( 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 .

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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 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 .

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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 ;

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

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 .

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

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 .

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

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 .

O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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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 .

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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 ) .

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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 .

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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 B-CONTROLLED_VAR 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 . 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 ) ) .

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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 ) . 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 - . )

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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 ) . 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 ) .

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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 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 - ) .

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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 plated on ibidi slides ( n = 4 ; control vs . Sema3C ( − VEGF ) at 20 × magnification ( Supplementary Movies S1 S2 ) ) . Representative pictures are shown at time points 0 1 h . Arrowheads indicate gaps between endothelial . 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 . Endothelial junctions were examined by whole - mount VE - cadherinimmunofluorescence 6 h later . Representative show VE - cadherin at junctions ( arrows ) . linear structure ( i ) was disrupted in Sema3C - treated retinae ( ii ) . Scale bar , 100 μm ( upper panel ) 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 vascular bed by CD31 apoptotic by caspase - 3 in retinae at P5 8 h after intravitreal injection of Sema3C or IgG - Fc . Representative show active caspase - 3 signals that co - localize with endothelial ( arrows ) . Scale bar , 100 μm . of caspase - 3 normalized to of CD31 - stained vessels ( mean ± s . e . m ; n = 5 ; * * P = 0 . 007 using paired Student ' s t - ) .

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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 S4 ) . Representative are shown at 0 2 h . Arrowheads indicate detached 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 migration was determined using a scratch wound with a co - culture model . A wound was generated in a confluent HUVEC monolayer , were cultured in basal medium with or without VEGF ( 25 ng / ml ) in a 24 - well plate . At same time , transwells containing Sema3C or GFP - expressing pericytes . Representative pictures show remaining gaps within HUVEC monolayer . Scale bar , 200 μm . C Migration was measured after 24 h normalized to control treatment ( mean ± s . e . m ; n = 3 independent experiments , control vs . Sema3C ( − VEGF ) , * P = 0 . 021 control vs . Sema3C ( + VEGF ) , * P = 0 . 039 using unpaired Student ' s t - ) .

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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 . lysates were subjected to SDS - PAGE probed with anti - phosphoT180 / Y182 p38MAPK antibody . of p38MAPK activation was normalized to total p38MAPK presented as to with control medium without VEGF treatment . Control vs . Sema3C ( − VEGF ) , * P = 0 . 046 control vs . Sema3C ( + VEGF ) , * P = 0 . 043 . ( E ) HUVECs were treated as described before activation of FAK detected by anti - phosphoY397 FAK antibody normalized to total amount of FAK . Control vs . Sema3C ( − VEGF ) , * P = 0 . 033 control vs . Sema3C ( + VEGF ) , * P = 0 . 047 . ( Mean ± s . e . m ; n = 3 independent experiments ; unpaired Student ' s t - . )

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

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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 Western blotting were performed after 48 h ( n = 3 ; paired Student ' s t - , NS siRNA vs . PlexinD1 siRNA , * * * P = 0 . 0004 ) . lysates were subjected to SDS - PAGE probed with anti - PlexinD1 .

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

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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).


Silencing of Nrp - 1 by lentiviral particles expressing shRNA . After culturing 72 h in selection medium , was harvested for qPCR . NS shRNA vs . Nrp - 1 shRNA : # 1 , * P = 0 . 017 # 2 , * P = 0 . 02 ; n = 3 ; mean ± s . e . m . lysates were subjected to SDS - PAGE probed with anti - Nrp - 1 .

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Silencing of Nrp-1 by lentiviral particles expressing shRNA. After culturing 72 h in selection medium, RNA was harvested for qPCR analysis. NS shRNA vs. Nrp-1 shRNA: #1, *P = 0.017 and #2, *P = 0.02; n = 3; mean ± s.e.m. Cell lysates were subjected to SDS-PAGE and probed with anti-Nrp-1.


Depletion of Nrp - 1 abolished response to Sema3C . Sema3Cp60 had no effect on HUVEC adhesion . Arrows indicate ruffling . Scale bar , 20 μm . repellence was quantified as number of with ruffling normalized to number of nuclei ( n = 5 ; mean ± s . e . m . ; paired Student ' s t - , * * * P = 0 . 0003 ) .

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Depletion of Nrp-1 abolished the response to Sema3C. Sema3Cp60 had no effect on HUVEC adhesion. Arrows indicate 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 = 0.0003).


HUVECs were treated with Sema3C - conditioned medium for 10 min lysed , Nrp - 1 was immunoprecipitated . Bound Sema3C was detected by Western blotting .

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HUVECs were treated with Sema3C-conditioned medium for 10 min and lysed, and Nrp-1 was immunoprecipitated. Bound Sema3C was detected by Western blotting.


scope analyses showed Sema3C PlexinD1 expression in pre - retinal tufts ( OIR model , P19 ) . retinae were subsequently stained against collagen IV . Sema3CmRNA was detected in peri - endothelial . Scale bar : 20 μm .

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RNA scope analyses showed Sema3C and PlexinD1 expression in pre-retinal tufts (OIR model, P19). The retinae were subsequently stained against collagen IV. Sema3CmRNA was detected in peri-endothelial cells. Scale bar: 20 μm.


Nrp - 1 expression in pre - retinal tufts ( OIR model , P17 ) . Abundant expression of Nrp1 in endothelial . Pericytes at vascular tufts were immunostained with anti - NG2 . Scale bar , 20 μm .

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Nrp-1 expression in pre-retinal tufts (OIR model, P17). Abundant expression of Nrp1 in endothelial cells. Pericytes at vascular tufts were immunostained with anti-NG2. Scale bar, 20 μm.


H & amp ; amp ; E shows neovascular tufts ( arrows ) . Representative of five retinae per group in two independent experiments . Scale bar , 50 μm .

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H&amp;E staining shows neovascular tufts (arrows). Representative of five retinae per group in two independent experiments. Scale bar, 50 μm.


Whole - mount CD31 immunostaining . defines whole retinal , red outlines vaso - obliterative zone . Inset image shows large numbers of peri - retinal tufts ( i , arrow heads ) in control IgG - Fc - treated retinae compared to Sema3C - treated retinae ( ii ) . Scale bar , 500 μm ( upper panel ) 100 μm ( lower panel ) . avascular is presented as of whole retinal ( n = 10 retinae in each group ; n . s . , non - significant using paired Student ' s t - ) . formation of pre - retinal tufts was quantified in superficial layer of retinal vasculature normalized to whole retinal ( n = 6 retinae in each group ; * P = 0 . 033 using paired Student ' s t - ) .

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Whole-mount CD31 immunostaining. The white line defines the whole retinal area, and red line outlines the vaso-obliterative zone. Inset image shows large numbers of peri-retinal tufts (i, arrow heads) in control IgG-Fc-treated retinae compared to Sema3C-treated retinae (ii). Scale bar, 500 μm (upper panel) and 100 μm (lower panel).The avascular area is presented as percentage of the whole retinal area (n = 10 retinae in each group; n.s., non-significant using paired Student's t-test).The formation of pre-retinal tufts was quantified in the superficial layer of the retinal vasculature and normalized to whole retinal area (n = 6 retinae in each group; *P = 0.033 using paired Student's t-test).


ISRE - luciferase reporter in 293T infected with BTV - 8 or mock - infected ( Control ) treated with 1000 U / ml of universal - IFN Luminescence fold induction was measured at different time points p . i . Mean luciferase activity fold inductions in control or BTV - infected in three experiments are presented . *

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ISRE-luciferase reporter assay in 293T cells infected with BTV-8 or mock-infected (Control) and treated with 1000 U/ml of universal-IFN Luminescence fold induction was measured at different time points p.i. Mean luciferase activity fold inductions in control or BTV-infected in three experiments are presented. *


- luciferase reporter in Vero infected with BTV - 8 or mock - infected ( Control ) treated wit 5 ng / ml of IFN - γ . Luminescence fold induction was measured at different time points p . i . Mean luciferase activity fold inductions in control or BTV - infected in three experiments are

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GAS-luciferase reporter assay in Vero cells infected with BTV-8 or mock-infected (Control) and treated wit 5 ng/ml of IFN-γ. Luminescence fold induction was measured at different time points p.i. Mean luciferase activity fold inductions in control or BTV-infected in three experiments are presented


qRT - PCR of ISGs induction in mock - or BTV - 8 - infected 293T after type I IFN treatment for 8 hr . Mean mRNA fold inductions for 3 experiments are presented .

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qRT-PCR analysis of ISGs induction in mock- or BTV-8-infected 293T cells after type I IFN treatment for 8 hr. Mean mRNA fold inductions for 3 experiments are presented.


qRT - PCR of ISGs induction in mock - or BTV - 8 - infected Vero after type II IFN treatment for 8 hr . Mean mRNA fold inductions for 3 experiments are presented .

qRT-PCR analysis of ISGs induction in mock- or BTV-8-infected Vero cells after type II IFN treatment for 8 hr. Mean mRNA fold inductions for 3 experiments are presented.


Immunoblot of Vero infected with BTV - 8 for 16 h , probed for total phosphorylated STAT1 / 2 .

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Immunoblot of Vero cells infected with BTV-8 for 16 h, and probed for total and phosphorylated STAT1/2 levels.


Vero infected with BTV - 8 were fixed at 16hpi , stained with anti - BTV - VP7 antibody .

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Vero cells infected with BTV-8 were fixed at 16hpi, and stained with anti-BTV-VP7 antibody.


STAT1 phosphorylation in IFN - I - treated Vero infected with BTV - 8 detected by ( C ) flow cytometry ( D ) confocal microscopy ( Scale bar = 30 µm ) . Means ± SD of P - STAT1 mean ( MFI ) in 3 experiments are shown .

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STAT1 phosphorylation in IFN-I-treated Vero cells infected with BTV-8 detected by (C) flow cytometry and (D) confocal microscopy (Scale bar = 30 µm). Means ± SD of P-STAT1 mean fluorescence intensity (MFI) in 3 experiments are shown.


STAT2 expression in BTV - 8 - infected Vero detected by ( E ) flow cytometry ( F ) confocal microscopy ( Scale bar = 30 µm ) . Means ± SD of STAT2 MFI in 3 experiments are shown .

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STAT2 expression in BTV-8-infected Vero cells detected by (E) flow cytometry and (F) confocal microscopy (Scale bar = 30 µm). Means ± SD of STAT2 MFI in 3 experiments are shown.


Vero were infected at MOI 1 ( or mock infected ) treated with proteasome inhibitors MG132 ( MG 20 µM ) , lactacystin ( LC 20 µM ) , chloroquine ( CQ 50 µM ) , 3 - MA ( 5 mM ) , or bafilomycin - A1 ( BAF - A1 0 . 2 µM ) for final 6h . Representative flow cytometry histogram of BTV - VP7 expression in inhibitor - treated .

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Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), or bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. Representative flow cytometry histogram of BTV-VP7 expression in inhibitor-treated cells.


Vero were infected at MOI 1 ( or mock infected ) treated with proteasome inhibitors MG132 ( MG 20 µM ) , lactacystin ( LC 20 µM ) , chloroquine ( CQ 50 µM ) , 3 - MA ( 5 mM ) , for final 6h . Immunoblots probed for STAT1 , STAT2 , LC3 GAPDH expression .

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Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), for the final 6h. Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression.


Vero were infected at MOI 1 ( or mock infected ) treated with bafilomycin - A1 ( BAF - A1 0 . 2 µM ) for final 6h . Immunoblots probed for STAT1 , STAT2 , LC3 GAPDH expression .

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Vero cells were infected at MOI 1 (or mock infected) and treated with bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression.


Immunofluorescence of BTV - 8 or mock - infected Vero ( treated for last 6h with BAF - A1 ) stained for BTV , STAT2 p62 . Inset shows detail of STAT2 p62 colocalization . Nuclei were counterstained with DAPI . Arrowheads in inset indicate p62 STAT2 colocalization . Scale bar = 20 μm . Inset scale bar = 3 μm .

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Immunofluorescence of BTV-8 or mock-infected Vero cells (treated for the last 6h with BAF-A1) stained for BTV proteins, STAT2 and p62. Inset shows detail of STAT2 and p62 colocalization. Nuclei were counterstained with DAPI. Arrowheads in inset indicate p62 and STAT2 colocalization. Scale bar = 20 μm. Inset scale bar = 3 μm.


ISRE - luciferase reporter assays in 293T stimulated with IFN - U co - transfected with BTV - VP6 , - VP7 , - NS1 , - NS2 , - NS3 , or - NS4 - expressing plasmids or empty expression plasmid ( Control ) . Representative mean fold inductions of luciferase activity of three independent experiments are shown .

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ISRE-luciferase reporter assays in 293T cells stimulated with IFN-U co-transfected with BTV -VP6, -VP7, -NS1, -NS2, -NS3, or -NS4-expressing plasmids or empty expression plasmid (Control). Representative mean fold inductions of luciferase activity of three independent experiments are shown.


- luciferase reporter assays in Vero stimulated with IFN - γ co - transfected with BTV - VP6 , - VP7 , - NS1 , - NS2 , - NS3 , or - NS4 - expressing plasmids or empty expression plasmid ( Control ) . Representative mean fold inductions of luciferase activity of three independent experiments are shown .

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GAS-luciferase reporter assays in Vero cells stimulated with IFN-γ co-transfected with BTV -VP6, -VP7, -NS1, -NS2, -NS3, or -NS4-expressing plasmids or empty expression plasmid (Control). Representative mean fold inductions of luciferase activity of three independent experiments are shown.


Immunoblot of 293T transfected with BTV - NS2 , - NS3 , - NS4 ( FLAG ) plasmids NiV - V ( HA ) WNV - NS5 ( HA ) used as control for STAT1 phosphorylation inhibition DENV - NS5 ( HA ) used as control for STAT2 degradation .

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Immunoblot of 293T cells transfected with BTV -NS2, -NS3, -NS4 (FLAG) plasmids and NiV-V (HA) and WNV-NS5 (HA) used as control for STAT1 phosphorylation inhibition and DENV-NS5 (HA) used as control for STAT2 degradation.


Three independent immunoblot experiments as in ( C ) were quantified for STAT1 , P - STAT1 , STAT2 P - STAT2 . Mean ± SD normalized to GAPDH non - transfected .

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Three independent immunoblot experiments as in (C) were quantified for STAT1, P-STAT1, STAT2 and P-STAT2. Mean ± SD normalized to GAPDH and non-transfected cells.


Immunoprecipitation ( IP ) for STAT1 / 2 binding using 293T transfected with BTV - NS2 , - NS3 , or - NS4 plasmids . NiV - V used as positive control for STAT1 STAT2 binding , DENV - NS5 used as positive control for STAT2 binding . were treated ( or left untreated ) with 1000 U / ml universal - IFN for final 16 h . Immunoblots of WCE IP were probed ( IB ) for STAT1 , STAT2 , HA ( NiV V , DENV NS5 ) , FLAG ( NS2 , NS3 , NS4 ) GAPDH .

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Immunoprecipitation (IP) for STAT1/2 binding using 293T transfected with BTV -NS2, -NS3, or -NS4 plasmids. NiV-V used as positive control for STAT1 and STAT2 binding, DENV-NS5 used as positive control for STAT2 binding. Cells were treated (or left untreated) with 1000 U/ml universal-IFN for the final 16 h. Immunoblots of WCE and IP were probed (IB) for STAT1, STAT2, HA (NiV V, DENV NS5), FLAG (NS2, NS3, NS4) and GAPDH.


STAT1 phosphorylation ( G ) STAT2 translocation in BTV - NS3 - transfected Vero detected by immunofluorescence confocal microscopy after 30 min IFN treatment . ( Scale bar = 20 µm ) . Arrowheads in merge indicate examples of transfected .

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STAT1 phosphorylation and (G) STAT2 nuclear translocation in BTV-NS3-transfected Vero cells detected by immunofluorescence and confocal microscopy after 30 min IFN treatment. (Scale bar = 20 µm). Arrowheads in merge images indicate examples of transfected cells.


Immunoblots of BTV - NS3 - transfected 293T treated with proteasome inhibitors MG or LC . Membranes were probed for STAT1 , STAT2 , BTV - NS3 ( FLAG ) GAPDH . ( B - D ) Immunoblots of BTV - NS3 - transfected 293T treated with increasing of lysosome acidification inhibitors ( B ) CQ or ( C ) NH4Cl or with increasing of ( D ) autophagosome formation inhibitor 3 - MA , probed ( IB ) for STAT1 , STAT2 , BTV - NS3 ( FLAG ) GAPDH expression .

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Immunoblots of BTV-NS3-transfected 293T cells treated with proteasome inhibitors MG or LC. Membranes were probed for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH. (B-D) Immunoblots of BTV-NS3-transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors (B) CQ or (C) NH4Cl or with increasing concentration of the (D) autophagosome formation inhibitor 3-MA, and probed (IB) for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH expression.


293T were co - transfected with NS3 expression plasmid ( or empty plasmid as control ) siRNA for Atg7 Lysates were obtained from FACS - sorted transfected . Immunoblots of sorted were probed for STAT1 ; STAT2 ; Atg7 FLAG - tagged NS3 ; LC3 GAPDH .

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293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Atg7 Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Atg7 FLAG-tagged NS3; LC3 and GAPDH.


293T were co - transfected with NS3 expression plasmid ( or empty plasmid as control ) siRNA for Beclin - 1 ( or siRNA control or no as controls ) . Lysates were obtained from FACS - sorted transfected . Immunoblots of sorted were probed for STAT1 ; STAT2 ; Beclin - 1 FLAG - tagged NS3 ; LC3 GAPDH .

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293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Beclin-1 (or siRNA control or no RNA as controls). Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Beclin-1 FLAG-tagged NS3; LC3 and GAPDH.


Representative flow cytometry dot plots of Vero infected with rgBTV - 8 or BTV - 8 ΔNS3 stained for BTV - VP7 . Gating on VP7 + was used for sorting to obtain lysates from infected .

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Representative flow cytometry dot plots of Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and stained for BTV-VP7. Gating on VP7+ cells was used for sorting to obtain lysates from infected cells.


Immunoblot of FACS - sorted Vero infected with rgBTV - 8 or BTV - 8 ΔNS3 treated with IFN - U for 30min prior to fixation , sorting . Membranes were probed for STAT1 , P - STAT1 , STAT2 GAPDH .

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Immunoblot of FACS-sorted Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and treated with IFN-U for 30min prior to fixation, staining and sorting. Membranes were probed for STAT1, P-STAT1, STAT2 and GAPDH.


Immunofluorescence confocal of Vero infected with rgBTV - 8 , BTV - 8 ΔNS3 or mock - infected treated with IFN - U for 30min prior to fixation . were stained for BTV - VP7 ( I ) P - STAT1 or ( J ) STAT2 . Nuclei were counterstained with DAPI . ( Scale bar = 30 μm ) . ( I ) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV - 8 ΔNS3 infected . ( J ) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV - 8 ΔNS3 - infected .

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Immunofluorescence confocal images of Vero cells infected with rgBTV-8, BTV-8 ΔNS3 or mock-infected and treated with IFN-U for 30min prior to fixation and staining. Cells were stained for BTV-VP7 and (I) P-STAT1 or (J) STAT2. Nuclei were counterstained with DAPI. (Scale bar = 30 μm). (I) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV-8 ΔNS3 infected cells. (J) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV-8 ΔNS3-infected cells.


WCE FLAG - tag NS3 IP immunoblots of 293T transfected with BTV - NS3 - FLAG plasmids increasing amounts of Ubiquitin - HA plasmids , probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 ( FLAG ) or GAPDH expression .

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WCE and FLAG-tag NS3 IP assay immunoblots of 293T transfected with BTV-NS3-FLAG plasmids and increasing amounts of Ubiquitin-HA plasmids, and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG) or GAPDH expression.


Immunofluorescence of Vero co - transfected with BTV - NS3 ubiquitin acquired with Airyscan super - resolution . Scale bar = 10 μm . Inset shows detailed arrowheads indicate NS3 ubiquitin signal co - incidence ( scale bar = 2 μm ) .

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Immunofluorescence staining of Vero cells co-transfected with BTV-NS3 and ubiquitin and acquired with Airyscan super-resolution. Scale bar = 10 μm. Inset shows detailed area and arrowheads indicate NS3 and ubiquitin signal co-incidence (scale bar = 2 μm).


profile of section indicated by ( blue : DAPI , green : NS3 ; red : Ub ) . Arrowheads on image arrows on profile indicate matching signal for NS3 Ub .

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Fluorescence intensity profile of cell section indicated by the white line (blue: DAPI, green: NS3; red: Ub). Arrowheads on image and arrows on intensity profile indicate matching fluorescence signal for NS3 and Ub.


FLAG - tag IP of BTV - NS3 WCE immunoblot of 293T co - transfected with BTV FLAG - NS3 - , - NS3 - K13R , - NS3 - K15R , - NS3 - K13 / 15R or - NS3 - PPRY / AARH plasmid Ubiquitin - HA plasmid probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 ( FLAG ) , STAT2 GAPDH expression .

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FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected with BTV FLAG-NS3-, -NS3-K13R, -NS3-K15R, -NS3-K13/15R or -NS3-PPRY/AARH plasmid and Ubiquitin-HA plasmid and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG), STAT2 and GAPDH expression.


WCE HA - tag Ubiquitin IP immunoblots of 293T transfected with Ubiquitin - HA plasmids increasing amounts of BTV - FLAG - NS3 - K13 / 15R , probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 - K13 / 15R ( FLAG ) or GAPDH expression .

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WCE and HA-tag Ubiquitin IP assay immunoblots of 293T transfected with Ubiquitin-HA plasmids and increasing amounts of BTV-FLAG-NS3-K13/15R, and probed (IB) for ubiquitin (HA), BTV-NS3-K13/15R (FLAG) or GAPDH expression.


ISRE - driven luciferase reporter in 293T transfected with empty expression plasmid ( Control ) , BTV - NS2 ( 300 ng ) , - NS3 ( 50 , 300 600 ng ) , or - NS3 - K13 / 15R ( 50 , 300 600 ng ) - expressing plasmids . Mean % inductions of luciferase activity of three independent experiments are shown .

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ISRE-driven luciferase reporter assay in 293T cells transfected with empty expression plasmid (Control), BTV -NS2 (300 ng), -NS3 (50, 300 and 600 ng), or -NS3-K13/15R (50, 300 and 600 ng)-expressing plasmids. Mean % inductions of luciferase activity of three independent experiments are shown.


FLAG - tag IP of BTV - NS3 WCE immunoblot of 293T co - transfected FLAG - BTV - NS3 plasmid HA - UB - WT or HA - UB lysine ( K to R ) mutants . IP were probed ( IB ) for ubiquitin ( HA ) BTV - NS3 ( FLAG ) expression . WCE were probed for STAT1 , STAT2 , ubiquitin ( HA ) , BTV - NS3 ( FLAG ) , GAPDH expression .

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FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected FLAG-BTV-NS3 plasmid and HA-UB-WT or HA-UB lysine (K to R) mutants. IP were probed (IB) for ubiquitin (HA) and BTV-NS3 (FLAG) expression. WCE were probed for STAT1, STAT2, ubiquitin (HA), BTV-NS3 (FLAG), and GAPDH expression.


( a , b ) in spleens of Slamf1 − / − Rag1 − / − Rag1 − / − BALB / c mice ( a ) or Slamf1 − / − Slamf1 + / + BALB / c mice ( b ) 48 h after intraperitoneal injection of virulent S . typhimurium 14028s or attenuated S . typhimurium SseB − . CFU , colony - forming units ; ND , not detectable . Data are representative of four independent experiments ( mean s . d . ) .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacteria in the spleens of Slamf1−/−Rag1−/− and Rag1−/− BALB/c mice (a) or Slamf1−/− and Slamf1+/+ BALB/c mice (b) 48 h after intraperitoneal injection of virulent S. typhimurium 14028s or attenuated S. typhimurium SseB−. CFU, colony-forming units; ND, not detectable. Data are representative of four independent experiments (mean and s.d.).


( c - e ) Killing of by peritoneal macrophages from Slamf1 + / + Slamf1 − / − BALB / c mice exposed to S . typhimurium SseB − ( c ) , E . coli F18 ( d ) or S . aureus ( e ) , assessed by gentamycin . Data are representative of five independent experiments ( mean s . d . ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-e) Killing of bacteria by peritoneal macrophages from Slamf1+/+ and Slamf1−/− BALB/c mice exposed to S. typhimurium SseB− (c), E. coli F18 (d) or S. aureus (e), assessed by gentamycin assay. Data are representative of five independent experiments (mean and s.d.).


( f ) Uptake of by Slamf1 + / + ( red solid lines ) or Slamf1 − / − ( black solid lines ) BALB / c peritoneal macrophages incubated at 37 ° C with E . coli - eGFP or S . typhimurium - eGFP or by Slamf1 + / + macrophages incubated for 60 min at 4 ° C with ( dotted lines ) . Right , mean ( MFI ) . Data are representative of three independent experiments ( mean s . d . ) .

O O O O O O B-CONTROLLED_VAR 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 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

(f) Uptake of bacteria by Slamf1+/+ (red solid lines) or Slamf1−/− (black solid lines) BALB/c peritoneal macrophages incubated at 37 °C with E. coli-eGFP or S. typhimurium-eGFP or by Slamf1+/+ macrophages incubated for 60 min at 4 °C with the bacteria (dotted lines). Right, mean fluorescence intensity (MFI). Data are representative of three independent experiments (mean and s.d.).


( a ) NOX2 activity in Slamf1 + / + Slamf1 - / - BALB / c peritoneal macrophages stimulated for 0 - 100 min with E . coli F18 , S . aureus or PMA , assessed with lucigenin . Data are representative of five independent experiments ( mean ± s . d . ) .

O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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) NOX2 activity in Slamf1+/+ and Slamf1-/- BALB/c peritoneal macrophages stimulated for 0-100 min with E. coli F18, S. aureus or PMA, assessed with lucigenin. Data are representative of five independent experiments (mean ± s.d.).


( b ) Phagosomal pH of Slamf1 + / + , Slamf1 - / - gp91phox - / - B6 macrophages loaded for 0 - 200 min with pHrodo - coated E . coli or S . aureus , analyzed by flow cytometry . Data are representative of three independent experiments ( mean ± s . d . ) .

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

(b) Phagosomal pH of Slamf1+/+, Slamf1-/- and gp91phox-/- B6 primary macrophages loaded for 0-200 min with pHrodo-coated E. coli or S. aureus, analyzed by flow cytometry. Data are representative of three independent experiments (mean ± s.d.).


( c ) NOX2 activity in macrophages in response to LPS , purified OmpC , peptidoglycan ( PGN ) or PMA , assessed with lucigenin . TLR4 - KO , TLR4 - deficient ( del / Jtht ; C3H ) . Data are representative of five independent experiments ( mean ± s . d . ) .

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

(c) NOX2 activity in primary macrophages in response to LPS, purified OmpC, peptidoglycan (PGN) or PMA, assessed with lucigenin. TLR4-KO, TLR4-deficient (strain del/Jtht; C3H). Data are representative of five independent experiments (mean ± s.d.).