were prepared from NSPCs treated with various concentrations of crotonate (A) and 1 μM MS-275 (B) for 24 h and subjected to western blotting analysis using anti-Kcr antibody, and the experiments were repeated three times.


C NSPCs were treated with 20 μM C646 , 10 mM acetate ( Ac ) , 20 μM C646 and 10 mM acetate ( Ac + C646 ) , 10 mM crotonate ( Cr ) , and 20 μM C646 and 10 mM crotonate ( Cr + C646 ) for 24 h , then core histones were extracted and subjected to western blotting analysis using anti - Kcr antibody , the experiments were repeated three times .

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

C NSPCs were treated with 20 μM C646, 10 mM acetate (Ac), 20 μM C646 and 10 mM acetate (Ac+C646), 10 mM crotonate (Cr), and 20 μM C646 and 10 mM crotonate (Cr+C646) for 24 h, then core histones were extracted and subjected to western blotting analysis using anti-Kcr antibody, the experiments were repeated three times.


E Quantification analysis of changes in histone Kcr levels in NSPCs treated with DMSO ( Control ) , 5 μM triptolide ( FP ) , 5 μM flavopiridol and 10 mM crotonate ( FP + CR ) , and 10 mM crotonate ( CR ) for 1 h . Data are presented as mean ± SEM of three independent biological replicates , n = 3 . One - way ANOVA with post hoc Tukey ' s test was used to analyze statistical significance ; NS : no significance , P < 0 . 01 , P < 0 . 001 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification analysis of changes in histone Kcr levels in NSPCs treated with DMSO (Control), 5 μM triptolide (FP), 5 μM flavopiridol and 10 mM crotonate (FP+CR), and 10 mM crotonate (CR) for 1 h. Data are presented as mean ± SEM of three independent biological replicates, n = 3. One-way ANOVA with post hoc Tukey's test was used to analyze statistical significance; NS: no significance, P < 0.01, P < 0.001.


F Co - IP analysis indicating RNAP2 binding in H3K9cr enriched regions in NSPCs . H3 was used as a loading control for the input nuclear proteins .

O O O O 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 Co-IP analysis indicating RNAP2 binding in H3K9cr enriched regions in NSPCs. H3 was used as a loading control for the input nuclear proteins.


G Quantification analysis of changes in chromatin openness at Tgfb1 and Notum promoter regions by FAIRE - qPCR among different groups of NSPCs . Data are presented as mean ± SEM of three independent biological replicates , n = 3 . Two - tailed unpaired Student ' s t - test was used to analyze statistical significance ; P < 0 . 05 , 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 O O O O O O O O O O O O O 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 Quantification analysis of changes in chromatin openness at Tgfb1 and Notum promoter regions by FAIRE-qPCR among different groups of NSPCs. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05, P < 0.01.


A , B Representative images ( A ) and quantification analysis ( B ) of changes in cell proliferation ability measured by BrdU incorporation rate among different groups of NSPCs . Scale bar , 50 μm . Data are presented as mean ± SEM of three independent biological replicates , n = 3 . Two - tailed unpaired Student ' s t - test was used to analyze statistical significance ; 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 O O O O O O O O O O O O O O O O O O O O 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 Representative images (A) and quantification analysis (B) of changes in cell proliferation ability measured by BrdU incorporation rate among different groups of NSPCs. Scale bar, 50 μm. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.01.


C , D Representative images ( C ) and quantification analysis ( D ) of changes in neuronal differentiation potential defined by TUJ1 positivity among different groups of NSPCs . Scale bar , 20 μm . Data are presented as mean ± SEM of three independent biological replicates , n = 4 . Two - tailed unpaired Student ' s t - test was used to analyze statistical significance ; P < 0 . 001 .

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C, D Representative images (C) and quantification analysis (D) of changes in neuronal differentiation potential defined by TUJ1 positivity among different groups of NSPCs. Scale bar, 20 μm. Data are presented as mean ± SEM of three independent biological replicates, n = 4. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.001.


I , J Quantification analysis of expression changes of Mir - 203 ( I ) and Bmi1 ( J ) by qRT - PCR among different groups of NSPCs . Data are presented as mean ± SEM of three independent biological replicates , n = 3 . Two - tailed unpaired Student ' s t - test was used to analyze statistical significance ; P < 0 . 05 , P < 0 . 01 .

I, J Quantification analysis of expression changes of Mir-203 (I) and Bmi1 (J) by qRT-PCR among different groups of NSPCs. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05, P < 0.01.


K Representative blots ( top panel ) and quantification analysis ( bottom panel ) of changes in BMI1 protein levels among different groups of NSPCs . Data are presented as mean ± SEM of three independent biological replicates , n = 3 . Two - tailed unpaired Student ' s t - test was used to analyze statistical significance ; P < 0 . 05 .

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K Representative blots (top panel) and quantification analysis (bottom panel) of changes in BMI1 protein levels among different groups of NSPCs. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05.


A , B Core histones were prepared from E13 . 5 forebrain explants treated with various concentrations of crotonate for 3 h and subjected to western blotting analysis using anti - H3K9cr ( A ) and anti - Kcr ( B ) antibodies . Naïve : isolated forebrains without subsequent culture and treatment . C , D Quantification analysis of changes in H3K9cr ( C ) and H3 - Kcr ( D ) levels by western blotting among different groups of explants . Data are presented as mean ± SEM of three independent biological replicates , n = 3 . Two - tailed unpaired Student ' s t - test was used to analyze statistical significance ; NS : no significance , P < 0 . 01 , P < 0 . 001 .

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

A, B Core histones were prepared from E13.5 forebrain explants treated with various concentrations of crotonate for 3 h and subjected to western blotting analysis using anti-H3K9cr (A) and anti-Kcr (B) antibodies. Naïve: isolated forebrains without subsequent culture and treatment. C, D Quantification analysis of changes in H3K9cr (C) and H3-Kcr (D) levels by western blotting among different groups of explants. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; NS: no significance, P < 0.01, P < 0.001.


A , B Core histones were prepared from E13 . 5 forebrain explants treated with various concentrations of crotonate for 3 h and subjected to western blotting analysis using anti - H3K9cr ( A ) and anti - Kcr ( B ) antibodies . Naïve : isolated forebrains without subsequent culture and treatment .

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

A, B Core histones were prepared from E13.5 forebrain explants treated with various concentrations of crotonate for 3 h and subjected to western blotting analysis using anti-H3K9cr (A) and anti-Kcr (B) antibodies. Naïve: isolated forebrains without subsequent culture and treatment.


G , H Quantitative analysis of changes in H3K9cr ( G , left panel of H ) and H3K14la ( right panel of H ) levels by western blotting among different groups of the embryonic forebrain . Data are presented as mean ± SEM of three independent biological replicates , n = 3 . Two - tailed unpaired Student ' s t - test was used to analyze statistical significance ; NS : no significance , P < 0 . 05 , P < 0 . 001 .

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G, H Quantitative analysis of changes in H3K9cr (G, left panel of H) and H3K14la (right panel of H) levels by western blotting among different groups of the embryonic forebrain. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; NS: no significance, P < 0.05, P <0.001.


A 3 - D reconstructions of propidium iodide ( PI ) - stained cell walls ( red ) and BCECF - stained vacuoles ( green ) of epidermal atrichoblasts in the early and late meristem and in the early and late elongation zone . Scale bars : 5 µm .

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A 3-D reconstructions of propidium iodide (PI)-stained cell walls (red) and BCECF-stained vacuoles (green) of epidermal atrichoblasts in the early and late meristem and in the early and late elongation zone. Scale bars: 5 µm.


B Boxplots showing vacuolar occupancy of cells in the defined zones ( n = 7 - 11 ) . Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

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B Boxplots showing vacuolar occupancy of cells in the defined zones (n=7-11). Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


c ) Graph depicts absolute values of cell volume and vacuolar volume in the depicted root zones ( data points display mean , error bars depict s . e . m . , n = 7 - 11 ) . " cytosol calc . " is approximated as the difference between cell volume and vacuolar volume .

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c) Graph depicts absolute values of cell volume and vacuolar volume in the depicted root zones (data points display mean, error bars depict s.e.m., n=7-11). "cytosol calc." is approximated as the difference between cell volume and vacuolar volume.


A Representative images and quantification of vacuolar morphology of late meristematic cells . PI ( green ) and pUBQ10 : : VAMP711 ( yellow ) depict cell wall and tonoplast , respectively . Seedlings were treated with DMSO ( solvent control ) or 5 µM FC ( Fusicoccin ) for 2 . 5 h in liquid medium ( n = 24 ) . Mann Whitney U test ( * * * p < 0 . 001 ) . Data information : Scale bars : 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative images and quantification of vacuolar morphology of late meristematic cells. PI (green) and pUBQ10::VAMP711 (yellow) depict cell wall and tonoplast, respectively. Seedlings were treated with DMSO (solvent control) or 5 µM FC (Fusicoccin) for 2.5 h in liquid medium (n=24). Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.


B Left : 3 - D reconstructions of PI - stained cell wall ( red ) and BCECF - stained vacuole ( green ) of late meristematic cells . Right : Boxplot depicts vacuolar occupancy of the cell treated with the solvent DMSO or 5 µM FC for 2 . 5 h in liquid medium ( n = 11 ) . Student ´ s t - test ( * p < 0 . 05 ) . Data information : Boxplots : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

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

B Left: 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Right: Boxplot depicts vacuolar occupancy of the cell treated with the solvent DMSO or 5 µM FC for 2.5 h in liquid medium (n = 11). Student´s t-test (*p < 0.05). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


C Cell wall and vacuolar membrane was visualized with PI ( green ) and MDY - 64 ( yellow ) . pER8 : : GFP - SAUR19 seedlings were treated with DMSO ( n = 60 ) or 10 µM β - estradiol ( n = 56 ) for 6 h in liquid medium . Mann Whitney U test ( * * * p < 0 . 001 ) . Data information : Scale bars : 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 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

C Cell wall and vacuolar membrane was visualized with PI (green) and MDY-64 (yellow). pER8::GFP-SAUR19 seedlings were treated with DMSO (n=60) or 10 µM β-estradiol (n=56) for 6 h in liquid medium. Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.


D Left : 3 - D cell reconstructions of PI - stained cell wall ( red ) and BCECF - stained vacuole ( green ) of late meristematic cells in pER8 : : GFP - SAUR19 lines . Right : Boxplot depicts vacuolar occupancy of the cell . Seedlings were treated with the solvent control DMSO ( n = 11 ) or 10 µM β - estradiol ( n = 8 ) for 6 h in liquid medium . Student ´ s t - test ( * * p < 0 . 01 ) . Data information : Boxplots : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

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

D Left: 3-D cell reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells in pER8::GFP-SAUR19 lines. Right: Boxplot depicts vacuolar occupancy of the cell. Seedlings were treated with the solvent control DMSO (n=11) or 10 µM β-estradiol (n=8) for 6 h in liquid medium. Student´s t-test (**p < 0.01). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


E Cell wall and vacuolar membrane was visualized with PI ( green ) and pUBQ10 : : VAMP711 ( E ) ( yellow ) . Col - 0 wild type seedlings were treated for 3 h in liquid medium adjusted to pH 5 . 7 ( n = 44 ) or pH 4 . 5 ( n = 40 ) . Mann Whitney U test ( * * * p < 0 . 001 ) . Data information : Scale bars : 5 µm .

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E Cell wall and vacuolar membrane was visualized with PI (green) and pUBQ10::VAMP711 (E) (yellow). Col-0 wild type seedlings were treated for 3 h in liquid medium adjusted to pH 5.7 (n=44) or pH 4.5 (n=40). Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.


F Left : 3 - D reconstructions of PI - stained cell wall ( red ) and BCECF - stained vacuole ( green ) of late meristematic cells . Right : Boxplot depicts vacuolar occupancy of cell . Seedlings were treated for 3 h in liquid medium adjusted to pH 5 . 7 or pH 4 . 5 ( n = 11 ) . Student ´ s t - test ( * * * p < 0 . 001 ) . Data information : Boxplots : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Left: 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Right: Boxplot depicts vacuolar occupancy of cell. Seedlings were treated for 3 h in liquid medium adjusted to pH 5.7 or pH 4.5 (n=11). Student´s t-test (***p < 0.001). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


A Representative images and quantification of vacuolar morphology of late meristematic cells . PI ( green ) and MDY - 64 ( yellow ) staining depict cell wall and vacuolar membrane , respectively . Seedlings were treated with DMSO solvent control or 50 µM EGCG for 22 h on solid medium ( n = 40 ) . Mann Whitney U test ( * * * p < 0 . 001 ) . Data information : Scale bars : 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 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

A Representative images and quantification of vacuolar morphology of late meristematic cells. PI (green) and MDY-64 (yellow) staining depict cell wall and vacuolar membrane, respectively. Seedlings were treated with DMSO solvent control or 50 µM EGCG for 22 h on solid medium (n=40). Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.


B 3 - D reconstructions of PI - stained cell wall ( red ) and BCECF - stained vacuole ( green ) of late meristematic cells . Boxplot depicts vacuolar occupancy of the cell . Seedlings were treated with DMSO ( n = 16 ) or 50 µM EGCG ( n = 15 ) for 22 h on solid medium . Student ´ s t - test ( * * p < 0 . 01 ) . Data information : Boxplots : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

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

B 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Boxplot depicts vacuolar occupancy of the cell. Seedlings were treated with DMSO (n=16) or 50 µM EGCG (n=15) for 22 h on solid medium. Student´s t-test (**p < 0.01). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


C Representative images and quantification of vacuolar morphology of late meristematic cells . PI ( green ) and MDY - 64 ( yellow ) staining depict cell wall and vacuolar membrane , respectively . Seedling roots were grown on the surface ( n = 28 ) or into the matrix ( n = 28 ) of 2 % agar - containing solid medium . Mann Whitney U test ( * * * p < 0 . 001 ) . Data information : Scale bars : 5 µm .

C Representative images and quantification of vacuolar morphology of late meristematic cells. PI (green) and MDY-64 (yellow) staining depict cell wall and vacuolar membrane, respectively. Seedling roots were grown on the surface (n=28) or into the matrix (n=28) of 2 % agar-containing solid medium. Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.


D 3 - D reconstructions of PI - stained cell wall ( red ) and BCECF - stained vacuole ( green ) of late meristematic cells . Boxplot depicts vacuolar occupancy of surface ( n = 10 ) and into the medium ( n = 11 ) grown seedling roots . Student ´ s t - test ( * * p < 0 . 01 ) . Data information : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Boxplot depicts vacuolar occupancy of surface (n=10) and into the medium (n=11) grown seedling roots. Student´s t-test (**p < 0.01). Data information: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells . PI ( green ) and MDY - 64 ( yellow ) staining depicts cell wall and vacuolar membrane , respectively . ( A ) Vacuolar morphology of Col - 0 ( n = 64 ) and fer - 4 ( n = 60 ) . Mann Whitney U test ( * * * p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (A) Vacuolar morphology of Col-0 (n=64) and fer-4 (n=60). Mann Whitney U test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells . ( B ) 3 - D reconstructions of PI - stained cell wall ( red ) and BCECF - stained vacuole ( green ) . Boxplot depicts vacuolar occupancy of Col - 0 control ( n = 12 ) and fer - 4 ( n = 10 ) mutant cells . Mann Whitney U test ( * * p < 0 . 01 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. (B) 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green). Boxplot depicts vacuolar occupancy of Col-0 control (n=12) and fer-4 (n=10) mutant cells. Mann Whitney U test (**p < 0.01). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells . PI ( green ) and MDY - 64 ( yellow ) staining depicts cell wall and vacuolar membrane , respectively . ( C ) Col - 0 ( n = 52 ) and fer - 4 ( n = 44 ) seedlings were treated with solvent DMSO or 50 µM EGCG for 22 h on solid medium . Kruskal Wallis test followed by Dunn ´ s multiple comparison ( b : p < 0 . 05 , c : p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (C) Col-0 (n=52) and fer-4 (n=44) seedlings were treated with solvent DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells . PI ( green ) and MDY - 64 ( yellow ) staining depicts cell wall and vacuolar membrane , respectively . ( D ) Col - 0 ( n = 28 ) and fer - 4 ( n = 28 ) mutant seedling roots were grown on the surface or into the matrix of 2 % agar - containing solid medium . Kruskal Wallis test followed by Dunn ´ s multiple comparison ( b : p < 0 . 05 , c : p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

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Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (D) Col-0 (n=28) and fer-4 (n=28) mutant seedling roots were grown on the surface or into the matrix of 2 % agar-containing solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells . PI ( green ) and MDY - 64 ( yellow ) staining depicts cell wall and vacuolar membrane , respectively . ( A ) Vacuolar morphology of Col - 0 control ( n = 52 ) and lrx3 / 4 / 5 triple mutants ( n = 48 ) . Mann Whitney U test ( * * * p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (A) Vacuolar morphology of Col-0 control (n=52) and lrx3/4/5 triple mutants (n=48). Mann Whitney U test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells . ( B ) 3 - D reconstructions of PI - stained cell wall ( red ) and BCECF - stained vacuole ( green ) of late meristematic cells . Boxplot depicts vacuolar occupancy of the cell in Col - 0 control ( n = 11 ) and lrx3 / 4 / 5 ( n = 10 ) . Student ´ s t - test ( * * * p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th and 75th percentile , horizontal line represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. (B) 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Boxplot depicts vacuolar occupancy of the cell in Col-0 control (n=11) and lrx3/4/5 (n=10). Student´s t-test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.