Control ( top ) centrinone - treated ( bottom ) RPE - 1 double stained for either AKAP450 , CDK5Rap2 , PCNT or CEP192 ( shown in green ) giantin ( red ) as a Golgi marker . was counterstained with DAPI is shown in blue . Single channel ( left , green ) merged ( right ) are shown in each case . arrows indicate centrosome ( CTR ) .

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Control (top) and centrinone-treated (bottom) RPE-1 cells double stained for either AKAP450, CDK5Rap2, PCNT or CEP192 (all shown in green) and giantin (red) as a Golgi marker. DNA was counterstained with DAPI and is shown in blue. Single channel (left, green) and merged images (right) are shown in each case. White arrows indicate the centrosome (CTR).


IF of control centrinone - treated RPE - 1 treated with NZ to induce GA fragmentation double stained with either anti - AKAP450 , anti - CDK5Rap2 or anti - PCNT ( shown in green ) anti - giantin antibodies ( ) . Enlarged views of boxed areas are presented at bottom with or without signal of Golgi marker . yellow dashed indicates contour of nucleus . Arrowheads point to accumulation of respective in Golgi surfaces .

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IF images of control and centrinone-treated RPE-1 cells treated with NZ to induce GA fragmentation and double stained with either anti-AKAP450, anti-CDK5Rap2 or anti-PCNT (all shown in green) and anti-giantin antibodies (white). Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker. The yellow dashed line indicates the contour of the nucleus. Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces.


Box - - whisker plots showing of association between indicated Golgi elements Top bottom ends of boxes represent 75th 25th percentiles , whiskers represent 90th 10th percentiles . median is depicted with a solid . Golgi elements from at least 14 were delineated ( > 500 elements / experiment ) of each spot associated with each Golgi element was measured . Data were collected from two independent experiments normalized to WT mean .

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Box-and-whisker plots showing quantification of association between the indicated protein and Golgi elements Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Individual Golgi elements from at least 14 cells were delineated (>500 elements/experiment) and the intensity of each protein spot associated with each Golgi element was measured. Data were collected from two independent experiments and normalized to WT mean.


MT - regrowth experiment after NZ treatment in centrinone - treated . High magnification at bottom are representative of a triple with α - tubulin ( green ) , GM130 ( blue ) indicated ( red ) show MTs growing from surface of GA . Double labelling without α - tubulin are also shown ( top ) to allow better visualization of PCM - spots on Golgi elements acting as MT nucleation sites ( arrowheads ) .

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MT-regrowth experiment after NZ treatment in centrinone-treated cells. High magnification images at the bottom are representative of a triple staining with α-tubulin (green), GM130 (blue) and the indicated protein (red) and show MTs growing from the surface of the GA. Double labelling without α-tubulin are also shown (top) to allow better visualization of PCM-protein spots on Golgi elements acting as MT nucleation sites (arrowheads).


Centrinone - treated pcnt KO , c5rap2 KO akap KO , were incubated with NZ for 3h , fixed double labelled for GM130 either CDK5Rap2 or PCNT as indicated ( n = 3 ) .

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Centrinone-treated pcnt KO, c5rap2 KO and akap KO cells, were incubated with NZ for 3h, fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated (n=3).


MT regrowth in centrinone - treated WT , pcnt KO akap KO RPE - 1 stained with antibodies to EB1 giantin . At right , of EB1 at Golgi membranes in WT pcnt KO treated with centrinone , as a measure of MT - nucleation from GA . Data were collected from two independent experiments normalized to WT mean .

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MT regrowth assay in centrinone-treated WT, pcnt KO and akap KO RPE-1 cells stained with antibodies to EB1 and giantin. At right, quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone, as a measure of MT-nucleation from the GA. Data were collected from two independent experiments and normalized to WT mean.


Representative MT network of a centrosome - less akap KO .

Representative MT network of a centrosome-less akap KO cell.


MT - regrowth experiment after NZ treatment in centrinone - treated akap KO . A representative confocal image of a double labelling for EB1 ( green ) giantin ( blue ) is shown . High magnification at right shows that growing MT asters are not associated to Golgi fragments .

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MT-regrowth experiment after NZ treatment in centrinone-treated akap KO cells. A representative confocal image of a double labelling for EB1 (green) and giantin (blue) is shown. High magnification at right shows that growing MT asters are not associated to Golgi fragments.


Centrinone - treated akap KO were subjected to a MT regrowth double stained with indicated antibodies . High magnification of boxed areas are shown as merge ( top ) or green or red labelling ( middle bottom panels ) .

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Centrinone-treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies. High magnification images of the boxed areas are shown as merge (top) or individual green or red labelling (middle and bottom panels).


lacking AKAP450 were treated with centrinone in order to induce formation of then transfected with siRNAs specific for CDK5Rap2 ( top panels ) or PCNT ( bottom panels ) . After 3 - min MT regrowth , were fixed stained with indicated antibodies .

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Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 (top panels) or PCNT (bottom panels). After 3-min MT regrowth, cells were fixed and stained with the indicated antibodies.


MT regrowth experiments in centrinone - treated akap KO transfected with either scramble or PCNT siRNAs . After a 3h NZ treatment , MTs were allowed to polymerize , at indicated time points after washout , were fixed labelled for α - tubulin ( green ) , PCNT ( red ) CAP350 ( blue ) .

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MT regrowth experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After a 3h NZ treatment, MTs were allowed to polymerize and, at the indicated time points after washout, cells were fixed and labelled for α-tubulin (green), PCNT (red) and CAP350 (blue).


MT repolymerization experiments in centrinone - treated akap KO transfected with either scramble or PCNT siRNAs . After NZ treatment removal , were incubated for 10 min on ice then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin . were finally fixed triple stained with EB1 ( green ) , PCNT ( red ) CAP350 ( blue ) antibodies . Enlarged views of boxed areas are presented at right . Scale bars , 5 μm .

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MT repolymerization experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After NZ treatment and removal, cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin. Cells were finally fixed and triple stained with EB1 (green), PCNT (red) and CAP350 (blue) antibodies. Enlarged views of the boxed areas are presented at right. Scale bars, 5 μm.


Confocal showing MT network in WT , akap KO , c5rap2 KO , pcnt KO pc - ak - 2KO RPE - 1 fixed stained with antibodies against α - tubulin ( left ) or EB1 ( right ) . red yellow lines indicate contour of .

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Confocal images showing MT network in WT, akap KO, c5rap2 KO, pcnt KO and pc-ak-2KO RPE-1 cells fixed and stained with antibodies against α-tubulin (left) or EB1 (right). The red and yellow lines indicate the contour of cells.


Confocal showing MT network in centrinone - treated WT , akap KO , c5rap2 KO pcnt KO backgrounds . containing extra centrosomes induced by centrinone washout were also quantified ( bottom panels ) .

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Confocal images showing MT network in centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO backgrounds. Cells containing extra centrosomes induced by centrinone washout were also quantified (bottom panels).


of α - tubulin ( as a measure of MT mass polymer , C ) , in control centrinone - treated WT , akap KO , c5rap2 KO pcnt KO . Non - centrinone treated double KO centrinone - treated WT after washout were also included . Scatter plots show each data point horizontal black lines bars represent mean .

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Quantification of α-tubulin fluorescence intensity (as a measure of MT mass polymer, C), in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


of number of EB1 comets per ( D ) in control centrinone - treated WT , akap KO , c5rap2 KO pcnt KO . Non - centrinone treated double KO centrinone - treated WT after washout were also included . Scatter plots show each data point horizontal black lines bars represent mean .

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Quantification of number of EB1 comets per cell (D) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


of α , ( E ) in control centrinone - treated WT , akap KO , c5rap2 KO pcnt KO . Non - centrinone treated double KO centrinone - treated WT after washout were also included . Scatter plots show each data point horizontal black lines bars represent mean .

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Quantification of α , cell area (E) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


of MT density ( MT mass polymer / ) ( F ) in control centrinone - treated WT , akap KO , c5rap2 KO pcnt KO . Non - centrinone treated double KO centrinone - treated WT after washout were also included . Scatter plots show each data point horizontal black lines bars represent mean .

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Quantification of MT density (MT mass polymer/cell area) (F) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


A Bisulfite methylation sequencing of Athila6ATE transcriptional start site ( TSS ) in genotypes with silenced TEs ( left of dashed ) or transcriptionally active TEs ( right of dashed ) . Methylation in CG ( red ) , CHG ( blue ) CHH ( green ) contexts are shown ( H = C , T , or A ) . Error bars represent Wilson score interval 95 % confidence limits . For multiple statistical , one ‐ way ANOVA was performed using Tukey ' s multiple comparison . ns , not significant .

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A Bisulfite DNA methylation sequencing of the Athila6ATE transcriptional start site (TSS) in genotypes with silenced TEs (left of the dashed line) or transcriptionally active TEs (right of the dashed line). Methylation in the CG (red), CHG (blue) and CHH (green) sequence contexts are shown (H = C, T, or A). Error bars represent Wilson score interval 95% confidence limits. For multiple statistical tests, one‐way ANOVA was performed using Tukey's multiple comparison test. ns, not significant.


B reads ‐ per ‐ million ‐ ( RPM ) ‐ normalized accumulation of Athila6ATE siRNAs from TE ‐ silenced epigenomes ( left ) ddm1TE ‐ active epigenomes ( right ) . Inset graphs reflecting same values are shown for select genotypes . graphs have same y ‐ axis scale are shown for increased resolution in genotypes with low siRNA .

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B The reads‐per‐million‐(RPM)‐normalized accumulation of Athila6ATE siRNAs from TE‐silenced epigenomes (left) and ddm1TE‐active epigenomes (right). Inset line graphs reflecting the same values are shown for select genotypes. All line graphs have the same y‐axis scale and are shown for increased resolution in genotypes with low siRNA levels.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . A A representative microRNA , miR158B . location of miR158B from this locus is shown in orange .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.A A representative microRNA, miR158B. The location of miR158B from this locus is shown in orange.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . B SimpleHat2 non ‐ autonomous TE targeted by Pol IV ‐ RdDM . C RDR6 ‐ RdDM target locus TAS3a .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.B The SimpleHat2 non‐autonomous TE targeted by Pol IV‐RdDM.C The RDR6‐RdDM target locus TAS3a.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . D Athila6A consensus element in wt Col epigenome . E same Athila6A consensus element as in ( D ) upon transcriptional activation in ddm1 epigenome .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.D The Athila6A consensus element in the wt Col epigenome.E The same Athila6A consensus element as in (D) upon transcriptional activation in the ddm1 epigenome.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . F total small RNAs produced in wt Col TE ‐ silenced epigenome . G total small RNAs in ddm1 mutants .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.F The total small RNAs produced in the wt Col TE‐silenced epigenome.G The total small RNAs in ddm1 mutants.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . H Bisulfite sequencing of different regions of Athila6A element . Error bars represent Wilson score interval 95 % confidence limits . Differences in methylation status were analyzed with a two ‐ unpaired Student ' s t ‐ .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.H Bisulfite sequencing of different regions of the Athila6A element. Error bars represent Wilson score interval 95% confidence limits. Differences in the methylation status were analyzed with a two‐tailed unpaired Student's t‐test.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . I A second biological replicate of FLAG ‐ AGO6 ‐ IP no ‐ antigen control followed by deep sequencing shown for Athila6A consensus element in ddm1 mutants . transcriptional start sites of LTR TSS ‐ IR ‐ driven transcripts are shown .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.I A second biological replicate of the FLAG‐AGO6‐IP and no‐antigen control followed by deep sequencing shown for the Athila6A consensus element in ddm1 mutants. The transcriptional start sites of the LTR TSS‐ and IR‐driven transcripts are shown.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . J of first base of AGO6 ‐ enriched siRNAs from wt Col TE ‐ silenced epigenome ( left ) ddm1 TE ‐ transcriptionally active epigenome ( right ) .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.J Analysis of the first base of AGO6‐enriched siRNAs from the wt Col TE‐silenced epigenome (left) and the ddm1 TE‐transcriptionally active epigenome (right).


B H3Ac control ChIP from same used in AGO6 ‐ IP for At1g08200 constitutively expressed gene Athila6A TSS . Enrichment of H3Ac at Athila6A TSS is increased upon transcriptional activation in ddm1 mutants , while enrichment of H3Ac at gene At1g08200 is consistently high in both wt Col ddm1 . Data are represented as mean ± SD of six biological replicates for each genotype .

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B H3Ac control ChIP from the same chromatin used in the AGO6‐IP for the At1g08200 constitutively expressed gene and the Athila6A TSS. Enrichment of H3Ac at the Athila6A TSS is increased upon transcriptional activation in ddm1 mutants, while enrichment of H3Ac at the gene At1g08200 is consistently high in both wt Col and ddm1. Data are represented as mean ± SD of six biological replicates for each genotype.


C - E AGO6 ‐ ChIP qPCR of ( C ) gene At1g08200 Pol IV ‐ RdDM target SimpleHat2 , ( D ) Athila6A TSS in wt Col TE ‐ silenced epigenomes ( left ) ddm1 TE ‐ active epigenomes ( right ) ( E ) TAS3a . ChIP signals in ( D ) are normalized to ago6 or ddm1 ago6 no ‐ antigen controls . ChIP signals in ( C ) ( E ) are normalized to ago6 no ‐ antigen control . In ( C - E ) , each biological replicate data point is shown as a gray dot . data are represented as mean ± SD . For multiple statistical , one ‐ way ANOVA was performed using Tukey ' s multiple comparison .

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C-E AGO6‐ChIP qPCR analysis of (C) the gene At1g08200 and the Pol IV‐RdDM target SimpleHat2, (D) the Athila6A TSS in wt Col TE‐silenced epigenomes (left) and ddm1 TE‐active epigenomes (right) and (E) TAS3a. ChIP signals in (D) are normalized to the ago6 or ddm1 ago6 no‐antigen controls. ChIP signals in (C) and (E) are normalized to the ago6 no‐antigen control. In (C-E), each biological replicate data point is shown as a gray dot. The data are represented as mean ± SD. For multiple statistical tests, one‐way ANOVA was performed using Tukey's multiple comparison test.


F ChIP qPCR of FLAG ‐ tagged POL V in wt Col ddm1 . ChIP signals are normalized to wt Col no ‐ antigen control .

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F ChIP qPCR analysis of the FLAG‐tagged POL V protein in wt Col and ddm1. ChIP signals are normalized to the wt Col no‐antigen control.


A AGO6 enrichment depletion of 21 - 22nt siRNAs for entire genome in 100 ‐ bp tiles for both wt Col ddm1 . Tiles were categorized by annotation feature .

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A AGO6 enrichment and depletion of 21-22nt siRNAs for the entire genome in 100‐bp tiles for both wt Col and ddm1. Tiles were categorized by annotation feature.


B Of tiles in ( A ) in both wt Col ddm1 , we determined number of tiles with ≥ 2 ‐ fold CHH methylation dependent on RDR6 .

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B Of the tiles in (A) in both wt Col and ddm1, we determined the number of tiles with ≥ 2‐fold CHH methylation dependent on RDR6.


C Distribution of AGO6 enrichment across TEs ( solid lines ) genes ( dashed lines ) for different sizes of small RNAs in either wt Col TE ‐ silenced epigenome or ddm1 TE ‐ transcriptionally active epigenome . Dashed lines have same color codes as solid lines . ' Start ' ' Stop ' represent edges of TE transcriptional start stop sites of genes .

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C Distribution of AGO6 enrichment across TEs (solid lines) and genes (dashed lines) for different sizes of small RNAs in either the wt Col TE‐silenced epigenome or the ddm1 TE‐transcriptionally active epigenome. Dashed lines have the same color codes as the solid lines. 'Start' and 'Stop' represent the edges of the TE and the transcriptional start and stop sites of the genes.


D - G Heatmaps of AGO6 enrichment of TE siRNA classes in wt Col ddm1 epigenomes . Analyzed were ( D ) position of TE on a chromosome TE , ( E ) copy number of TE , ( F ) TE type ( G ) TE family name . color of TE family name in ( G ) corresponds to TE type in ( F ) .

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D-G Heatmaps of AGO6 enrichment of TE siRNA size classes in wt Col and ddm1 epigenomes. Analyzed were (D) the position of the TE on a chromosome and TE size, (E) the copy number of the TE, (F) the TE type and (G) the TE family name. The color of the TE family name in (G) corresponds to the TE type in (F).


H Bisulfite sequencing of TE targets . Error bars represent Wilson score interval 95 % confidence limits .

H Bisulfite sequencing of TE targets. Error bars represent Wilson score interval 95% confidence limits.


A Bisulfite sequencing of Athila6A TSS in juvenile leaf tissue .

A Bisulfite sequencing of the Athila6A TSS in juvenile leaf tissue.


B qRT - PCR of Athila6A gag / pol steady ‐ state mRNA transcript in inflorescence leaf tissue .

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B qRT-PCR analysis of Athila6A gag/pol steady‐state mRNA transcript levels in inflorescence and leaf tissue.


C qRT - PCR of AGO6 AGO4 mRNA transcript in inflorescence juvenile leaf tissue .

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C qRT-PCR analysis of AGO6 and AGO4 mRNA transcript levels in inflorescence and juvenile leaf tissue.


D Western blot of wt Col leaf inflorescence . arrow denotes AGO6 ; higher band is a previously observed cross ‐ reactive band ( see also Supplementary Fig S3A ) .

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D Western blot analysis of wt Col leaf and inflorescence. The arrow denotes the AGO6 protein; the higher band is a previously observed cross‐reactive band (see also Supplementary Fig S3A).


E , F Side view of an inflorescence with ( E ) or without ( F ) AGO6 ‐ GFP transgene . G , H Top ‐ down view of an inflorescence with ( G ) or without ( H ) AGO6 ‐ GFP transgene .

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E, F Side view of an inflorescence with (E) or without (F) the AGO6‐GFP transgene.G, H Top‐down view of an inflorescence with (G) or without (H) the AGO6‐GFP transgene.


J fractionation of inflorescence . cytoplasm ‐ enriched represents 2 . 5 % of total ‐ enriched . H3Ac is a marker , while PEPC is a .

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J Nuclear and cytoplasmic fractionation of inflorescence protein. The cytoplasm‐enriched protein represents 2.5% of the total nuclear‐enriched protein. H3Ac is a nuclear protein marker, while PEPC is a cytoplasmic protein.


K CHH methylation of RDR6 ‐ RdDM targets TAS3a Athila6A TSS in whole ‐ inflorescence ‐ tissue samples compared to dissected young buds corresponding to floral stages 6 - 8 .

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K CHH methylation levels of the RDR6‐RdDM targets TAS3a and Athila6A TSS in whole‐inflorescence‐tissue samples compared to dissected young buds corresponding to floral stages 6-8.


( A ) Pearson correlation between ZEB1 MITF mRNA expression in 61 melanomacell lines available through CCLE .

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(A) Pearson correlation between ZEB1 and MITF mRNA expression in 61 melanomacell lines available through the CCLE.


( B ) ZEB1 , ZEB2 , TWIST1 MITF expression in a panel of BRAFV600 - mutated melanomacells assessed by Western blot . GLO C - 09 . 10cells are - derived short - term cultures . Actin was used as a loading control .

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(B) ZEB1, ZEB2, TWIST1 and MITF expression in a panel of BRAFV600-mutated melanomacells assessed by Western blot. GLO and C-09.10cells are patient-derived short-term cultures. Actin was used as a loading control.


( C ) Quantitative PCR analyses of ZEB1 , ZEB2 , TWIST1and MITF in same panel of lines . mRNA expression are represented relatively to C - 09 . 10 , in which were arbitrarily fixed at 1 ( n = 3 , mean ± SD ) . dotted separates ZEB1high ( left ) ZEB1low ( right ) lines .

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(C) Quantitative PCR analyses of ZEB1, ZEB2, TWIST1and MITF in the same panel of cell lines. mRNA expression levels are represented relatively to C-09.10 cells, in which the levels were arbitrarily fixed at 1 (n=3, mean ± SD). The dotted line separates ZEB1high (left) and ZEB1low (right) cell lines.


( D ) Tukey box plot of ZEB1 , ZEB2 , TWIST1 , MITF mRNA expression according to IC50 of drug ( µM ) administered ( BRAFi / MEKi ) , in melanoma lines from CCLE ( n = 28 ) ( Student ' s t - ) . High ZEB1 , low ZEB2 , low MITF expression were correlated with BRAFi ( PLX4720 ) MEKi ( AZD6244 ) resistance . PLX4720 is an analog of PLX4032 .

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(D) Tukey box plot of ZEB1, ZEB2, TWIST1, and MITF mRNA expression according to the IC50 of the drug (µM) administered (BRAFi/MEKi), in melanoma cell lines from the CCLE (n=28) (Student's t-Test). High ZEB1, low ZEB2, and low MITF expression levels were correlated with BRAFi (PLX4720) and MEKi (AZD6244) resistance. PLX4720 is an analog of PLX4032.


( E ) IC50 values of PLX4032 ( µM ) in panel of BRAFV600 melanoma as determined by ATP ( n = 3 , mean ± SD ) . For SKMEL24 WM793 , IC50 was > 8µM .

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(E) IC50 values of PLX4032 (µM) in the panel of BRAFV600 melanoma cells as determined by ATP assay (n=3, mean ± SD). For SKMEL24 and WM793, IC50 was >8µM.


( A ) Pearson correlation between ZEB1 MITF mRNA expression in 467 melanomatumors from TCGA data set .

(A) Pearson correlation between ZEB1 and MITF mRNA expression levels in 467 melanomatumors from TCGA data set.


( B ) Representative pictures of ZEB1 MITF immunostaining in melanomas . Scale bar = 40 µm . aberrant activation of ZEB1 in melanomas is correlated with a MITFlow phenotype .

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(B) Representative pictures of ZEB1 and MITF immunostaining in primary melanomas. Scale bar = 40 µm. The aberrant activation of ZEB1 in melanomas is correlated with a MITFlow phenotype.


( C ) Representative pictures of ZEB1 immunostaining in BRAFV600 tumors from , classified in ZEB1 high , int low subgroups , on of ZEB1 on of positive for ZEB1 . Scale bar = 80 µm . ( D ) Pie charts representing distribution of ZEB1 alone ( upper part ) , or ZEB1 TWIST1 ( lower part ) immunohistochemistry in tumors according to their initial response to vemurafenib + / - cobimetinib treatment . ZEB1 + / - TWIST1 are higher in MAPKi resistant melanomas ( initial non - responders ) compared to tumors that initially respond to treatment ( n = 70 , Fisher ' s exact ) .

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(C) Representative pictures of ZEB1 immunostaining in BRAFV600 tumors from patients, classified in ZEB1 high, int and low subgroups, based on the intensity of ZEB1 staining and on the percentage of cells positive for ZEB1. Scale bar = 80 µm. (D) Pie charts representing the distribution of ZEB1 alone (upper part), or ZEB1 and TWIST1 (lower part) immunohistochemistry staining in tumors according to their initial response to vemurafenib +/- cobimetinib treatment. ZEB1 +/- TWIST1 levels are higher in MAPKi primary resistant melanomas (initial non-responders) compared to tumors that initially respond to treatment (n=70, Fisher's exact test).


( E ) Representative pictures of ZEB1 MITF immunostainings , before after vemurafenib treatment , in from 1 , exhibiting resistance to BRAFi . Scale bar = 80 µm . Right : Magnification of MITFhigh MITFlow clones in resistant under treatment . Arrows indicate endothelial stromal that also show positive for ZEB1 , besides .

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(E) Representative pictures of ZEB1 and MITF immunostainings, before and after vemurafenib treatment, in the tumor from patient 1, exhibiting primary resistance to BRAFi. Scale bar = 80 µm. Right: Magnification of MITFhigh and MITFlow clones in the resistant tumor under treatment. Arrows indicate endothelial and stromal cells that also show positive staining for ZEB1, besides tumor cells.


( A ) PLX4032 IC50 ( µM ) of sensitive A375 SKMEL5 resistant ( A375 - R , SKMEL5 - R ) lines , as well as of GOKA ESPcells , two BRAFi - resistant - derived short - term cultures , as determined by ATP ( n = 3 , mean ± SD ) . For ESP , IC50 was > 8µM .

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(A) PLX4032 IC50 (µM) of sensitive A375 and SKMEL5 and resistant (A375-R, SKMEL5-R) cell lines, as well as of GOKA and ESPcells, two BRAFi-resistant patient-derived short-term cultures, as determined by ATP assay (n=3, mean ± SD). For ESP, IC50 was >8µM.


( B ) Western blot analyses of ZEB1 , TWIST1 FRA1 in A375 - R SKMEL5 - Rversus parental naive , in GOKA ESP . GAPDH was used as a loading control .

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(B) Western blot analyses of ZEB1, TWIST1 and FRA1 in A375-R and SKMEL5-Rversus the parental naive cells, and in GOKA and ESP cells. GAPDH was used as a loading control.


( C ) Quantitative PCR analyses of ZEB1and MITF in A375 - R SKMEL5 - Rversus parental naive , in GOKA ESP . mRNA expression are represented as arbitrary units ( a . u ) . Statistical difference relative to sensitive A375 is shown ( n = 3 , mean ± SD , Student ' s t - ) .

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(C) Quantitative PCR analyses of ZEB1and MITF in A375-R and SKMEL5-Rversus the parental naive cells, and in GOKA and ESP cells. mRNA expression levels are represented as arbitrary units (a.u). Statistical difference relative to sensitive A375 cells is shown (n=3, mean ± SD, Student's t-test).


( D ) Representative pictures of ZEB1 MITF immunostainings in tumors from 2 , 3 4 , before after vemurafenib treatment . Scale bar = 40 µm . For ZEB1 in 4 , inset shows a magnification . Arrows point at stromal ( s ) . other positive for ZEB1 are .

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(D) Representative pictures of ZEB1 and MITF immunostainings in tumors from patients 2, 3 and 4, before and after vemurafenib treatment. Scale bar = 40 µm. For ZEB1 staining in patient 4, the inset shows a magnification. Arrows point at stromal cells (s). All other cells positive for ZEB1 are tumor cells.


( A ) A375 were infected with retroviruses expressing ZEB1 . Western blot analyses of ZEB1 p75 . GAPDH was used as a loading control .

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(A) A375 cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1 and p75. GAPDH was used as a loading control.


( B ) Quantitative PCR analyses of MITF , p75 , ABCB5 JARID1B upon ZEB1 expression . mRNA expression are represented relatively to control , in which were fixed at 1 ( mean ± SD , n = 3 , Student ' s t - ) .

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(B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 expression. mRNA expression levels are represented relatively to control cells, in which the levels were fixed at 1 (mean ± SD, n=3, Student's t-test).


( C ) Soft agar colony formation upon ZEB1 expression . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - ) .

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(C) Soft agar colony formation assay upon ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).


( D ) 2 x 106 Control or ZEB1 - overexpressing A375 were injected subcutaneously into nude mice . mean volume for 5 mice is represented ± ( Student ' s t - ) .

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(D) 2 x 106 Control or ZEB1-overexpressing A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented ± SEM (Student's t-test).


( E ) Western blot analyses of ZEB1 , MITF , P - ERK , TWIST1 in control or ZEB1 - expressing + / - 150 nM PLX4032 treatment for 24 h . GAPDH was used as a loading control .

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(E) Western blot analyses of ZEB1, MITF, P-ERK, and TWIST1 levels in control or ZEB1-expressing cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control.


( F ) FACS analyses of p75 surface expression upon ZEB1 overexpression , after 10 days treatment with or without 150 nM PLX4032 . Bar chart representing mean of p75 - high , int low from 2 independent experiments ( Fisher ' s Exact ) .

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(F) FACS analyses of p75 cell surface expression upon ZEB1 overexpression, after 10 days treatment with or without 150 nM PLX4032. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher's Exact Test).


( G ) Control or ZEB1 - overexpressing A375 were transfected with control or p75 - siRNA . p75 MITF expression were was analyzed by quantitative PCR after 48 h . mRNA expression are represented relatively to control transfected with siRNA Control , in which were fixed at 1 ( mean ± SD , n = 2 ) .

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(G) Control or ZEB1-overexpressing A375 cells were transfected with control or p75-siRNA. p75 and MITF expression levels were was analyzed by quantitative PCR after 48 h. mRNA expression levels are represented relatively to control cells transfected with siRNA Control, in which the levels were fixed at 1 (mean ± SD, n=2).


( H ) Clonogenic + / - PLX4032 ( 150 nM ) , + / - GDC - 0973 ( 5 nM ) treatment for 10 days . graphs represent mean number of colonies ( ± SD ) in 3 independent experiments ( Student ' s t - ) . ( I ) Number of of chronic exposure to PLX4032 before emergence of resistance in control or ZEB1 - expressing ( n = 3 , Student ' s t - ) .

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(H) Clonogenic assay +/- PLX4032 (150 nM), +/- GDC-0973 (5 nM) treatment for 10 days. The graphs represent the mean number of colonies (± SD) in 3 independent experiments (Student's t-test). (I) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or ZEB1-expressing cells (n=3, Student's t-test).


( A ) C - 09 . 10 short - term culture were infected with retroviruses expressing ZEB1 . Western blot analyses of ZEB1 p75 . GAPDH was used as a loading control .

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(A) C-09.10 short-term culture cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1 and p75. GAPDH was used as a loading control.


( B ) Quantitative PCR analyses of MITF p75 upon ZEB1 expression . mRNA expression are represented relatively to control ( mean ± SD , n = 3 , Student ' s t - ) .

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(B) Quantitative PCR analyses of MITF and p75 upon ZEB1 expression. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test).


( C ) Soft agar colony formation following ZEB1 expression . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - ) .

(C) Soft agar colony formation assay following ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).


( D ) FACS analyses of p75 surface expression upon ZEB1 overexpression . Bar chart representing mean of p75 - high , int , low negative from 2 independent experiments ( Fisher ' s exact ) .

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(D) FACS analyses of p75 cell surface expression upon ZEB1 overexpression. Bar chart representing the mean percentage of p75-high, int, low and negative cells from 2 independent experiments (Fisher's exact test).


( E ) Clonogenic + / - PLX4032 ( 100 nM ) treatment for 10 days . graph represents mean number of colonies in 3 independent experiments ( Student ' s t - ) .

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(E) Clonogenic assay +/- PLX4032 (100 nM) treatment for 10 days. The graph represents the mean number of colonies in 3 independent experiments (Student's t-test).


( A ) A375 were infected with retroviruses expressing a control or ZEB1 - shRNA . Western blot analyses of ZEB1 p75 upon ZEB1 knock - down . GAPDH was used as a loading control . High low exposures ( exp ) for p75 are shown .

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(A) A375 cells were infected with retroviruses expressing a control or ZEB1-shRNA. Western blot analyses of ZEB1 and p75 upon ZEB1 knock-down. GAPDH was used as a loading control. High and low exposures (exp) for p75 are shown.


( B ) Quantitative PCR analyses of MITF , p75 , ABCB5 JARID1B upon ZEB1 knock - down . mRNA expression are represented relatively to shRNA control ( mean ± SD , n = 3 , Student ' s t - ) .

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(B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 knock-down. mRNA expression levels are represented relatively to shRNA control cells (mean ± SD, n=3, Student's t-test).


( C ) FACS analyses of p75 expression upon ZEB1 knock - down . Bar chart representing mean of p75 - high , int low from 2 independent experiments ( Fisher ' s exact ) .

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(C) FACS analyses of p75 expression upon ZEB1 knock-down. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher's exact test).


( D ) Soft agar colony formation upon ZEB1 knock - down . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - ) .

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(D) Soft agar colony formation assay upon ZEB1 knock-down. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).


( E ) 2 x 106 Control or ZEB1 - shRNA A375 were injected subcutaneously into nude mice . mean volume for 5 mice is represented ( ± ) . ( Student ' s t - ) .

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(E) 2 x 106 Control or ZEB1-shRNA A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).


( F ) A375 were infected with an IPTG - inducible ZEB1 - shRNA . Left panel : Western blot analyses of ZEB1 expression ± IPTG ( 100µM ) treatment for 6 days . Actin was used as a loading control . Right panel : 2 x 106 IPTG - inducible shRNA - Control or shRNA - ZEB1A375 were injected subcutaneously into nude mice . When reached 5 mm in diameter , ZEB1 expression was silenced by adding IPTG ( 10 mM ) into drinking water for 20 days . mean volume for 5 mice is represented ( ± ) . ( Student ' s t - ) .

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(F) A375 cells were infected with an IPTG-inducible ZEB1-shRNA. Left panel: Western blot analyses of ZEB1 expression ± IPTG (100µM) treatment for 6 days. Actin was used as a loading control. Right panel: 2 x 106 IPTG-inducible shRNA-Control or shRNA-ZEB1A375 cells were injected subcutaneously into nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by adding IPTG (10 mM) into the drinking water for 20 days. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).


( G ) SKMEL5 expressing an IPTG - inducible ZEB1 - shRNA were treated with IPTG ( 100µM ) for 10 days ( + IPTG ) , then IPTG was removed ( - IPTG ) ZEB1 , MITF p75 expression were analyzed by Western blot / or quantitative PCR analyses . GAPDH was used as a loading control , mRNA expression are represented relatively to untreated , in which were fixed at 1 ( mean ± SD , n = 2 ) .

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(G) SKMEL5 cells expressing an IPTG-inducible ZEB1-shRNA were treated with IPTG (100µM) for 10 days (+IPTG), then IPTG was removed (-IPTG) and ZEB1, MITF and p75 expression levels were analyzed by Western blot and/or quantitative PCR analyses. GAPDH was used as a protein loading control, mRNA expression levels are represented relatively to untreated cells, in which the levels were fixed at 1 (mean ± SD, n=2).


( A ) Western blot analyses of ZEB1 P - ERK in shRNA - control or shRNA - ZEB1 - expressing A375 + / - 150 nM PLX4032 treatment for 24 h . GAPDH was used as a loading control .

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(A) Western blot analyses of ZEB1 and P-ERK in shRNA-control or shRNA-ZEB1-expressing A375 cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control.


( B ) Soft agar colony formation in A375 in presence or absence of PLX4032 ( 150 nM ) . Scale bar = 100 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - ) . ( C ) Number of of chronic exposure to PLX4032 before emergence of resistance in control or shRNA - ZEB1 - expressing ( n = 3 , Student ' s t - ) .

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(B) Soft agar colony formation assay in A375 cells in the presence or absence of PLX4032 (150 nM). Scale bar = 100 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test). (C) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or shRNA-ZEB1-expressing cells (n=3, Student's t-test).


( D ) RPMI7951 , A375 - R SKMEL5 - R were infected with a retrovirus encoding a constitutive shRNA - ZEB1 . Short - term cultures of GOKA ESP , derived from vemurafenib - resistant , were infected with a lentivirus encoding an IPTG - inducible shRNA - ZEB1 . Western blot analyses showing efficient ZEB1 knock - down in different models , ± IPTG ( 200µM ) , ± PLX4032 ( 3µM ) as indicated . Induction of was assessed by PARP cleavage . GAPDH or actin were used as loading control .

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(D) RPMI7951, A375-R and SKMEL5-R were infected with a retrovirus encoding a constitutive shRNA-ZEB1. Short-term cultures of GOKA and ESP cells, derived from vemurafenib-resistant patients, were infected with a lentivirus encoding an IPTG-inducible shRNA-ZEB1. Western blot analyses showing efficient ZEB1 knock-down in the different models, ±IPTG (200µM), ±PLX4032 (3µM) as indicated. Induction of cell death was assessed by PARP cleavage. GAPDH or actin were used as loading control.


( E ) Clonogenic assays in presence of 3 µM PLX4032 , with or without IPTG ( 200 µM ) as indicated . graphs represent mean number of colonies in 3 independent experiments ( Student ' s t - ) .

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(E) Clonogenic assays in the presence of 3 µM PLX4032, and with or without IPTG (200 µM) as indicated. The graphs represent the mean number of colonies in 3 independent experiments (Student's t-test).


( F ) Western blot analyses of ZEB1 , p75 quantitative PCR analyses of MITF in shRNA - Control or shRNA - ZEB1ESPvemurafenib - resistant - derived short - term culture . Actin was used as a loading control . mRNA expression are represented relatively to control ( mean ± SD , n = 3 , Student ' s t - ) .

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(F) Western blot analyses of ZEB1, p75 and quantitative PCR analyses of MITF in shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant patient-derived short-term culture cells. Actin was used as a loading control. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test).


( G ) 2 , 5 x 106 shRNA - Control or shRNA - ZEB1ESPvemurafenib - resistant were injected subcutaneously in nude mice . When reached 5 mm in diameter , ZEB1 expression was silenced by providing mice with IPTG ( 10 mM ) in their drinking water orally administering vemurafenib ( 50 mg / kg ) daily for 4 . mean volume for 5 mice is represented ( ± ) . ( Student ' s t - ) .

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(G) 2,5 x 106 shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant cells were injected subcutaneously in nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by providing mice with IPTG (10 mM) in their drinking water and orally administering vemurafenib (50 mg/kg) daily for 4 weeks. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).


( H ) Upper part : Western blot analyses of ZEB1 in shRNA - Control ( 1 to 4 ) or shRNA - ZEB1 ( 5 to 8 ) ESPxenografttumors , showing efficient ZEB1 knock - down directly in tumors , after IPTG ± PLX4032 treatment in vivo . Lower part : Representative pictures of ZEB1 immunostaining in shRNA - Control or shRNA - ZEB1tumors , after IPTG treatment in vivo . Scale bar = 40µm .

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(H) Upper part: Western blot analyses of ZEB1 in shRNA-Control (1 to 4) or shRNA-ZEB1 (5 to 8) ESPxenografttumors, showing efficient ZEB1 knock-down directly in the tumors, after IPTG ±PLX4032 treatment in vivo. Lower part: Representative pictures of ZEB1 immunostaining in shRNA-Control or shRNA-ZEB1tumors, after IPTG treatment in vivo. Scale bar = 40µm.


A Frequency of MN leukemic expressing immature marker cKit mature myeloid marker Ly6G in peripheral blood following 5 days of treatment ( n = 9 - 10 mice / group ; mice with < 2 % burden in any condition were censored from ) . Data information : data in A are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

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A Frequency of MN leukemic cells expressing the immature marker cKit and mature myeloid marker Ly6G in the peripheral blood following 5 days of treatment (n = 9-10 mice/group; mice with < 2% tumor burden in any condition were censored from the analysis). Data information: data in A are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


B Absolute number of MN in peripheral blood quantified by flow cytometry . Grey bars denote treatment ( n = 11 - 12 mice / group ) . Data information : Dox - doxycycline .

B Absolute number of MN cells in the peripheral blood quantified by flow cytometry. Grey bars denote treatment (n = 11-12 mice/group). Data information: Dox - doxycycline.


C Kaplan - Meier survival curve of leukemic mice ( n = 11 - 12 mice / group , median survival is 16 . 5 for vehicle not reached for AG636 doxycycline , P < 0 . 0001 by Log - rank ) . Data information : Dox - doxycycline .

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C Kaplan-Meier survival curve of leukemic mice (n = 11-12 mice/group, median survival is 16.5 for vehicle and not reached for AG636 and doxycycline, P < 0.0001 by Log-rank test). Data information: Dox - doxycycline.


D Representative FACS plots showing differentiation induced by AG636 doxycycline . Data information : Dox - doxycycline .

D Representative FACS plots showing differentiation induced by AG636 and doxycycline. Data information: Dox - doxycycline.


E Spleen weights of MN - bearing mice ( n = 3 - 6 mice / group ) . Data information : data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

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E Spleen weights of MN tumor-bearing mice (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


F Absolute number of MN in bone marrow ( n = 3 - 6 mice / group ) . Data information : data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

F Absolute number of MN cells in the bone marrow (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


G Frequency ( as of MN ) of LSCs ( CD11blowcKithighFcgR + ) differentiated ( CD182 + Ly6G + ) in bone marrow ( n = 3 - 6 mice / group ) . Data information : data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

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G Frequency (as percentage of all MN cells) of LSCs (CD11blowcKithighFcgR+) and differentiated cells (CD182+Ly6G+) in the bone marrow (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


H May - Grunwald - Giemsa - stained cytospins of sorted MN showing myeloid differentiation . Data information : Dox - doxycycline .

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H May-Grunwald-Giemsa-stained cytospins of sorted MN cells showing myeloid differentiation. Data information: Dox - doxycycline.


I of functionally - define LSCs using a limiting dilution calculated by ELDA ( n = 4 - 8 mice / group ) .

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I Quantification of functionally-define LSCs using a limiting dilution assay calculated by ELDA (n = 4-8 mice/group).


A Spleen weights of RUNX1 - RUNX1T1 or I1DN bearing mice treated with AG636 or vehicle for 4 days . Data information : n = 5 - 6 mice / group for RUNX1 - RUNX1T1 model , n = 4 mice / group for I1DN model ; data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 .

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A Spleen weights of RUNX1-RUNX1T1 or I1DN tumor bearing mice treated with AG636 or vehicle for 4 days. Data information: n = 5-6 mice/group for RUNX1-RUNX1T1 model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.


B - C Absolute number of leukemic in bone marrow ( B ) spleens ( C ) of RUNX1 - RUNX1T1 or I1DN bearing mice treated with AG636 or vehicle for 4 days . Data information : n = 5 - 6 mice / group for RUNX1 - RUNX1T1 model , n = 4 mice / group for I1DN model ; data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 .

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B-C Absolute number of leukemic cells in the bone marrow (B) and spleens (C) of RUNX1-RUNX1T1 or I1DN tumor bearing mice treated with AG636 or vehicle for 4 days. Data information: n = 5-6 mice/group for RUNX1-RUNX1T1 model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.


D - E Frequency of leukemic expressing immature marker cKit mature myeloid marker CD11b in bone marrow spleens of RUNX1 - RUNX1T1 ( D ) or I1DN ( E ) bearing mice treated with AG636 or vehicle for 4 days ( n = 6 mice / group ) . Data information : n = 5 - 6 mice / group for RUNX1 - RUNX1T1 model , n = 4 mice / group for I1DN model ; data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 .

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D-E Frequency of leukemic cells expressing the immature marker cKit and mature myeloid marker CD11b in the bone marrow and spleens of RUNX1-RUNX1T1 (D) or I1DN (E) tumor bearing mice treated with AG636 or vehicle for 4 days (n = 6 mice/group). Data information: n = 5-6 mice/group for RUNX1-RUNX1T1 model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.


Various bone marrow populations were quantified by flow cytometry in mice treated with AG636 or vehicle for 1 or 4 days ( acute treatment ) or for 4 cycles followed by 4 off treatment ( post - recovery ) . Data information : n = 4 mice / group for acute treatment , n = 5 - 6 mice for prolonged treatment recovery ; data are presented as mean ± s . d . ; P values were calculated using a two - Student ' s unpaired t - ; only comparisons that meet threshold of P < 0 . 05 are shown . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 .

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Various bone marrow populations were quantified by flow cytometry in mice treated with AG636 or vehicle for 1 or 4 days (acute treatment) or for 4 cycles followed by 4 weeks off treatment (post-recovery). Data information: n = 4 mice/group for acute treatment, n = 5-6 mice for prolonged treatment and recovery; data are presented as mean ± s.d.; P values were calculated using a two-tailed Student's unpaired t-test; only comparisons that meet the threshold of P < 0.05 are shown. *P < 0.05, **P < 0.01, ***P < 0.001


E - H Peripheral blood red blood ( E ) , platelets ( F ) , myeloid ( G ) lymphoid ( H ) quantified in mice treated with AG636 or vehicle . Grey bars denote treatment .

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E-H Peripheral blood red blood cells (E), platelets (F), myeloid cells (G) and lymphoid cells (H) quantified in mice treated with AG636 or vehicle. Grey bars denote treatment.


sequencing performed on cKithighCD11blow MN sorted from AG636 , doxycycline or vehicle treated mice ( n = 3 mice / group ) . Gene set enrichment showing common differential enrichment of biological pathways in gene expression data from AG636 or doxycycline treated . Gene sets are from C2 : CGP Reactome sub - collections in MSigDB database ( B ) .

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RNA sequencing performed on cKithighCD11blow MN cells sorted from AG636, doxycycline or vehicle treated mice (n = 3 mice/group). Gene set enrichment analysis showing common and differential enrichment of biological pathways in gene expression data from AG636 or doxycycline treated animals. Gene sets are from C2:CGP and Reactome sub-collections in MSigDB database (B).


C sequencing performed on cKit + CD11b - RUNX1 - RUNX1T1 or I1DN sorted from mice treated with AG636 or vehicle for 1 day ( n = 3 mice / group ) . Bar code plots showing enrichment of selected pathways .

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C RNA sequencing performed on cKit+CD11b- RUNX1-RUNX1T1 or I1DN cells sorted from mice treated with AG636 or vehicle for 1 day (n = 3 mice/group). Bar code plots showing enrichment of selected pathways.


E - F qPCR showing down - regulation of genes encoding ribosomal in MN ( E ) human AML lines ( F ) treated for 24 hours with AG636 ( n = 3 biological replicates for each ) . Data information : data in E , F are presented as mean ± s . e . m . ; P values were calculated using a one - Student ' s unpaired t - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ;

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E-F qPCR showing down-regulation of genes encoding ribosomal proteins in MN cells (E) and human AML cell lines (F) treated for 24 hours with AG636 (n = 3 biological replicates for each cell line). Data information: data in E, F are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;


G - H Polysome profiling of MN treated in vitro with AG636 or vehicle for 24h . Representative trace ( G ) of sub - polysome heavy polysome fractions ( H ) determined by measuring under curve ( AUC ) ( n = 4 biological replicates ) . Data information : data in are presented as mean ± s . e . m . ; P values were calculated using a one - Student ' s unpaired t - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ;

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G-H Polysome profiling of MN cells treated in vitro with AG636 or vehicle for 24h. Representative trace (G) and quantification of sub-polysome and heavy polysome fractions (H) determined by measuring area under the curve (AUC) (n = 4 biological replicates). Data information: data in are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;


I Nascent synthesis quantified using AHA incorporation in MN or MOLM13 treated with AG636 for 24 hours or cycloheximide for 1 hour . that were cultured in absence of AHA served as a negative control . ( n = 3 biological replicates ) . Data information : data in I are presented as mean ± s . e . m . ; P values were calculated using a one - Student ' s unpaired t - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; CHX - cycloheximide .

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I Nascent protein synthesis quantified using the AHA incorporation assay in MN or MOLM13 cells treated with AG636 for 24 hours or cycloheximide for 1 hour. Cells that were cultured in the absence of AHA served as a negative control. (n = 3 biological replicates). Data information: data in I are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; CHX - cycloheximide.


C Rank plots of ChIPseq enrichment scores of transcription factors at promoter regions ( + 1000 bp to - 50 bp from TSS ) of genes within indicated gene sets extracted from ENCODE 3 ( n = 24 genes for translation genes , n = 707 genes for AG636 down - regulated genes , n = 4237 for other genes ) .

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C Rank plots of ChIPseq enrichment scores of transcription factors at the promoter regions (+1000 bp to -50 bp from TSS) of genes within the indicated gene sets extracted from ENCODE 3 (n = 24 genes for translation genes, n = 707 genes for AG636 down-regulated genes, n = 4237 for the other genes).


D ChIPqPCR showing binding of YY1 at promoter regions of selected translation genes in MN treated with AG636 or DMSO . For each replicate enrichment was normalized to YY1 pull - down in DMSO treated ( n = 2 biological replicates ) . Data information : data in D are presented as mean ± s . e . m . ; P values were calculated using a two - Student ' s unpaired t - in D ; * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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D ChIPqPCR showing the binding of YY1 at the promoter regions of selected translation genes in MN cells treated with AG636 or DMSO. For each replicate enrichment was normalized to YY1 pull-down in DMSO treated cells (n = 2 biological replicates). Data information: data in D are presented as mean ± s.e.m.; P values were calculated using a two-tailed Student's unpaired t-test in D ; *P < 0.05, ***P < 0.001, ****P < 0.0001.


E Time - course showing global downregulation of O - GlcNAcylation YY1 expression in MN treated with AG636 . experiment was repeated twice with similar results .

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E Time-course showing global downregulation of O-GlcNAcylation and YY1 expression in MN cells treated with AG636. The experiment was repeated twice with similar results.


F Western blot of YY1 expression in human AML lines treated with AG636 for 24 hours .

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F Western blot of YY1 expression in human AML cell lines treated with AG636 for 24 hours.


G qPCR showing down - regulation of genes encoding ribosomal that are putative YY1 targets in Cas9 expressing MN treated with AG636 or DMSO ( left ) ; or transduced with sgRNAs targeting YY1 or control sgRNAs ( right ) ( n = 2 biological replicates ) . Data information : data in G are presented as mean ± s . e . m . ; P values were calculated using a one - Student ' s unpaired t - in G ; * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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G qPCR showing down-regulation of genes encoding ribosomal proteins that are putative YY1 targets in Cas9 expressing MN cells treated with AG636 or DMSO (left); or transduced with sgRNAs targeting YY1 or control sgRNAs (right) (n = 2 biological replicates). Data information: data in G are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test in G; *P < 0.05, ***P < 0.001, ****P < 0.0001.


H Barcode plots showing downregulation of Reactome Translation gene set in YY1 knockout pro B human melanoma upon YY1

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H Barcode plots showing downregulation of the Reactome Translation gene set in YY1 knockout pro B cells and human melanoma cells upon YY1 knockdown


G Proliferative competition assays in MN transduced with sgRNAs cultured in AG636 or DMSO ( n = 2 biological replicates ) . Data information : data in G represented as mean ± s . e . m . ; P values were calculated using one - Student ' s unpaired t - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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G Proliferative competition assays in MN cells transduced with individual sgRNAs and cultured in AG636 or DMSO (n = 2 biological replicates). Data information: data in G represented as mean ± s.e.m.; P values were calculated using one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001.


A - B Proliferative competition assays in AML transduced with indicated sgRNAs cultured in AG636 or DMSO ( n = 2 biological replicates ) . Data information : data represented as mean ± s . e . m ; P values were calculated using one - Student ' s unpaired t - in A B ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * P < 0 . 0001 .

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A-B Proliferative competition assays in AML cells transduced with indicated sgRNAs and cultured in AG636 or DMSO (n = 2 biological replicates). Data information: data represented as mean ± s.e.m; P values were calculated using one-tailed Student's unpaired t-test in A and B ; *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.


F AML were treated with AG636 , Roscovitine or combination for 17 days . Cumulative live are shown ( n = 2 biological replicates ) . Data information : data represented as mean ± s . e . m ; P values were calculated using Brown - Forsythe Welch ANOVA in F ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * P < 0 . 0001 .

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F AML cells were treated with AG636, Roscovitine or the combination for 17 days. Cumulative live cell counts are shown (n = 2 biological replicates). Data information: data represented as mean ± s.e.m; P values were calculated using Brown-Forsythe and Welch ANOVA test in F; *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.


B ( left ) SRM of paraspeckles in HAP1 NEAT1 ∆ 3 ′ mutant ( ∆ 3 ′ ) detected by NEAT1 _ 5 ′ ( green ) NEAT1 _ 15k ( magenta ) FISH probes in presence of MG132 ( 5 μM for 6 h ) . Scale bar , 500 nm . ( right ) Graph showing proportion of paraspeckles with localization of NEAT1 3 ′ ends in or in shell ( n = 44 ) .

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

B (left) SRM images of paraspeckles in HAP1 NEAT1 ∆3′ mutant cells (∆3′) detected by NEAT1_5′ (green) and NEAT1_15k (magenta) FISH probes in the presence of MG132 (5 μM for 6 h). Scale bar, 500 nm. (right) Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends in the core or in the core and shell (n = 44).


B SRM of paraspeckles in ∆ 0 - 0 . 8kb ∆ 0 - 1 . 9kb ( ∆ 5 ′ ) treated with MG132 ( 5 μM for 6 h ) detected by NEAT1 _ 5 ′ , 1k , 2k ( green ) NEAT1 _ 3 ′ ( magenta ) FISH probes . Scale bar , 500 nm .

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

B SRM images of the paraspeckles in ∆0-0.8kb and ∆0-1.9kb (∆5′) cells treated with MG132 (5 μM for 6 h) detected by NEAT1_5′, 1k, and 2k (green) and NEAT1_3′ (magenta) FISH probes. Scale bar, 500 nm.


D ( left ) EM observation of paraspeckles in ∆ 5 ′ treated with MG132 ( 5 μM for 17 h ) using NEAT1 _ 5 ′ probes . Scale bar , 100 nm . ( middle ) Graph showing proportion of localization of NEAT1 _ 5 ′ probes ( 347 gold particles ) within paraspeckles in ∆ 5 ′ . ( right ) Graph showing proportion of localization of NEAT1 _ 5 ′ probes in each paraspeckle in ∆ 5 ′ ( n = 20 ) . box plot shows median ( inside ) , 25 - 75 percentiles ( box bottom to top ) , 10 - 90 percentiles ( whisker bottom to top ) .

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

D (left) EM observation of the paraspeckles in ∆5′ cells treated with MG132 (5 μM for 17 h) using NEAT1_5′ probes. Scale bar, 100 nm. (middle) Graph showing the proportion of localization of NEAT1_5′ probes (347 gold particles) within the paraspeckles in ∆5′ cells. (right) Graph showing the proportion of the localization of NEAT1_5′ probes in each paraspeckle in ∆5′ cells (n = 20). The box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).


B SRM of paraspeckles in MG132 - treated ( 5 μM for 6 h ) HAP1 NEAT1 ∆ 5 ′ / ∆ 3 ′ detected by NEAT1 _ 2k ( green ) NEAT1 _ 15k FISH probes ( left , magenta ) or NONO immunofluorescence ( IF ) ( right , magenta ) . Scale bar , 500 nm .

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 B-MEASURED_VAR O O O O O B-MEASURED_VAR 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

B SRM images of the paraspeckles in MG132-treated (5 μM for 6 h) HAP1 NEAT1 ∆5′/∆3′ cells detected by NEAT1_2k (green) and NEAT1_15k FISH probes (left, magenta) or NONO immunofluorescence (IF) (right, magenta). Scale bar, 500 nm.


D ( upper ) EM observation of paraspeckles in MG132 - treated ( 5 μM for 17 h ) ∆ 5 ′ / ∆ 3 ′ using NEAT1 _ 5 ′ ( left ) NEAT1 _ D2 probes ( right ) . Scale bar , 100 nm . ( middle ) Graph showing proportion of localization of NEAT1 _ 5 ′ ( left , 310 gold particles ) NEAT1 _ D2 ( right , 294 gold particles ) probes within paraspeckles in HAP1 NEAT1 ∆ 5 ′ / ∆ 3 ′ . ( lower ) Graph showing proportion of localization of NEAT1 _ 5 ′ ( left , 19 ) D2 ( right , 22 ) probes in each paraspeckle in ∆ 5 ′ / ∆ 3 ′ . Each box plot shows median ( inside ) , 25 - 75 percentiles ( box bottom to top ) , 10 - 90 percentiles ( whisker bottom to top ) .

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

D (upper) EM observation of the paraspeckles in MG132-treated (5 μM for 17 h) ∆5′/∆3′ cells using NEAT1_5′ (left) and NEAT1_D2 probes (right). Scale bar, 100 nm. (middle) Graph showing the proportion of the localization of NEAT1_5′ (left, 310 gold particles) and NEAT1_D2 (right, 294 gold particles) probes within the paraspeckles in HAP1 NEAT1 ∆5′/∆3′ cells. (lower) Graph showing the proportion of the localization of NEAT1_5′ (left, 19 cells) and D2 (right, 22 cells) probes in each paraspeckle in ∆5′/∆3′ cells. Each box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).


C Diameters ( Sx ) of paraspeckles in WT , ∆ 3 ′ , ∆ 5 ′ , ∆ 5 ′ / ∆ 3 ′ treated with MG132 ( 5 μM for 6 h ) determined by SRM ( WT : n = 129 , ∆ 3 ′ : n = 106 , ∆ 5 ′ : n = 100 , ∆ 5 ′ / ∆ 3 ′ : n = 292 ) . WT ( mean : 387 . 5 nm ) , ∆ 3 ′ ( mean : 492 . 4 nm ) , ∆ 5 ′ ( mean : 497 . 7 nm ) , ∆ 5 ′ / ∆ 3 ′ mutant ( mean : 753 . 3 nm ) . ( * * * * : P < 0 . 0001 , compared with WT : Kruskal - Wallis with Dunn ' s multiple comparison ) . Each box plot shows median ( inside ) , 25 - 75 percentiles ( box bottom to top ) , 10 - 90 percentiles ( whisker bottom to top ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Diameters (Sx) of the paraspeckles in WT, ∆3′, ∆5′, and ∆5′/∆3′ cells treated with MG132 (5 μM for 6 h) determined by SRM (WT: n = 129, ∆3′: n = 106, ∆5′: n = 100, ∆5′/∆3′: n = 292). WT (mean size: 387.5 nm), ∆3′ (mean size: 492.4 nm), ∆5′ (mean size: 497.7 nm), and ∆5′/∆3′ mutant (mean size: 753.3 nm). (****: P < 0.0001, compared with WT: Kruskal-Wallis test with Dunn's multiple comparison test). Each box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).


B Quantitation of NEAT1 _ 2 by RT - qPCR in WT ∆ 5 ′ / ∆ 3 ′ / ∆ PAS with or without MG132 treatment ( 6 h ) . Data are represented as mean ± SD ( n = 3 ) .

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

B Quantitation of NEAT1_2 by RT-qPCR in WT and ∆5′/∆3′/∆PAS cells with or without MG132 treatment (6 h). Data are represented as mean ± SD (n = 3).


C paraspeckles in WT ∆ 5 ′ / ∆ 3 ′ / ∆ PAS detected by SRM using NEAT1 _ 5 ′ 2k FISH probes ( green ) , NONO IF ( magenta ) under conditions shown in ( B ) . Scale bar , 500 nm .

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C The paraspeckles in WT and ∆5′/∆3′/∆PAS cells detected by SRM using NEAT1_5′ and 2k FISH probes (green), and NONO IF (magenta) under the conditions shown in (B). Scale bar, 500 nm.


D . Global measurement of severing of filaments exposed to 20 nM mCherry - cofilin - 1 from time t = 0 onwards . N = 20 ( from 1 representative experiment ) 50 ( 1 experiment ) for non - oxidized oxidized actin filaments , respectively . P - value = 8 . 10 - 8 ( log - rank ) . Data information : Thick solid dashed lines are survival fractions calculated from experimental data . Thin grey lines are single exponential fits . 95 % confidence intervals are shown as shaded surfaces .

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

D. Global measurement of the severing of filaments exposed to 20 nM mCherry-cofilin-1 from time t=0 onwards. N = 20 (from 1 representative experiment) and 50 (1 experiment) for non-oxidized and oxidized actin filaments, respectively. P-value = 8.10-8 (log-rank test). Data information: Thick solid and dashed lines are survival fractions calculated from the experimental data. Thin grey lines are single exponential fits. 95% confidence intervals are shown as shaded surfaces.


B . Global of severing events on oxidized non - oxidized filaments exposed to unlabelled S3D - cofilin1 from time t = 0 onwards . From top to bottom , N = 50 ( from 1 experiment ) , 50 ( 1 experiment ) , 100 ( 2 experiments ) filaments . Data information lines are survival fractions calculated from experimental data 95 % confidence intervals are shown as shaded surfaces .

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

B. Global quantification of the severing events on oxidized and non-oxidized filaments exposed to unlabelled S3D-cofilin1 from time t=0 onwards. From top to bottom, N = 50 (from 1 experiment), 50 (1 experiment), 100 (2 experiments) filaments. Data information All lines are survival fractions calculated from the experimental data 95% confidence intervals are shown as shaded surfaces.


A . Time - lapse of Tpm - saturated actin filament depolymerizing from their barbed end ( BE ) , with or without MICAL1 . Depolymerization is initiated at time t = 0 from a non - oxidized actin filament . increase in depolymerization rate reflects oxidation of actin with time . Tpm is labeled with neonGreen ( nGr ) , actin is unlabeled .

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

A. Time-lapse images of Tpm-saturated actin filament depolymerizing from their barbed end (BE), with or without MICAL1. Depolymerization is initiated at time t=0 from a non-oxidized actin filament. The increase in the depolymerization rate reflects the oxidation of actin with time. Tpm is labeled with neonGreen (nGr), and actin is unlabeled.


C . Time - lapse showing disassembly of Tpm domains . Filaments were saturated with 100 nM nGr - Tpm1 . 8 before experiment Tpm was removed from solution at time t = 0 ( solution contains 0 . 6 µM unlabeled G - actin to prevent filament depolymerization ) . PE BE indicate F - actin pointed barbed end , respectively .

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C. Time-lapse images showing the disassembly of Tpm domains. Filaments were saturated with 100 nM nGr-Tpm1.8 before the experiment and Tpm was removed from solution at time t=0 (the solution contains 0.6 µM unlabeled G-actin to prevent filament depolymerization). PE and BE indicate F-actin pointed and barbed end, respectively.


A . Time - lapse of filaments , oxidized or not , bare or Tpm - saturated , exposed to 1 µM mCherry - cofilin - 1 , from time t = 0 onwards .

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

A. Time-lapse images of filaments, oxidized or not, bare or Tpm-saturated, exposed to 1 µM mCherry-cofilin-1, from time t=0 onwards.


A . Time - lapse of actin filaments constantly exposed to nGr - Tpm1 . 8 ( yellow ) , WT mCh - cofilin - 1 ( blue ) S3D - cofilin - 1 , to MICAL1 from time t = 100s onwards . Here we mixed WT with an excess of S3D - cofilin - 1 to mimic conditions in which most of cofilin - 1 is inhibited by phosphorylation . While Tpm prevents cofilin from binding to non - oxidized filaments , MICAL1 rapidly oxidizes actin , allowing cofilin to bind sever filaments .

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

A. Time-lapse of actin filaments constantly exposed to nGr-Tpm1.8 (yellow), WT mCh-cofilin-1 (blue) and S3D-cofilin-1, and to MICAL1 from time t=100s onwards. Here we mixed WT with an excess of S3D-cofilin-1 to mimic cellular conditions in which most of cofilin-1 is inhibited by phosphorylation. While Tpm prevents cofilin from binding to non-oxidized filaments, MICAL1 rapidly oxidizes actin, allowing cofilin to bind and sever filaments.


Conditioned medium was collected from pericytes 48 h after adenoviral transduction . Western blotting verified secretion of Sema3CΔ13 ( 81 kDa ) Sema3Cp60 ( 60 kDa ) isoforms .

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

Conditioned medium was collected from pericytes 48 h after adenoviral transduction. Western blotting verified the secretion of Sema3CΔ13 (81 kDa) and Sema3Cp60 (60 kDa) isoforms.


HUVECs were suspended in conditioned medium with VEGF ( 25 ng / ml ) plated on Matrigel . Branch points of capillary - like network were counted after 18 h . n = 4 independent experiments ; scale bar , 200 μm . Control vs . Sema3C , * P = 0 . 0375 control vs . Sema3CΔ13 , * P = 0 . 0162 .

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HUVECs were suspended in conditioned medium with VEGF (25 ng/ml) and plated on Matrigel. Branch points of the capillary-like network were counted after 18 h. n = 4 independent experiments; scale bar, 200 μm. Control vs. Sema3C, *P = 0.0375 and control vs. Sema3CΔ13, *P = 0.0162.


HUVEC spheroids in collagen were treated with conditioned medium with or without VEGF ( 25 ng / ml ) . After 24 h , cumulative sprout per spheroid was quantified . Data are representative of three independent experiments with ten spheroids per group . Scale bar , 100 μm . Control vs . Sema3C ( − VEGF ) , * P = 0 . 0276 ; control vs . Sema3CΔ13 ( − VEGF ) , * P = 0 . 0443 ; control vs . Sema3C ( + VEGF ) , * P = 0 . 028 control vs . Sema3CΔ13 ( + VEGF ) , * P = 0 . 032 .

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

HUVEC spheroids in collagen were treated with conditioned medium with or without VEGF (25 ng/ml). After 24 h, the cumulative sprout length per spheroid was quantified. Data are representative of three independent experiments with ten spheroids per group. Scale bar, 100 μm. Control vs. Sema3C (− VEGF), *P = 0.0276; control vs. Sema3CΔ13 (− VEGF), *P = 0.0443; control vs. Sema3C (+ VEGF), *P = 0.028 and control vs. Sema3CΔ13 (+ VEGF), *P = 0.032.


Microvessels within plugs stained against human - specific CD34 . nuclei were stained with DAPI . Scale bar , 100 μm . of CD34 - positive microvessels was quantified normalized to total of plug . Results represent means ± s . e . m of 5 plugs ( control ) 7 plugs ( Sema3C ) , * P = 0 . 018 .

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

Microvessels within the plugs stained against human-specific CD34. Cell nuclei were stained with DAPI. Scale bar, 100 μm.The area of CD34-positive microvessels was quantified and normalized to the total area of plug. Results represent the means ± s.e.m of 5 plugs (control) and 7 plugs (Sema3C), *P = 0.018.


Vessel coverage was assessed by αSMA . Scale bar , 100 μm . Vessel coverage was quantified as of endothelial - associated mural to total amount of vessels ( n = 5 ( control ) n = 7 ( Sema3C ) ) . Vessel perfusion was evaluated by i . v . injection of FITC - lectin ( 0 . 15 mg ) 30 min before mice were sacrificed . data show of perfused vessels in plug normalized to total vessel ( n = 5 ( control ) n = 7 ( Sema3C ) ) .

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

Vessel coverage was assessed by αSMA staining. Scale bar, 100 μm.Vessel coverage was quantified as the ratio of endothelial cell-associated mural cells to total amount of vessels (n = 5 (control) and n = 7 (Sema3C)).Vessel perfusion was evaluated by i.v. injection of FITC-lectin (0.15 mg) 30 min before mice were sacrificed. The data show the percentage of perfused vessels in the plug and normalized to total vessel area (n = 5 (control) and n = 7 (Sema3C)).


A C57BL / 6N mice pups at P3 received 1 μg of mouse Sema3C - Fc ( right eye ) or control IgG - Fc ( left eye ) . retinal vasculature was examined at P5 by whole - mount CD31immunofluorescence . Representative show total retinal vasculature sprouting tips at vascular front ( iii iv , arrows ) enlarged intercapillary space ( ii iv , asterisks ) . Scale bar , 100 μm 20 μm ( iii iv ) . B , C ( B ) Retinal vessel outgrowth measured as total from central optic disk to vascular front normalized to control IgG - Fc treatment , * * * P = 0 . 0009 . ( C ) Vessel density was quantified by CD31 - stained vessels normalized to total vessel , * * * P = 0 . 0002 . ( Mean ± s . e . m ; n = 23 retinae in each group from four independent experiments ; unpaired Student ' s t - . ) D , E ( D ) Number of sprouting tips per retina , * P < 0 . 0001 . ( E ) Branch points in capillary plexus represented as to control IgG - treated retinae , * * * P < 0 . 0001 . ( Mean ± s . e . m ; n = 18 retinae in each group from three independent experiments ; unpaired Student ' s t - . )

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

A C57BL/6N mice pups at P3 received 1 μg of mouse Sema3C-Fc (right eye) or control IgG-Fc (left eye). The retinal vasculature was examined at P5 by whole-mount CD31immunofluorescence staining. Representative images show the total retinal vasculature and sprouting tips at the vascular front (iii and iv, arrows) and the enlarged intercapillary space (ii and iv, asterisks). Scale bar, 100 μm and 20 μm (iii and iv).B,C (B) Retinal vessel outgrowth measured as the total area from the central optic disk to the vascular front normalized to control IgG-Fc treatment, ***P = 0.0009. (C) Vessel density was quantified by CD31-stained vessels and normalized to the total vessel area, ***P = 0.0002. (Mean ± s.e.m; n = 23 retinae in each group from four independent experiments; unpaired Student's t-test.)D,E (D) Number of sprouting tips per retina, *P < 0.0001. (E) Branch points in the capillary plexus represented as the ratio to the control IgG-treated retinae, ***P < 0.0001. (Mean ± s.e.m; n = 18 retinae in each group from three independent experiments; unpaired Student's t-test.)


F Pups received same treatment as described in ( A ) . astrocyte layer was examined at P5 by GFAP . Scale bar , 100 μm ( upper panel ) 20 μm ( lower panel ) . Representative of six retinae per group .

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F Pups received same treatment as described in (A). The astrocyte layer was examined at P5 by GFAP staining. Scale bar, 100 μm (upper panel) and 20 μm (lower panel). Representative images of six retinae per group.


G Six - week - old C57BL / 6N mice were intravitreally injected with Sema3C - Fc ( right eye ) or control IgG - Fc ( left eye ) , retinae were collected 2 days later . retinal vessel network was examined with CD31immunostaining . Scale bar , 100 μm ( upper panel ) 20 μm ( lower panel ) . Representative of four retinae per group .

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G Six-week-old C57BL/6N male mice were intravitreally injected with Sema3C-Fc (right eye) or control IgG-Fc (left eye), and retinae were collected 2 days later. The retinal vessel network was examined with CD31immunostaining. Scale bar, 100 μm (upper panel) and 20 μm (lower panel). Representative images of four retinae per group.


HUVECs were treated with Sema3C - or GFP - conditioned medium for 24 h . proliferation was quantified by BrdU incorporation ( mean ± s . e . m ; n = 3 ; n . s . , non - significant ) .

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

HUVECs were treated with Sema3C- or GFP-conditioned medium for 24 h. Cell proliferation was quantified by BrdU incorporation (mean ± s.e.m; n = 3; n.s., non-significant).


Apoptosis of HUVEC was measured by caspase - 3 caspase - 7 activities after 3 h of incubation with Sema3C - or control - conditioned medium ( mean ± s . e . m ; n = 3 , * P = 0 . 018 using unpaired Student ' s t - ) .

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Apoptosis of HUVEC was measured by caspase-3 and caspase-7 activities after 3 h of incubation with Sema3C- or control-conditioned medium (mean ± s.e.m; n = 3, *P = 0.018 using unpaired Student's t-test).


Serum - starved HUVECs were incubated with conditioned medium or conditioned medium + VEGF ( 50 ng / ml ) for 15 min . lysates were subjected to SDS - PAGE immunoblotted with anti - phospho - AKT antibody . of phospho - AKT normalized to total AKT ( mean ± s . e . m ; n = 4 ; control vs . Sema3C ( − VEGF ) , * P = 0 . 034 control vs . Sema3C ( + VEGF ) , * P = 0 . 024 using unpaired Student ' s t - ) .

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

Serum-starved HUVECs were incubated with conditioned medium or conditioned medium + VEGF (50 ng/ml) for 15 min. Cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-AKT antibody.Quantification of phospho-AKT normalized to total AKT levels (mean ± s.e.m; n = 4; control vs. Sema3C (− VEGF), *P = 0.034 and control vs. Sema3C (+ VEGF), *P = 0.024 using unpaired Student's t-test).