( 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 ,

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(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-GENEPROD O O O O O O O B-TISSUE O O B-GENEPROD O O O B-GENEPROD O O O O O B-ORGANISM O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O B-GENEPROD O O B-ORGANISM O B-TISSUE B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(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 ) .

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 ) .

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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 .

O O O B-GENEPROD B-EXP_ASSAY O O B-ORGANISM O B-TISSUE O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

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 × .

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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 ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O

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 .

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

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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O

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 .

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

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 .

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

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 .

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

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 .

O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O

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 .

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

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


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

O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O

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 .

O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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

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 .

O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

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 .

O O O O O O B-GENEPROD O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O B-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-TISSUE O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O B-GENEPROD O O O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O B-TISSUE O

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 .

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

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 .

O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O B-ORGANISM O B-ORGANISM O O O O

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 .

O O O O O O O O B-TISSUE B-GENEPROD O O O B-ORGANISM I-ORGANISM O B-ORGANISM O O

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 .

O O O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-TISSUE O 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-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O B-TISSUE O O O O O O

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.


( B ) Negatively stained electron micrographs of fractions 3 - 7 and 27 directly derived from size exclusion chromatography showing the structural signatures and their structural integrity within the fractions . Decreasing molecular weight correlates with increased protein concentration as a function of protein complex elution is highlighted .

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(B) Negatively stained electron micrographs of fractions 3-7 and 27 directly derived from size exclusion chromatography showing the structural signatures and their structural integrity within the fractions. Decreasing molecular weight correlates with increased protein concentration as a function of protein complex elution is highlighted.


( C ) Abundance profiles as determined by quantitative mass spectrometry correlate with the number of observed single particles of the corresponding structural signature within the negative - staining electron micrographs ; shown for fatty acid synthase , 20S proteasome , 60S and 40S ribosome ( the number of particles per image per fraction is indicated below the class averages ) .

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(C) Abundance profiles as determined by quantitative mass spectrometry correlate with the number of observed single particles of the corresponding structural signature within the negative-staining electron micrographs; shown for fatty acid synthase, 20S proteasome, 60S and 40S ribosome (the number of particles per image per fraction is indicated below the class averages).


( D ) Simplification of lysate ( collecting only the flow - through from anion exchange chromatography ) prior to SEC separation allows class - averaging of structural signatures from complex fractions that were previously too low abundant . Proteins identified in this simplified fraction are found in Table S4 .

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(D) Simplification of lysate (collecting only the flow-through from anion exchange chromatography) prior to SEC separation allows class-averaging of structural signatures from complex fractions that were previously too low abundant. Proteins identified in this simplified fraction are found in Table S4.


Fig . 5 Visualization of transient interactions in fatty acid synthesis . ( A ) Communities in fatty acid metabolism and the quantification of intra - community distances within cryo - electron micrographs . Fatty acid synthase ( FAS ) frequently interacts with other sizeable protein complexes in a linear ' pearl - string - like ' arrangement and usually localizes at the edges of the community . Scale bars correspond to 25 nm . FAS particles ( circles ) and their nearest neighbors ( arrow heads ) are indicated .

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Fig. 5 Visualization of transient interactions in fatty acid synthesis. (A) Communities in fatty acid metabolism and the quantification of intra-community distances within cryo-electron micrographs. Fatty acid synthase (FAS) frequently interacts with other sizeable protein complexes in a linear 'pearl-string-like' arrangement and usually localizes at the edges of the community. Scale bars correspond to 25 nm. FAS particles (circles) and their nearest neighbors (arrow heads) are indicated.


( C ) Related to ( A ) . Calculation of minimum distances between pairs of FAS molecules as well as FAS molecules and their closest non - FAS neighbors in comparison to random distributions . Whereas FAS molecules are randomly distributed , their binders are not , confirming physical interactions . Supervised picking means that all single - particles were manually picked from the images . Randomized distance means that these manually picked particles were assigned random coordinates in each image ( randomization of x , y coordinates considering image borders ) and then their distance is calculated .

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(C) Related to (A). Calculation of minimum distances between pairs of FAS molecules as well as FAS molecules and their closest non-FAS neighbors in comparison to random distributions. Whereas FAS molecules are randomly distributed, their binders are not, confirming physical interactions. Supervised picking means that all single-particles were manually picked from the images. Randomized distance means that these manually picked particles were assigned random coordinates in each image (randomization of x,y coordinates considering image borders) and then their distance is calculated.


( D ) Cross - linking mass spectrometry data show that the binder is a carboxylase that is bound to the malonyl transacylase domain and acyl carrier protein ( ACP ) is in the vicinity , considering cross - link length and the positions of the lysine on the ctFAS structure . Cross - links come from both affinity - purified and fractionated cell extracts .

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(D) Cross-linking mass spectrometry data show that the binder is a carboxylase that is bound to the malonyl transacylase domain and acyl carrier protein (ACP) is in the vicinity, considering cross-link length and the positions of the lysine on the ctFAS structure. Cross-links come from both affinity-purified and fractionated cell extracts.


Fig . 6 CryoEM structure of fatty acid synthase resolved to 4 . 7 Å as obtained from cryo electron micrographs of fraction numbers 7 - 9 . ( A ) The cryoEM of C . thermophilum fatty acid synthase ( ctFAS ) map is shown isosurface rendered and superimposed with the fitted X - ray structure of yeast FAS ( Jenni et al , 2007 ) . The domes and the cap show the unambiguous fit of α - helices and β - strands .

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Fig. 6 CryoEM structure of fatty acid synthase resolved to 4.7 Å as obtained from cryo electron micrographs of fraction numbers 7-9. (A) The cryoEM of C. thermophilum fatty acid synthase (ctFAS) map is shown isosurface rendered and superimposed with the fitted X-ray structure of yeast FAS (Jenni et al, 2007). The domes and the cap show the unambiguous fit of α-helices and β-strands.


( B ) A slice through the central wheel of fungal FAS . The pitch of α - helices is resolved .

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(B) A slice through the central wheel of fungal FAS. The pitch of α-helices is resolved.


( C ) As for ( B ) but sliced through the dome structure .

(C) As for (B) but sliced through the dome structure.


( D ) Location of acyl carrier protein ( ACP ) within the cryoEM map of ctFAS and the position of additional density outside the dome .

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(D) Location of acyl carrier protein (ACP) within the cryoEM map of ctFAS and the position of additional density outside the dome.


( E ) Northern blot analysis of circBtnl1 in different tissues of two - month - old mice , 18S RNA served as a loading control .

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(E) Northern blot analysis of circBtnl1 in different tissues of two-month-old mice, 18S RNA served as a loading control.


( F ， G ) RNA fluorescence in situ hybridization of circBtnl1 in small intestine tissues ( F ) and organoid ( G ) . Green region shows the distribution of circBtnl1 using antisense probe , gray region shows the nuclei staining by DAPI . For ( F ) ， scale bars ， 5um ; for ( G ) ， scale bars ， 20 um .

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(F， G) RNA fluorescence in situ hybridization of circBtnl1 in small intestine tissues (F) and organoid (G). Green region shows the distribution of circBtnl1 using antisense probe, gray region shows the nuclei staining by DAPI. For (F)，scale bars，5um; for (G)，scale bars，20 um.


( C ) Representative images of Ki67 staining of in small intestinal tissues from circBtnl1 + / + and circBtnl1 - / - mice . For all representative images , at least three independent experiments were performed with similar results . For every microscope field , average Ki67 + ISC numbers per crypt are shown as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . Scale bar , 50 μm .

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(C) Representative images of Ki67 staining of in small intestinal tissues from circBtnl1+/+ and circBtnl1-/- mice. For all representative images, at least three independent experiments were performed with similar results. For every microscope field, average Ki67+ ISC numbers per crypt are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel. Scale bar, 50 μm.


( E ) H & E staining of small intestine tissues from indicated mice at different time points ( D0 - D7 ) after 8 Gy radiation damage . Number of crypts were calculated as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . n = 5 fields were calculated for each group . Scale bar , 50 μm .

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(E) H&E staining of small intestine tissues from indicated mice at different time points (D0 - D7) after 8 Gy radiation damage. Number of crypts were calculated as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel. n = 5 fields were calculated for each group. Scale bar, 50 μm.


( G ) Confocal microscopy of ISCs makers Olmf4 in organoids from circBtnl1 + / + and circBtnl1 - / - mice . Scale bars , 30 μm . Three independent experiments were performed with similar results , and representative experiments are shown . For every microscope field , average Olmf4 + ISC numbers per crypt are shown as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel in right panel .

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(G) Confocal microscopy of ISCs makers Olmf4 in organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 30 μm. Three independent experiments were performed with similar results, and representative experiments are shown. For every microscope field, average Olmf4+ ISC numbers per crypt are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel in right panel.


( B , C ) CircBtnl1 was co - localized with Ddx3y protein in small intestine tissue ( B ) and organoid ( C ) by RNA FISH , followed by immunofluorescence staining . Scale bars , 5 μm .

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(B, C) CircBtnl1 was co-localized with Ddx3y protein in small intestine tissue (B) and organoid (C) by RNA FISH, followed by immunofluorescence staining. Scale bars, 5 μm.


( G ) Electrophoretic mobility shift assay ( EMSA ) using biotin - labeled circBtnl1 and recombinant Ddx3y .

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(G) Electrophoretic mobility shift assay (EMSA) using biotin-labeled circBtnl1 and recombinant Ddx3y.


( H ) Truncated fragments of biotinylated circBtnl1 were incubated with small intestine crypts lysates , followed by RNA pulldown assay and Western blot .

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(H) Truncated fragments of biotinylated circBtnl1 were incubated with small intestine crypts lysates, followed by RNA pulldown assay and Western blot.


( B ) Small intestine crypt cells were isolated from circBtnl1 + / + and circBtnl1 - / - two - month - old mice , followed by RNA extraction and q - PCR analysis for top ten upregulated transcription factors . n = 3 independent samples . Results of relative fold changes are shown as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test .

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(B) Small intestine crypt cells were isolated from circBtnl1+/+ and circBtnl1-/- two-month-old mice, followed by RNA extraction and q-PCR analysis for top ten upregulated transcription factors. n = 3 independent samples. Results of relative fold changes are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test.


( D ) Immunoblotting analysis of Ddx3y in RNA pull - down samples by Atf4 sense , antisense and control probes in mice ISCs . ( E ) Immunoblotting analysis of Ddx3y in RNA pull - down samples by Atf4 and control probes in circBtnl1 + / + and circBtnl1 - / - mice ISCs .

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(D) Immunoblotting analysis of Ddx3y in RNA pull-down samples by Atf4 sense, antisense and control probes in mice ISCs. (E) Immunoblotting analysis of Ddx3y in RNA pull-down samples by Atf4 and control probes in circBtnl1+/+ and circBtnl1-/- mice ISCs.


( J ) Confocal microscopy of ATF4 in Ddx3y knockdown and overexpression organoids from circBtnl1 + / + and circBtnl1 - / - mice . Scale bars , 20 μm .

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(J) Confocal microscopy of ATF4 in Ddx3y knockdown and overexpression organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 20 μm.


( K , L ) Expression levels of Ddx3y and Atf4 in circBtnl1 + / + and circBtnl1 - / - ISCs were examined by real - time PCR ( K ) and Western blot ( L ) , three independent experiments were performed . Results of relative fold changes are shown as means ± SD . * * P < 0 . 01 , NS , not significant , by two - tailed Student ' s t - test .

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(K, L) Expression levels of Ddx3y and Atf4 in circBtnl1+/+ and circBtnl1-/- ISCs were examined by real-time PCR (K) and Western blot (L), three independent experiments were performed. Results of relative fold changes are shown as means ± SD. ** P < 0.01, NS, not significant, by two-tailed Student's t-test.


( D , E ) ChIP assay was performed using anti - H3K4me3 ( D ) or anti - H3K27me3 ( E ) with circBtnl1 + / + and circBtnl1 - / - ISCs lysates . * * P < 0 . 01 by two - tailed Student ' s t - test . Data are representative of at least three independent experiments .

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(D, E) ChIP assay was performed using anti-H3K4me3 (D) or anti-H3K27me3 (E) with circBtnl1+/+ and circBtnl1-/- ISCs lysates. **P < 0.01 by two-tailed Student's t-test. Data are representative of at least three independent experiments.


( H ) Confocal microscopy of Sox9 in Atf4 knockdown ( Atf4 KD ) organoids from circBtnl1 + / + and circBtnl1 - / - mice . Scale bars , 30 μm .

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(H) Confocal microscopy of Sox9 in Atf4 knockdown (Atf4 KD) organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 30 μm.


( D ) H & E staining of small intestinal tissues from indicated mice . Two - month - old mice were used and n = 3 fields were taken for each group . Scale bar , 100 μm . Number of crypts were calculated as means ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t - test in right panel .

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(D) H&E staining of small intestinal tissues from indicated mice. Two-month-old mice were used and n = 3 fields were taken for each group. Scale bar, 100 μm. Number of crypts were calculated as means ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t-test in right panel.


( E ) H & E staining of intestinal tissues from indicated mice at different time points after 8 Gy of radiation damage . Two - month - old mice were used and n = 3 fields were taken for each group . Number of crypts were calculated as means ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . Scale bar , 50 μm .

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(E) H&E staining of intestinal tissues from indicated mice at different time points after 8 Gy of radiation damage. Two-month-old mice were used and n = 3 fields were taken for each group. Number of crypts were calculated as means ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t-test in right panel. Scale bar, 50 μm.


( F ) Representative organoid images of circBtnl1 + / + ; sgSox9 and circBtnl1 - / - ; sgSox9 mice , sgCtrl served as control . Organoid formation ratios ( n = 3 ) are shown . Organoid formation ratios are shown as mean ± s . d . * P < 0 . 05 by two - tailed Student ' s t - test . Scale bars , 200 μm .

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(F) Representative organoid images of circBtnl1+/+; sgSox9 and circBtnl1-/-; sgSox9 mice, sgCtrl served as control. Organoid formation ratios (n = 3) are shown. Organoid formation ratios are shown as mean ± s. d. *P < 0.05 by two-tailed Student's t-test. Scale bars, 200 μm.


BATs were isolated from cold - exposed mice ( 4 ℃ , 72 h ) or control mice ( 30 ℃ , 72 h ) , then lysed and subjected to western blotting for analysis of the indicated proteins ( A ) . Relative protein expression levels , normalized to Actin , are shown in ( B ) .

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BATs were isolated from cold-exposed mice (4℃, 72 h) or control mice (30℃, 72 h), then lysed and subjected to western blotting for analysis of the indicated proteins (A). Relative protein expression levels, normalized to Actin, are shown in (B).


mRNA expression levels of the indicated genes were assayed by real - time PCR and normalized to Actb ( C ) .

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mRNA expression levels of the indicated genes were assayed by real-time PCR and normalized to Actb (C).


Mitochondrial DNA copy numbers were determined by real - time PCR and normalized to nuclear DNA standards ( D ) .

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Mitochondrial DNA copy numbers were determined by real-time PCR and normalized to nuclear DNA standards (D).


Relative increase of mitochondrial proteins was normalized to mtDNA copy numbers and plotted ( E ) .

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Relative increase of mitochondrial proteins was normalized to mtDNA copy numbers and plotted (E).


( F - G ) Representative images showing immunohistochemical detection of FUNDC1 in sections of BATs from mice kept at 30 ℃ or 4 ℃ for 72 h . Scale bar , 50 μm . The expression of FUNDC1 was quantified by analyzing and normalizing the average optical density of FUNDC1 staining .

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(F-G) Representative images showing immunohistochemical detection of FUNDC1 in sections of BATs from mice kept at 30℃ or 4℃ for 72 h. Scale bar, 50 μm. The expression of FUNDC1 was quantified by analyzing and normalizing the average optical density of FUNDC1 staining.


Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30 ℃ ( Normal ) or following 72 h at 4 ℃ ( Cold ) ( A ) . Relative protein expression levels were normalized to Actin ( B ) .

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Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30℃ (Normal) or following 72 h at 4℃ (Cold) (A). Relative protein expression levels were normalized to Actin (B).


Flag - PGC - 1α was introduced into PGC - 1α knockdown brown preadipocytes for 24 h and samples were collected for western blotting analyses of the indicated proteins ( D ) . Expression levels of the indicated proteins were quantified ( E ) ( mean ± SEM ; n = 3 ) . Scramble , scramble shRNA .

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Flag-PGC-1α was introduced into PGC-1α knockdown brown preadipocytes for 24 h and samples were collected for western blotting analyses of the indicated proteins (D). Expression levels of the indicated proteins were quantified (E) (mean ± SEM; n=3). Scramble, scramble shRNA.


Samples were collected for real - time PCR analysis . The expression of Fundc1 was normalized ( F ) .

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Samples were collected for real-time PCR analysis. The expression of Fundc1 was normalized (F).


( G - H ) Scramble and PGC - 1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses ( G ) and quantification of the indicated proteins ( H )

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(G-H) Scramble and PGC-1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses (G) and quantification of the indicated proteins (H)


( B ) Luciferase activity stimulated by the human Fundc1 promoter . The activity of Fundc1 promoter fragments , either wild type ( WT ) or with a mutation in the potential NRF1 binding site , was assayed using a dual luciferase reporter system .

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(B) Luciferase activity stimulated by the human Fundc1 promoter. The activity of Fundc1 promoter fragments, either wild type (WT) or with a mutation in the potential NRF1 binding site, was assayed using a dual luciferase reporter system.


( C , D ) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 ( F1 probe or mut F1 probe ) , the confirmed NRF1 binding site in the promoter of VSNL1 ( positive probe ) . The probes were incubated with HeLa nuclear extracts , then run on a gel ( C ) . A super - shifted band ( asterisk ) is detected in the presence of an anti - NRF1 antibody ( D ) . Labeled probe : biotin labeled wild type Fundc1 probe ; Cold probe : wild type Fundc1 probe ; Mut probe : NRF1 binding site mutant Fundc1 probe ; asterisk indicates the supershifted band generated with antibody to NRF1 .

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(C,D) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 (F1 probe or mut F1 probe) ,the confirmed NRF1 binding site in the promoter of VSNL1 (positive probe). The probes were incubated with HeLa nuclear extracts, then run on a gel (C). A super-shifted band (asterisk) is detected in the presence of an anti-NRF1 antibody (D). Labeled probe: biotin labeled wild type Fundc1 probe; Cold probe: wild type Fundc1 probe; Mut probe: NRF1 binding site mutant Fundc1 probe; asterisk indicates the supershifted band generated with antibody to NRF1.


( E , F ) ChIP assay to analyze the interaction of NRF1 ( E ) or PGC - 1α ( F ) proteins with the Fundc1 promoter .

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(E,F) ChIP assay to analyze the interaction of NRF1 (E) or PGC-1α (F) proteins with the Fundc1 promoter.


( G ) NRF1 - myc was introduced into NRF1 shRNA knockdown cells for 24 h and scramble cells were used as control and samples were collected for western blotting with the indicated antibodies . ( H ) Scramble and NRF1 shRNA knockdown cells were transfected with vector and PGC - 1α - Flag for 24 h and samples were collected for western blotting with the indicated antibodies .

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(G) NRF1-myc was introduced into NRF1 shRNA knockdown cells for 24 h and scramble cells were used as control and samples were collected for western blotting with the indicated antibodies. (H) Scramble and NRF1 shRNA knockdown cells were transfected with vector and PGC-1α-Flag for 24 h and samples were collected for western blotting with the indicated antibodies.


( I ) ChIP analysis of the interaction of PGC - 1α protein with the Fundc1 promoter in scramble or NRF1 shRNA knockdown cells .

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(I) ChIP analysis of the interaction of PGC-1α protein with the Fundc1 promoter in scramble or NRF1 shRNA knockdown cells.


( A , B ) Western blotting analyses of the levels of autophagy - related proteins in BAT from mice at the normal temperature ( 30 ℃ ) or at 4 ℃ for 72 h ( A ) . Expression of the indicated proteins was analyzed before and after 72 h cold exposure ( 4 ℃ ) ( B ) .

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(A,B) Western blotting analyses of the levels of autophagy-related proteins in BAT from mice at the normal temperature (30℃) or at 4℃ for 72 h (A). Expression of the indicated proteins was analyzed before and after 72 h cold exposure (4℃) (B).


( C ) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature ( 30 ℃ ) or 4 ℃ for 72 h .

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(C) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature (30 ℃) or 4℃ for 72 h.


( D Representative transmission electron microscope ( TEM ) images of BAT cells isolated from mice at the normal temperature ( 30 ℃ ) or 4 ℃ for 72 h . Scale bar , 1

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(D Representative transmission electron microscope (TEM) images of BAT cells isolated from mice at the normal temperature (30℃) or 4℃ for 72 h. Scale bar, 1 μm


( D ) . The number of mitophagosomes per 100 mitochondria was counted and normalized ( E ) .

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(D).The number of mitophagosomes per 100 mitochondria was counted and normalized (E).


( F , G ) BATs from mice exposed to normal conditions ( 30 ℃ ) or cold ( 4 ℃ ) were lysed and immunoprecipitated with antibodies against FUNDC1 , LC3B or IgG , then immunoblotted with the indicated antibodies . The ratio between immunoprecipitated LC3B to FUNDC1 or FUNDC1 to LC3B were quantified and plotted ( G ) .

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(F,G) BATs from mice exposed to normal conditions (30℃) or cold (4℃) were lysed and immunoprecipitated with antibodies against FUNDC1, LC3B or IgG, then immunoblotted with the indicated antibodies. The ratio between immunoprecipitated LC3B to FUNDC1 or FUNDC1 to LC3B were quantified and plotted (G).


( A , B ) BATs isolated from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) mice at 30 ℃ or following 72 h at 4 ℃ were analyzed by transmission electron microscopy . Representative images are shown , scale bar , 1μm ( A ) . The number of mitophagosomes per 100 mitochondria were counted and normalized ( B ) .

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(A,B) BATs isolated from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) mice at 30℃ or following 72 h at 4℃ were analyzed by transmission electron microscopy. Representative images are shown, scale bar, 1μm (A). The number of mitophagosomes per 100 mitochondria were counted and normalized (B).


( C , D ) . Representative fluorescence images of BAT sections from Fundc1fl / fl / Adiponectincre - ( Cre - ) and Fundc1fl / fl / Adiponectincre + ( Cre + ) mito - Keima mice at normal conditions ( 30 ℃ ) or 4 ℃ for 72 h . Scale bar , 50 μm ( C ) . Quantification of the ratio between red and green signals ( D ) .

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(C,D).Representative fluorescence images of BAT sections from Fundc1fl/fl/Adiponectincre- (Cre-) and Fundc1fl/fl/Adiponectincre+ (Cre+) mito-Keima mice at normal conditions (30℃) or 4℃ for 72 h. Scale bar, 50 μm (C). Quantification of the ratio between red and green signals (D).


( E ) Fundcfl / fl / Ucp1cre - ( Cre - ) and Fundcfl / fl / Ucpcre + ( Cre + ) mice were maintained at 30 ℃ ( Normal ) or exposed to 4 ℃ for 72 h ( Cold ) . Mitochondrial DNA copy numbers were analyzed by null and normalized to nuclear DNA standards .

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(E) Fundcfl/fl/Ucp1cre- (Cre-) and Fundcfl/fl/Ucpcre+ (Cre+) mice were maintained at 30℃ (Normal) or exposed to 4℃ for 72 h (Cold). Mitochondrial DNA copy numbers were analyzed by null and normalized to nuclear DNA standards.


( F ) The relative total mito - keima signal ( green signal ) in Fundc1 deficiency and control mito - keima mice under normal condition or cold exposure were quantified and plotted .

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(F) The relative total mito-keima signal (green signal ) in Fundc1 deficiency and control mito-keima mice under normal condition or cold exposure were quantified and plotted.


( G ) BATs isolated from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) at 30 ℃ or following 72 h at 4 ℃ were analyzed by transmission electron microscopy . The percent of mitochondria without cristae is shown in the histogram .

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(G) BATs isolated from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) at 30℃ or following 72 h at 4℃ were analyzed by transmission electron microscopy. The percent of mitochondria without cristae is shown in the histogram.


Oxygen consumption rate ( OCR ) ( H ) were measured in BAT from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucpcre + ( Cre + ) mice kept at 30 ℃ or at 4 ℃ for 72 h .

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Oxygen consumption rate (OCR) (H) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.


mitochondrial superoxide ( I ) were measured in BAT from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucpcre + ( Cre + ) mice kept at 30 ℃ or at 4 ℃ for 72 h .

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mitochondrial superoxide (I) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.


( A ) Body temperature measurements in Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) fasting male mice before and during cold exposure ( 4 ° C ) for the indicated time . Body temperature ( B ) before and after 72 h cold exposure ( 4 ° C ) of Cre - and Cre + male mice .

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(A) Body temperature measurements in Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) fasting male mice before and during cold exposure (4°C) for the indicated time. Body temperature (B) before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice.


BAT tissue weight ( C ) measurements before and after 72 h cold exposure ( 4 ° C ) of Cre - and Cre + male mice .

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BAT tissue weight (C) measurements before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice.


Fundc1fl / fl / Ucpcre - and Fundc1fl / fl / Ucp1cre + mice were exposed to 4 ℃ for 72 h ( Cold ) , and control mice were kept at 30 ℃ ( Normal ) . Western blotting analysis of BAT protein levels ( D ) . Protein expression levels were normalized to Actin ( E ) .

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Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Western blotting analysis of BAT protein levels (D). Protein expression levels were normalized to Actin (E).


Fundc1fl / fl / Ucpcre - and Fundc1fl / fl / Ucp1cre + mice were exposed to 4 ℃ for 72 h ( Cold ) , and control mice were kept at 30 ℃ ( Normal ) . Levels of mRNAs encoding Fundc1 and Ucp1 were measured by real - time PCR and normalized to Actb ( F ) .

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Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Levels of mRNAs encoding Fundc1 and Ucp1 were measured by real-time PCR and normalized to Actb (F).


( G ) Representative images of UCP1 immunohistochemistry of BAT sections from Cre - or Cre + mice in normal conditions ( 30 ℃ ) or following 72 h at 4 ℃ . Scale bar , 50 μm .

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(G) Representative images of UCP1 immunohistochemistry of BAT sections from Cre- or Cre+ mice in normal conditions (30℃) or following 72 h at 4℃. Scale bar, 50 μm.


( I ) Normalized death rates in L929 wt cells plotted with pMLKL protein levels measured via

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(I) Normalized death rates in L929 wt cells plotted with pMLKL protein levels measured via immunoblot


( M ) Death rates in TNF - treated cell lines including clonal RelA - knockout population ( mean of three independent experiments ± standard deviation ) .

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(M) Death rates in TNF-treated cell lines including clonal RelA-knockout population (mean of three independent experiments ± standard deviation).


( A ) TNF - induced mRNA expression in indicated L929 cell lines measured via qRT - PCR ( mean of three independent experiments ± standard deviation , two - tailed Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 ) .

O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) TNF-induced mRNA expression in indicated L929 cell lines measured via qRT-PCR (mean of three independent experiments ± standard deviation, two-tailed Student's t-test *P<0.05, **P<0.01).


( B ) Immunoblot after co - immunoprecipitation ( IP ) of RIPK3 . FT , flow through . ( C ) Relative quantification of A20 in RIPK3 - IP fraction ( means and statistical significance established for 0 , 2 , 4 . 5 and 8 hour time points from three independent experiments ± standard deviation ; two - tailed Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 , n . s . , P > 0 . 05 ) .

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

(B) Immunoblot after co-immunoprecipitation (IP) of RIPK3. FT, flow through. (C) Relative quantification of A20 in RIPK3-IP fraction (means and statistical significance established for 0, 2, 4.5 and 8 hour time points from three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05, **P<0.01, n.s., P>0.05).


( I , J ) ( I ) TNF - induced distributions of death times ( representative data of three independent experiments ) , and ( J ) death rates obtained by live - cell microscopy ( mean of three independent experiments ± standard deviation ) .

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

(I,J) (I) TNF-induced distributions of death times (representative data of three independent experiments), and (J) death rates obtained by live-cell microscopy (mean of three independent experiments ± standard deviation).