( b ) Spore viability assay . Individual tetrads were micromanipulated on a agar surface and spores were dissected and arrayed . Genotype was determined by PCR genotyping of the DAD1 locus . Quantification from all tetrad dissections performed , total are shown below . The one - tailed probability of observing 51 % spore viability in DAD1E50D SK1 over a total of 139 tetrads is p < 0 . 000001 , assuming a spore viability of ~ 90 % from wild type SK1 .

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(b) Spore viability assay. Individual tetrads were micromanipulated on a agar surface and spores were dissected and arrayed. Genotype was determined by PCR genotyping of the DAD1 locus. Quantification from all tetrad dissections performed, total are shown below. The one-tailed probability of observing 51% spore viability in DAD1E50D SK1 over a total of 139 tetrads is p < 0.000001, assuming a spore viability of ~90% from wild type SK1.


Double fluorescent whole mount in situ hybridization ( WM - ISH ) to detect her6 ( green ) and her9 ( magenta ) ; coronal view ( left panel ) and transversal section ( right panel ) , scale bar 20μm ; 30 - 32hpf ; annotations denote anterior ( A ) , posterior ( P ) , otic vesicle ( ov ) , dorsal ( D ) and ventral ( V ) .

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Double fluorescent whole mount in situ hybridization (WM-ISH) to detect her6 (green) and her9 (magenta); coronal view (left panel) and transversal section (right panel), scale bar 20μm; 30-32hpf; annotations denote anterior (A), posterior (P), otic vesicle (ov), dorsal (D) and ventral (V).


Representative time series example of Her6 : : Venus expression during development , in the midbrain and hindbrain . Confocal images represented as 2D Maximum projection ; longitudinal view ; scale bar 50μm ; otic vesicle ( ov ) ; also included in Movie EV1 . r1 : rhombomere 1 , r2 : rhombomere 2 , r3 : rhombomere 3 , r4 : rhombomere 4 , r5 : rhombomere 5 , r6 : rhombomere 6 .

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Representative time series example of Her6::Venus expression during development, in the midbrain and hindbrain. Confocal images represented as 2D Maximum projection; longitudinal view; scale bar 50μm; otic vesicle (ov); also included in Movie EV1. r1: rhombomere 1, r2: rhombomere 2, r3: rhombomere 3, r4: rhombomere 4, r5: rhombomere 5, r6: rhombomere 6.


Intensity mean of Her6 : : Venus per rhombomere area over development grouped by expression level , related to the r1 - r6 regions in panel ( c ) : ( d ) r1 and r2 ; ( e ) r3 and r4 ; ( f ) r5 and r6 .

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Intensity mean of Her6::Venus per rhombomere area over development grouped by expression level, related to the r1-r6 regions in panel (c) : (d) r1 and r2; (e) r3 and r4; (f) r5 and r6.


Transversal view of r6 in Her6 : : Venus embryos over time ; Her6 : : Venus protein expression domains : a ventral domain ( arrows ) and a more dorsal lateral domain ( arrowheads ) ; the caax - mRFP was used as membrane marker ( magenta ) ; scale bars 20μm ; images at 30 - 40hpf are maximum projection of 4 z - stacks from Movie EV2 .

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Transversal view of r6 in Her6::Venus embryos over time; Her6::Venus protein expression domains: a ventral domain (arrows) and a more dorsal lateral domain (arrowheads); the caax-mRFP was used as membrane marker (magenta); scale bars 20μm; images at 30-40hpf are maximum projection of 4 z-stacks from Movie EV2.


Quantification of Her6 : : Venus ( + ) cell number ( green ) compared to total cell number ( black ) over development .

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Quantification of Her6::Venus(+) cell number (green) compared to total cell number (black) over development.


Proportional changes in Her6 : : Venus ( + ) cell numbers during development ; bars indicate median and interquartile range of counts collected from 3 different z - stacks per embryo and one embryo per condition ; one - way ANOVA with Bonferroni multiple comparison test with significance p < 0 .

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Proportional changes in Her6::Venus(+) cell numbers during development; bars indicate median and interquartile range of counts collected from 3 different z-stacks per embryo and one embryo per condition; one-way ANOVA with Bonferroni multiple comparison test with significance p<0.01


Nuclear abundance and nuclear concentration of Her6 : : Venus protein in homozygous embryos at different stages during development measured by fluorescence correlation spectroscopy ( FCS ) ; bars indicate median and interquartile range of ( 19hpf : 6 embryos , 50 cells ) , ( 29hpf : 6 embryos , 86 cells ) , ( 33hpf : 5 embryos , 76 cells ) , ( 35hpf : 6 embryos , 59 cells ) and ( 48hpf : 6 embryos , 89 cells ) ; Kruskal - Wallis with Dunn ' s multiple comparison test , significance p < 0 . 05 * , p < 0 . 001 * * * , p < 0 . 0001 * * * * .

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Nuclear abundance and nuclear concentration of Her6::Venus protein in homozygous embryos at different stages during development measured by fluorescence correlation spectroscopy (FCS); bars indicate median and interquartile range of (19hpf: 6 embryos, 50 cells), (29hpf: 6 embryos, 86 cells), (33hpf: 5 embryos, 76 cells), (35hpf: 6 embryos, 59 cells) and (48hpf: 6 embryos, 89 cells); Kruskal-Wallis with Dunn's multiple comparison test, significance p<0.05*, p<0.001***, p<0.0001****.


3D confocal representative images of a single neural progenitor cell ( red arrow ) tracked over time , data starting from 34hpf ; scale bar 10μm , images from Movie EV3 .

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3D confocal representative images of a single neural progenitor cell (red arrow) tracked over time, data starting from 34hpf; scale bar 10μm, images from Movie EV3.


Single cell time series of Her6 : : Venus , mKeima - H2B and Her6 : : Venus signal normalized by mKeima - H2B corresponding to the cell in ( c ) .

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Single cell time series of Her6::Venus, mKeima-H2B and Her6::Venus signal normalized by mKeima-H2B corresponding to the cell in (c).


Chromogenic WM - ISH of miR - 9 using miR - 9 LNA 5 ' - Dig observed at different stages during development ; longitudinal view , anterior to the left .

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Chromogenic WM-ISH of miR-9 using miR-9 LNA 5'-Dig observed at different stages during development; longitudinal view, anterior to the left.


Transverse section of double fluorescent WM - ISH for her6 ( green ) and mir - 9 - 4 ( magenta ) imaged in fixed embryo at 31hpf ; scale bar 30μm .

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Transverse section of double fluorescent WM-ISH for her6 (green) and mir-9-4 (magenta) imaged in fixed embryo at 31hpf; scale bar 30μm.


Confocal imaging of CTRL and MBSm embryo showing Her6 : : Venus expression in the hindbrain , rhombomeres 3 to 6 ( r3 - r6 ) over the course of development ; longitudinal view , scale 30μm , otic vesicle ( ov ) . Images are representing 2D maximum projection .

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Confocal imaging of CTRL and MBSm embryo showing Her6::Venus expression in the hindbrain, rhombomeres 3 to 6 (r3-r6) over the course of development; longitudinal view, scale 30μm, otic vesicle (ov). Images are representing 2D maximum projection.


Her6 : : Venus expression ( red arrows ) in hindbrain , rhombomere 6 of live CTRL and MBSm embryos at 52hpf ; edge of ventricular zone shown in yellow ; transversal view ; scale 30μm .

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Her6::Venus expression (red arrows) in hindbrain, rhombomere 6 of live CTRL and MBSm embryos at 52hpf; edge of ventricular zone shown in yellow; transversal view; scale 30μm.


Her6 : : Venus protein abundance in CTRL versus MBSm homozygous embryos observed in hindbrain , r6 between 34hpf and 69hpf ; bars indicate median and interquartile range of ( 34hpf CTRL : 36 cells , 1 embryo ) , ( 34hpf MBSm : 35 cells , 1 embryo ) , ( 48 ± 2hpf CTRL : 172 cells , 7 embryos ) , ( 48 ± 2hpf MBSm : 124 cells , 5 embryos ) , ( 56 ± 2hpf CTRL : 69 cells , 4 embryos ) , ( 56 ± 2hpf MBSm : 31 cells , 2 embryos ) , ( 69hpf CTRL : 12 cells , 1 embryo ) , ( 69hpf MBSm : 6 cells , 2 embryos ) ; Kruskal - Wallis with Dunn ' s multiple comparison test , significance : p < 0 . 05 * , p < 0 . 001 * * * , p < 0 . 0001 * * * * .

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Her6::Venus protein abundance in CTRL versus MBSm homozygous embryos observed in hindbrain, r6 between 34hpf and 69hpf; bars indicate median and interquartile range of (34hpf CTRL: 36 cells, 1 embryo), (34hpf MBSm: 35 cells, 1 embryo), (48±2hpf CTRL: 172 cells, 7 embryos), (48±2hpf MBSm: 124 cells, 5 embryos), (56±2hpf CTRL: 69 cells, 4 embryos), (56±2hpf MBSm: 31 cells, 2 embryos), (69hpf CTRL: 12 cells, 1 embryo), (69hpf MBSm: 6 cells, 2 embryos); Kruskal-Wallis with Dunn's multiple comparison test, significance: p<0.05*, p<0.001***, p<0.0001****.


Comparative profiles of Her6 protein abundance over development in CTRL and MBSm embryos quantified as median of single cell abundance data in ( f ) .

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Comparative profiles of Her6 protein abundance over development in CTRL and MBSm embryos quantified as median of single cell abundance data in (f).


Representative examples of single cell oscillators observed in CTRL ( a ) and MBSm ( b ) embryos imaged from 34hpf onwards ; time series represent Her6 : : Venus expression relative to mKeima - H2B ( top panel ) and detrended relative signal ( bottom panel ) ; parameters reported for log - likelihood ratio ( LLR ) and period correspond to KOUosc .

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Representative examples of single cell oscillators observed in CTRL (a) and MBSm (b) embryos imaged from 34hpf onwards; time series represent Her6::Venus expression relative to mKeima-H2B (top panel) and detrended relative signal (bottom panel); parameters reported for log-likelihood ratio (LLR) and period correspond to KOUosc .


Pairwise analysis comparing proportion of oscillators in CTRL versus MBSm embryos indicated as dots ; paired t test with two tail significance p < 0 . 01 * * .

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Pairwise analysis comparing proportion of oscillators in CTRL versus MBSm embryos indicated as dots; paired t test with two tail significance p<0.01**.


Pairwise analysis comparing periods observed in CTRL versus MBSm oscillatory cells ; dots indicate median per experiment per condition ; paired t test with two tail not significant .

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Pairwise analysis comparing periods observed in CTRL versus MBSm oscillatory cells; dots indicate median per experiment per condition; paired t test with two tail not significant.


Representative examples of detrended Her6 : : Venus relative to mKeima - H2B in non - oscillatory single cells observed in CTRL ( f ) and MBSm ( g ) embryos imaged from 34hpf onwards .

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Representative examples of detrended Her6::Venus relative to mKeima-H2B in non-oscillatory single cells observed in CTRL (f) and MBSm (g) embryos imaged from 34hpf onwards.


Representative examples of triple fluorescent whole mount WM - ISH labelling of gfap ( green ) , elavl3 ( magenta ) and her6 ( grey ) domains of expression ( top panels ) in hindbrain rhombomere6 ( r6 ) in wild - type embryo observed at 34hpf ; merged images indicate how the her6 expression domain overlaps with the progenitor zone ( NP = gfap ( + ) / elavl3 ( - ) ) , transition zone ( T = gfap ( + ) / elavl3 ( + ) ) and neurogenic zone ( N = gfap ( - ) / elavl3 ( + ) ) ; ( bottom - right panel ) schematic representation showing the her6 domain spanning the NP , T and N zones with quantification of distances from dorsal ; transversal view ; annotations denote dorsal ( D ) and ventral ( V ) ; scale bar 30μm .

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Representative examples of triple fluorescent whole mount WM-ISH labelling of gfap (green), elavl3 (magenta) and her6 (grey) domains of expression (top panels) in hindbrain rhombomere6 (r6) in wild-type embryo observed at 34hpf; merged images indicate how the her6 expression domain overlaps with the progenitor zone (NP=gfap(+)/elavl3(-)), transition zone (T=gfap(+)/elavl3(+)) and neurogenic zone (N=gfap(-)/elavl3(+)); (bottom-right panel) schematic representation showing the her6 domain spanning the NP, T and N zones with quantification of distances from dorsal; transversal view; annotations denote dorsal (D) and ventral (V); scale bar 30μm.


Single molecule fluorescent in situ hybridization ( smFISH ) showing her6 ( green ) , elavl3 ( magenta ) and dapi nuclear staining ( blue ) obtained from hindbrain ( r6 ) sections of wild type embryo at 34hpf ; head arrows indicates examples of co - existence of transcriptional active sites for her6 and elavl3 ; scale bar 3μm .

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Single molecule fluorescent in situ hybridization (smFISH) showing her6 (green), elavl3 (magenta) and dapi nuclear staining (blue) obtained from hindbrain (r6) sections of wild type embryo at 34hpf; head arrows indicates examples of co-existence of transcriptional active sites for her6 and elavl3; scale bar 3μm.


Triple fluorescent WM - ISH for gfap ( green ) and elavl3 ( magenta ) and her6 ( grey ) in the hindbrain ( r6 ) of CTRL ( top ) and MBSm ( bottom ) embryos at 52hpf ; dorsal ( D ) ; ventral ( V ) ; scale bar 30μm . Magnification of inset from merged gfap / elavl3 in ( c ) showing CTRL ( top ) and MBSm ( bottom ) embryos with corresponding NP / T / N and NP / T zones respectively ; dorsal ( D ) ; ventral ( V ) ; scale bar 30μm . Normalized intensity mean of elavl3 and gfap along the DV axis spanning the NP / T / N zones in CTRL and NP / T zones in MBSm respectively ; region of interest ( ROI ) delineates high elavl3 versus gfap in CTRL ( T and N zones ) while in MBSm only the T zone is observed , overlap of elavl3 and gfap intensity mean peaks .

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Triple fluorescent WM-ISH for gfap (green) and elavl3 (magenta) and her6 (grey) in the hindbrain (r6) of CTRL (top) and MBSm (bottom) embryos at 52hpf; dorsal (D); ventral (V); scale bar 30μm. Magnification of inset from merged gfap/elavl3 in (c) showing CTRL (top) and MBSm (bottom) embryos with corresponding NP/T/ N and NP/T zones respectively; dorsal (D); ventral (V); scale bar 30μm. Normalized intensity mean of elavl3 and gfap along the DV axis spanning the NP/T/N zones in CTRL and NP/T zones in MBSm respectively; region of interest (ROI) delineates high elavl3 versus gfap in CTRL (T and N zones) while in MBSm only the T zone is observed, overlap of elavl3 and gfap intensity mean peaks.


Normalized mean of elavl3 and gfap intensities in ROI observed in : ( f ) uninjected ( UI ) ( 5 embryos , 62 slices ) , ( g ) control ( CTRL ) ( 5 embryos , 35 slices ) and ( h ) MBSm ( 8 embryos , 62 slices ) conditions ; bars represent mean and SEM ; multiple t - test with Benjamini , Krieger and Yekuteli discovery , significance : p < 0 . 05 * , p < 0 . 001 * * * , p < 0 . 0001 * * * * .

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Normalized mean of elavl3 and gfap intensities in ROI observed in: (f) uninjected (UI) (5 embryos, 62 slices), (g) control (CTRL) (5 embryos, 35 slices) and (h) MBSm (8 embryos, 62 slices) conditions; bars represent mean and SEM; multiple t-test with Benjamini, Krieger and Yekuteli discovery, significance: p<0.05*, p<0.001***, p<0.0001****.


Transverse sections of double fluorescent WM - ISH for gfap ( green ) and neuroD4 ( blue ) in the hindbrain ( r6 ) of CTRL ( top ) and MBSm ( bottom ) embryos at 52hpf ; dorsal ( D ) ; ventral ( V ) ; scale bar 30μm .

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Transverse sections of double fluorescent WM-ISH for gfap (green) and neuroD4 (blue) in the hindbrain (r6) of CTRL (top) and MBSm (bottom) embryos at 52hpf; dorsal (D); ventral (V); scale bar 30μm.


Chromogenic WM - ISH showing neuroD4 expression intensity in : uninjected ( UI ) 51 / 56 embryos ( 91 % ) , CTRL 50 / 54 embryos ( 93 % ) and mutant ( MBSm ) ; 36 / 65 embryos ( 55 % ) ; longitudinal view , scale bar 100μm .

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Chromogenic WM-ISH showing neuroD4 expression intensity in: uninjected (UI) 51/56 embryos (91%), CTRL 50/54 embryos (93%) and mutant (MBSm); 36/65 embryos (55 %); longitudinal view, scale bar 100μm.


( A ) Murine Ntras enrichment in endothelial cells versus other heart - derived cell types ( data acquired from GSE95755 ( n = 4 mice ) . Data information : In ( A , data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( A ) One - way

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(A) Murine Ntras enrichment in endothelial cells versus other heart-derived cell types (data acquired from GSE95755 (n = 4 mice). Data information: In (A, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (A) One-way ANOVA


( B ) Changes in lncRNA expression from HUVECs exposed to hypoxia ( 0 . 2 % O2 for 12 h or 24 h ) , determined by ribo - minus RNA deep sequencing ( data acquired from GSE107033 ; NTRAS is highlighted ( n = 2 independent biological replicates ) .

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(B) Changes in lncRNA expression from HUVECs exposed to hypoxia (0.2 % O2 for 12 h or 24 h), determined by ribo-minus RNA deep sequencing (data acquired from GSE107033 ; NTRAS is highlighted (n = 2 independent biological replicates).


( C ) Validation of hypoxia - induced NTRAS expression at the indicated time points by RT - qPCR ( n = 4 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . two - tailed unpaired t -

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(C) Validation of hypoxia-induced NTRAS expression at the indicated time points by RT-qPCR (n = 4 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test


( D ) Subcellular localization of NTRAS and control transcripts , assayed by nuclear - cytoplasmic fractionation of HUVECs and RT - qPCR ( n = 3 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . two - tailed unpaired t -

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(D) Subcellular localization of NTRAS and control transcripts, assayed by nuclear-cytoplasmic fractionation of HUVECs and RT-qPCR (n = 3 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test


( E ) FITC - dextran - based in vitro permeability comparing control and NTRAS - silenced HUVECs ( n = 5 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . two - tailed unpaired t - test ,

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(E) FITC-dextran-based in vitro permeability comparing control and NTRAS-silenced HUVECs (n = 5 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test,


( F ) Cumulative in vitro sprout lengths of control and NTRAS - silenced HUVECs under basal conditions and VEGFA stimulation ( n = 3 - 10 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(F) Cumulative in vitro sprout lengths of control and NTRAS-silenced HUVECs under basal conditions and VEGFA stimulation (n = 3-10 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( G ) Cell cycle analysis in Ntras - silenced H5V cells ( n = 3 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( G ) two - way ANOVA ,

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

(G) Cell cycle analysis in Ntras-silenced H5V cells (n = 3 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (G) two-way ANOVA,


( H ) TMR - dextran - based assessment of vascular permeability in vivo , comparing heart homogenates from control and Ntras - silenced mice . Data normalized to organ and body weight ( n = 11 - 12 mice per group ) . Experimental outline on the left . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . H ) two - tailed unpaired t -

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 O O O O O O O

(H) TMR-dextran-based assessment of vascular permeability in vivo, comparing heart homogenates from control and Ntras-silenced mice. Data normalized to organ and body weight (n = 11-12 mice per group). Experimental outline on the left. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. H) two-tailed unpaired t-test


( I ) Survival of control and Ntras - silenced mice under basal conditions ( black , n = 21 - 23 mice per group ) and in hind limb ischemia ( HLI ; red , n = 8 mice per group ) . Number of survivors is indicated ; experimental outline on the left . Data information : ( I ) Mantel - Cox - test .

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

(I) Survival of control and Ntras-silenced mice under basal conditions (black, n = 21-23 mice per group) and in hind limb ischemia (HLI; red, n = 8 mice per group). Number of survivors is indicated; experimental outline on the left. Data information: (I) Mantel-Cox-test.


( A ) Separation of mock and proteinase K - treated HeLa nuclear extracts by sucrose density gradient ultracentrifugation followed by NTRAS detection using RT - qPCR . Fractions 1 and 14 represent top and bottom of the gradient , respectively ( n = 1 ) .

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

(A) Separation of mock and proteinase K-treated HeLa nuclear extracts by sucrose density gradient ultracentrifugation followed by NTRAS detection using RT-qPCR. Fractions 1 and 14 represent top and bottom of the gradient, respectively (n = 1).


( B ) Enrichment of NTRAS by antisense affinity selection from HeLa cell lysate followed by RT - qPCR , comparing a control and an NTRAS - specific probe ( n = 5 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - tailed unpaired t - test .

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

(B) Enrichment of NTRAS by antisense affinity selection from HeLa cell lysate followed by RT-qPCR, comparing a control and an NTRAS-specific probe (n = 5 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( D ) RT - qPCR - based validation of NTRAS - hnRNPL - interaction by anti - hnRNPL RIPs from HUVEC nuclear fractions ( n = 6 independent biological replicates ) . Representative western blot on the right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - tailed unpaired t - test .

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

(D) RT-qPCR-based validation of NTRAS-hnRNPL-interaction by anti-hnRNPL RIPs from HUVEC nuclear fractions (n = 6 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( E ) RT - qPCR analysis of NTRAS expression in HUVECs upon silencing of hnRNPL ( n = 4 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - tailed unpaired t - test .

O O O O O O O O B-MEASURED_VAR 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

(E) RT-qPCR analysis of NTRAS expression in HUVECs upon silencing of hnRNPL (n = 4 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( F ) RPLP0 - normalized NTRAS - CA16 motif expression after hnRNPL silencing determined by RT - PCR ( n = 4 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - tailed unpaired t - test .

O O O O O O B-MEASURED_VAR 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

(F) RPLP0-normalized NTRAS-CA16 motif expression after hnRNPL silencing determined by RT-PCR (n = 4 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( H ) NTRAS and hnRNPL co - regulated alternative splicing ( AS ) events . Data points with an FDR < 0 . 05 are circled ( n = 2 independent 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

(H) NTRAS and hnRNPL co-regulated alternative splicing (AS) events. Data points with an FDR < 0.05 are circled (n = 2 independent biological replicates).


( I ) RT - PCR - based analysis of TJP1 exon 20 inclusion upon silencing of NTRAS in HUVECs ( n = 7 independent biological replicates ) . Representative agarose gels on the right . Data information : , I , data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . I , two - tailed unpaired t - test .

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

(I) RT-PCR-based analysis of TJP1 exon 20 inclusion upon silencing of NTRAS in HUVECs (n = 7 independent biological replicates). Representative agarose gels on the right. Data information: , I, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. I, two-tailed unpaired t-test.


( J ) RT - PCR - based analysis of TJP1 exon 20 inclusion upon silencing of hnRNPL in HUVECs ( n = 4 independent biological replicates ) . Representative agarose gels on the right . Data information : , J ) data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . J ) two - tailed unpaired t - test .

(J) RT-PCR-based analysis of TJP1 exon 20 inclusion upon silencing of hnRNPL in HUVECs (n = 4 independent biological replicates). Representative agarose gels on the right. Data information: , J) data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. J) two-tailed unpaired t-test.


( B ) In vitro splicing efficiency of the TJP1 splice substrate , comparing mock , NTRAS - depleted , and NTRAS - CA16 motif add - back conditions ( n = 7 - 12 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . ( B two - tailed unpaired t - test ,

O O O O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro splicing efficiency of the TJP1 splice substrate, comparing mock, NTRAS-depleted, and NTRAS-CA16 motif add-back conditions (n = 7-12 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. (B two-tailed unpaired t-test,


( C ) Co - precipitation of TJP1 pre - mRNA in anti - hnRNPL RIPs , using nuclear lysates from control and NTRAS - silenced HUVECs ( n = 6 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed paired t -

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

(C) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-silenced HUVECs (n = 6 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test


( D ) Co - precipitation of TJP1 pre - mRNA in anti - hnRNPL RIPs , using nuclear lysates from control and NTRAS - overexpressing cells ( n = 5 independent biological replicates ) . Representative western blot on the right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed paired t - test ,

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

(D) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-overexpressing cells (n = 5 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test,


( E ) Co - precipitation of TJP1 pre - mRNA in anti - hnRNPL RIPs , using nuclear lysates from control and NTRAS - CA16 motif overexpressing cells ( n = 5 independent biological replicates ) . Representative western blot on the right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed paired t - test ,

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

(E) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-CA16 motif overexpressing cells (n = 5 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test,


( F ) RT - PCR - based analysis of TJP1 exon 20 inclusion upon NTRAS overexpression and overexpression of the NTRAS - CA16 motif ( n = 8 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . one - way ANOVA .

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

(F) RT-PCR-based analysis of TJP1 exon 20 inclusion upon NTRAS overexpression and overexpression of the NTRAS-CA16 motif (n = 8 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. one-way ANOVA.


( G ) Endothelial resistance of NTRAS - silenced HUVECs ( n = 3 - 5 independent biological replicates ) , analyzed by electrical cell - substrate impedance sensing ( ECIS ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed unpaired t -

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

(G) Endothelial resistance of NTRAS-silenced HUVECs (n = 3-5 independent biological replicates), analyzed by electrical cell-substrate impedance sensing (ECIS). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test


( H ) Endothelial resistance of hnRNPL - silenced HUVECs ( n = 3 independent biological replicates ) , analyzed by ECIS . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed unpaired t - test ,

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

(H) Endothelial resistance of hnRNPL-silenced HUVECs (n = 3 independent biological replicates), analyzed by ECIS. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,


( I ) Analysis of TJP1 exon 20 inclusion by RT - PCR upon transfection of HUVECs with a control SSO or an SSO masking the exon 20 - intron 20 boundary ( E20 SSO ) ( n = 9 independent biological replicates ) . Representative agarose gel on the right . Schematic outline at the top right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed unpaired t - test ,

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

(I) Analysis of TJP1 exon 20 inclusion by RT-PCR upon transfection of HUVECs with a control SSO or an SSO masking the exon 20 - intron 20 boundary (E20 SSO) (n = 9 independent biological replicates). Representative agarose gel on the right. Schematic outline at the top right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,


( J ) Endothelial resistance of control SSO - or E20 SSO - transfected HUVECs ( n = 4 independent biological replicates ) , analyzed by ECIS . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - tailed unpaired t - test ,

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

(J) Endothelial resistance of control SSO- or E20 SSO-transfected HUVECs (n = 4 independent biological replicates), analyzed by ECIS. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,


( A ) Validation of Ntras - hnRNPL - interaction by anti - hnRNPL RIPs from H5V lysates , followed by RT - qPCR ( n = 4 independent biological replicates ) . Representative western blot on the right . Data information : In ( A , data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( A ) two - tailed paired t -

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

(A) Validation of Ntras-hnRNPL-interaction by anti-hnRNPL RIPs from H5V lysates, followed by RT-qPCR (n = 4 independent biological replicates). Representative western blot on the right. Data information: In (A, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. (A) two-tailed paired t-test


( B ) RT - PCR - based analysis of murine Tjp1 exon 20 inclusion in cardiac tissue from control and Ntras - silenced mice ( n = 8 mice per group ) . Experimental outline on the left . Data information : In data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( B , two - tailed unpaired t - test ,

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-PCR-based analysis of murine Tjp1 exon 20 inclusion in cardiac tissue from control and Ntras-silenced mice (n = 8 mice per group). Experimental outline on the left. Data information: In data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. (B, two-tailed unpaired t-test,


( D ) Co - precipitation of Ntras in anti - hnRNPL RIPs using whole heart lysates from NtrasCA / CA and Ntras ∆ CA / ∆ CA mice ( n = 3 - 4 mice per group ) . Representative western blot on the right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - tailed unpaired t - test ,

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-precipitation of Ntras in anti-hnRNPL RIPs using whole heart lysates from NtrasCA/CA and Ntras∆CA/∆CA mice (n = 3-4 mice per group). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( E ) RT - PCR - based analysis of TJP1 exon 20 inclusion in hearts from NtrasCA / CA and Ntras ∆ CA / ∆ CA mice ( n = 9 - 14 mice per group ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - tailed unpaired t - test ,

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

(E) RT-PCR-based analysis of TJP1 exon 20 inclusion in hearts from NtrasCA/CA and Ntras∆CA/∆CA mice (n = 9-14 mice per group). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( F ) Retinal angiogenesis assessed in P7 NtrasCA / CA and Ntras ∆ CA / ∆ CA pups by immunostaining of isolectin B4 . Vascularized areas were normalized to total retinal area ( n = 4 - 8 mice per group ) . Representative micrographs are shown . Scale bars are 200 µm . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - tailed unpaired t - test ,

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Retinal angiogenesis assessed in P7 NtrasCA/CA and Ntras∆CA/∆CA pups by immunostaining of isolectin B4. Vascularized areas were normalized to total retinal area (n = 4-8 mice per group). Representative micrographs are shown. Scale bars are 200 µm. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( G ) FTSC and TMR - dextran in vivo permeability assays , comparing homogenates of hearts from NtrasCA / CA and Ntras ∆ CA / ∆ CA mice . Data normalized to organ and body weight ( n = 11 - 18 mice per group ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - tailed unpaired t - test ,

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FTSC and TMR-dextran in vivo permeability assays, comparing homogenates of hearts from NtrasCA/CA and Ntras∆CA/∆CA mice. Data normalized to organ and body weight (n = 11-18 mice per group). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( H ) Quantification of CD45 + cell ( red ) infiltration into cardiac tissue from NtrasCA / CA and Ntras ∆ CA / ∆ CA mice normalized to DAPI . Isolectin B4 ( green ) was used to label endothelial cells ( n = 4 - 5 mice per group ) . Representative micrographs are shown . Scale bars are 50 µm . Insets and arrows indicate sites of CD45 + cell infiltration . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - tailed unpaired t - test ,

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

(H) Quantification of CD45+ cell (red) infiltration into cardiac tissue from NtrasCA/CA and Ntras∆CA/∆CA mice normalized to DAPI. Isolectin B4 (green) was used to label endothelial cells (n = 4-5 mice per group). Representative micrographs are shown. Scale bars are 50 µm. Insets and arrows indicate sites of CD45+ cell infiltration. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( I ) Percentage of control and Ntras - silenced mice showing cardiac pathologies ( n = 15 - 17 mice per group ) . Prevalence is indicated above the bars . H & E micrographs of murine heart sections from control and Ntras - silenced mice are shown . Insets and arrows indicate sites of lymphocytic infiltration . Scale bars are 100 µm . Data information : ( I ) Chi - square test .

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(I) Percentage of control and Ntras-silenced mice showing cardiac pathologies (n = 15-17 mice per group). Prevalence is indicated above the bars. H&E micrographs of murine heart sections from control and Ntras-silenced mice are shown. Insets and arrows indicate sites of lymphocytic infiltration. Scale bars are 100 µm. Data information: (I) Chi-square test.


A EN qRT - PCR of murine liver sinusoidal endothelial cells ( LSEC ) , Kupffer cells ( KC ) , hepatocytes ( Hep ) , and HSC ( n = 3 per cell type ) .

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A EN qRT-PCR of murine liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), hepatocytes (Hep), and HSC (n = 3 per cell type).


B EN qRT - PCR of human hepatocytes ( n = 1 ) and HSC cell line LX - 2 ( n = 3 ) .

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

B EN qRT-PCR of human hepatocytes (n = 1) and HSC cell line LX-2 (n = 3).


C EN immunofluorescence staining of LX - 2 cells . Scale bar : 25 μm .

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C EN immunofluorescence staining of LX-2 cells. Scale bar: 25 μm.


D EN qRT - PCR of normal liver ( NL ) and fibrotic liver ( FL , n = 8 each ) , and active or inactive cirrhotic liver ( CL + / − , n = 10 / n = 2 ) .

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D EN qRT-PCR of normal liver (NL) and fibrotic liver (FL,n = 8 each), and active or inactive cirrhotic liver (CL+/−, n = 10/ n = 2).


E - H EN immunohistochemistry staining of human normal liver ( E ) , fibrotic liver ( F ) , and cirrhotic liver ( G , H ) . Weak EN expression in portal tracts of normal liver ( E ) . Abundant EN expression detected in activated HSC in fibrous septa of fibrotic ( F ) , active cirrhotic ( G ) , and inactive cirrhotic liver ( H ) . Scale bars : 500 μm . I - N Higher magnification of EN - stained normal liver ( I ) , fibrotic liver ( K ) , and ( active ) cirrhotic liver ( M ) compared to alpha - smooth muscle actin ( αSMA ) immunohistochemistry on serial slides ( J , L and N ) . Scale bars : 100 μm .

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E-H EN immunohistochemistry staining of human normal liver (E), fibrotic liver (F), and cirrhotic liver (G, H). Weak EN expression in portal tracts of normal liver (E). Abundant EN expression detected in activated HSC in fibrous septa of fibrotic (F), active cirrhotic (G), and inactive cirrhotic liver (H). Scale bars: 500 μm.I-N Higher magnification of EN-stained normal liver (I), fibrotic liver (K), and (active) cirrhotic liver (M) compared to alpha-smooth muscle actin (αSMA) immunohistochemistry on serial slides (J, L and N). Scale bars: 100 μm.


O EN / αSMA mRNA ratio in normal liver , fibrotic liver , and active cirrhotic liver .

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O EN/αSMA mRNA ratio in normal liver, fibrotic liver, and active cirrhotic liver.


P Collagen 1a ( Col1a ) expression in normal , fibrotic , and active cirrhotic liver determined by qRT - PCR .

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P Collagen 1a (Col1a) expression in normal, fibrotic, and active cirrhotic liver determined by qRT-PCR.


A , B qRT - PCR for EN ( A ) and αSMA ( B ) of liver lysates from single high - dose CCl4 - or oil - treated wild - type ( WT ) mice ( n = 2 per time point ) .

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A, B qRT-PCR for EN (A) and αSMA (B) of liver lysates from single high-dose CCl4- or oil-treated wild-type (WT) mice (n = 2 per time point).


C Early activated and long - term - activated HSC isolated by differential density gradient centrifugation ( 8 . 26 % versus 11 . 5 % density layer ) . qRT - PCR for EN from early activated ( 8 . 26 % ) and long - term - activated HSC ( 11 . 5 % ) from 4 - week CCL4 - treated mice and vehicle control mice ( n = 3 - 4 per data point ) .

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C Early activated and long-term-activated HSC isolated by differential density gradient centrifugation (8.26% versus 11.5% density layer). qRT-PCR for EN from early activated (8.26%) and long-term-activated HSC (11.5%) from 4-week CCL4-treated mice and vehicle control mice (n = 3-4 per data point).


D EN expression in liver lysates from 2 - , 4 - , and 6 - week CCl4 - treated mice ( n = 5 - 8 ) and vehicle control mice ( n = 3 ) determined by qRT - PCR .

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D EN expression in liver lysates from 2-, 4-, and 6-week CCl4-treated mice (n = 5-8) and vehicle control mice (n = 3) determined by qRT-PCR.


E - G METAVIR score of 2 - , 4 - , and 6 - week CCl4 - treated mice ( F - G ) Col1a and F4 / 80 immunofluorescence of 6 - week CCl4 - treated and vehicle control WT ( F ) and ENKO ( G ) mice . H Mean Col1a + area per high power field ( HPF ) shown as percentage of the total area ( n = 5 - 8 ) .

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E-G METAVIR score of 2-, 4-, and 6-week CCl4-treated mice (F-G) Col1a and F4/80 immunofluorescence of 6-week CCl4-treated and vehicle control WT (F) and ENKO (G) mice.H Mean Col1a+ area per high power field (HPF) shown as percentage of the total area (n = 5-8).


I Western blot analysis for collagen 1a ( Col1a ) , platelet - derived growth factor receptor beta ( PDGFRβ ) and β - actin of total liver protein lysates from 6 - week CCl4 - treated or control vehicle WT and ENKO mice ( n = 3 - 5 ) .

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I Western blot analysis for collagen 1a (Col1a), platelet-derived growth factor receptor beta (PDGFRβ) and β-actin of total liver protein lysates from 6-week CCl4-treated or control vehicle WT and ENKO mice (n = 3-5).


J - L Double immunofluorescence of 6 - week CCl4 - treated and vehicle control WT ( J ) and ENKO ( K ) mice for desmin and αSMA ( L ) shown as percentage of the total area ( n = 5 - 8 ) .

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J-L Double immunofluorescence of 6-week CCl4-treated and vehicle control WT (J) and ENKO (K) mice for desmin and αSMA (L) shown as percentage of the total area (n = 5-8).


M Serum liver enzymes ( GPT / ALT and GOT / AST ) from 2 - , 4 - , and 6 - week CCl4 - treated WT and ENKO mice ( n = 5 - 8 ) .

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M Serum liver enzymes (GPT/ALT and GOT/AST) from 2-, 4-, and 6-week CCl4-treated WT and ENKO mice (n = 5-8).


N qRT - PCR of endosialin ( EN ) , αSMA , and collagen 1a1 / 6a1 from lentiviral - mediated endosialin knockdown ( shEN ) and control - transfected ( CTL ) human hepatic stellate cells ( LX - 2 ) .

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N qRT-PCR of endosialin (EN), αSMA, and collagen 1a1/6a1 from lentiviral-mediated endosialin knockdown (shEN) and control-transfected (CTL) human hepatic stellate cells (LX-2).


O Lentiviral - mediated endosialin knockdown ( shEN ) and control ( CTL ) in LX - 2 cells ( n = 3 ) . Magnification 10 × .

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 Lentiviral-mediated endosialin knockdown (shEN) and control (CTL) in LX-2 cells (n = 3). Magnification 10×.


A Representative images of Ki - 67 immunohistochemistry after 2 , 4 , and 6 weeks of CCl4 treatment of WT and ENKO mice ( n = 5 - 8 mice per group and time point ) . Scale bars : 75 μm . B Quantitation of Ki67 + hepatocytes / HPF after 2 , 4 , and 6 weeks of CCl4 treatment .

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A Representative images of Ki-67 immunohistochemistry after 2, 4, and 6 weeks of CCl4 treatment of WT and ENKO mice (n = 5-8 mice per group and time point). Scale bars: 75 μm.B Quantitation of Ki67+ hepatocytes/HPF after 2, 4, and 6 weeks of CCl4 treatment.


C Time course of EN expression after partial hepatectomy ( 2 / 3 PHx ) determined by qRT - PCR of total liver lysates ( n = 5 / time point , sham : n = 3 ) .

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C Time course of EN expression after partial hepatectomy (2/3 PHx) determined by qRT-PCR of total liver lysates (n = 5/time point, sham: n = 3).


D Representative images of Ki - 67immunohistochemistry 2 days after 2 / 3 PHx of WT and ENKOmice ( n = 5 - 9 mice per group and time point ) . E Quantitation of Ki - 67 + hepatocytes / HPF determined 1 , 2 , 4 , and 8 days after 2 / 3 PHx . F Number of mitotic figures 2 days after 2 / 3 PHx of WT and ENKOmice ( n = 5 - 9 mice per group and time point ) .

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D Representative images of Ki-67immunohistochemistry 2 days after 2/3 PHx of WT and ENKOmice (n = 5-9 mice per group and time point).E Quantitation of Ki-67+hepatocytes/HPF determined 1, 2, 4, and 8 days after 2/3 PHx.F Number of mitotic figures 2 days after 2/3 PHx of WT and ENKOmice (n = 5-9 mice per group and time point).


G qRT - PCR analysis of total liver lysates 1 day after PHx for IGF2 .

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G qRT-PCR analysis of total liver lysates 1 day after PHx for IGF2.


H Western blot analysis of phosphorylated insulin - like growth factor receptor 1 ( pIGFR1 ) , phosphorylated insulin receptor substrate 1 ( pIRS1 ) and β - actin 2 days after PHx .

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H Western blot analysis of phosphorylated insulin-like growth factor receptor 1 (pIGFR1), phosphorylated insulin receptor substrate 1 (pIRS1) and β-actin 2 days after PHx.


I Western blot analysis of phosphorylated and total extracellular signal - regulated kinase ( ERK ) and AKT 2 days after PHx .

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I Western blot analysis of phosphorylated and total extracellular signal-regulated kinase (ERK) and AKT 2 days after PHx.


J , K qRT - PCR of IGF2 in isolated liver sinusoidal endothelial cells ( LSEC ) ( J ) or hepatocytes ( HEP ) ( K ) 1 day after PHx .

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J, K qRT-PCR of IGF2 in isolated liver sinusoidal endothelial cells (LSEC)(J) or hepatocytes (HEP)(K) 1 day after PHx.


L EdU - positive hepatocytes ( in % ) after 24 - h stimulation with conditioned medium ( CM ) from either WT or ENKO HSC ( 3 , 5 , or 7 days of activation ) . HGF ( 40 ng / ml ) and TGF - β ( 10 ng / ml ) serve as positive or negative control , respectively .

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L EdU-positive hepatocytes (in %) after 24-h stimulation with conditioned medium (CM) from either WT or ENKO HSC (3, 5, or 7 days of activation). HGF (40 ng/ml) and TGF-β (10 ng/ml) serve as positive or negative control, respectively.


M EdU - positive WT and ENKO hepatocytes ( in % ) after stimulation with IGF2 ( 100 ng / ml ) . HGF ( 40 or 10 ng / ml ) and TGF - β ( 10 ng / ml ) serve as positive or negative control , respectively .

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M EdU-positive WT and ENKO hepatocytes (in %) after stimulation with IGF2 (100 ng/ml). HGF (40 or 10 ng/ml) and TGF-β (10 ng/ml) serve as positive or negative control, respectively.


A Up , Representative western blot from whole cell extracts showing H2AX protein levels in wt and junD - / - fibroblasts ( Left ) or in wt and Nfe2l2 - / - fibroblasts ( Right ) . Two representative clones , with their corresponding controls , are shown per genotype . H2B was used as negative control . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) . n ≥ 3 independent experiments .

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A Up, Representative western blot from whole cell extracts showing H2AX protein levels in wt and junD-/- fibroblasts (Left) or in wt and Nfe2l2-/- fibroblasts (Right). Two representative clones, with their corresponding controls, are shown per genotype. H2B was used as negative control. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). n ≥ 3 independent experiments.


B Up , Representative western blot from kidney whole cell extracts showing H2AX protein levels in age - matched wt and junD - / - mice . Mice are 2 months old ( young ) and 18 months old ( old ) mice , respectively . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) . N ≥ 5 mice per age and per genotype .

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B Up, Representative western blot from kidney whole cell extracts showing H2AX protein levels in age-matched wt and junD-/- mice. Mice are 2 months old (young) and 18 months old (old) mice, respectively. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 5 mice per age and per genotype.


C Up , Representative western blot from kidney whole cell extracts , showing H2AX protein levels in 24 months old wt and junD - / - mice , either untreated ( - ) or treated with the anti - oxidant agent , N - acetyl - cysteine ( NAC ) . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) . N ≥ 4 mice per treatment and per genotype .

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C Up, Representative western blot from kidney whole cell extracts, showing H2AX protein levels in 24 months old wt and junD-/- mice, either untreated (-) or treated with the anti-oxidant agent, N-acetyl-cysteine (NAC). Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 4 mice per treatment and per genotype.


D , E Left , Representative western blots from whole cell extract showing γ - H2AX , H2AX protein levels and Kap1 phosphorylation ( P - Kap1 ) in wt and junD - / - fibroblasts ( D ) or in wt and Nfe2l2 - / - fibroblasts ( E ) , after H2O2 exposure for the indicated times ( hours , h ) . Right , Bar plots showing γ - H2AX and H2AX protein levels , as well as γ - H2AX / H2AX ratio as assessed by densitometry analysis of western blots ( as shown on the left ) . n = 3 independent experiments .

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D,E Left, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and Kap1 phosphorylation (P-Kap1) in wt and junD-/- fibroblasts (D) or in wt and Nfe2l2-/- fibroblasts (E), after H2O2 exposure for the indicated times (hours, h). Right, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown on the left). n = 3 independent experiments.


F , H Up , Representative western blots from whole cell extract showing γ - H2AX , H2AX protein levels and P - Kap1 in wt and junD - / - fibroblasts , following 2 Gy of γ - irradiation ( F ) for the indicated times ( hours , h ) . Down , Bar plots showing γ - H2AX and H2AX protein levels , as well as γ - H2AX / H2AX ratio as assessed by densitometry analysis of western blots ( as shown upper ) . n = 3 independent experiments .

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F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, following 2 Gy of γ-irradiation (F) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments.


G , I Up , Representative γ - H2AX immunofluorescence ( green ) in wt and junD - / - fibroblasts before ( - ) or after γ - irradiation ( 2Gy , 45 min ) ( G ) . Blue signal corresponds to DAPI staining . Down , box - plots of large γ - H2AX foci per nuclei ( diameter > 0 . 8 μm ) . At least 50 nuclei per genotype have been used for quantification .

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G,I Up, Representative γ-H2AX immunofluorescence (green) in wt and junD-/- fibroblasts before (-) or after γ-irradiation (2Gy, 45 min) (G). Blue signal corresponds to DAPI staining. Down, box-plots of large γ-H2AX foci per nuclei (diameter > 0.8 μm). At least 50 nuclei per genotype have been used for quantification.


F , H Up , Representative western blots from whole cell extract showing γ - H2AX , H2AX protein levels and P - Kap1 in wt and junD - / - fibroblasts , after camptothecin ( CPT ) treatment ( H ) for the indicated times ( hours , h ) . Down , Bar plots showing γ - H2AX and H2AX protein levels , as well as γ - H2AX / H2AX ratio as assessed by densitometry analysis of western blots ( as shown upper ) . n = 3 independent experiments .

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F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, after camptothecin (CPT) treatment (H) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments.


G , I Up , Representative γ - H2AXimmunofluorescence ( green ) in wt and junD - / - fibroblasts before ( - ) or after after camptothecin ( CPT ) treatment for 1h ( I ) . . Blue signal corresponds to DAPI staining . Down , box - plots of large γ - H2AX foci per nuclei ( diameter > 0 . 8 μm ) . At least 50 nuclei per genotype have been used for quantification .

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G,I Up, Representative γ-H2AXimmunofluorescence (green) in wt and junD-/- fibroblasts before (-) or after after camptothecin (CPT) treatment for 1h (I).. Blue signal corresponds to DAPI staining. Down, box-plots of large γ-H2AX foci per nuclei (diameter > 0.8 μm). At least 50 nuclei per genotype have been used for quantification.


A Bar plot showing the doubling time of wt and junD - / - fibroblasts .

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A Bar plot showing the doubling time of wt and junD-/- fibroblasts.


B Representative cell cycle distribution of asynchronous wt and junD - / - fibroblasts .

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B Representative cell cycle distribution of asynchronous wt and junD-/- fibroblasts.


C Bar plots showing the percentage of wt and junD - / - fibroblasts in G1 , S and G2 phases of the cell cycle , following serum stimulation for the indicated times ( hours , h ) , after 48h serum starvation .

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C Bar plots showing the percentage of wt and junD-/- fibroblasts in G1, S and G2 phases of the cell cycle, following serum stimulation for the indicated times (hours, h), after 48h serum starvation.


D Representative views of alkaline comet assays from wt and junD - / - fibroblasts either untreated or after 10Gy γ - irradiation for the indicated times ( minutes , min ) . E Alkaline comet assays from wt and junD - / - fibroblasts at basal state . Left , Box - plots showing tail moments at basal state in wt and junD - / - fibroblasts . Right , Distribution of wt and junD - / - fibroblasts expressed as percentage of cells ( % ) according to categorized tail moments . At basal state , 16 % of junD - / - cells exhibit tail moment higher than 5 , while it is the case for only 3 % of wt cells . F Alkaline comet assays from wt and junD - / - fibroblasts after 10Gy γ - irradiation . Box - plots showing tail moments at basal state in wt and junD - / - fibroblasts , following 10Gy γ - irradiation for the indicated times ( minutes , min ) . G Distribution of wt and junD - / - fibroblasts expressed as percentage of cells ( % ) according to categorized tail moments , following 10Gy γ - irradiation for the indicated times ( minutes , min ) .

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D Representative views of alkaline comet assays from wt and junD-/- fibroblasts either untreated or after 10Gy γ-irradiation for the indicated times (minutes, min).E Alkaline comet assays from wt and junD-/- fibroblasts at basal state. Left, Box-plots showing tail moments at basal state in wt and junD-/- fibroblasts. Right, Distribution of wt and junD-/- fibroblasts expressed as percentage of cells (%) according to categorized tail moments. At basal state, 16% of junD-/- cells exhibit tail moment higher than 5, while it is the case for only 3% of wt cells.F Alkaline comet assays from wt and junD-/- fibroblasts after 10Gy γ-irradiation. Box-plots showing tail moments at basal state in wt and junD-/- fibroblasts, following 10Gy γ-irradiation for the indicated times (minutes, min).G Distribution of wt and junD-/- fibroblasts expressed as percentage of cells (%) according to categorized tail moments, following 10Gy γ-irradiation for the indicated times (minutes, min).


H Surviving fraction of wt and junD - / - fibroblasts 7 days after 2Gy or 5Gy γ - irradiation , as assessed by clonogenic assay . Surviving fraction is expressed as percentage ( % ) of number of colonies after irradiation normalized with respect to untreated conditions .

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H Surviving fraction of wt and junD-/- fibroblasts 7 days after 2Gy or 5Gy γ-irradiation, as assessed by clonogenic assay. Surviving fraction is expressed as percentage (%) of number of colonies after irradiation normalized with respect to untreated conditions.


I Surviving fraction of wt and junD - / - fibroblasts either transfected with an empty vector ( + Ctl ) or H2AX - expressing ( + H2AX ) vector . Surviving fraction is expressed as percentage ( % ) of alive cells , 72 hours post - transfection .

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I Surviving fraction of wt and junD-/- fibroblasts either transfected with an empty vector (+ Ctl) or H2AX-expressing (+ H2AX) vector. Surviving fraction is expressed as percentage (%) of alive cells, 72 hours post-transfection.


A Up , Representative western blot from chromatin - free fraction or whole cell extracts , as indicated , showing H2AX protein levels in wt and junD - / - fibroblasts . Different amounts of loaded proteins and exposures ( low - or high - exp ) are presented , H2AX being easier to detect when associated with chromatin . Down , Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown upper ) .

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A Up, Representative western blot from chromatin-free fraction or whole cell extracts, as indicated, showing H2AX protein levels in wt and junD-/- fibroblasts. Different amounts of loaded proteins and exposures (low- or high-exp) are presented, H2AX being easier to detect when associated with chromatin. Down, Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown upper).


B Up , Representative western blot from chromatin - free fraction extracts showing H2AX protein levels in wt and junD - / - fibroblasts either untreated ( - ) or after 8h treatment ( + ) with proteasome inhibitor MG132 . Jun is used as positive control for treatment efficiency . Down , Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown upper ) . C Bar plot showing stabilization of H2AX protein in wt and junD - / - fibroblasts , as assessed by densitrometry analysis of western blots ( as shown in B ) . Data are expressed as percentage ( % ) of H2AX protein levels after treatment with MG132 with respect to untreated conditions .

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B Up, Representative western blot from chromatin-free fraction extracts showing H2AX protein levels in wt and junD-/- fibroblasts either untreated (-) or after 8h treatment (+) with proteasome inhibitor MG132. Jun is used as positive control for treatment efficiency. Down, Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown upper).C Bar plot showing stabilization of H2AX protein in wt and junD-/- fibroblasts, as assessed by densitrometry analysis of western blots (as shown in B). Data are expressed as percentage (%) of H2AX protein levels after treatment with MG132 with respect to untreated conditions.


D Representative western blots from whole cell extracts showing HA - tagged ubiquitin ( Left ) or Flag - tagged H2AX ( Right ) protein levels . wt and junD - / - fibroblasts were co - transfected with vectors encoding HA - tagged ubiquitin and Flag - tagged wild - type H2AX ( WT ) , or K13 - , K15 - , K119 - , or 9K - H2AX mutants . Flag - tagged H2AX proteins were then immunoprecipitated with Flag - specific antibody and incubated either with HA - specific antibody ( Left ) or Flag - specific antibody ( Right ) . Two different exposures ( low or high exp ) are presented . * stands for the immunoglobulins .

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D Representative western blots from whole cell extracts showing HA-tagged ubiquitin (Left) or Flag-tagged H2AX (Right) protein levels. wt and junD-/- fibroblasts were co-transfected with vectors encoding HA-tagged ubiquitin and Flag-tagged wild-type H2AX (WT), or K13-, K15-, K119-, or 9K-H2AX mutants. Flag-tagged H2AX proteins were then immunoprecipitated with Flag-specific antibody and incubated either with HA-specific antibody (Left) or Flag-specific antibody (Right). Two different exposures (low or high exp) are presented. * stands for the immunoglobulins.


E Left , Representative western blots showing H2AX - WT or H2AX - K119 protein levels ( revealed using Flag - specific antibody ) from whole cell extracts in wt and junD - / - fibroblasts . Cells were first transfected with Flag - tagged wild - type H2AX ( H2AX - WT ) or K119 H2AX mutant ( H2AX - K119 ) and next treated with cycloheximide ( CHX ) for the indicated times ( hours , h ) . Right , Bar graph showing H2AX - WT and H2AX - K119 protein half - life in wt and junD - / - fibroblasts , as indicated . Protein half - life has been calculated from the degradation curve of H2AX protein ( based on densitometry analysis of western blots , as shown on the Left ) by extrapolating its linear part .

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E Left, Representative western blots showing H2AX-WT or H2AX-K119 protein levels (revealed using Flag-specific antibody) from whole cell extracts in wt and junD-/- fibroblasts. Cells were first transfected with Flag-tagged wild-type H2AX (H2AX-WT) or K119 H2AX mutant (H2AX-K119) and next treated with cycloheximide (CHX) for the indicated times (hours, h). Right, Bar graph showing H2AX-WT and H2AX-K119 protein half-life in wt and junD-/- fibroblasts, as indicated. Protein half-life has been calculated from the degradation curve of H2AX protein (based on densitometry analysis of western blots, as shown on the Left) by extrapolating its linear part.


F Left , Representative western blots showing H2AX - WT protein levels ( revealed using Flag - specific antibody ) from whole cell extracts in junD - / - fibroblasts co - transfected with Flag - tagged H2AX - WT construct and either non - targeting siRNA ( siCtrl ) or siRNA targeting RNF168 ( siRNF168 ) , RNF8 ( siRNF8 ) or BMI1 ( siBMI1 ) . Efficiency of each siRNA has been verified and reached 60 % of inhibition , in average , as shown for RNF168 ( Up ) . Protein extracts were processed following cycloheximide ( CHX ) treatment for the indicated times ( hours , h ) . Right , Bar graph showing the percentage ( % ) of decrease of H2AX - WT protein levels after 4h treatment with CHX ( t = 4 ) with respect to untreated conditions ( t = 0 ) in junD - / - fibroblasts transfected with specific siRNA , as indicated .

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F Left, Representative western blots showing H2AX-WT protein levels (revealed using Flag-specific antibody) from whole cell extracts in junD-/- fibroblasts co-transfected with Flag-tagged H2AX-WT construct and either non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168), RNF8 (siRNF8) or BMI1 (siBMI1). Efficiency of each siRNA has been verified and reached 60% of inhibition, in average, as shown for RNF168 (Up). Protein extracts were processed following cycloheximide (CHX) treatment for the indicated times (hours, h). Right, Bar graph showing the percentage (%) of decrease of H2AX-WT protein levels after 4h treatment with CHX (t=4) with respect to untreated conditions (t=0) in junD-/- fibroblasts transfected with specific siRNA, as indicated.


G Left , Representative western blots showing H2AX - WT or H2AX - K119 protein levels from whole cell extracts in junD - / - fibroblasts transfected either with Flag - tagged wild - type H2AX ( H2AX - WT ) or K119 H2AX mutant ( H2AX - K119 ) and co - transfected with non - targeting siRNA ( siCtrl ) or siRNA targeting RNF168 ( siRNF168 ) . Cells were treated with cycloheximide ( CHX ) for the indicated times ( hours , h ) before protein extraction . Right , Bar graph showing H2AX - WT and H2AX - K119 protein half - life in junD - / - fibroblasts transfected with non - targeting siRNA ( siCtrl ) or siRNA targeting RNF168 ( siRNF168 ) , as indicated . Protein half - life has been calculated from the degradation curve of H2AX protein ( based on densitometry analysis of western blots , as shown on the Left ) by extrapolating its linear part .

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G Left, Representative western blots showing H2AX-WT or H2AX-K119 protein levels from whole cell extracts in junD-/- fibroblasts transfected either with Flag-tagged wild-type H2AX (H2AX-WT) or K119 H2AX mutant (H2AX-K119) and co-transfected with non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168). Cells were treated with cycloheximide (CHX) for the indicated times (hours, h) before protein extraction. Right, Bar graph showing H2AX-WT and H2AX-K119 protein half-life in junD-/- fibroblasts transfected with non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168), as indicated. Protein half-life has been calculated from the degradation curve of H2AX protein (based on densitometry analysis of western blots, as shown on the Left) by extrapolating its linear part.


H Representative western blot showing RNF168 , Flag - tagged H2AX ( H2AX - FLAG ) and endogenous H2AX ( H2AX ) protein levels from whole cell extracts in wt and junD - / - fibroblasts . Cells were first co - transfected with vectors encoding HA - tagged ubiquitin and Flag - tagged wild - type H2AX ( WT ) , or K13 - , K15 - , K119 - , or 9K - H2AX mutants . Flag - tagged H2AX proteins were next immunoprecipitated with Flag - specific antibody and incubated either with RNF168 - specific antibody ( Up ) or H2AX - specific antibody ( Down ) .

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H Representative western blot showing RNF168, Flag-tagged H2AX (H2AX-FLAG) and endogenous H2AX (H2AX) protein levels from whole cell extracts in wt and junD-/- fibroblasts. Cells were first co-transfected with vectors encoding HA-tagged ubiquitin and Flag-tagged wild-type H2AX (WT), or K13-, K15-, K119-, or 9K-H2AX mutants. Flag-tagged H2AX proteins were next immunoprecipitated with Flag-specific antibody and incubated either with RNF168-specific antibody (Up) or H2AX-specific antibody (Down).


A Up , Representative views of H2AXimmunostaining from 117 BC patients of Luminal - A ( LumA , N = 33 ) , HER2 ( N = 37 ) and Triple - Negative ( TN , N = 47 ) subtypes . Scale bars = 100 μm . Down , representative views at high magnification of H2AXimmunostaining focused on the Fibroblasts ( Left ) or Epithelium ( Right ) cells . Scale bars = 25 μm . B Scatter plots of H2AX histological score ( Hscore = staining intensity ( 0 to 4 ) x % of positive cells quantified from H2AXimmunostaining ( as shown in A ) in Fibroblasts ( Left ) or Epithelial ( Right ) cells from LumA , HER2 and TNbreasttumours . C Correlation plots between H2AX Hscores evaluated in fibroblasts and in epithelial cells ( as shown in B ) , in HER2 ( left ) and TN ( right ) breasttumours .

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A Up, Representative views of H2AXimmunostaining from 117 BC patients of Luminal-A (LumA, N=33), HER2 (N=37) and Triple-Negative (TN, N=47) subtypes. Scale bars = 100 μm. Down, representative views at high magnification of H2AXimmunostaining focused on the Fibroblasts (Left) or Epithelium (Right) cells. Scale bars = 25 μm.B Scatter plots of H2AX histological score (Hscore = staining intensity (0 to 4) x % of positive cells quantified from H2AXimmunostaining (as shown in A) in Fibroblasts (Left) or Epithelial (Right) cells from LumA, HER2 and TNbreasttumours.C Correlation plots between H2AX Hscores evaluated in fibroblasts and in epithelial cells (as shown in B), in HER2 (left) and TN (right) breasttumours.


D Representative views of smooth muscle actin ( SMA ) immunostaining from BC patients ( N = 29 ) of Luminal - A ( LumA , N = 10 ) , HER2 ( N = 10 ) and TN ( N = 9 ) subtypes . Scale bars = 100 μm . Zoom views are focused on the fibroblast compartment . Scale bars = 25 μm . E Scatter plots of SMA histological score ( Hscore ) evaluated from SMA immunostaining ( as shown in D ) . F Correlation plot between SMA and H2AX Hscores in stromal compartment .

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D Representative views of smooth muscle actin (SMA) immunostaining from BC patients (N=29) of Luminal-A (LumA, N=10), HER2 (N=10) and TN (N=9) subtypes. Scale bars = 100 μm. Zoom views are focused on the fibroblast compartment. Scale bars = 25 μm.E Scatter plots of SMA histological score (Hscore) evaluated from SMA immunostaining (as shown in D).F Correlation plot between SMA and H2AX Hscores in stromal compartment.


G Scatter plot of the mitotic index , evaluated by pathologists , in LumA , HER2 , and TN BC patients , as indicated .

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G Scatter plot of the mitotic index, evaluated by pathologists, in LumA, HER2, and TN BC patients, as indicated.


H Absence of correlation between mitotic index and epithelial H2AX Hscore , in LumA HER2 and TN BC .

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H Absence of correlation between mitotic index and epithelial H2AX Hscore, in LumA HER2 and TN BC.


B Left , Representative views of H2AX and γ - H2AX immunostaining in TN tumours ( N = 22 ) before and after successive cycles of chemotherapy . Scale bars = 100 μm . Zoom views are focused on the epithelial cells . Scale bars = 25 μm . Right , Scatter plots of H2AX , γ - H2AX histological scores ( Hscores ) and γ - H2AX / H2AX ratio evaluated from immunostaining in the epithelium , before and after chemotherapy .

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B Left, Representative views of H2AX and γ-H2AX immunostaining in TN tumours (N = 22) before and after successive cycles of chemotherapy. Scale bars = 100 μm. Zoom views are focused on the epithelial cells. Scale bars = 25 μm. Right, Scatter plots of H2AX, γ-H2AX histological scores (Hscores) and γ-H2AX/H2AX ratio evaluated from immunostaining in the epithelium, before and after chemotherapy.


C Correlation plot between Cisplatin IC50 ( half maximal inhibitory concentration ) and H2AX protein level defined by western blots in BC cell lines ( MCF7 , BT474 , MDA361 , SKBr3 , MDA453 , MDA231 , HCC70 ) . Correlation coefficients σ and P - value are based on Spearman ' s rank correlation test .

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C Correlation plot between Cisplatin IC50 (half maximal inhibitory concentration) and H2AX protein level defined by western blots in BC cell lines (MCF7, BT474, MDA361, SKBr3, MDA453, MDA231, HCC70). Correlation coefficients σ and P-value are based on Spearman's rank correlation test.


D Up , Representative western - blot showing H2AX protein levels from whole cell extracts in HCC70 TN BC cell line transfected with non - targeting siRNA ( siCtrl ) or an siRNA directed against H2AX ( siH2AX ) . Actin is used as internal control for protein loading . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) .

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D Up, Representative western-blot showing H2AX protein levels from whole cell extracts in HCC70 TN BC cell line transfected with non-targeting siRNA (siCtrl) or an siRNA directed against H2AX (siH2AX). Actin is used as internal control for protein loading. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above).


E Bar plot showing the percentage ( % ) of apoptotic cells as assessed by flow - cytometry analysis of annexin V and DAPI staining in HCC70 cell line transfected with non - targeting siRNA ( siCtrl ) or an siRNA directed against H2AX ( siH2AX ) , and next treated with cisplatin for 72 hrs .

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E Bar plot showing the percentage (%) of apoptotic cells as assessed by flow-cytometry analysis of annexin V and DAPI staining in HCC70 cell line transfected with non-targeting siRNA (siCtrl) or an siRNA directed against H2AX (siH2AX), and next treated with cisplatin for 72 hrs.


F Box plot showing the impact of H2AX decrease on the therapeutic response of TN BC patients ( N = 22 ) . Reduction of H2AX protein level was assessed by the difference of H2AX Hscores before and after chemotherapy ( as evaluated in B ) , for each patient ( values in Log2 ) . Only patients who have experienced an incomplete response have been taken into account . After treatment H2AX staining was evaluated only on residual tumour cells . The therapeutic response was monitored by pathologists through evaluation of tumour residual mass and detection of tumour cells inside lymph nodes .

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F Box plot showing the impact of H2AX decrease on the therapeutic response of TN BC patients (N = 22). Reduction of H2AX protein level was assessed by the difference of H2AX Hscores before and after chemotherapy (as evaluated in B), for each patient (values in Log2). Only patients who have experienced an incomplete response have been taken into account. After treatment H2AX staining was evaluated only on residual tumour cells. The therapeutic response was monitored by pathologists through evaluation of tumour residual mass and detection of tumour cells inside lymph nodes.


G Kaplan - Meier curves showing specific overall survival ( Left ) and progression - free survival ( Right ) of TN patients with respect to major ( N = 15 ) or minor ( N = 7 ) H2AX protein level decrease , observed upon chemotherapy . Iterative analyses were performed on ΔLog2 H2AX Hscore to find optimal threshold that maximally discriminates the major and minor H2AX decrease ( respectively 2 / 3 and 1 / 3 of patients ) .

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G Kaplan-Meier curves showing specific overall survival (Left) and progression-free survival (Right) of TN patients with respect to major (N=15) or minor (N=7) H2AX protein level decrease, observed upon chemotherapy. Iterative analyses were performed on ΔLog2 H2AX Hscore to find optimal threshold that maximally discriminates the major and minor H2AX decrease (respectively 2/3 and 1/3 of patients).


H Scatter plots showing the number of cleaved caspase 3 - positive cells / mm2 in TN BC , categorized according to major - or minor - H2AX protein level decrease after chemotherapy .

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H Scatter plots showing the number of cleaved caspase 3-positive cells / mm2 in TN BC, categorized according to major- or minor-H2AX protein level decrease after chemotherapy.


I Scatter plots showing variation of NRF2 histological score in patients with major - H2AX decrease before and after chemotherapy .

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I Scatter plots showing variation of NRF2 histological score in patients with major-H2AX decrease before and after chemotherapy.


J Box plots showing expression levels of representative NRF2 - target genes in TN tumours with minor - or major - H2AX decrease , as indicated . Lines indicate Means and whiskers show 10 - 90 percentiles . ACOT7 : Acyl - CoA Thioesterase 7 ; PGD : Phosphogluconate Dehydrogenase . AHR : Aryl Hydrocarbon Receptor ; JAG1 : Jagged1 . P - values are from t - test .

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J Box plots showing expression levels of representative NRF2-target genes in TN tumours with minor- or major-H2AX decrease, as indicated. Lines indicate Means and whiskers show 10-90 percentiles. ACOT7: Acyl-CoA Thioesterase 7; PGD: Phosphogluconate Dehydrogenase. AHR: Aryl Hydrocarbon Receptor; JAG1: Jagged1. P-values are from t-test.


Fig . 2 Identification of protein complexes and communities in the cellular extracts . ( A ) Elution of selected protein complexes as a function of their retention times ( see Appendix Fig . S4 for their corresponding subunit elutions ) .

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Fig. 2 Identification of protein complexes and communities in the cellular extracts. (A) Elution of selected protein complexes as a function of their retention times (see Appendix Fig. S4 for their corresponding subunit elutions).


( B ) Scatter plot indicating discrepancies in the expected and measured molecular weights of 102 protein complexes that elute as a single peak ; 50 % of protein complexes are observed to have higher molecular weights than structurally characterized , indicating that they are organized in higher - order assemblies .

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(B) Scatter plot indicating discrepancies in the expected and measured molecular weights of 102 protein complexes that elute as a single peak; 50% of protein complexes are observed to have higher molecular weights than structurally characterized, indicating that they are organized in higher-order assemblies.


Fig . 4 Higher - order assemblies identified by proteome - wide cross - linking mass spectrometry , biomolecular modeling and negative - stain electron microscopy . ( A ) Distance distributions of identified cross - links on top of the modeled protein complexes and identification of novel interactions . Satisfied distances are shown in blue and over - length cross - links are shown in red .

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Fig. 4 Higher-order assemblies identified by proteome-wide cross-linking mass spectrometry, biomolecular modeling and negative-stain electron microscopy. (A) Distance distributions of identified cross-links on top of the modeled protein complexes and identification of novel interactions. Satisfied distances are shown in blue and over-length cross-links are shown in red.