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

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


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

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

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 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

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

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


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

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


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

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


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

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


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

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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 ) .

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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 ) .

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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 ) .

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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 .

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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 .

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 .

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 ) ) .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 - ) .

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

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


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

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

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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 - ) .

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

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 .

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 - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 - . )

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


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 .

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

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 ) .

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

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 .

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

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 .

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 .

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

H&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 - ) .

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

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 . *

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

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

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

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 .

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

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 .

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 .

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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 .

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 .

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

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 .

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 . ) .

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(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 . ) .

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(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 . ) .

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(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 . ) .

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(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 . ) .

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(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 . ) .

(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.).


( a ) microscopy of E . coli - containing phagosomes in macrophages transfected with RFP - conjugated LAMP - 1 , showing colocalization with E . coli - eGFP or S . aureus - eGFP . Right , of LAMP - 1 + phagosomes in microscopy at left .

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(a) Fluorescence microscopy of E. coli-containing phagosomes in primary macrophages transfected with RFP-conjugated LAMP-1, showing colocalization with E. coli-eGFP or S. aureus-eGFP. Right, quantification of LAMP-1+ phagosomes in the microscopy at left.


( b ) Immunoblot of LAMP - 1 in phagosomes isolated by sucrose - gradient flotation from RAW264 . 7 macrophages after phagocytosis by Slamf1 - or mock - transfected RAW264 . 7 macrophages of beads coated with E . coli outer extract . WCL , whole - lysate . Right , of LAMP - 1 in immunoblot at left .

(b) Immunoblot analysis of LAMP-1 in phagosomes isolated by sucrose-gradient flotation from RAW264.7 macrophages after phagocytosis by Slamf1- or mock-transfected RAW264.7 macrophages of beads coated with E. coli outer membrane extract. WCL, whole-cell lysate. Right, quantification of LAMP-1 in the immunoblot at left.


( c ) Localization of MHC class II - eGFP ( MHCII GFP ) in phagosomes of macrophages from Slamf1 + / + or Slamf1 − / − MHC class II - eGFP ( B6 ) mice 3 - μm beads coated with E . coli outer extract . ( d ) Entry of 3 - μm beads coated with E . coli outer extract into lysosomes loaded with Texas red - dextran . Right ( c , d ) , of in microscopy at left . Numbers in bottom right corners ( a , c , d ) indicate time ( in min ) . Original magnification ( a - c ) , × 60 . DIC , differential interference contrast . Data are representative of three combined experiments ( a ) or at least three independent experiments ( b - d ) with at least 100 beads or 80 in each ( error bars , s . d . ) .

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(c) Localization of MHC class II-eGFP (MHCII GFP) in phagosomes of primary macrophages from Slamf1+/+ or Slamf1−/− MHC class II-eGFP (B6) mice and 3-μm beads coated with E. coli outer membrane extract. (d) Entry of 3-μm beads coated with E. coli outer membrane extract into lysosomes loaded with Texas red-dextran. Right (c,d), quantification of fluorescence in the microscopy at left. Numbers in bottom right corners (a,c,d) indicate time (in min). Original magnification (a-c), ×60. DIC, differential interference contrast. Data are representative of three combined experiments (a) or at least three independent experiments (b-d) with at least 100 beads or 80 bacteria in each (error bars, s.d.).


( a , b ) Association of EEA - 1 ( a ) or Rab5 ( b ) with phagosomes generated in macrophages by 3 - μm beads coated with E . coli outer extract ; numbers in bottom corners indicate time ( in min ) . α - , anti - . Original magnification , × 60 . Right ( a , b ) , of . Data are representative of at least three independent experiments ( error bars , s . d . ) .

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(a,b) Association of EEA-1 (a) or Rab5 (b) with phagosomes generated in primary macrophages by 3-μm beads coated with E. coli outer membrane extract; numbers in bottom corners indicate time (in min). α-, anti-. Original magnification, ×60. Right (a,b), quantification of fluorescence. Data are representative of at least three independent experiments (error bars, s.d.).


( a ) Colocalization of SLAM in RAW264 . 7 macrophages transfected with SLAM - mCherry allowed to phagocytose E . coli - eGFP or S . aureus - eGFP for 0 - 120 min ( time ( in min ) , bottom right corners at left ) . Original magnification , × 60 . Right , of at left . Data are representative of two independent experiments with a minimum of 40 per time point ( error bars , s . d . ) .

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(a) Colocalization of SLAM and bacteria in RAW264.7 macrophages transfected with SLAM-mCherry and allowed to phagocytose E. coli-eGFP or S. aureus-eGFP for 0-120 min (time (in min), bottom right corners at left). Original magnification, ×60. Right, quantification of fluorescence at left. Data are representative of two independent experiments with a minimum of 40 bacteria per time point (error bars, s.d.).


( b ) Immunoblot of phagosome isolates from RAW264 . 7 macrophages transiently transfected with cDNA encoding Myc - tagged SLAM ( Slamf1 - Myc ) or mock transfected allowed to phagocytose beads coated with E . coli outer extract for 60 or 120 min . kDa , kilodaltons . Data are representative of at least three independent experiments .

(b) Immunoblot analysis of phagosome isolates from RAW264.7 macrophages transiently transfected with cDNA encoding Myc-tagged SLAM (Slamf1-Myc) or mock transfected and allowed to phagocytose beads coated with E. coli outer membrane extract for 60 or 120 min. kDa, kilodaltons. Data are representative of at least three independent experiments.


( a ) Luciferase activity in Jurkat transfected with a fusion of SLAM CD3ζ a luciferase reporter ( as described in Results ) , plus a renilla luciferase reporter , then exposed to heat - killed ( top ) .

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(a) Luciferase activity in Jurkat cells transfected with a fusion of SLAM and CD3ζ and a luciferase reporter (as described in Results), plus a renilla luciferase reporter, then exposed to heat-killed bacteria (top).


( b ) Luciferase activity in Jurkat transfected with fusion in a ( Slamf1 - CD3ζ ) or a fusion of SLAM ectodomain construct lacking immunoglobulin V domain CD3ζ ( ΔIgV - Slamf1 - CD3ζ ) , luciferase reporters as in a , then exposed to heat - killed E . coli F18 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luciferase activity in Jurkat cells transfected with the fusion in a (Slamf1-CD3ζ) or a fusion of SLAM ectodomain construct lacking the immunoglobulin V domain and CD3ζ (ΔIgV-Slamf1-CD3ζ), and luciferase reporters as in a, then exposed to heat-killed E. coli F18.


( c ) Luciferase activity in Jurkat transfected as in a , then left uninoculated ( − ) or inoculated with 10 × 108 E . coli F18 ( + ) , followed by addition of monoclonal antibody ( mAb ; amount , under graph ) 9D1 to SLAM ( α - SLAM ) or rat immunoglobulin G2b isotype - matched control antibody ( Rat IgG2b ) .

(c) Luciferase activity in Jurkat cells transfected as in a, then left uninoculated (−) or inoculated with 10 × 108 E. coli F18 (+), followed by the addition of monoclonal antibody (mAb; amount, under graph) 9D1 to SLAM (α-SLAM) or rat immunoglobulin G2b isotype-matched control antibody (Rat IgG2b).


( d ) Luciferase activity in Jurkat transfected as in a , then exposed to medium alone ( Med ) , wild - type E . coli ( JM101 ) , or JM101 E . coli mutants lacking both Omp C Omp F ( HN705 ) or lacking either OmpC ( ΔOmpC ) or OmpF ( ΔOmpF ) . Data are representative of at least three independent experiments ( error bars ( a ) , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luciferase activity in Jurkat cells transfected as in a, then exposed to medium alone (Med), wild-type E. coli (JM101), or JM101 E. coli mutants lacking both Omp C and Omp F (HN705) or lacking either OmpC (ΔOmpC) or OmpF (ΔOmpF). Data are representative of at least three independent experiments (error bars (a), s.d.).


( a , b ) Production of phagosomal PtdIns ( 3 ) P in E . coli - containing phagosomes of peritoneal macrophages transfected with reporter cDNA encoding an eGFP - tagged PX domain of p40phox ( p40 PX - eGFP ) treated with DsRed - expressing E . coli ( E . coli - DsRed ; a ) or beads coated with E . coli outer extract ( b ) . Numbers in bottom right corners indicate time ( in min ) . Original magnification , × 60 . Right , of in microscopy at left .

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(a,b) Production of phagosomal PtdIns(3)P in E. coli-containing phagosomes of primary peritoneal macrophages transfected with reporter cDNA encoding an eGFP-tagged PX domain of p40phox (p40 PX-eGFP) and treated with DsRed-expressing E. coli (E. coli-DsRed; a) or beads coated with E. coli outer membrane extract (b). Numbers in bottom right corners indicate time (in min). Original magnification, ×60. Right, quantification of fluorescence in microscopy at left.


( c ) HPLC of production of PtdIns ( 3 ) P in macrophages labeled with 3H - tagged myoinositol treated with beads coated with E . coli outer extract .

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(c) HPLC analysis of the production of PtdIns(3)P in primary macrophages labeled with 3H-tagged myoinositol and treated with beads coated with E. coli outer membrane extract.


( d ) PtdIns ( 3 ) P production in RAW264 . 7 stably expressing SLAM or a mock construct , transfected with eGFP - tagged p40 PX treated with beads coated with E . coli outer extract . Data are from three combined experiments ( a ) or are representative of at least three independent experiments ( b , d ) or two independent experiments ( c ) with at least 100 beads or per experiment ( error bars , s . d . ) .

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(d) PtdIns(3)P production in RAW264.7 cells stably expressing SLAM or a mock construct, transfected with eGFP-tagged p40 PX and treated with beads coated with E. coli outer membrane extract. Data are from three combined experiments (a) or are representative of at least three independent experiments (b,d) or two independent experiments (c) with at least 100 beads or bacteria per experiment (error bars, s.d.).


( a , b ) Immunoassay of 293 transfected with various combinations of full - SLAM ( a ) or hemagglutinin - tagged ( - HA ) tail - less SLAM ( b ) , EAT - 2A , V5 - tagged ( - V5 ) Vps34 - Vps15 , beclin - 1 ; immunoprecipitated ( IP ) from lysates with monoclonal antibody to SLAM , as well as whole - lysates ( WCL ) , were analyzed by immunoblot ( IB ) with anti - beclin - 1 , anti - Vps34 , anti - V5 , anti - hemagglutinin , anti - SLAM or anti - EAT2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoassay of 293 cells transfected with various combinations of full-length SLAM (a) or hemagglutinin-tagged (-HA) tail-less SLAM (b), EAT-2A, V5-tagged (-V5) Vps34-Vps15, and beclin-1; proteins immunoprecipitated (IP) from lysates with monoclonal antibody to SLAM, as well as whole-cell lysates (WCL), were analyzed by immunoblot (IB) with anti-beclin-1, anti-Vps34, anti-V5, anti-hemagglutinin, anti-SLAM or anti-EAT2.


( c ) Microscopy of RAW264 . 7 transiently transfected with cDNA encoding GFP - tagged beclin - 1 SLAM - mCherry , then treated with beads coated with E . coli outer extract ( at a of 10 : 1 , beads / ) fixed after 60 min . Data are representative of six ( a ) or two ( b , c ) independent experiments .

(c) Microscopy of RAW264.7 cells transiently transfected with cDNA encoding GFP-tagged beclin-1 and SLAM-mCherry, then treated with beads coated with E. coli outer membrane extract (at a ratio of 10:1, beads/cells) and fixed after 60 min. Data are representative of six (a) or two (b,c) independent experiments.


Representative still of control , Rod depleted Rod depleted supplemented with RNAi resistant Venus - Rod treated with nocodazole time in minutes indicated . Scale bar , 5 μm .

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Representative still images of control, Rod depleted cells and Rod depleted cells supplemented with RNAi resistant Venus-Rod treated with nocodazole and time in minutes indicated. Scale bar, 5 μm.


Time from NEBD to mitotic exit for indicated conditions with each circle representing a single analyzed mean time indicated by red standard error of mean shown . number of counted mean time indicated above .

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Time from NEBD to mitotic exit for the indicated conditions with each circle representing a single cell analyzed and mean time indicated by red line and standard error of mean shown. The number of cells counted and mean time indicated above.


Immunofluorescence of ZW10 Mad1 upon depletion of Rod . Scale bar , 5 μm .

Immunofluorescence analysis of ZW10 and Mad1 upon depletion of Rod. Scale bar, 5 μm.


of kinetochore intensities of indicated in control or Rod depleted with signal normalized to CREST Bub1 pSpT normalized to Bub1 . Bar indicates mean standard error of mean is shown by . At least 200 kinetochores from 10 were analyzed representative result from at least 2 independent experiments is shown .

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

Quantification of kinetochore intensities of the indicated proteins in control or Rod depleted cells with the signal normalized to CREST levels and Bub1 pSpT normalized to Bub1. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Time from NEBD to mitotic exit in control depleted , Rod depleted , Bub1 depleted Rod Bub1 co - depleted HeLa , U2OS RPE1 . were treated with nocodazole .

Time from NEBD to mitotic exit in control depleted, Rod depleted, Bub1 depleted and Rod and Bub1 co-depleted HeLa, U2OS and RPE1 cells. Cells were treated with nocodazole.


Mad1 kinetochore in indicated conditions normalized to level of CREST in HeLa . were fixed at 45 min after releasing from RO3306 arrest into nocodazole . Bar indicates mean standard error of mean is shown by . At least 200 kinetochores from 10 were analyzed representative result from at least 2 independent experiments is shown .

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Mad1 kinetochore levels in the indicated conditions normalized to level of CREST in HeLa cells. Cells were fixed at 45 min after releasing from RO3306 arrest into nocodazole. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Western blot of whole extract for indicated conditions . Quantitative of Rod using LiCor technology is indicated below . asterisk indicates an unspecific band .

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

Western blot analysis of whole cell extract for the indicated conditions. Quantitative analysis of Rod levels using LiCor technology is indicated below. The asterisk indicates an unspecific band.


of ZW10 Mad1 kinetochore in indicated conditions . Kinetochore signal is normalized to CREST signal . Bar indicates mean standard error of mean is shown by . At least 200 kinetochores from 10 were analyzed representative result from at least 2 independent experiments shown .

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Analysis of ZW10 and Mad1 kinetochore levels in the indicated cells and conditions. Kinetochore signal is normalized to CREST signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown.


Representative still of unperturbed mitosis for parental , Rod C Rod CR with time in minutes indicated . CFP - Histone 3 was used here as chromosome marker . Scale bar , 5 μm .

Representative still images of unperturbed mitosis for parental cells, Rod C cells and Rod CR cells with time in minutes indicated. CFP-Histone 3 was used here as the chromosome marker. Scale bar, 5 μm.


Time from NEBD to exit of indicated conditions with treated with nocodazole Red circles represent still arrested in mitosis when filming stopped .

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

Time from NEBD to exit of indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit of indicated conditions with treated with taxol . Red circles represent still arrested in mitosis when filming stopped .

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

Time from NEBD to exit of indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit in indicated conditions with treated with nocodazole Red circles represent still arrested in mitosis when filming stopped .

Time from NEBD to exit in indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit in indicated conditions with treated with taxol . Red circles represent still arrested in mitosis when filming stopped .

Time from NEBD to exit in indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.


Mass spectrometry of BubR1 IPs from indicated lines . Relative values ( MaxLFQ , log10 ) are plotted across conditions . Data from of 3 technical repeats of BubR1 purifications with standard deviation indicated .

Mass spectrometry analysis of BubR1 IPs from the indicated cell lines. Relative protein quantification values (MaxLFQ, log10) are plotted across conditions. Data from analysis of 3 technical repeats of BubR1 purifications with standard deviation indicated.


Estimated of Bub1 in indicated conditions relative to parental lines . In Bub1 CR we only detected one peptide from three purifications . Standard deviation indicated .

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

Estimated protein levels of Bub1 in the indicated conditions relative to parental cell lines. In Bub1 CR we only detected one peptide from three purifications. Standard deviation indicated.


Representative still of unperturbed mitosis for parental , Bub1 C Bub1 CR with time in minutes indicated . CFP - Histone 3 was used as chromosome marker . Scale bar , 5 μm .

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

Representative still images of unperturbed mitosis for parental cells, Bub1 C cells and Bub1 CR cells with time in minutes indicated. CFP-Histone 3 was used as the chromosome marker. Scale bar, 5 μm.


Tethering of Mad1 ( Mad1 485 - 718 ) to Ndc80 time from NEBD to exit was measured in each condition . Red circles represent still arrested in mitosis when filming stopped .

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

Tethering of Mad1 (Mad1 485-718) to Ndc80 and the time from NEBD to exit was measured in each condition. Red circles represent cells still arrested in mitosis when the filming stopped.


Tethering of Mad1 ( Mad1 485 - 718 ) to Ndc80 , KNL1 ( Zhang et al , 2017 ) or Bub1 1 - 553ΔCD1 time from NEBD to exit was measured in each condition . Red circles represent still arrested in mitosis when filming stopped .

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

Tethering of Mad1 (Mad1 485-718) to Ndc80, KNL1 (Zhang et al, 2017) or Bub1 1-553ΔCD1 and the time from NEBD to exit was measured in each condition. Red circles represent cells still arrested in mitosis when the filming stopped.


Localization of Venus - tagged Mad1 in parental HeLa , Bub1 CR Rod CR in presence of nocodazole . Scale bar , 5 μm .

Localization of Venus-tagged Mad1 in parental HeLa cells, Bub1 CR cells and Rod CR cells in the presence of nocodazole. Scale bar, 5 μm.


Still from FRAP experiments using Venus - tagged Mad1 in indicated conditions . Image before bleach is shown then time following bleach . Arrowheads indicate kinetochore pairs . Scale bar , 5 μm for whole image 1 μm for zoom in on kinetochores .

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Still images from FRAP experiments using Venus-tagged Mad1 in the indicated conditions. Image before bleach is shown and then time following bleach. Arrowheads indicate kinetochore pairs. Scale bar, 5 μm for whole cell image and 1 μm for the zoom in on kinetochores.


Analysis of Rod levels in cells used for FRAP experiment.


Venus - tagged Mad1 kinetochore versus time from bleaching shortly after NEBD in unperturbed ( left ) or in arrested with nocodazole ( right ) . Data combined from 3 independent experiments standard deviation indicated .

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

Venus-tagged Mad1 kinetochore intensity versus time from bleaching shortly after NEBD in unperturbed cells (left) or in cells arrested with nocodazole (right). Data combined from 3 independent experiments and standard deviation indicated.


A HeLa stably expressing Mad1 - BirA was depleted of Rod synchronized in mitosis using thymidine nocodazole . Biotin was added to media where indicated biotinylated were purified analyzed by western blot with indicated antibodies . Representative of 2 independent experiments .

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

A HeLa cell line stably expressing Mad1-BirA was depleted of Rod and synchronized in mitosis using thymidine and nocodazole. Biotin was added to media where indicated and biotinylated proteins were purified and analyzed by western blot with the indicated antibodies. Representative of 2 independent experiments.


duration from NEBD to exit in each indicated conditions was recorded by time - lapse microscopy .

The duration from NEBD to exit in each indicated conditions was recorded by time-lapse microscopy.


Mitotic duration in indicated conditions expressing Mad1 - Bub1 ( Mad1 485 - 718 fused to Bub1 1 - 553ΔCD1 ) fusion . time from NEBD to exit is indicated . Red circles represent still arrested in mitosis when filming stopped .

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

Mitotic duration in the indicated conditions expressing Mad1-Bub1 (Mad1 485-718 fused to Bub1 1-553ΔCD1) fusion protein. The time from NEBD to exit is indicated. Red circles represent cells still arrested in mitosis when the filming stopped.


depleted of Rod using RNAi transfected with indicated Bub1 constructs . ( D ) Representative immunofluorescence of stained for YFP Mad1 in each condition . Scale bar , 5 μm .

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

Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. (D) Representative immunofluorescence images of cells stained for YFP and Mad1 in each condition. Scale bar, 5 μm.


depleted of Rod using RNAi transfected with indicated Bub1 constructs . Mad1 kinetochore were measured normalized to Bub1 ( YFP ) level . Bar indicates mean standard error of mean is shown by . At least 200 kinetochores from 10 were analyzed representative result from at least 2 independent experiments is shown .

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

Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. Mad1 kinetochore levels were measured and normalized to Bub1 (YFP) level. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Bub1 CR complemented with indicated Bub1 constructs time from NEBD to exit in nocodazole measured by time - lapse microscopy . Mean ( red ) standard error of mean ( black bar ) indicated .

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

Bub1 CR cells complemented with the indicated Bub1 constructs and time from NEBD to exit in nocodazole measured by time-lapse microscopy. Mean (red line) and standard error of mean (black bar) indicated.


Kinetochore of Mad1 in Bub1 CR complemented with indicated YFP tagged Bub1 constructs . Mad1 are normalized to YFP signal . Bar indicates mean standard error of mean is shown by . At least 200 kinetochores from 10 were analyzed representative result from at least 2 independent experiments shown .

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

Kinetochore levels of Mad1 in Bub1 CR cells complemented with the indicated YFP tagged Bub1 constructs. Mad1 levels are normalized to YFP signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown.


Still of unperturbed mitosis of HeLa transfected with Venus - Bub1 4xCD1 . CFP - Histone 3 was used as chromosome marker ( middle panel ) Venus - Bub1 4xCD1 was shown in bottom panel . Scale bar , 5 μm .

Still images of unperturbed mitosis of HeLa cells transfected with Venus-Bub1 4xCD1. CFP-Histone 3 was used as the chromosome marker (the middle panel) and Venus-Bub1 4xCD1 was shown in the bottom panel. Scale bar, 5 μm.


HeLa transfected with indicated Bub1 constructs time from NEBD to exit determined during an unperturbed mitosis . time from NEBD to exit was analyzed in each condition . Mean ( red ) standard error of mean ( black bar ) indicated . ( Mann Whitney , ns : non - significant ) . A representative result from at least 3 independent experiments is shown .

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HeLa cells transfected with the indicated Bub1 constructs and time from NEBD to exit determined during an unperturbed mitosis. The time from NEBD to exit was analyzed in each condition. Mean (red line) and standard error of mean (black bar) indicated. (Mann Whitney test, ns: non-significant). A representative result from at least 3 independent experiments is shown.


Localization of Venus - Rod during unperturbed mitosis in control depleted or Bub1 depleted .

Localization of Venus-Rod during unperturbed mitosis in control depleted or Bub1 depleted cells.


HeLa were depleted of Bub1 using RNAi complemented with indicated Venus - Bub1 constructs . Immunofluorescence were shown for Bub1 ZW10 localization . of Bub1 ZW10 kinetochore normalized to CREST in indicated conditions .

HeLa cells were depleted of Bub1 using RNAi and complemented with indicated Venus-Bub1 constructs. Immunofluorescence images were shown for Bub1 and ZW10 localization. Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions.


HeLa were depleted of Bub1 using RNAi Immunofluorescence were shown for Bub1 ZW10 localization . of Bub1 ZW10 kinetochore normalized to CREST in indicated conditions . with indicated Bub1 fusion

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

HeLa cells were depleted of Bub1 using RNAi Immunofluorescence images were shown for Bub1 and ZW10 localization. Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions. with the indicated Bub1 fusion proteins


A Neurosphere assays were performed using mouse E14 . 5 neural progenitors treated with CRH with or without antalarmin ( Ant ) ( n = 3 biological replicates ) .

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

A Neurosphere assays were performed using mouse E14.5 primary neural progenitors treated with CRH with or without antalarmin (Ant) (n = 3 biological replicates).


B Effects of CRH treatment on stemness of neural progenitor at 2 days post - treatment were analyzed by Western blotting using an anti - SOX2 antibody ( n = 3 biological replicates ) .

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

B Effects of CRH treatment on the stemness of neural progenitor cells at 2 days post-treatment were analyzed by Western blotting using an anti-SOX2 antibody (n = 3 biological replicates).


Effects of CRHR1 overexpression on ( C ) neurosphere

Effects of CRHR1 overexpression on the (C) neurosphere formation


Effects of CRHR1 overexpression on ( D ) SOX2 expression ( n = 3 biological replicates ) .

Effects of CRHR1 overexpression on the (D) SOX2 expression (n = 3 biological replicates).


E CRHR1 knockdown efficiency of shRNAs used in this study ( n = 4 biological replicates ) .

E CRHR1 knockdown efficiency of shRNAs used in this study (n = 4 biological replicates).


Effects of CRHR1 knockdown on ( F ) neurosphere formation ,

Effects of CRHR1 knockdown on the (F) neurosphere formation,


Effects of CRHR1 knockdown on ( G ) SOX2 expression n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

Effects of CRHR1 knockdown on the (G) SOX2 expression n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


Effects of CRHR1 knockdown on ( H ) neurosphere , n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

Effects of CRHR1 knockdown on the (H) neurosphere morphology, n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


Effects of CRHR1 knockdown on ( I ) βIII - tubulin expression . n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

Effects of CRHR1 knockdown on the (I) βIII-tubulin expression. n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


J , K Immunostaining using anti - GFP ( green ) together with ( J ) anti - βIII - tubulin ( clone TuJ1 ; red ) ( n = 5 biological replicates ) or ( K ) anti - GFAP ( red ) antibodies ( n = 3 biological replicates ) after in vitro differentiation of E14 . 5 neural progenitor transduced with a lentiviral vector expressing shCRHR1 .

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J, K Immunostaining using anti-GFP (green) together with (J) anti-βIII-tubulin (clone TuJ1; red) (n = 5 biological replicates) or (K) anti-GFAP (red) antibodies (n = 3 biological replicates) after in vitro differentiation of E14.5 neural progenitor cells transduced with a lentiviral vector expressing shCRHR1.


A Embryonic brains that were intraventricularly electroporated CRHR1 - expressing plasmids at E13 . 5 were harvested at E15 . 5 immunostained using an anti - GFP ( green ) SOX2 ( red ) antibodies . B of fraction in each laminar layer in ( A ) ( n = 3 biological replicates ) . C of SOX2 + among GFP + in ( A ) ( n = 4 biological replicates ) .

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

A Embryonic brains that were intraventricularly electroporated CRHR1-expressing plasmids at E13.5 were harvested at E15.5 and immunostained using an anti-GFP (green) and SOX2 (red) antibodies. B Quantification of the cell fraction in each laminar layer in (A) (n = 3 biological replicates). C Percentage of SOX2+ cells among GFP+ cells in (A) (n = 4 biological replicates).