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 .

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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 analysis of the FLAG ‐ tagged POL V protein in wt Col and ddm1 . ChIP signals are normalized to the 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 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 .

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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 the tiles in ( A ) in both wt Col and ddm1 , we determined the 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 ) 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 .

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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 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 ) .

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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 the Athila6A TSS in juvenile leaf tissue .

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


B qRT - PCR analysis of Athila6A gag / pol steady ‐ state mRNA transcript levels in inflorescence and 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 analysis of AGO6 and AGO4 mRNA transcript levels in inflorescence and 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 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 ) .

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

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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 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 .

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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 and MITF mRNA expression in 61 melanomacell lines available through the 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 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 .

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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 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 .

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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 , 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 .

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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 the panel of BRAFV600 melanoma cells as determined by ATP assay ( n = 3 , mean ± SD ) . For SKMEL24 and 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 and MITF mRNA expression levels in 467 melanomatumors from TCGA data set .

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


( 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 .

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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 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 ) .

O O O O O O B-GENEPROD O O B-GENEPROD B-TISSUE O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O

(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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 cells were infected with retroviruses expressing ZEB1 . Western blot analyses of ZEB1 and 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 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 ) .

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

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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 cells were injected subcutaneously into nude mice . The mean tumor volume for 5 mice is represented ± SEM ( Student ' s t - test ) .

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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 , and TWIST1 levels in control or ZEB1 - expressing cells + / - 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 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 ) .

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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 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 ) .

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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 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 ) .

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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 cells were infected with retroviruses expressing ZEB1 . Western blot analyses of ZEB1 and 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 and p75 upon ZEB1 expression . mRNA expression levels are represented relatively to control cells ( mean ± SD , n = 3 , Student ' s t - test ) .

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

(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 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 ) .

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

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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 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 .

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

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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 the mean percentage of p75 - high , int and low cells from 2 independent experiments ( Fisher ' s exact test ) .

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

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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 cells were injected subcutaneously into nude mice . The mean tumor volume for 5 mice is represented ( ± SEM ) . ( Student ' s t - test ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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

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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 and not reached for AG636 and doxycycline , P < 0 . 0001 by Log - rank test ) . 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 and doxycycline . Data information : Dox - doxycycline .

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


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 .

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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 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 .

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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 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 .

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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 cells showing myeloid differentiation . Data information : Dox - doxycycline .

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


I Quantification of functionally - define LSCs using a limiting dilution assay 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells ( E ) , platelets ( F ) , myeloid cells ( G ) and lymphoid cells ( 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.


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 ) .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-ORGANISM O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

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 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 ;

O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O O B-ORGANISM B-DISEASE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-F 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 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 ;

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O

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 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 ) .

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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 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 .

O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 and YY1 expression in MN cells treated with AG636 . The experiment was repeated twice with similar results .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O

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 cell 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 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 .

O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


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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 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 .

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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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-DISEASE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O B-DISEASE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-CELL_LINE B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O B-GENEPROD O O O O O 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 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 .

O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY 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 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 ) .

O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O 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 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 .

O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY 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 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 ) .

O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_LINE B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and ∆ 5 ′ / ∆ 3 ′ / ∆ PAS cells with or without MG132 treatment ( 6 h ) . Data are represented as mean ± SD ( n = 3 ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 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 .

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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 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 .

O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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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 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 .

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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 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 .

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

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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 ) 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 .

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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 the secretion of Sema3CΔ13 ( 81 kDa ) and Sema3Cp60 ( 60 kDa ) isoforms .

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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 ) 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 .

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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 , 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 .

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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 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 .

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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 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 ) ) .

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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 ) . 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 . )

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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 ) . 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 .

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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 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 .

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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 . Cell proliferation was quantified by BrdU incorporation ( mean ± s . e . m ; n = 3 ; n . s . , non - significant ) .

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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 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 ) .

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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 . 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 ) .

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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).


HUVECs were adenovirally transduced with VE - cadherin - GFP and plated on ibidi slides ( n = 4 ; control vs . Sema3C ( − VEGF ) at 20 × magnification ( Supplementary Movies S1 and S2 ) ) . Representative pictures are shown at time points 0 and 1 h . Arrowheads indicate gaps between endothelial cells . Scale bar , 100 μm .

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HUVECs were adenovirally transduced with VE-cadherin-GFP and plated on ibidi slides (n = 4; control vs. Sema3C (− VEGF) at 20× magnification (Supplementary Movies S1 and S2)). Representative pictures are shown at time points 0 and 1 h. Arrowheads indicate gaps between endothelial cells. Scale bar, 100 μm.


C57BL / 6Nmicepups at P5 received 1 μg of recombinant Sema3C or IgG - Fc proteins . Endothelial cell junctions were examined by whole - mount VE - cadherinimmunofluorescence staining 6 h later . Representative images show VE - cadherin at cell junctions ( arrows ) . The linear structure ( i ) was disrupted in Sema3C - treated retinae ( ii ) . Scale bar , 100 μm ( upper panel ) and 20 μm ( lower panel ) .

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C57BL/6Nmicepups at P5 received 1 μg of recombinant Sema3C or IgG-Fc proteins. Endothelial cell junctions were examined by whole-mount VE-cadherinimmunofluorescence staining 6 h later. Representative images show VE-cadherin at cell junctions (arrows). The linear structure (i) was disrupted in Sema3C-treated retinae (ii). Scale bar, 100 μm (upper panel) and 20 μm (lower panel).


Whole - mount immunostaining of the vascular bed by CD31 and apoptotic cells by cleaved caspase - 3 in the retinae at P5 8 h after intravitreal injection of Sema3C or IgG - Fc . Representative images show active caspase - 3 signals that co - localize with endothelial cells ( arrows ) . Scale bar , 100 μm . Quantification of cleaved caspase - 3 normalized to area of CD31 - stained vessels ( mean ± s . e . m ; n = 5 ; * * P = 0 . 007 using paired Student ' s t - test ) .

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Whole-mount immunostaining of the vascular bed by CD31 and apoptotic cells by cleaved caspase-3 in the retinae at P5 8 h after intravitreal injection of Sema3C or IgG-Fc. Representative images show active caspase-3 signals that co-localize with endothelial cells (arrows). Scale bar, 100 μm.Quantification of cleaved caspase-3 normalized to area of CD31-stained vessels (mean ± s.e.m; n = 5; **P = 0.007 using paired Student's t-test).


A HUVECs were transduced with adenoviral vector expressing RFP - conjugated F - actin binding peptide ( LifeAct ) . Time - lapse imaging of F - actin in HUVEC spheroids was obtained at 20 × magnification ( Supplementary Movies S3 and S4 ) . Representative images are shown at 0 and 2 h . Arrowheads indicate detached cells after Sema3C treatment . Scale bar , 100 μm .

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A HUVECs were transduced with adenoviral vector expressing RFP-conjugated F-actin binding peptide (LifeAct). Time-lapse imaging of F-actin in HUVEC spheroids was obtained at 20× magnification (Supplementary Movies S3 and S4). Representative images are shown at 0 and 2 h. Arrowheads indicate detached cells after Sema3C treatment. Scale bar, 100 μm.


B Endothelial cell migration was determined using a scratch wound assay with a co - culture model . A wound was generated in a confluent HUVEC monolayer , and cells were cultured in basal medium with or without VEGF ( 25 ng / ml ) in a 24 - well plate . At the same time , the transwells containing Sema3C or GFP - expressing pericytes . Representative pictures show remaining gaps within the HUVEC monolayer . Scale bar , 200 μm . C Migration distance was measured after 24 h and normalized to control treatment ( mean ± s . e . m ; n = 3 independent experiments , control vs . Sema3C ( − VEGF ) , * P = 0 . 021 and control vs . Sema3C ( + VEGF ) , * P = 0 . 039 using unpaired Student ' s t - test ) .

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B Endothelial cell migration was determined using a scratch wound assay with a co-culture model. A wound was generated in a confluent HUVEC monolayer, and cells were cultured in basal medium with or without VEGF (25 ng/ml) in a 24-well plate. At the same time, the transwells containing Sema3C or GFP-expressing pericytes. Representative pictures show remaining gaps within the HUVEC monolayer. Scale bar, 200 μm.C Migration distance was measured after 24 h and normalized to control treatment (mean ± s.e.m; n = 3 independent experiments, control vs. Sema3C (− VEGF), *P = 0.021 and control vs. Sema3C (+ VEGF), *P = 0.039 using unpaired Student's t-test).


D , E ( D ) Serum - starved HUVECs were treated with Sema3C - or control - conditioned medium with or without VEGF ( 50 ng / ml ) for 10 min . Cell lysates were subjected to SDS - PAGE and probed with anti - phosphoT180 / Y182 p38MAPK antibody . Quantification of p38MAPK activation was normalized to total p38MAPK and presented as the ratio to the cells with control medium without VEGF treatment . Control vs . Sema3C ( − VEGF ) , * P = 0 . 046 and control vs . Sema3C ( + VEGF ) , * P = 0 . 043 . ( E ) HUVECs were treated as described before and the activation of FAK detected by anti - phosphoY397 FAK antibody and normalized to the total amount of FAK . Control vs . Sema3C ( − VEGF ) , * P = 0 . 033 and control vs . Sema3C ( + VEGF ) , * P = 0 . 047 . ( Mean ± s . e . m ; n = 3 independent experiments ; unpaired Student ' s t - test . )

O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O 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 (D) Serum-starved HUVECs were treated with Sema3C- or control-conditioned medium with or without VEGF (50 ng/ml) for 10 min. Cell lysates were subjected to SDS-PAGE and probed with anti-phosphoT180/Y182 p38MAPK antibody. Quantification of p38MAPK activation was normalized to total p38MAPK and presented as the ratio to the cells with control medium without VEGF treatment. Control vs. Sema3C (− VEGF), *P = 0.046 and control vs. Sema3C (+ VEGF), *P = 0.043. (E) HUVECs were treated as described before and the activation of FAK detected by anti-phosphoY397 FAK antibody and normalized to the total amount of FAK. Control vs. Sema3C (− VEGF), *P = 0.033 and control vs. Sema3C (+ VEGF), *P = 0.047. (Mean ± s.e.m; n = 3 independent experiments; unpaired Student's t-test.)


F Serum - starved HUVECs were treated with conditioned medium for 15 min . Focal adhesion proteins were examined with anti - phosphoY397FAK and anti - vinculin antibodies . F - actin was stained with Alexa Fluor 546 - conjugated phalloidin . Merged images show the coupling of stress fibers with focal adhesion complexes in the cells treated with control medium . Inset , phospho - Y397FAK and vinculin formed focal adhesions that were stained in an elongated dot - like structure ( i ) . Lower panel ( ii ) , Sema3C treatment induced F - actin disassembly and membrane ruffling . Phospho - Y397FAK and vinculin were randomly distributed at the peripheral membrane . Scale bars , 10 μm .

O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

F Serum-starved HUVECs were treated with conditioned medium for 15 min. Focal adhesion proteins were examined with anti-phosphoY397FAK and anti-vinculin antibodies. F-actin was stained with Alexa Fluor 546-conjugated phalloidin. Merged images show the coupling of stress fibers with focal adhesion complexes in the cells treated with control medium. Inset, phospho-Y397FAK and vinculin formed focal adhesions that were stained in an elongated dot-like structure (i). Lower panel (ii), Sema3C treatment induced F-actin disassembly and membrane ruffling. Phospho-Y397FAK and vinculin were randomly distributed at the peripheral membrane. Scale bars, 10 μm.


HUVECs were transfected with siRNA against PlexinD1 or control NS ( non - silence ) siRNA . qPCR analyses and Western blotting were performed after 48 h ( n = 3 ; paired Student ' s t - test , NS siRNA vs . PlexinD1 siRNA , * * * P = 0 . 0004 ) . Cell lysates were subjected to SDS - PAGE and probed with anti - PlexinD1 .

B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O

HUVECs were transfected with siRNA against PlexinD1 or control NS (non-silence) siRNA. qPCR analyses and Western blotting were performed after 48 h (n = 3; paired Student's t-test, NS siRNA vs. PlexinD1 siRNA, ***P = 0.0004). Cell lysates were subjected to SDS-PAGE and probed with anti-PlexinD1.


HUVECs were transfected with siRNA against PlexinD1 and plated on gelatin - coated coverslips . Confluent monolayers were treated with Sema3C - , Sema3Cp60 - or control - conditioned medium for 30 min . Cells were fixed and stained with Alexa - Fluor 488 - conjugated phalloidin to visualize F - actin . Arrows indicate Sema3C - induced cell membrane ruffling . Scale bar , 20 μm . Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei ( n = 5 ; mean ± s . e . m . , paired Student ' s t - test , * * P = 0 . 001 ) .

B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O 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 transfected with siRNA against PlexinD1 and plated on gelatin-coated coverslips. Confluent monolayers were treated with Sema3C-, Sema3Cp60- or control-conditioned medium for 30 min. Cells were fixed and stained with Alexa-Fluor 488-conjugated phalloidin to visualize F-actin. Arrows indicate Sema3C-induced cell membrane ruffling. Scale bar, 20 μm. Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei (n = 5; mean ± s.e.m., paired Student's t-test, **P = 0.001).