( D ) Quantification of Ki - 67 + and AURKB + double positive cells was performed using five confocal images per mice in control and TGFα mice . Images were obtained at 40X magnification . Scale bar : 50µm . * * * * P < 0 . 00005 , unpaired t - test , ( n = 4 ) .

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(D) Quantification of Ki-67+ and AURKB+ double positive cells was performed using five confocal images per mice in control and TGFα mice. Images were obtained at 40X magnification. Scale bar: 50µm. ****P < 0.00005, unpaired t-test, (n=4).


( E ) Proliferation was measured in primary lung resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA . * * * * P < 0 . 00005 , unpaired t - test , ( n = 9 - 11 ) .

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(E) Proliferation was measured in primary lung resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA. ****P < 0.00005, unpaired t-test, (n=9-11).


( F ) Proliferation was measured in primary lung - resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα ( 20ng / mL ) for 24hrs . * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(F) Proliferation was measured in primary lung-resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα (20ng/mL) for 24hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).


( G ) Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs . * * P < 0 . 005 , * * * P < 0 . 0005 , unpaired t - test , ( n = 3 ) .

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(G) Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).


( H ) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB ( green ) and αSMA ( red ) . Images were obtained at 40X magnification . Scale bar : 50µm . ( n = 4 ) .

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(H) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB (green) and αSMA (red). Images were obtained at 40X magnification. Scale bar: 50µm. (n=4).


( I ) Quantification of apoptotic cells using Incucyte ZOOM ( Caspase - 3 / 7 - positive cells ) in lung - resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h . * * P < 0 . 005 , * * * * P < 0 . 00005 , 2 - way ANOVA , ( n = 4 ) .

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(I) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in lung-resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h. **P < 0.005, ****P < 0.00005, 2-way ANOVA, (n=4).


( J ) Quantification of BAK , BAX and FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs . * * P < 0 . 005 , * * * P < 0 . 0005 , unpaired t - test , ( n = 3 ) .

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(J) Quantification of BAK, BAX and FAS gene transcripts in human IPF lung resident fibroblasts treated with either control or AURKB siRNA for 72hrs. **P < 0.005, ***P < 0.0005, unpaired t-test, (n=3).


( K ) Quantification of Bak , Bax and Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72hrs . * P < 0 . 005 , * * P < 0 . 005 , unpaired t - test , ( n = 3 ) .

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(K) Quantification of Bak, Bax and Fas gene transcripts in lung resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72hrs. *P < 0.005, **P < 0.005, unpaired t-test, (n=3).


( A ) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs . * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(A) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).


( B ) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs . * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(B) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs. ***P < 0.0005, 1-way ANOVA, (n=9-11).


( C ) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody . Images were obtained at 40X magnification . Scale bar : 50µm . The number of PCNA - positive cells and total DAPI - positive cells were quantified using MetaMorph image analysis software . Proliferation is indicated as the percentage of proliferating cells in total DAPI - positive cells . * * * * P < 0 . 00005 , unpaired t - test , ( n = 3 - 4 ) .

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(C) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody. Images were obtained at 40X magnification. Scale bar: 50µm. The number of PCNA-positive cells and total DAPI-positive cells were quantified using MetaMorph image analysis software. Proliferation is indicated as the percentage of proliferating cells in total DAPI-positive cells. ****P < 0.00005, unpaired t-test, (n=3-4).


( D ) Proliferation was measured in primary fibroblasts treated with TGFα ( 20ng / mL ) and indicated doses of barasertib for 48hrs . . * * P < 0 . 005 , 1 - way ANOVA , ( n = 9 - 11 ) .

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(D) Proliferation was measured in primary fibroblasts treated with TGFα (20ng/mL) and indicated doses of barasertib for 48hrs.. **P<0.005, 1-way ANOVA, (n=9-11).


( E ) Quantification of apoptotic cells using Incucyte ZOOM ( Caspase - 3 / 7 - positive cells ) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib . * * * * P < 0 . 00005 , 2 - way ANOVA , ( n = 4 ) .

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(E) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib. ****P < 0.00005, 2-way ANOVA, (n=4).


( B ) Representative images of Masson ' s trichrome - stained lung sections from the vehicle and barasertib treated mice . Images were obtained at 10X magnification . Scale bar : 200μm .

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(B) Representative images of Masson's trichrome-stained lung sections from the vehicle and barasertib treated mice. Images were obtained at 10X magnification. Scale bar: 200μm.


( C ) Quantification of right lung weight of mice treated with vehicle or barasertib . * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 - 10 mice / group ) .

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(C) Quantification of right lung weight of mice treated with vehicle or barasertib. ***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8-10 mice/group).


( D ) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib . * P < 0 . 05 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 8 - 10 mice / group ) .

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(D) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=8-10 mice/group).


( E ) Quantification of Col1α , Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib . * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 mice / group ) .

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(E) Quantification of Col1α, Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib.***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).


( A ) Lung sections from vehicle and barasertib treated mice were immunostained using Ki - 67 antibody . Representative images were obtained at 20X magnification . Scale bar : 50μm .

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(A) Lung sections from vehicle and barasertib treated mice were immunostained using Ki-67 antibody. Representative images were obtained at 20X magnification. Scale bar: 50μm.


( B ) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody . Gapdh is used as loading control . * * P < 0 . 005 , 1 - way ANOVA , ( n = 4 - 5 mice / group ) .

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(B) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody. Gapdh is used as loading control. **P< 0.005, 1-way ANOVA, (n=4-5 mice/group).


( C ) Quantification of Aurkb , Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib . * * P < 0 . 005 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 8 mice / group ) .

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(C) Quantification of Aurkb, Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib. **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).


( B ) Quantification of right lung weight of mice treated with vehicle and barasertib . * * P < 0 . 005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 10 mice / group ) .

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(B) Quantification of right lung weight of mice treated with vehicle and barasertib. **P< 0.005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).


( C ) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies . Gapdh is used as loading control . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 4 - 5 mice / group ) .

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(C) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies. Gapdh is used as loading control. *P< 0.05, **P<0.005, ***P < 0.0005, 1-way ANOVA, (n=4-5 mice/group).


( D ) Representative images of Masson ' s trichrome stained lung section from mice treated with vehicle and barasertib . Images were obtained at 10X magnification . Scale bar : 200μm .

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(D) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.


( E ) Quantification of lung mechanics in mice treated with vehicle and barasertib . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 9 - 10 mice / group ) .

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(E) Quantification of lung mechanics in mice treated with vehicle and barasertib. *P<0.05, **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).


( B ) Representative images of Masson ' s trichrome stained lung section from mice treated with vehicle and barasertib . Images were obtained at 10X magnification . Scale bar : 200μm .

(B) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.


( C ) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib . * P < 0 . 05 , * * * P < 0 . 0005 , 1 - way ANOVA , ( n = 7 - 8 mice / group ) .

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(C) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=7-8 mice/group).


( D ) Quantification of Col1α1 , Col3α , Col5α , Col14 , Col15 , αSma , Ccna2 , and Fas gene transcripts in total lungs of mice treated with vehicle or barasertib . * P < 0 . 05 , * * P < 0 . 005 * * * P < 0 . 0005 , * * * * P < 0 . 00005 , 1 - way ANOVA , ( n = 6 mice / group ) .

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(D) Quantification of Col1α1, Col3α, Col5α, Col14, Col15, αSma, Ccna2, and Fas gene transcripts in total lungs of mice treated with vehicle or barasertib. *P< 0.05, **P<0.005 ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=6 mice/group).


( E ) Quantification of αSma protein levels in lung lysates from vehicle and barasertib treated mice . Gapdh is used as loading control . Data are presented as mean ± SEM . * * P < 0 . 005 , unpaired t - test ( n = 5mice / group ) .

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(E) Quantification of αSma protein levels in lung lysates from vehicle and barasertib treated mice. Gapdh is used as loading control. Data are presented as mean ± SEM. **P<0.005, unpaired t-test (n=5mice /group).


A CD4 + CD127 - CD25hiFoxP3 + CTLA4 + Treg induction from PBMCs incubated with CVF , CLys and CSN ( 10µg / ml ) after 4h , 24h , 48h , 72h ( plots and graphs shown ) , 120h and 168h .

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A CD4+CD127-CD25hiFoxP3+CTLA4+ Treg induction from PBMCs incubated with CVF, CLys and CSN (10µg/ml) after 4h, 24h, 48h, 72h (plots and graphs shown), 120h and 168h.


B Trafficking receptors ( CXCR3 , CCR4 , CCR6 and CCR7 ) evaluation on induced Treg ( CD25hiCD127 - ) and non - Treg cells ( CD127 + CD25 - ) .

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B Trafficking receptors (CXCR3, CCR4, CCR6 and CCR7) evaluation on induced Treg (CD25hiCD127-) and non-Treg cells (CD127+CD25-).


C Induced Treg proliferation with Ki67 staining and Treg expression of IL - 10 and IFNγ .

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C Induced Treg proliferation with Ki67 staining and Treg expression of IL-10 and IFNγ.


A Levels of pro - and anti - inflammatory cytokines in PBMC culture supernatants with CVF , CLys and CSN ( 10µg / ml ) after 72h determined using a LUMINEX - based technology .

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A Levels of pro- and anti-inflammatory cytokines in PBMC culture supernatants with CVF, CLys and CSN (10µg/ml) after 72h determined using a LUMINEX-based technology.


B Composition and levels of bioactive lipid mediators ( LMs ) , including prostaglandins , leukotrienes , lipoxins , thromboxanes , and their precursors in culture with CVF , CLys and CSN quantified by LC - MS / MS - based profiling . Heat maps show relative amounts of LMs detected by LC - MS / MS in supernatants . Asterisks show statistical differences analyzed using two - way ANOVA followed by Turkey ' s multiple comparison test .

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B Composition and levels of bioactive lipid mediators (LMs), including prostaglandins, leukotrienes, lipoxins, thromboxanes, and their precursors in culture with CVF, CLys and CSN quantified by LC-MS/MS-based profiling. Heat maps show relative amounts of LMs detected by LC-MS/MS in supernatants. Asterisks show statistical differences analyzed using two-way ANOVA followed by Turkey's multiple comparison test.


C Dynamic of PGE2 release correlated with Treg induction by CLys ( upper panel ) and CSN ( lower panel ) using a Pearson correlation .

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C Dynamic of PGE2 release correlated with Treg induction by CLys (upper panel) and CSN (lower panel) using a Pearson correlation.


D Treg induction by CLys or CSN following PBMC pre - incubation with 10µM mPGES1 inhibitor 934 ( or DMSO as a control ) .

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D Treg induction by CLys or CSN following PBMC pre-incubation with 10µM mPGES1 inhibitor 934 (or DMSO as a control).


E Induction of Tregs with PGE2 analog ( DINP ) ( 5µM ) and / or recombinant human IL - 10 ( rhIL - 10 ) ( 10ng / ml ) as compared to CLys .

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E Induction of Tregs with PGE2 analog (DINP) (5µM) and/or recombinant human IL-10 (rhIL-10) (10ng/ml) as compared to CLys.


A Levels of bioactive LMs and IL - 10 released from monocyte and non - monocyte fractions .

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A Levels of bioactive LMs and IL-10 released from monocyte and non-monocyte fractions.


B Non - monocyte fractions were pre - incubated with IL - 10R inhibitor and PGE2 receptor ( EP2 and EP4 ) antagonists ( or DMSO as a control ) and incubated with CLys ( left upper panel ) or CSN ( 10µg / ml ) ( left lower panel ) in presence or absence of monocytes and Treg induction was analysed after 72h . On the right panel , PGE2 receptors were first blocked on monocytes ( 2 ) or non - monocytes ( 3 ) or both ( 4 ) before addition of non - monocytes ( 2 ) or monocytes ( 3 ) and incubation with CSN for 72h and analysis of Treg induction compared to control condition ( 1 ) .

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B Non-monocyte fractions were pre-incubated with IL-10R inhibitor and PGE2 receptor (EP2 and EP4) antagonists (or DMSO as a control) and incubated with CLys (left upper panel) or CSN (10µg/ml) (left lower panel) in presence or absence of monocytes and Treg induction was analysed after 72h. On the right panel, PGE2 receptors were first blocked on monocytes (2) or non-monocytes (3) or both (4) before addition of non-monocytes (2) or monocytes (3) and incubation with CSN for 72h and analysis of Treg induction compared to control condition (1).


C Treg induction by CLys and CSN in presence of monocyte / non - monocyte fractions and naïve CD4 + CD45RA + CD45RO - T cells .

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C Treg induction by CLys and CSN in presence of monocyte/non-monocyte fractions and naïve CD4+CD45RA+CD45RO- T cells.


D Monocyte phenotyping in presence of CVF , CLys and CSN by FACS using CD68 , CD80 , CD163 and CD206 surface expression .

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D Monocyte phenotyping in presence of CVF, CLys and CSN by FACS using CD68, CD80, CD163 and CD206 surface expression.


E Levels of LMs and PGE2 released by macrophages in presence of CVF , CLys and CSN Additionally , macrophages were lysed and gene expression of PTGS2 , PTGES , ALOX5 , ALOX15 and MaR1 quantified by RT - PCR .

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E Levels of LMs and PGE2 released by macrophages in presence of CVF, CLys and CSN Additionally, macrophages were lysed and gene expression of PTGS2, PTGES, ALOX5, ALOX15 and MaR1 quantified by RT-PCR.


F Treg induction following naïve CD4 + CD45RA + CD45RO - T cells incubation with CLys - , CSN - monocyte conditioned media .

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F Treg induction following naïve CD4+CD45RA+CD45RO- T cells incubation with CLys-, CSN-monocyte conditioned media.


A Induction of Treg following PBMC treatment with either native or proteinase K - treated CLys or CSN ( 10µg / ml ) for 72h .

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A Induction of Treg following PBMC treatment with either native or proteinase K-treated CLys or CSN (10µg/ml) for 72h.


C PBMCs were treated with GDH inhibitor bithionol ( BT ) ( 20µM ) or IDH inhibitor IDH - 305 ( 50nM ) alone or in combination before incubation with CLys or CSN . Percentage of Treg was determined and PGE2 and IL - 10 levels were evaluated .

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C PBMCs were treated with GDH inhibitor bithionol (BT) (20µM) or IDH inhibitor IDH-305 (50nM) alone or in combination before incubation with CLys or CSN. Percentage of Treg was determined and PGE2 and IL-10 levels were evaluated.


D Treg induction from PBMC by CLys , CSN and TsGDH ( 5µg / ml ) and levels of PGE2 and IL - 10 in culture supernatants .

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D Treg induction from PBMC by CLys, CSN and TsGDH (5µg/ml) and levels of PGE2 and IL-10 in culture supernatants.


E Treg induction from monocyte - naïve CD4 + - TsGDH culture and levels of PGE2 and IL - 10 in monocyte - CD4 + - TsGDH and monocyte - TsGDH culture supernatants .

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E Treg induction from monocyte-naïve CD4+-TsGDH culture and levels of PGE2 and IL-10 in monocyte-CD4+-TsGDH and monocyte-TsGDH culture supernatants.


F Treg induction from PBMC by CLys , CSN and rhGDH ( 10µg / ml ) .

F Treg induction from PBMC by CLys, CSN and rhGDH (10µg/ml).


( A ) In vitro Methyltransferase activity . Recombinant proteins Nsp10 and Nsp16 wt or Nsp16mut were combined with cap0 RNA and Adenosyl - L - methionine ( [ methyl - 3H ] SAM ) for 90 min at 37 ° C . Methylation of cap0 RNA was quantified using a scintillation counter ( Beckmann ) . ( - ) control did not contain any recombinant protein . Results are plotted as mean of triplicate reactions with error bars representing SD . One out of three independent biological repeats is shown . Data information : Statistical analysis was done using a one - way ANOVA followed by Tukey ' s multiple comparisons test .

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(A) In vitro Methyltransferase activity. Recombinant proteins Nsp10 and Nsp16 wt or Nsp16mut were combined with cap0 RNA and Adenosyl-L-methionine ([methyl-3H] SAM) for 90 min at 37°C. Methylation of cap0 RNA was quantified using a scintillation counter (Beckmann).(-) control did not contain any recombinant protein. Results are plotted as mean of triplicate reactions with error bars representing SD. One out of three independent biological repeats is shown. Data information: Statistical analysis was done using a one-way ANOVA followed by Tukey's multiple comparisons test.


( B ) MTase activity of Nsp16 upon viral infection . Cellular mRNA was purified 24 h postinfection with SARS - CoV - 2 wt or Nsp16mut ( triplicate infections , biological replicates ) . Purified mRNA was incubated with 100U of the recombinant MTase VACV VP39 ( NEB ) and [ methyl - 3H ] SAM for 90 min at 37 ° C . Methylation of mRNA from SARS - CoV - 2 infected cells was quantified using a scintillation counter ( DPM3H ; Beckmann ) . ( - ) control did not contain cellular RNA . Results are plotted as mean of triplicate infections with error bars representing SD . One out of three independent experiments is shown ( n = 3 ) . Data information : Statistical analysis was done using a one - way ANOVA followed by Tukey ' s multiple comparisons test .

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(B) MTase activity of Nsp16 upon viral infection. Cellular mRNA was purified 24 h postinfection with SARS-CoV-2 wt or Nsp16mut (triplicate infections, biological replicates). Purified mRNA was incubated with 100U of the recombinant MTase VACV VP39 (NEB) and [methyl-3H] SAM for 90 min at 37 °C. Methylation of mRNA from SARS-CoV-2 infected cells was quantified using a scintillation counter (DPM3H; Beckmann). (-) control did not contain cellular RNA. Results are plotted as mean of triplicate infections with error bars representing SD. One out of three independent experiments is shown (n=3). Data information: Statistical analysis was done using a one-way ANOVA followed by Tukey's multiple comparisons test.


( A ) Calu - 3 cells were incubated with the indicated amounts of type I IFN 24 h prior to infection with normalized amounts of SARS - CoV - 2 wt ( WT ) or Nsp16mut at 0 . 015 RNA copies / cell . Viral load in the supernatant was quantified at the indicated time points by RT - qPCR targeting viral polymerase RdRp . Results are plotted as mean of triplicate infections with error bars representing the SD ( biological replicates ) . One out of three independent experiments is shown ( n = 3 ) . Three days postinfection , protein expression of IFIT1 was analyzed by immunoblot to control for type I IFN treatment . Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90 . Data information : Statistical analysis was done using two - way ANOVA followed by Bonferroni ' s multiple comparisons test . ns = not significant ( p > 0 . 05 ) .

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(A) Calu-3 cells were incubated with the indicated amounts of type I IFN 24 h prior to infection with normalized amounts of SARS-CoV-2 wt (WT) or Nsp16mut at 0.015 RNA copies/cell. Viral load in the supernatant was quantified at the indicated time points by RT-qPCR targeting viral polymerase RdRp. Results are plotted as mean of triplicate infections with error bars representing the SD (biological replicates). One out of three independent experiments is shown (n=3). Three days postinfection, protein expression of IFIT1 was analyzed by immunoblot to control for type I IFN treatment. Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90. Data information: Statistical analysis was done using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).


( C ) Calu - 3 cells lacking MDA5 ( MDA5 KO ) , RIG - I ( RIG - I KO ) , or control KO cells ( Ctrl ) were seeded into 96well plates and infected with increasing amounts of SARS - CoV - 2 wt ( WT ) or Nsp16mut in triplicates ( biological replicates ) . Type I IFN release was quantified after 48 h by incubating supernatants with HEK - Blue IFN - α / β reporter cells . SEAP activity in the supernatant of the reporter cells is shown as mean of quadruplicates with error bars representing SD . ( D ) Calu - 3 cells ( Ctrl ) were seeded into 96 well plates and infected with 10 RNA copies / cell of SARS - CoV - 2 wt ( WT ) or Nsp16mut in the presence of increasing amounts of Remdesivir ( RDV ) . Type I IFN release was quantified after 48 h by IFN bioassay on HEK - Blue IFN - α / β and is depicted as mean of quadruplicates with error bars representing SD . In parallel , infectivity of SARS - CoV - 2 wt and Nsp16 in the presence of increasing amounts of RDV was determined at the indicated time points by RT - qPCR targeting viral polymerase RdRp transcripts . Data information : Statistical analysis was performed using two - way ANOVA followed by Bonferroni ' s multiple comparisons test . ns = not significant ( p > 0 . 05 ) .

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(C) Calu-3 cells lacking MDA5 (MDA5 KO), RIG-I (RIG-I KO), or control KO cells (Ctrl) were seeded into 96well plates and infected with increasing amounts of SARS-CoV-2 wt (WT) or Nsp16mut in triplicates (biological replicates). Type I IFN release was quantified after 48 h by incubating supernatants with HEK-Blue IFN-α/β reporter cells. SEAP activity in the supernatant of the reporter cells is shown as mean of quadruplicates with error bars representing SD. (D) Calu-3 cells (Ctrl) were seeded into 96 well plates and infected with 10 RNA copies/cell of SARS-CoV-2 wt (WT) or Nsp16mut in the presence of increasing amounts of Remdesivir (RDV). Type I IFN release was quantified after 48 h by IFN bioassay on HEK-Blue IFN-α/β and is depicted as mean of quadruplicates with error bars representing SD. In parallel, infectivity of SARS-CoV-2 wt and Nsp16 in the presence of increasing amounts of RDV was determined at the indicated time points by RT-qPCR targeting viral polymerase RdRp transcripts. Data information: Statistical analysis was performed using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).


( B ) Three days after SARS - CoV - 2 Nsp16mut infection of A549 cells described in ( A ) , expression of IFIT1 and viral nucleocapsid protein N was analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 , SARS - CoV - 2 N protein , and the housekeeping gene HSP90 .

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(B) Three days after SARS-CoV-2 Nsp16mut infection of A549 cells described in (A), expression of IFIT1 and viral nucleocapsid protein N was analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1, SARS-CoV-2 N protein, and the housekeeping gene HSP90.


( D ) IFIT1 expression levels in Calu - 3 Ctrl , IFIT1 KO # 1 and # 2 lines treated with type I IFN ( 24 h ; 100 U / ml ) were analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 or the housekeeping gene HSP90 .

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(D) IFIT1 expression levels in Calu-3 Ctrl, IFIT1 KO #1 and #2 lines treated with type I IFN (24 h; 100 U/ml) were analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1 or the housekeeping gene HSP90.


( F ) Three days postinfection , A549 cells described in ( E ) were analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 , SARS - CoV - 2 N protein , or the housekeeping gene HSP90 .

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(F) Three days postinfection, A549 cells described in (E) were analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1, SARS-CoV-2 N protein, or the housekeeping gene HSP90.


( H ) IFIT1 expression was analyzed in mock treated , SARS - CoV - 2 wt ( WT ) or Nsp16mut infected cells at 48 hpi by immunoblot . Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90 .

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(H) IFIT1 expression was analyzed in mock treated, SARS-CoV-2 wt (WT) or Nsp16mut infected cells at 48 hpi by immunoblot. Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90.


. MEFs from δWD and littermate control mice were incubated with HBSS , chloroquine or monensin for 2 hours as indicated . Cells were fixed and permeabilized and stained for LC3 . Arrows indicate LC3 vacuoles where a ring of LC3 signal surrounds vacuoles ranging between 2 and 8 μm diameter . B . Numbers of fluorescent LC3 puncta and LC3 vacuoles in each cell were quantified by fluorescence microscopy . LC3 puncta were identified using spot function software to locate puncta ranging from 0 . 5 - 1 . 0mm diameter . LC3 vacuoles were identified by eye as rings of fluorescence ranging between 2 and 8 μm diameter .

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. MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 hours as indicated. Cells were fixed and permeabilized and stained for LC3. Arrows indicate LC3 vacuoles where a ring of LC3 signal surrounds vacuoles ranging between 2 and 8 μm diameter. B. Numbers of fluorescent LC3 puncta and LC3 vacuoles in each cell were quantified by fluorescence microscopy. LC3 puncta were identified using spot function software to locate puncta ranging from 0.5-1.0mm diameter. LC3 vacuoles were identified by eye as rings of fluorescence ranging between 2 and 8 μm diameter.


MEFs from δWD and littermate control mice were incubated with HBSS , chloroquine or monensin for 2 h as indicated and cell lysates analysed by western blot for ATG16L1 , δWD and LC3 as indicated . Control MEFS express α and β isoforms of ATG16L1 at 70 kDa , and MEFs from δWD mice express a truncated ATG16L1 at 30kDa . ( D ) shows the level of conversion of LC3 to LC3II estimated by densitometry from a mean ( ± SD ) of three replicate blots .

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MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 h as indicated and cell lysates analysed by western blot for ATG16L1, δWD and LC3 as indicated. Control MEFS express α and β isoforms of ATG16L1 at 70 kDa, and MEFs from δWD mice express a truncated ATG16L1 at 30kDa. (D) shows the level of conversion of LC3 to LC3II estimated by densitometry from a mean (±SD) of three replicate blots.


E ) fluorescence images of phagosomes following engulfment of Zymosan ( red ) by bone marrow - derived macrophages ( BMDM ) from control and δWD mice . White line indicates track used for line profile analysis to compare the LC3 signal on the limiting membrane of the phagosome with the center . F shows examples of line profile analysis G , H . ( G ) shows the percentage of LC3 positive phagosomes per cell and ( H ) shows line profile analysis of data from 10 cells , bars represent the mean ± SD and were compared by Student ' s t - test ( * * * * P < 0 . 0001 ) .

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E) fluorescence images of phagosomes following engulfment of Zymosan (red) by bone marrow-derived macrophages (BMDM) from control and δWD mice. White line indicates track used for line profile analysis to compare the LC3 signal on the limiting membrane of the phagosome with the center. F shows examples of line profile analysis G, H. (G) shows the percentage of LC3 positive phagosomes per cell and (H) shows line profile analysis of data from 10 cells, bars represent the mean ± SD and were compared by Student's t-test (****P<0.0001).


Littermate control and δWD mice were challenged intranasally with IAV strain X31 ( 103 pfu ) . ( A ) Mice were monitored for weight loss at indicated time - points . ( n = 8 ) .

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Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points. (n = 8).


Littermate control and δWD mice were challenged intranasally with IAV strain X31 ( 103 pfu ) . ( B ) Survival was assessed at indicated time points ( n = 15 ) .

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Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (B) Survival was assessed at indicated time points (n = 15).


Littermate control and δWD mice were challenged intranasally with IAV strain X31 ( 103 pfu ) . ( C ) IAV titre in lungs was determined by plaque assay at 5 d . p . i . ( n = 6 ) .

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Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (C) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6).


Littermate control and δWD mice were challenged intranasally with IAV strain X31 ( 103 pfu ) . ( D ) The presence of IAV antigen was assessed by IH at 7 d . p . i . ( representative images from n = 6 ) .

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Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (D) The presence of IAV antigen was assessed by IH at 7 d.p.i. (representative images from n = 6).


Littermate control and δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . ( A ) At the indicated time points , cytokine mRNA transcripts in lung tissue ( n = 5 ) were evaluated by qPCR .

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Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). (A) At the indicated time points, cytokine mRNA transcripts in lung tissue (n = 5) were evaluated by qPCR.


Littermate control and δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . ( B ) Representative lung sections from animals ( n = 6 ) taken at 3 d . p . i . were stained by IH for neutrophils ( Ly6G ) or neutrophil extracellular traps ( NET ; anti - H3 ) . Sections at 7 d . p . i . were stained for macrophages ( Iba - 1 ) .

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Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). (B) Representative lung sections from animals (n = 6) taken at 3 d.p.i. were stained by IH for neutrophils (Ly6G) or neutrophil extracellular traps (NET; anti-H3). Sections at 7 d.p.i. were stained for macrophages (Iba-1).


Littermate control and δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . C ) BAL ( n = 5 ) was taken at 2 d . p . i and evaluated by flow cytometry , with pre - gating on CD45 + . The percentage of neutrophils ( CD11b + , Ly6G + ) cells is shown ± SEM and were compared using Mann - Whitney U test ( * P < 0 . 05 ) .

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Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). C) BAL (n = 5) was taken at 2 d.p.i and evaluated by flow cytometry, with pre-gating on CD45+. The percentage of neutrophils (CD11b+, Ly6G+) cells is shown ±SEM and were compared using Mann-Whitney U test (*P<0.05).


Littermate control and δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . ( D ) Single cell suspensions were prepared from lungs taken at 5 d . p . i and evaluated by flow cytometry , with pre - gating on CD45 + . The percentage of macrophage / monocytes ( CD11b + , F4 / 80 + ) cells is shown ± SEM and were compared using Mann - Whitney U test ( * P < 0 . 05 ) .

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Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). (D) Single cell suspensions were prepared from lungs taken at 5 d.p.i and evaluated by flow cytometry, with pre-gating on CD45+. The percentage of macrophage/monocytes (CD11b+, F4/80+) cells is shown ±SEM and were compared using Mann-Whitney U test (*P<0.05).


( E ) δWD mice were treated with either anti - PCDA - 1 ( to deplete plasmacytoid DC ) or an isotype - matched control . Litter - mate control mice were used as comparator . Weight loss was measured at the indicated days p . i . ( n = 5 ) .

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(E) δWD mice were treated with either anti-PCDA-1 (to deplete plasmacytoid DC) or an isotype-matched control. Litter-mate control mice were used as comparator. Weight loss was measured at the indicated days p.i. (n = 5).


A - D . Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 weeks , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( A ) Mice were monitored for weight loss at indicated time - points .

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A-D. Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points.


Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 weeks , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( B ) Survival was assessed at indicated time points .

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Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (B) Survival was assessed at indicated time points.


Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 weeks , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( C ) IAV titre in lungs was determined by plaque assay at 5 d . p . i . ( n = 6 ) .

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Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (C) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6).


Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 weeks , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( D ) Lungs taken at 5 d . p . i . were analysed for neutrophils ( Ly6G ) , neutrophil extracellular traps ( NET ; anti - H3 ) and macrophages ( Iba - 1 ) .

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Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (D) Lungs taken at 5 d.p.i. were analysed for neutrophils (Ly6G), neutrophil extracellular traps (NET; anti-H3) and macrophages (Iba-1) .


δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) cells Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( A ) Mice were monitored for weight loss at indicated time - points .

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δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points.


δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) cells Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( B ) IAV titre in lungs was determined by plaque assay at 5 d . p . i . ( n = 6 ) .

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δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (B) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6).


δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) cells Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( C ) IL - 1β mRNA transcripts in lung at 5 d . p . i . were determined by qPCR .

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δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (C) IL-1β mRNA transcripts in lung at 5 d.p.i. were determined by qPCR.


δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) cells Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( D ) Precision - cut lung slices from control and δWD mice were infected with IAV . Virus titres were determined at indicated time points .

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δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (D) Precision-cut lung slices from control and δWD mice were infected with IAV. Virus titres were determined at indicated time points.


A ) . The WD domain of ATG16L1 reduces IAV replication in MEFs . Cells were infected with IAV and virus titres were estimated at 8 hours post infection by plaque assay .

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A). The WD domain of ATG16L1 reduces IAV replication in MEFs. Cells were infected with IAV and virus titres were estimated at 8 hours post infection by plaque assay.


B ) . IAV binding is similar between control and δWD MEFs . Cells were bound with IAV X31 labeled with AF488 on ice for 1 h , and fixed immediately . The cells were stained with anti - HA ( H3SKE , red ) without permeabilisation to visualise extracellular IAV particles , counter stained with Hoechst ( nuclei ) and WGA - AF647 to label the cell membrane . Cells were imaged by automated confocal microscopy using a 40x objective , and the maximum intensity projection images were analysed to quantify the number of bound IAV particles per cell . The results and means ( horizontal line ) of n = 3 independent experiments ( 50 - 100 cells per experiment ) are shown .

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B). IAV binding is similar between control and δWD MEFs. Cells were bound with IAV X31 labeled with AF488 on ice for 1 h, and fixed immediately. The cells were stained with anti-HA (H3SKE, red) without permeabilisation to visualise extracellular IAV particles, counter stained with Hoechst (nuclei) and WGA-AF647 to label the cell membrane. Cells were imaged by automated confocal microscopy using a 40x objective, and the maximum intensity projection images were analysed to quantify the number of bound IAV particles per cell. The results and means (horizontal line) of n = 3 independent experiments (50-100 cells per experiment) are shown.


C ) IAV infection following acid bypass is comparable between control and δWD MEFs . IAV was bound on ice for 1 h , and warmed at either neutral pH ( 6 . 8 ) or low pH ( pH 5 . 0 ) for 2 min , followed by incubation in STOP medium containing NH4Cl for 18 hours . Cells were fixed and stained for NP and Hoechst , imaged by automated confocal microscopy and maximum intensity projection images analysed to quantify viral infectivity . The results from N = 3 independent experiments ( 5 , 000 - 10 , 000 cells quantified per experiment ) , and the means are shown . Scale bar ; 500 µm .

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C) IAV infection following acid bypass is comparable between control and δWD MEFs. IAV was bound on ice for 1 h, and warmed at either neutral pH (6.8) or low pH (pH 5.0) for 2 min, followed by incubation in STOP medium containing NH4Cl for 18 hours. Cells were fixed and stained for NP and Hoechst, imaged by automated confocal microscopy and maximum intensity projection images analysed to quantify viral infectivity. The results from N=3 independent experiments (5,000-10,000 cells quantified per experiment), and the means are shown. Scale bar; 500 µm.


D ) . IAV fusion with endosomes is increased in δWD MEFs . Cells were infected with dual labeled ( R18 / SP - DiOC18 ) IAV for 1 h , fixed , counterstained with Hoechst ( nuclei blue ) and WGA - AF647 ( cell perimeter in white ) . Cells were imaged by automated confocal microscopy and the number of fusion events per cell ( represented by number of SP - DiOC18 puncta ) was quantified . Fused and non - fused viral particles are shown as green and red spots respectively . The results and means ( horizontal lines ) of n = 3 independent experiments ( 30 - 60 cells per experiment ) are shown . Representative cells overlayed with the cell boundary segmented from the WGA staining are shown on the right .

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D). IAV fusion with endosomes is increased in δWD MEFs. Cells were infected with dual labeled (R18/SP-DiOC18) IAV for 1 h, fixed, counterstained with Hoechst (nuclei blue) and WGA-AF647 (cell perimeter in white). Cells were imaged by automated confocal microscopy and the number of fusion events per cell (represented by number of SP-DiOC18 puncta) was quantified. Fused and non-fused viral particles are shown as green and red spots respectively. The results and means (horizontal lines) of n = 3 independent experiments (30-60 cells per experiment) are shown. Representative cells overlayed with the cell boundary segmented from the WGA staining are shown on the right.


E , F ) Numbers of fusion positive cells is increased for δWD MEFs . MEFs from δWD ( white circles ) or littermate control mice ( black circles ) were incubated with dual - labelled ( SP - DiOC18 / R18 ) IAV at 40C for 45 min and warmed to 37 ° C for 30 ad 60 minutes . Cells were harvested by trypsinisation , fixed in PFA and analysed by flow cytometry to determine to determine percentage of cells positive for fusion ( E ) and median fluorescence intensity ( MFI ) of dequenched SP - DiOC18 signa ( F ) . Graphs show individual replicates ( n = 3 ) with a horizontal line at the mean and were compared using two - way ANOVA with Bonferroni post - tests * * P < 0 . 01 , * * * P < 0 . 001 ) .

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E, F) Numbers of fusion positive cells is increased for δWD MEFs. MEFs from δWD (white circles) or littermate control mice (black circles) were incubated with dual-labelled (SP-DiOC18/R18) IAV at 40C for 45 min and warmed to 37°C for 30 ad 60 minutes. Cells were harvested by trypsinisation, fixed in PFA and analysed by flow cytometry to determine to determine percentage of cells positive for fusion (E) and median fluorescence intensity (MFI) of dequenched SP-DiOC18 signa (F). Graphs show individual replicates (n = 3) with a horizontal line at the mean and were compared using two-way ANOVA with Bonferroni post-tests **P<0.01, ***P<0.001).


( G , H ) Endocytosis of IAV increases in δWD MEFs . MEFs from δWD or littermate control mice were incubated with AlexFluor 488 - labelled IAV at 40C for 1hr and warmed to 37 ° C for 15 Cells were fixed and permeabilised and stained with antibody against HA ( red ) . The blue line indicates the plasma membrane of the cell . Green arrows indicate examples of viruses only labelled with green fluorescence . Double - labelled virus particles ( green - red ) represent non - endocytosed particles bound at the cell surface , whereas green particles represent endocytosed particles . The results and means of n = 3 independent experiments ( 50 - 100 cells per experiment ) are shown .

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(G, H) Endocytosis of IAV increases in δWD MEFs. MEFs from δWD or littermate control mice were incubated with AlexFluor 488-labelled IAV at 40C for 1hr and warmed to 37°C for 15 Cells were fixed and permeabilised and stained with antibody against HA (red). The blue line indicates the plasma membrane of the cell. Green arrows indicate examples of viruses only labelled with green fluorescence. Double-labelled virus particles (green-red) represent non-endocytosed particles bound at the cell surface, whereas green particles represent endocytosed particles. The results and means of n = 3 independent experiments (50-100 cells per experiment) are shown.


( I , J ) Interferon signalling increases in δWD MEFs . MEFs from δWD or littermate control mice were infected with IAV . At the indicated time points , mRNA transcripts were evaluated by qPCR for IFIT1 ( I ) and ISG15 ( J ) .

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(I, J) Interferon signalling increases in δWD MEFs. MEFs from δWD or littermate control mice were infected with IAV. At the indicated time points, mRNA transcripts were evaluated by qPCR for IFIT1 (I) and ISG15 (J).


( A ) Western blot analysis showing the expression of the WRNIP1 protein in wild - type cells ( shWRNIP1WT ) and WRNIP1 - deficient ( shWRNIP1 ) or mutant ( shWRNIP1T294A ) cells . MRC5SV fibroblasts were used as a positive control . The membrane was probed with an anti - FLAG or anti - WRNIP1 . GAPDH was used as a loading control . Below each lane of the blot the ratio of WRNIP1 protein to total protein , then normalised to MRC5SV , is reported .

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(A) Western blot analysis showing the expression of the WRNIP1 protein in wild-type cells (shWRNIP1WT) and WRNIP1-deficient (shWRNIP1) or mutant (shWRNIP1T294A) cells. MRC5SV fibroblasts were used as a positive control. The membrane was probed with an anti-FLAG or anti-WRNIP1. GAPDH was used as a loading control. Below each lane of the blot the ratio of WRNIP1 protein to total protein, then normalised to MRC5SV, is reported.


( B ) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells . Cells were pulse - labelled with CldU , and then subjected to a pulse - labelling with IdU . ( C ) Analysis of replication fork velocity ( fork speed ) in the cells under unperturbed conditions . The length of the green tracks were measured . Mean values are represented as horizontal black lines ( ns , not significant ; Student ' s t - test ) .

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(B) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were pulse-labelled with CldU, and then subjected to a pulse-labelling with IdU.(C) Analysis of replication fork velocity (fork speed) in the cells under unperturbed conditions. The length of the green tracks were measured. Mean values are represented as horizontal black lines (ns, not significant; Student's t-test).


( D ) Cells were treated as in ( B ) . For each replication origin , the length of the right - fork signal was measured and plotted against the length of the left - fork signal . A schematic representation of symmetric and asymmetric forks is given . If the ratio between the left - fork length and the right - fork length deviated by more than 33 % from 1 ( that is , outside the violet dashed lines in the graphs ) , the fork was considered asymmetric . The percentage of asymmetric forks was calculated for all cell lines . N = number of forks counted for each cell line . R represents linear correlation coefficient .

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(D) Cells were treated as in (B). For each replication origin, the length of the right-fork signal was measured and plotted against the length of the left-fork signal. A schematic representation of symmetric and asymmetric forks is given. If the ratio between the left-fork length and the right-fork length deviated by more than 33% from 1 (that is, outside the violet dashed lines in the graphs), the fork was considered asymmetric. The percentage of asymmetric forks was calculated for all cell lines. N= number of forks counted for each cell line. R represents linear correlation coefficient.


( E ) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells . Cells were pulse - labelled with CldU , treated with 4mM HU and then subjected to a pulse - labelling with IdU . ( F ) Graphs show the percentage of red ( CldU ) tracts ( stalled forks ) or red - green ( CldU - IdU ) contiguous tracts ( restarting forks ) in the cells . Mean shown , n = 3 . Error bars represent standard error . ( * , p < 0 . 05 ; * * , p < 0 . 01 ; Student ' s t test ) . Representative DNA fiber images are shown . Scale bars , 10 µm .

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(E) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were pulse-labelled with CldU, treated with 4mM HU and then subjected to a pulse-labelling with IdU.(F) Graphs show the percentage of red (CldU) tracts (stalled forks) or red-green (CldU-IdU) contiguous tracts (restarting forks) in the cells. Mean shown, n = 3. Error bars represent standard error. (*, p < 0.05; **, p < 0.01; Student's t test). Representative DNA fiber images are shown. Scale bars, 10 µm.


( G ) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells . Cells were sequentially pulse - labelled with CldU and IdU as indicated , then treated or not with 4 mM HU . ( H ) Representative IdU tract length distributions in all cell lines under unperturbed conditions ( left graph ) or after HU treatment ( right graph ) . Median tract lengths are given in parentheses . See also Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are shown . Scale bars , 10 µm .

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(G) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were sequentially pulse-labelled with CldU and IdU as indicated, then treated or not with 4 mM HU.(H) Representative IdU tract length distributions in all cell lines under unperturbed conditions (left graph) or after HU treatment (right graph). Median tract lengths are given in parentheses. See also Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are shown. Scale bars, 10 µm.


( A ) Experimental scheme of dual labelling of DNA fibers in wild - type cells ( shWRNIP1WT ) or WRNIP1 - deficient cells ( shWRNIP1 ) . Cells were sequentially pulse - labelled with CldU and IdU as indicated , then left untreated or treated with 4 mM HU in combination or not with 50 µM Mirin . ( B ) Representative IdU tract length distributions in shWRNIP1WT ( left graph ) or shWRNIP1 cells ( right graph ) after treatment . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are shown . Scale bars , 10 µm .

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(A) Experimental scheme of dual labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were sequentially pulse-labelled with CldU and IdU as indicated, then left untreated or treated with 4 mM HU in combination or not with 50 µM Mirin.(B) Representative IdU tract length distributions in shWRNIP1WT (left graph) or shWRNIP1 cells (right graph) after treatment. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are shown. Scale bars, 10 µm.


( A ) Evaluation of ssDNA accumulation at parental - strand by immunofluorescence microscopy analysis in wild - type ( shWRNIP1WT ) or WRNIP1 - deficient ( shWRNIP1 ) cells . Experimental design of ssDNA assay is shown . Cells were labelled with IdU for 24 h , as indicated , washed and left to recover for 2 h , then treated or not with 4 mM HU . In parallel samples , the MRE11 activity is chemically inhibited with 50 µM Mirin , alone or in combination with HU - induced replication stress . After treatment , cells were fixed and stained with an anti - IdU antibody without denaturing the DNA to specifically detect parental ssDNA . Horizontal black lines represent the mean SE ; n = 3 . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ; two - tailed Student ' s t test ) . Representative images are shown . DNA was counterstained with DAPI ( blue ) .

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(A) Evaluation of ssDNA accumulation at parental-strand by immunofluorescence microscopy analysis in wild-type (shWRNIP1WT) or WRNIP1-deficient (shWRNIP1) cells. Experimental design of ssDNA assay is shown. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU. In parallel samples, the MRE11 activity is chemically inhibited with 50 µM Mirin, alone or in combination with HU-induced replication stress. After treatment, cells were fixed and stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental ssDNA. Horizontal black lines represent the mean SE; n = 3. Error bars represent standard error (ns, not significant; **, p<0.01; ****, p<0.0001; two-tailed Student's t test). Representative images are shown. DNA was counterstained with DAPI (blue).


( B ) Analysis of chromatin binding of MRE11 and RAD51 in shWRNIP1WT and shWRNIP1 cells . Chromatin fractions of cells , treated or not with 4 mM HU , were analysed by immunoblotting . The membrane was probed with the anti - WRNIP1 , anti - MRE11 and anti - RAD51 antibodies . LAMIN B1 was used as a loading for the chromatin fraction . Total amount of RAD51 and MRE11 ( Input ) in the cells was determined with the relevant antibodies . LAMIN B1 was used as a loading control . In the graph , the fold increase respect to the wild - type untreated of the normalized ratio of the chromatin - bound RAD51 ( or MRE11 ) / total RAD51 ( or MRE11 ) is reported for each cell line .

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(B) Analysis of chromatin binding of MRE11 and RAD51 in shWRNIP1WT and shWRNIP1 cells. Chromatin fractions of cells, treated or not with 4 mM HU, were analysed by immunoblotting. The membrane was probed with the anti-WRNIP1, anti-MRE11 and anti-RAD51 antibodies. LAMIN B1 was used as a loading for the chromatin fraction. Total amount of RAD51 and MRE11 (Input) in the cells was determined with the relevant antibodies. LAMIN B1 was used as a loading control. In the graph, the fold increase respect to the wild-type untreated of the normalized ratio of the chromatin-bound RAD51 (or MRE11)/ total RAD51 (or MRE11) is reported for each cell line.


( C ) Analysis of DNA - protein interactions between ssDNA and endogenous RAD51 in shWRNIP1WT and shWRNIP1 cells by in situ PLA assay . Experimental designed used for the assay is given . Cells were labelled with IdU for 24 h , as indicated , washed and left to recover for 2 h , then treated or not with 4 mM HU for 4 h . Next , cells were fixed , stained with an anti - IdU antibody without denaturing the DNA to specifically detect parental - strand ssDNA , and subjected to PLA assay as described in the " Experimental procedures " section . Antibodies raised against IdU or RAD51 were used to reveal ssDNA or endogenous RAD51 respectively . Each red spot represents a single interaction between ssDNA and RAD51 . No spot has been revealed in cells stained with each single antibody ( negative control ) . DNA was counterstained with DAPI ( blue ) . Representative images of the PLA assay are given . Graph shows the number of PLA spots per cell . Horizontal black lines represent the mean value ( ns , not significant ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) ; n = 3 .

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

(C) Analysis of DNA-protein interactions between ssDNA and endogenous RAD51 in shWRNIP1WT and shWRNIP1 cells by in situ PLA assay. Experimental designed used for the assay is given. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU for 4 h. Next, cells were fixed, stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental-strand ssDNA, and subjected to PLA assay as described in the "Experimental procedures" section. Antibodies raised against IdU or RAD51 were used to reveal ssDNA or endogenous RAD51 respectively. Each red spot represents a single interaction between ssDNA and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are given. Graph shows the number of PLA spots per cell. Horizontal black lines represent the mean value (ns, not significant; **, p < 0.01; two-tailed Student's t test); n = 3.


( D ) Localization of WRNIP1 , MRE11 and RAD51 to stalled replication forks . Forks were isolated by CldU - co - immunoprecipitation ( CldU - IP ) . shWRNIP1WT or shWRNIP1 cells were pulse - labelled with CldU , then fixed or treated with HU . Cells were cross - linked , and the nuclear extracts were isolated ( Input ) and subjected to CldU - IP using an anti - CldU antibody ( CldU - IP ) . The membranes were probed with the anti - WRNIP1 or anti - RAD51 antibodies . After stripping , the membranes were probed with an anti - MRE11 antibody . LAMIN B1 and GAPDH were used as loading controls ( Input ) . Ponceau S was used as a loading control of CldU - IP . Dot blot analysis was performed to confirm that equal amounts of immunoprecipitatedDNA from each sample . 10 % of each IP was loaded on a nitrocellulose membrane . The membrane was probed with an anti - CldU antibody . The graph shows the normalized ratio of the proteins co - immunoprecipitated with CldU ( CldUCo - IP proteins ) / the total of labelled DNAimmunoprecipitated with CldU ( CldUIP ) for each cell line after replication stress from two independent experiments . The dots in the graph represent the individual data points from each single experiment . Horizontal black line represents the mean value from two replicates ; n = 2 .

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

(D) Localization of WRNIP1, MRE11 and RAD51 to stalled replication forks. Forks were isolated by CldU-co-immunoprecipitation (CldU-IP). shWRNIP1WT or shWRNIP1 cells were pulse-labelled with CldU, then fixed or treated with HU. Cells were cross-linked, and the nuclear extracts were isolated (Input) and subjected to CldU-IP using an anti-CldU antibody (CldU-IP). The membranes were probed with the anti-WRNIP1 or anti-RAD51 antibodies. After stripping, the membranes were probed with an anti-MRE11 antibody. LAMIN B1 and GAPDH were used as loading controls (Input). Ponceau S was used as a loading control of CldU-IP. Dot blot analysis was performed to confirm that equal amounts of immunoprecipitatedDNA from each sample. 10% of each IP was loaded on a nitrocellulose membrane. The membrane was probed with an anti-CldU antibody. The graph shows the normalized ratio of the proteins co-immunoprecipitated with CldU (CldUCo-IP proteins)/ the total of labelled DNAimmunoprecipitated with CldU (CldUIP) for each cell line after replication stress from two independent experiments. The dots in the graph represent the individual data points from each single experiment. Horizontal black line represents the mean value from two replicates; n = 2.


( A ) Experimental scheme of pulse - labelling of DNA fibers in wild - type cells ( shWRNIP1WT ) or WRNIP1 - deficient cells ( shWRNIP1 ) . Cells were labelled with IdU and exposed or not to 25 μM RAD51 inhibitor , then treated or not with 4 mM HU . ( B ) Representative IdU tract length distributions in shWRNIP1WT cells ( left graph ) or shWRNIP1 cells ( right graph ) . Median tract lengths are reported in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are reported . Scale bars , 10 µm .

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

(A) Experimental scheme of pulse-labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were labelled with IdU and exposed or not to 25 μM RAD51 inhibitor, then treated or not with 4 mM HU.(B) Representative IdU tract length distributions in shWRNIP1WT cells (left graph) or shWRNIP1 cells (right graph). Median tract lengths are reported in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are reported. Scale bars, 10 µm.


( C ) Scheme of DNA fiber tract analysis in shWRNIP1 cells . Cells were transfected with an empty vector or a plasmid expressing a wild - type human RAD51 , and 48 h thereafter labelled with IdU and treated or not with 4 mM HU . ( D ) Representative IdU tract length distributions in shWRNIP1 cells or shWRNIP1 cells expressing exogenous wild - type RAD51 after HU exposure . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are given . Scale bars , 10 µm . Western blot shows the expression of the RAD51 protein in shWRNIP1 cells . The membrane was probed with an anti - RAD51 . LAMIN B1 was used as a loading control .

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

(C) Scheme of DNA fiber tract analysis in shWRNIP1 cells. Cells were transfected with an empty vector or a plasmid expressing a wild-type human RAD51, and 48 h thereafter labelled with IdU and treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1 cells or shWRNIP1 cells expressing exogenous wild-type RAD51 after HU exposure. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are given. Scale bars, 10 µm. Western blot shows the expression of the RAD51 protein in shWRNIP1 cells. The membrane was probed with an anti-RAD51. LAMIN B1 was used as a loading control.


( A ) Co - immunoprecipitation experiments in HEK293T cells transfected with empty vector or FLAG - WRNIP1 plasmid . Cells were treated or not with HU . After treatment , cell lysates were immunoprecipitated ( FLAG - IP ) using anti - FLAG antibody . The presence of WRNIP1 , BRCA2 and RAD51 was assessed by immunoblotting using the anti - FLAG , anti - RAD51 and anti - BRCA2 antibodies , respectively . Whole cell extracts were analysed ( Input ) . The membrane was probed with the same antibodies used for IP . GAPDH was used as a loading control .

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

(A) Co-immunoprecipitation experiments in HEK293T cells transfected with empty vector or FLAG-WRNIP1 plasmid. Cells were treated or not with HU. After treatment, cell lysates were immunoprecipitated (FLAG-IP) using anti-FLAG antibody. The presence of WRNIP1, BRCA2 and RAD51 was assessed by immunoblotting using the anti-FLAG, anti-RAD51 and anti-BRCA2 antibodies, respectively. Whole cell extracts were analysed (Input). The membrane was probed with the same antibodies used for IP. GAPDH was used as a loading control.


( B ) Analysis of protein - protein interactions between WRNIP1 and endogenous RAD51 in wild - type ( shWRNIP1WT ) or WRNIP1 - mutant ( shWRNIP1T294A ) cells by in situ PLA assay . Cells were labelled with IdU for 24 h , washed and left to recover for 2 h , then treated or not with 4 mM HU . Antibodies raised against FLAG - Tag and RAD51 were used to reveal FLAG - WRNIP1 or endogenous RAD51 respectively . Each red spot represents a single interaction between WRNIP1 and RAD51 . No spot has been revealed in cells stained with each single antibody ( negative control ) . DNA was counterstained with DAPI ( blue ) . Representative images of the PLA assay are shown . Graph shows the mean number of PLA spots per cell SE . Error bars represent standard error ( ns , not significant ; two - tailed Student ' s t test ) ; n = 3 .

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

(B) Analysis of protein-protein interactions between WRNIP1 and endogenous RAD51 in wild-type (shWRNIP1WT) or WRNIP1-mutant (shWRNIP1T294A) cells by in situ PLA assay. Cells were labelled with IdU for 24 h, washed and left to recover for 2 h, then treated or not with 4 mM HU. Antibodies raised against FLAG-Tag and RAD51 were used to reveal FLAG-WRNIP1 or endogenous RAD51 respectively. Each red spot represents a single interaction between WRNIP1 and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are shown. Graph shows the mean number of PLA spots per cell SE. Error bars represent standard error (ns, not significant; two-tailed Student's t test); n = 3.


( C ) Experimental scheme of pulse - labelling of DNA fibers in wild - type cells ( shWRNIP1WT ) or WRNIP1 - deficient cells ( shWRNIP1 ) . Cells were transfected with BRCA2 siRNA ( siBRCA2 ) , and 48 h thereafter labelled with IdU , then treated or not with 4 mM HU . ( D ) Representative IdU tract length distributions in shWRNIP1WT / siBRCA2 or shWRNIP1siBRCA2 cells treated or not with HU . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are reported . Scale bars , 10 µm . Western blot shows BRCA2 depletion in shWRNIP1WT and shWRNIP1 cells . The membrane was probed with an anti - BRCA2 or anti - WRNIP1 . GAPDH was used as a loading control .

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

(C) Experimental scheme of pulse-labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were transfected with BRCA2 siRNA (siBRCA2), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1WT/siBRCA2 or shWRNIP1siBRCA2 cells treated or not with HU. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are reported. Scale bars, 10 µm. Western blot shows BRCA2 depletion in shWRNIP1WT and shWRNIP1 cells. The membrane was probed with an anti-BRCA2 or anti-WRNIP1. GAPDH was used as a loading control.


( E ) Experimental scheme of pulse - labelling of DNA fibers in shWRNIP1 cells . Cells were transfected with control siRNA ( shWRNIP1siCtrl ) or FBH1 siRNA ( shWRNIP1siFBH1 ) , and 48 h thereafter labelled with IdU , then treated or not with 4 mM HU . ( F ) Representative IdU tract length distributions in shWRNIP1siCtrl or shWRNIP1siFBH1 cells with or without HU treatment . Representative DNA fiber images are reported . Scale bars , 10 µm . Western blot shows FBH1 depletion in the cells . The membrane was probed with an anti - FBH1 . GAPDH was used as a loading control . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test .

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

(E) Experimental scheme of pulse-labelling of DNA fibers in shWRNIP1 cells. Cells were transfected with control siRNA (shWRNIP1siCtrl) or FBH1 siRNA (shWRNIP1siFBH1), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.(F) Representative IdU tract length distributions in shWRNIP1siCtrl or shWRNIP1siFBH1 cells with or without HU treatment. Representative DNA fiber images are reported. Scale bars, 10 µm. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti-FBH1. GAPDH was used as a loading control. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test.


( G ) Analysis of chromatin binding of RAD51 in shWRNIP1 cells depleted for FBH1 . Cells were transfected with control siRNA ( shWRNIP1siCtrl ) or FBH1 siRNA ( shWRNIP1siFBH1 ) , and 48 h treated or not with HU for 4h . Chromatin fractions of cells were analysed by immunoblotting . The membrane was probed with the anti - FBH1 and anti - RAD51 antibodies . LAMIN B1 was used as a loading for the chromatin fraction . Total amount of RAD51 ( Input ) in the cells was determined with the relevant antibodies . GAPDH was used as a loading control . The ratio of the RAD51 / LAMIN B1 signal ( chromatin ) is reported below each lane .

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

(G) Analysis of chromatin binding of RAD51 in shWRNIP1 cells depleted for FBH1. Cells were transfected with control siRNA (shWRNIP1siCtrl) or FBH1 siRNA (shWRNIP1siFBH1), and 48 h treated or not with HU for 4h. Chromatin fractions of cells were analysed by immunoblotting. The membrane was probed with the anti-FBH1 and anti-RAD51 antibodies. LAMIN B1 was used as a loading for the chromatin fraction. Total amount of RAD51 (Input) in the cells was determined with the relevant antibodies. GAPDH was used as a loading control. The ratio of the RAD51/LAMIN B1 signal (chromatin) is reported below each lane.


( A ) Analysis of DNA damage accumulation . Wild - type ( shWRNIP1WT ) , WRNIP1 - deficient ( shWRNIP1 ) or mutant ( shWRNIP1T294A ) cells were treated or not with 4 mM HU for 4 h , then subjected to - H2AXimmunofluorescence microscopy . Graph shows data presented as mean of - H2AX - positive cells SE from three independent experiments ; n = 3 . Error bars represent standard error ( * , p < 0 . 1 ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative images of nuclei showing the different number of foci per nucleus are reported .

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 B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of DNA damage accumulation. Wild-type (shWRNIP1WT), WRNIP1-deficient (shWRNIP1) or mutant (shWRNIP1T294A) cells were treated or not with 4 mM HU for 4 h, then subjected to -H2AXimmunofluorescence microscopy. Graph shows data presented as mean of -H2AX-positive cells SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images of nuclei showing the different number of foci per nucleus are reported.


( B ) Analysis of DNA breakage accumulation . shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells were treated as in ( A ) , then subjected to alkaline Comet assay . Graph shows data presented as mean tail moment SE from three independent experiments ; n = 3 . Error bars represent standard error ( * , p < 0 . 1 ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative images are shown .

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(B) Analysis of DNA breakage accumulation. shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells were treated as in (A), then subjected to alkaline Comet assay. Graph shows data presented as mean tail moment SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images are shown.


( C ) Evaluation of cell death . shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU for 16 h . Cell viability was evaluated by LIVE / DEAD fluorescent assay . Data are expressed as mean of dead cells SE from three independent experiments ; n = 3 . Error bars represent standard error ( * , p < 0 . 1 ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative images of double - staining of viable ( green ) and dead ( red ) cells are shown .

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(C) Evaluation of cell death. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU for 16 h. Cell viability was evaluated by LIVE/DEAD fluorescent assay. Data are expressed as mean of dead cells SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images of double-staining of viable (green) and dead (red) cells are shown.


( D ) Experimental scheme for evaluation of the chromosomal aberrations is shown . shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU , then left to recover for 16h in drug - free medium and metaphases collected with colcemid . Next , cells were fixed and processed as reported in " Supplemental information " section . Dot plot shows the number of chromosomal aberrations per cell . Horizontal black lines represent the mean SE . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative Giemsa - stained metaphases of cells treated or not with 4 mM HU . Arrows indicate chromosomal aberrations .

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

(D) Experimental scheme for evaluation of the chromosomal aberrations is shown. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU, then left to recover for 16h in drug-free medium and metaphases collected with colcemid. Next, cells were fixed and processed as reported in "Supplemental information" section. Dot plot shows the number of chromosomal aberrations per cell. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of cells treated or not with 4 mM HU. Arrows indicate chromosomal aberrations.


( E ) Experimental scheme of the Mirin aberration analysis is given . The experiment was carried out as in ( D ) but cells were pre - treated or not with 50 µM Mirin . Dot plot shows the effect of Mirin exposure on the number of chromosome aberrations per cell in shWRNIP1 cells . Horizontal black lines represent the mean SE . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) . Representative Giemsa - stained metaphases of shWRNIP1 cells treated with Mirin alone or in combination with HU . Arrows indicate chromosomal aberrations .

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

(E) Experimental scheme of the Mirin aberration analysis is given. The experiment was carried out as in (D) but cells were pre-treated or not with 50 µM Mirin. Dot plot shows the effect of Mirin exposure on the number of chromosome aberrations per cell in shWRNIP1 cells. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of shWRNIP1 cells treated with Mirin alone or in combination with HU. Arrows indicate chromosomal aberrations.