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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( b ) Experimental validation of predicted synergistic SR - based combinational therapies in head and neck cancer : A table summarizing the experimentally observed synergism between primary drugs and their predicted rescuer - targeting treatments in 5 HNSC cell lines , based on drug treatment experiments . Synergism was estimated using standard Fa - CI analysis . The table displays the average combination index ( CI ; synergism CI < 1 , additivity effect CI = 1 , antagonism CI > 1 , NAN indeterminate CI ) at 50 % growth inhibition ( Fraction affected ) . Combinations that are synergistic are colored blue ( black otherwise ) for each cell lines tested . The inset shows an example of CI calculation for BYL719 and Dasatinib combination in HN12 cell lines based on the corresponding dose matrix ( number indicates % cell viability at 48h , n = 3 ) , and Fa - CI curve .

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(b) Experimental validation of predicted synergistic SR-based combinational therapies in head and neck cancer: A table summarizing the experimentally observed synergism between primary drugs and their predicted rescuer-targeting treatments in 5 HNSC cell lines, based on drug treatment experiments. Synergism was estimated using standard Fa-CI analysis. The table displays the average combination index (CI; synergism CI < 1, additivity effect CI = 1, antagonism CI > 1, NAN indeterminate CI) at 50% growth inhibition (Fraction affected). Combinations that are synergistic are colored blue (black otherwise) for each cell lines tested. The inset shows an example of CI calculation for BYL719 and Dasatinib combination in HN12 cell lines based on the corresponding dose matrix (number indicates % cell viability at 48h, n = 3), and Fa-CI curve.


( a ) Experimental testing of the predicted SR ( DU ) rescuers of DNMT1 via drug combination experiments in 18 NSCLC cell lines insensitive to Decitabine . The matrix displays drug interactions between Decitabine , a DNMT1 inhibitor , and inhibitors of its predicted rescuer genes ( X - axis ) across 18 NSCLC cell lines ( Y - axis ) that are insensitive to Decitabine . Row labels present rescuer genes and their inhibitors . Colors in the matrix show whether the interactions found are significantly synergistic ( red ) , antagonistic ( green ) or non - significant ( in gray ) . Values in the matrix show average synergism ( < 1 synergism and > 1 antagonism Thirteen predicted DU - SR rescuers ( red lines ) , two predicted DD SR rescuers ( green lines ) of DNMT1 and one random control ( JAK3i ) were tested .

(a) Experimental testing of the predicted SR (DU) rescuers of DNMT1 via drug combination experiments in 18 NSCLC cell lines insensitive to Decitabine. The matrix displays drug interactions between Decitabine, a DNMT1 inhibitor, and inhibitors of its predicted rescuer genes (X-axis) across 18 NSCLC cell lines (Y-axis) that are insensitive to Decitabine. Row labels present rescuer genes and their inhibitors. Colors in the matrix show whether the interactions found are significantly synergistic (red), antagonistic (green) or non-significant (in gray). Values in the matrix show average synergism (<1 synergism and >1 antagonism Thirteen predicted DU-SR rescuers (red lines), two predicted DD SR rescuers (green lines) of DNMT1 and one random control (JAK3i) were tested.


The transcriptomic alterations of rescuer genes post PD1 / PDL1 blockade in patient tumor biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after anti - PD1 ( b ) Each panel displays the expression fold change of each predicted rescuer gene ( rows ) for different tumor samples ( columns ) and the P - value of over - all paired Wilcoxon test of the expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models .

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The transcriptomic alterations of rescuer genes post PD1/PDL1 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-PD1 (b) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.


The transcriptomic alterations of rescuer genes post CTLA4 blockade in patient tumor biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after anti - CTLA4 ( c ) Each panel displays the expression fold change of each predicted rescuer gene ( rows ) for different tumor samples ( columns ) and the P - value of over - all paired Wilcoxon test of the expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models .

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The transcriptomic alterations of rescuer genes post CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-CTLA4 (c) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.


The transcriptomic alterations of rescuer genes post PD1 / PDL1 and CTLA4 blockade in patient tumor biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after PD1 + CTLA4 combination therapies ( d ) . Each panel displays the expression fold change of each predicted rescuer gene ( rows ) for different tumor samples ( columns ) and the P - value of over - all paired Wilcoxon test of the expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models .

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The transcriptomic alterations of rescuer genes post PD1/PDL1 and CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after PD1 + CTLA4 combination therapies (d). Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.


A Representative pictures of embryos at 18 . 5 days postcoitum ( E18 . 5 ) and hematoxylin and eosin staining of whole - mount embryo sections from PR - SET7loxp / Alfp - Cre ( PR - SET7ΔHepE ) mice and control littermates ( WT ) .

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A Representative pictures of embryos at 18.5 days postcoitum (E18.5) and hematoxylin and eosin staining of whole-mount embryo sections from PR-SET7loxp/Alfp-Cre (PR-SET7ΔHepE) mice and control littermates (WT).


B Hematoxylin and eosin staining of liver sections .

B Hematoxylin and eosin staining of liver sections.


C Immunohistochemical staining of liver sections with albumin antibody .

C Immunohistochemical staining of liver sections with albumin antibody.


D qPCR analysis of Albumin , Afp and HNF4 mRNA levels . Bars represent mean values of mRNA levels normalized to glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) mRNA and SEM from samples of four individual mice . * P - value < 0 . 01 .

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D qPCR analysis of Albumin,Afp and HNF4 mRNA levels. Bars represent mean values of mRNA levels normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and SEM from samples of four individual mice. *P-value < 0.01.


E Immunohistochemical staining of liver sections with cyclin B1 antibody .

E Immunohistochemical staining of liver sections with cyclin B1 antibody.


F Immunohistochemical staining of liver sections with γH2AX antibody .

F Immunohistochemical staining of liver sections with γH2AX antibody.


A Macroscopic appearance of livers in 120 - day - old ( P120 ) wild - type ( WT ) and PR - SET7loxp / Alb - Cre ( KO ) mice . Note , small adenomatous foci in KO livers .

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A Macroscopic appearance of livers in 120-day-old (P120) wild-type (WT) and PR-SET7loxp/Alb-Cre (KO) mice. Note, small adenomatous foci in KO livers.


B Representative hematoxylin and eosin staining of liver sections from P120 wild - type ( WT ) and PR - SET7loxp / Alb - Cre ( PR - SET7ΔHepA ) mice . Arrows show three areas containing morphologically different hepatocytes . Right panels : zoom - in to Area - A - ' normal zone ' , to Area - B - ' necrotic zone ' and to Area - C - ' regenerative zone ' .

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B Representative hematoxylin and eosin staining of liver sections from P120 wild-type (WT) and PR-SET7loxp/Alb-Cre (PR-SET7ΔHepA) mice. Arrows show three areas containing morphologically different hepatocytes. Right panels: zoom-in to Area-A-'normal zone', to Area-B-'necrotic zone' and to Area-C-'regenerative zone'.


C Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with HNF4 antibody .

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C Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with HNF4 antibody.


D TUNEL staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) . Note that cells containing enlarged nuclei ( white arrows ) are TUNEL negative .

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D TUNEL staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT). Note that cells containing enlarged nuclei (white arrows) are TUNEL negative.


E Immunohistological staining with γH2AX and albumin ( Alb ) antibodies .

E Immunohistological staining with γH2AX and albumin (Alb) antibodies.


A Electronmicroscopic images of cells containing enlarged nuclei in P120 PR - SET7ΔHepA livers and normal hepatocytes in control littermates ( WT ) . White arrows indicate : ( Nuc ) nuclei ; ( ER ) endoplasmic reticulum ; ( Mit ) mitochondrium ; ( PM ) plasma membrane ; ( a ) swollen mitochondria ; ( b ) swollen endoplasmic reticulum ; ( c ) disorganized endoplasmic reticulum ; ( d ) disrupted plasma membrane ; and ( e ) disorganized pieces of endoplasmic reticulum encircling mitochondria to form autophagosomes .

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A Electronmicroscopic images of cells containing enlarged nuclei in P120 PR-SET7ΔHepA livers and normal hepatocytes in control littermates (WT). White arrows indicate: (Nuc) nuclei; (ER) endoplasmic reticulum; (Mit) mitochondrium; (PM) plasma membrane; (a) swollen mitochondria; (b) swollen endoplasmic reticulum; (c) disorganized endoplasmic reticulum; (d) disrupted plasma membrane; and (e) disorganized pieces of endoplasmic reticulum encircling mitochondria to form autophagosomes.


B Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with H4K20Me1 and H4K20Me3 antibodies . Normal ( Area - A ) , Necrotic ( Area - B ) and Regenerative ( Area - C ) hepatocyte - containing areas are indicated . Arrows show the loss of H4K20Me1 signal from the large necrotic nuclei .

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B Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with H4K20Me1 and H4K20Me3 antibodies. Normal (Area-A), Necrotic (Area-B) and Regenerative (Area-C) hepatocyte-containing areas are indicated. Arrows show the loss of H4K20Me1 signal from the large necrotic nuclei.


C - F Partial ( 2 / 3rd ) hepatectomy was performed in 45 - day - old PR - SET7ΔHepA mice and control littermates ( WT ) . Two weeks later , at postnatal day 60 ( P60 ) liver sections were stained with hematoxylin and eosin ( C ) or γH2AX ( D ) or H4K20Me1 ( E ) or HNF4 and F4 / 80 ( F ) antibodies .

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C-F Partial (2/3rd) hepatectomy was performed in 45-day-old PR-SET7ΔHepA mice and control littermates (WT). Two weeks later, at postnatal day 60 (P60) liver sections were stained with hematoxylin and eosin (C) or γH2AX (D) or H4K20Me1 (E) or HNF4 and F4/80 (F) antibodies.


A , B Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with Ki67 , albumin ( Alb ) and A6 or HNF4 antibodies as indicated . Arrows depict the different areas in the sections as indicated above ( Fig . 1B ) . Asterisks ( * ) indicate Ki67 + hepatocytes . Small arrows ( > ) indicate Ki67 + non - hepatic cells . Note the lack of co - staining of cells with HNF4 and A6 antibodies .

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A, B Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with Ki67, albumin (Alb) and A6 or HNF4 antibodies as indicated. Arrows depict the different areas in the sections as indicated above (Fig.1B). Asterisks (*) indicate Ki67+ hepatocytes. Small arrows (>) indicate Ki67+ non-hepatic cells. Note the lack of co-staining of cells with HNF4 and A6 antibodies.


C Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with FGF7 and A6 antibodies .

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C Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with FGF7 and A6 antibodies.


D Relative mRNA levels of FGF7 . Bars represent mean FGF7 mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 45 days or 120 - day - old mice . The data are presented as fold over values obtained with wild - type P45 samples . * P - value < 0 . 01 .

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D Relative mRNA levels of FGF7. Bars represent mean FGF7 mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 45 days or 120-day-old mice. The data are presented as fold over values obtained with wild-type P45 samples. *P-value < 0.01.


A Analysis of reactive oxygen species ( ROS ) accumulation in frozen liver sections from 120 - day - old ( P120 ) and 240 - day - old ( P240 ) PR - SET7ΔHepA mice and control littermates ( WT ) .

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A Analysis of reactive oxygen species (ROS) accumulation in frozen liver sections from 120-day-old (P120) and 240-day-old (P240) PR-SET7ΔHepA mice and control littermates (WT).


B Immunohistological staining of liver sections from P240 animals with phospho - STAT3 antibody .

B Immunohistological staining of liver sections from P240 animals with phospho-STAT3 antibody.


C Western blot analysis of liver extracts from P45 , P120 and P240 mice with STAT3 and phospho - STAT3 antibodies .

C Western blot analysis of liver extracts from P45, P120 and P240 mice with STAT3 and phospho-STAT3 antibodies.


D Macroscopic appearance of livers in P240 PR - SET7ΔHepA mice and control littermates ( WT ) .

D Macroscopic appearance of livers in P240 PR-SET7ΔHepA mice and control littermates (WT).


E Serum alanine aminotransferase ( ALT ) levels in P45 , P120 and P240 mice . Bars represent mean values of ALT levels and SEM from liver extracts of five individual mice . * P - value < 0 . 01 .

E Serum alanine aminotransferase (ALT) levels in P45, P120 and P240 mice. Bars represent mean values of ALT levels and SEM from liver extracts of five individual mice. *P-value < 0.01.


F Representative hematoxylin and eosin staining of liver sections from P240 PR - SET7ΔHepA mice and control littermates ( WT ) . Two different magnifications are shown .

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F Representative hematoxylin and eosin staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT). Two different magnifications are shown.


G Relative mRNA levels of selected oncofetal genes . Bars represent mean Afp , H19 , GPC - 3 and CTGF mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 240 - day - old mice . The data are presented as fold over values obtained with wild - type samples . * P - value < 0 . 01 .

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G Relative mRNA levels of selected oncofetal genes. Bars represent mean Afp, H19, GPC-3 and CTGF mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 240-day-old mice. The data are presented as fold over values obtained with wild-type samples. *P-value < 0.01.


A , B Immunohistological staining of liver sections from P240 PR - SET7ΔHepA mice and control littermates ( WT ) with Afp and HNF4 ( A ) or Ki - 67 and albumin ( Alb ) ( B ) antibodies .

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A, B Immunohistological staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT) with Afp and HNF4 (A) or Ki-67 and albumin (Alb) (B) antibodies.


C - E Double staining of liver sections with A6 and HNF4 ( C ) or Sox9 and Alb ( D ) or CD133 and HNF4 ( E ) antibodies . Right panels show larger magnifications of stainings performed in PR - SET7ΔHepA liver sections .

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C-E Double staining of liver sections with A6 and HNF4 (C) or Sox9 and Alb (D) or CD133 and HNF4 (E) antibodies. Right panels show larger magnifications of stainings performed in PR-SET7ΔHepA liver sections.


A P240 PR - SET7ΔHepA liver sections were double - stained with A6 and Sox9 ( upper panel ) or CD133 and Sox9 ( lower panel ) . Pie charts at the right represent average percentages of cells stained positively with the indicated markers after counting DAPI - stained cells in 5 high - power fields ( HPF ) in liver sections of 3 different mice .

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A P240 PR-SET7ΔHepA liver sections were double-stained with A6 and Sox9 (upper panel) or CD133 and Sox9 (lower panel). Pie charts at the right represent average percentages of cells stained positively with the indicated markers after counting DAPI-stained cells in 5 high-power fields (HPF) in liver sections of 3 different mice.


B Cell duplication rate of primary hepatocytes from P240 PR - SET7ΔHepA livers in culture .

B Cell duplication rate of primary hepatocytes from P240 PR-SET7ΔHepA livers in culture.


C Pictures of representative subcutaneous xenograft tumors dissected from immunodeficient mice 20 days after injection with primary hepatocytes from P240 PR - SET7ΔHepA livers or Hepa 1 - 6 cells . Right panel shows average tumor volumes ( n = 3 ) dissected at the indicated time points following injection .

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C Pictures of representative subcutaneous xenograft tumors dissected from immunodeficient mice 20 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers or Hepa 1-6 cells. Right panel shows average tumor volumes (n = 3) dissected at the indicated time points following injection.


D , E Hematoxylin and eosin and immunohistological staining with A6 , HNF4 , Sox9 and CD133 antibodies of tumor sections from xenografts dissected 20 or 30 days after injection with primary hepatocytes from P240 PR - SET7ΔHepA livers .

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D, E Hematoxylin and eosin and immunohistological staining with A6, HNF4, Sox9 and CD133 antibodies of tumor sections from xenografts dissected 20 or 30 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers.


A Age , gender and BMI information of the Ashkenazi Jewish Centenarian cohort . Table showing the age , gender and BMI information in control and centenarian groups . Pie chart showing the percentage of male / female individuals in control and centenarian groups . Right panel showing the age and BMI distribution of all individuals recruited in targeted capture - seq . Data were presented as mean and SD , Students t - test , two tailed , * * * p < 0 . 001 .

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A Age, gender and BMI information of the Ashkenazi Jewish Centenarian cohort. Table showing the age, gender and BMI information in control and centenarian groups. Pie chart showing the percentage of male/female individuals in control and centenarian groups. Right panel showing the age and BMI distribution of all individuals recruited in targeted capture-seq. Data were presented as mean and SD, Students t-test, two tailed, *** p<0.001.


B , C Histograms of normalized allele counts in individuals 75 + years old compared to all age groups ( N = 125 , 748 ) across ( B ) all chromosome 19 SNPs and small insertions / deletions , and ( C ) missense SNPs and indels only . Dotted vertical lines show centSIRT6 .

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B, C Histograms of normalized allele counts in individuals 75+ years old compared to all age groups (N=125,748) across (B) all chromosome 19 SNPs and small insertions/deletions, and (C) missense SNPs and indels only. Dotted vertical lines show centSIRT6.


E Michaelis - Menten kinetic parameters as calculated by saturation curves using differential concentrations of myristoylated peptide or NAD + . n = 3 technical replicates ( TR ) ; error bars = SD .

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E Michaelis-Menten kinetic parameters as calculated by saturation curves using differential concentrations of myristoylated peptide or NAD+. n=3 technical replicates (TR); error bars=SD.


A , B Deacetylase activity on H3K9 ( A ) and H3K18 ( B ) residues shows reduced activity in centSIRT6 allele . Designer histones were incubated with purified SIRT6 and 5 mM NAD + , then resolved by SDS - PAGE and analyzed by immunoblotting with acetyl - specific histone antibodies . n = 3 TR ; error bars = SD . Students t - test , two tailed . Asterisks indicate p < 0 . 05 compared to WT , color corresponds to allele . H3K9ac , H3K18ac and H3 are blots , SIRT6 loading Coomassie stain HeLa histone preps were also

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A, B Deacetylase activity on H3K9 (A) and H3K18 (B) residues shows reduced activity in centSIRT6 allele. Designer histones were incubated with purified SIRT6 and 5 mM NAD+, then resolved by SDS-PAGE and analyzed by immunoblotting with acetyl-specific histone antibodies. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisks indicate p<0.05 compared to WT, color corresponds to allele. H3K9ac, H3K18ac and H3 are blots, SIRT6 loading Coomassie stain HeLa histone preps were also probed


C Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels . Relative fraction represents the portion of the peptide encompassing H3K9 - 17 compared to the summed total of all of the peptide quantitation values for the same region . n = 3 biological replicates ( BR ) ; error bars = SD . Students t - test , two

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C Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels. Relative fraction represents the portion of the peptide encompassing H3K9-17 compared to the summed total of all of the peptide quantitation values for the same region. n=3 biological replicates (BR); error bars=SD. Students t-test, two tailed


D Whole cell histone H3 acetylation levels in cumate - inducible SIRT6 human fibroblasts , assessed by Western blot . Cu , cumate ; PQ , paraquat . Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot , and administered accordingly to respective cell lines Cells were incubated with appropriate cumate dose for 48hrs prior to harvest , with PQ - treated cells receiving PQ 24hrs after initial cumate induction .

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D Whole cell histone H3 acetylation levels in cumate-inducible SIRT6 human fibroblasts, assessed by Western blot. Cu, cumate; PQ, paraquat. Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot, and administered accordingly to respective cell lines Cells were incubated with appropriate cumate dose for 48hrs prior to harvest, with PQ-treated cells receiving PQ 24hrs after initial cumate induction.


E - H Relative abundance of H3K9ac at SIRT6 - dependent NF - κB target genes . Anti - H3K9ac antibodies were used to perform ChIP in the absence ( - Cu ) or presence of ( + Cu ) cumate - induced SIRT6 expression . Samples were normalized using 10 % input . n = 3 BR ; error bars = SD . Students t - test , two tailed . Black asterisk indicate p < 0 . 05 compared to - Cu condition . Red asterisk indicated p < 0 . 05 compared to + Cu WT .

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E-H Relative abundance of H3K9ac at SIRT6-dependent NF-κB target genes. Anti-H3K9ac antibodies were used to perform ChIP in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6 expression. Samples were normalized using 10% input. n=3 BR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition. Red asterisk indicated p<0.05 compared to +Cu WT.


I , J Relative abundance of H3K18ac at satellite repeats associated with SIRT6 deacetylase activity . Anti - H3K18ac antibodies were used to perform ChIP in the absence ( - Cu ) or presence of ( + Cu ) cumate - induced SIRT6 expression . Samples were normalized using 10 % input . n = 3 TR ; error bars = SD . Students t - test , two tailed . Black asterisk indicate p < 0 . 05 compared to - Cu condition .

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I, J Relative abundance of H3K18ac at satellite repeats associated with SIRT6 deacetylase activity. Anti-H3K18ac antibodies were used to perform ChIP in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6 expression. Samples were normalized using 10% input. n=3 TR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition.


A Self - ribosylation of SIRT6 using biotin - labeled NAD + . Recombinant SIRT6 was incubated with NAD + conjugated with a biotin residue and then run on an SDS - PAGE . Each allele was assessed relative to its 0 hr time point and normalized to SIRT6 total protein loading controls . n = 3 TR ; error bars = SD . Students t - test , two tailed . Asterisk indicate p < 0 . 05 . Black asterisks indicate significance over 0 hr control . Red asterisks indicated significance over time - matched WT SIRT6 .

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A Self-ribosylation of SIRT6 using biotin-labeled NAD+. Recombinant SIRT6 was incubated with NAD+ conjugated with a biotin residue and then run on an SDS-PAGE. Each allele was assessed relative to its 0 hr time point and normalized to SIRT6 total protein loading controls. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05. Black asterisks indicate significance over 0 hr control. Red asterisks indicated significance over time-matched WT SIRT6.


B Self - ribosylation of SIRT6 with titration of NAD + . mADPr - specific antibody was used to detect ribosylated wild type SIRT6 and centSIRT6 proteins . Plot was fit the Michaelis - Menten best - fit with GraphPad software . Km NAD was 142 + 29 for centSIRT6 and 106 + 45 for wild type SIRT6 and maximal signal was ~ 2x greater for centSIRT6 compared to the wild type SIRT6 . Two separate batches of recombinant protein of each SIRT6 allele were prepared and assayed . n = 3 TR ; error bars = SD .

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B Self-ribosylation of SIRT6 with titration of NAD+. mADPr-specific antibody was used to detect ribosylated wild type SIRT6 and centSIRT6 proteins. Plot was fit the Michaelis-Menten best-fit with GraphPad software. Km NAD was 142+29 for centSIRT6 and 106+45 for wild type SIRT6 and maximal signal was ~2x greater for centSIRT6 compared to the wild type SIRT6. Two separate batches of recombinant protein of each SIRT6 allele were prepared and assayed. n=3 TR; error bars=SD.


C Activation of PARP1 by SIRT6 variants . SIRT6 protein was incubated with human PARP1 protein and then analyzed by immunoblotting with poly - ADPr antibody . Poly - ADPr activity of PARP1 results in a wide range of product size . Activity was assessed by quantifying poly - ADPr signal in whole lanes for each sample . n = 3 TR ; error bars = SD . Two - way ANOVA . Asterisk indicate p < 0 . 05 . Black asterisks indicate significance over HPRT control . Red asterisks indicated significance over wild type SIRT6 .

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C Activation of PARP1 by SIRT6 variants. SIRT6 protein was incubated with human PARP1 protein and then analyzed by immunoblotting with poly-ADPr antibody. Poly-ADPr activity of PARP1 results in a wide range of product size. Activity was assessed by quantifying poly-ADPr signal in whole lanes for each sample. n=3 TR; error bars=SD. Two-way ANOVA. Asterisk indicate p<0.05. Black asterisks indicate significance over HPRT control. Red asterisks indicated significance over wild type SIRT6.


A qRT - PCR analysis of LINE1 expression in cumate - inducible SIRT6 fibroblasts . Primers assessed both 5 ' ( ORF1 ) and 3 ' ( ORF2 ) LINE1 sequences from the L1HS family of evolutionarily active LINE1 retrotransposons . Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes . Asterisk indicate p < 0 . 05 . n = 3 TR ; error bars = SD . Students t - test , two tailed .

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A qRT-PCR analysis of LINE1 expression in cumate-inducible SIRT6 fibroblasts. Primers assessed both 5' (ORF1) and 3' (ORF2) LINE1 sequences from the L1HS family of evolutionarily active LINE1 retrotransposons. Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes. Asterisk indicate p<0.05. n=3 TR; error bars=SD. Students t-test, two tailed.


B LINE1 EGFP retrotransposition assay . Three independent transfections with LINE1 EGFP reporter plasmid were conducted and quantified by flow cytometry . Asterisk indicate p < 0 . 05 . Red asterisk indicated significance over SIRT6 KO control . Black asterisk indicate significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way ANOVA .

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B LINE1 EGFP retrotransposition assay. Three independent transfections with LINE1 EGFP reporter plasmid were conducted and quantified by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicated significance over SIRT6 KO control. Black asterisk indicate significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA.


C In vitro KAP1 ribosylation by SIRT6 . Asterisk indicate p < 0 . 05 . Red asterisk indicate significance over 0 hr , black asterisk indicate significance over compared to WT SIRT6 . n = 3 BR ; error bars = SD . Students t - test , two tailed .

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C In vitro KAP1 ribosylation by SIRT6. Asterisk indicate p<0.05. Red asterisk indicate significance over 0 hr, black asterisk indicate significance over compared to WT SIRT6. n=3 BR; error bars=SD. Students t-test, two tailed.


D , E Stimulation of NHEJ and HR by SIRT6 variants . Reporter cell lines were co - transfected with SIRT6 - expressing plasmid , I - Sce1 plasmid , and DsRed transfection control . After 72 hr recovery , reactivation of the GFP reporter was measured by flow cytometry . Stimulation of NHEJ or HR was calculated as radio of GFP + / DsRed + positive cells . Blots demonstrate equivalent SIRT6 abundance in transfected cells . Asterisk indicate p < 0 . 05 Black asterisk indicate significance over control , red asterisk indicate significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way

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D, E Stimulation of NHEJ and HR by SIRT6 variants. Reporter cell lines were co-transfected with SIRT6-expressing plasmid, I-Sce1 plasmid, and DsRed transfection control. After 72 hr recovery, reactivation of the GFP reporter was measured by flow cytometry. Stimulation of NHEJ or HR was calculated as radio of GFP+/DsRed+ positive cells. Blots demonstrate equivalent SIRT6 abundance in transfected cells. Asterisk indicate p<0.05 Black asterisk indicate significance over control, red asterisk indicate significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA


F Basal γH2AX foci in cumate - inducible SIRT6 fibroblasts . Foci were scored in at least 80 cells per condition . Asterisk indicate p < 0 . 05 . n = 3 BR ; error bars = SD . Students t - test , two tailed .

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F Basal γH2AX foci in cumate-inducible SIRT6 fibroblasts. Foci were scored in at least 80 cells per condition. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.


G DNA repair kinetics in cumate - inducible SIRT6 fibroblasts . Cells were grown on slides and irradiated with 2 Gy gamma radiation , followed by immunostaining for γH2AX . Irradiation was conducted when the cells were at 75 % confluency on slides . Cells were fixed and foci scored at t = 0 . 5 hr , 2 hr , 4 hr , 6 hr , and 24 hr post - irradiation . Foci were scored in at least 80 cells per genotype per time point . Asterisk indicate p < 0 . 05 . Asterisks indicate significance over WT SIRT6 . Color of the asterisk corresponds to the SIRT6 allele . n = 3 BR ; error bars = SD . Students t - test , two tailed .

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G DNA repair kinetics in cumate-inducible SIRT6 fibroblasts. Cells were grown on slides and irradiated with 2 Gy gamma radiation, followed by immunostaining for γH2AX. Irradiation was conducted when the cells were at 75% confluency on slides. Cells were fixed and foci scored at t=0.5 hr, 2 hr, 4 hr, 6 hr, and 24 hr post-irradiation. Foci were scored in at least 80 cells per genotype per time point. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n=3 BR; error bars=SD. Students t-test, two tailed.


H , I Oxidative stress resistance . Cumate - inducible SIRT6 fibroblasts were induced for SIRT6 expression and exposed to paraquat for 24 hours . Resistance was determined by apoptosis staining 48 hours after exposure . Asterisk indicate p < 0 . 05 . Asterisks indicate significance over WT SIRT6 . Color of the asterisk corresponds to the SIRT6 allele . n = 3 BR ; error bars = SD . Students t - test , two tailed .

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H, I Oxidative stress resistance. Cumate-inducible SIRT6 fibroblasts were induced for SIRT6 expression and exposed to paraquat for 24 hours. Resistance was determined by apoptosis staining 48 hours after exposure. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6 allele. n=3 BR; error bars=SD. Students t-test, two tailed.


A Number of adherent cells after transfection with SIRT6 variants . Cells were transfected with SIRT6 plasmids encoding different SIRT6 alleles and cell numbers were counted after 72 hours . HCA2 are normal human foreskin fibroblasts . Asterisk indicate p < 0 . 05 . n = 3 BR ; error bars = SD . Students t - test , two tailed .

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A Number of adherent cells after transfection with SIRT6 variants. Cells were transfected with SIRT6 plasmids encoding different SIRT6 alleles and cell numbers were counted after 72 hours. HCA2 are normal human foreskin fibroblasts. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.


B , C Apoptosis staining of cancer cell lines 48 hours after transfection . Cells were stained with Annexin V / PI and analyzed by flow cytometry . Asterisk indicate p < 0 . 05 . Red asterisk indicates significance over control , black asterisk indicates significance over WT SIRT6 . n = 3 BR ; error bars = SD . Two - way ANOVA .

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B, C Apoptosis staining of cancer cell lines 48 hours after transfection. Cells were stained with Annexin V/PI and analyzed by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicates significance over control, black asterisk indicates significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA.


B IP experiments on lysates from cumate - induced fibroblasts expressing wild type or centSIRT6 alleles with antibodies to SIRT6 , LMNA , and mADPr . SIRT6 expression was induced 48 hours prior to IP . n = 3 BR . One representative set of IPs is shown .

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B IP experiments on lysates from cumate-induced fibroblasts expressing wild type or centSIRT6 alleles with antibodies to SIRT6, LMNA, and mADPr. SIRT6 expression was induced 48 hours prior to IP. n=3 BR. One representative set of IPs is shown.


C CentSIRT6 shows stronger interaction with LMNA compared to the wild type SIRT6 . Quantification of the IP experiment shown in ( B ) . SIRT6 IP followed by Western blot with antibodies to LMNA . D LMNA shows enhanced interaction with centSIRT6 compared to the wild type . Quantification of the IP experiment shown in ( B ) . LMNA IP from cumate - inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6 . Data information : n = 3 BR ; error bars = SD . Students t - test , two tailed . Asterisk indicate p < 0 .

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C CentSIRT6 shows stronger interaction with LMNA compared to the wild type SIRT6. Quantification of the IP experiment shown in (B). SIRT6 IP followed by Western blot with antibodies to LMNA. D LMNA shows enhanced interaction with centSIRT6 compared to the wild type. Quantification of the IP experiment shown in (B). LMNA IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibodies to SIRT6. Data information: n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05


E centSIRT6 shows enhanced mADPr . Quantification of the IP experiment shown in ( B ) . SIRT6 IP from cumate - inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues F LMNA shows enhanced mADPr signal in cells expressing centSIRT6 . Quantification of the IP experiment shown in ( B ) . IP with mADPr antibody using extract from cumate - induced SIRT6 fibroblasts , followed by Western blot with antibodies to LMNA . Data information : n = 3 BR ; error bars = SD . Students t - test , two tailed . Asterisk indicate p < 0 .

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

E centSIRT6 shows enhanced mADPr. Quantification of the IP experiment shown in (B). SIRT6 IP from cumate-inducible SIRT6 fibroblasts followed by Western blot with antibody to mADPr residues F LMNA shows enhanced mADPr signal in cells expressing centSIRT6. Quantification of the IP experiment shown in (B). IP with mADPr antibody using extract from cumate-induced SIRT6 fibroblasts, followed by Western blot with antibodies to LMNA. Data information: n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05


( A ) 22 Tie1 - binding antibodies were screened for phospho - AKT levels in an in vitro Ang1 - stimulation experiment . Only one clone ( AB - Tie1 - 39 ) reduced AKT phosphorylation indicating suppressed Tie2 signaling ( n = 1 experiment ) .

(A) 22 Tie1-binding antibodies were screened for phospho-AKT levels in an in vitro Ang1-stimulation experiment. Only one clone (AB-Tie1-39) reduced AKT phosphorylation indicating suppressed Tie2 signaling (n = 1 experiment).


( C ) Tumor growth curves show delayed primary tumor growth upon treatment with AB - Tie1 - 39 antibody as compared to IgG treatment ( mean ± SEM , n = 9 mice ) . * , P < 0 . 05 ; * * * , P < 0 . 001 ( 2 - way ANNOVA test ) .

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(C) Tumor growth curves show delayed primary tumor growth upon treatment with AB-Tie1-39 antibody as compared to IgG treatment (mean ± SEM, n = 9 mice). *, P<0.05; ***, P<0.001 (2-way ANNOVA test).


( D ) Representative immunofluorescence images of tumor sections stained with CD31 ( EC - specific marker ) and Desmin ( mural cell - specific marker ) . Scale bars = 200 μm .

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(D) Representative immunofluorescence images of tumor sections stained with CD31 (EC-specific marker) and Desmin (mural cell-specific marker). Scale bars = 200 μm.


( E ) Dot plots show quantitation of intratumoral vessel density , mural cell coverage using Desmin and aSMA co - staining , and tumor necrosis ( mean ± SD , n = 9 mice ) . All comparisons were rendered non - significant according to two - tailed Mann - Whitney U test .

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(E) Dot plots show quantitation of intratumoral vessel density, mural cell coverage using Desmin and aSMA co-staining, and tumor necrosis (mean ± SD, n = 9 mice). All comparisons were rendered non-significant according to two-tailed Mann-Whitney U test.


( A ) Following a neoadjuvant treatment regimen with IgG or AB - Tie1 - 39 , mice were monitored by whole - body bioluminescence imaging . Shown are the images acquired two weeks after primary tumor resection .

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(A) Following a neoadjuvant treatment regimen with IgG or AB-Tie1-39, mice were monitored by whole-body bioluminescence imaging. Shown are the images acquired two weeks after primary tumor resection.


( B ) Dot plots quantifying total photon flux during in vivo whole body as well as ex vivo lung and lymph node bioluminescence imaging ( mean ± SD , nIgG = 8 , nAB - Tie1 - 39 = 9 mice ) . * , P < 0 . 05 ; * * , P < 0 . 01 ( two - tailed Mann - Whitney U test ) .

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(B) Dot plots quantifying total photon flux during in vivo whole body as well as ex vivo lung and lymph node bioluminescence imaging (mean ± SD, nIgG = 8, nAB-Tie1-39 = 9 mice). *, P<0.05; **, P<0.01 (two-tailed Mann-Whitney U test).


( D ) Kaplan - Meier graph comparing percent survival between mice treated with either IgG or AB - Tie1 - 39 ( nIgG = 10 , nAB - Tie1 - 39 = 9 mice ) . * * , P < 0 . 01 ( Log - rank ( Mantel - Cox ) test ) .

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(D) Kaplan-Meier graph comparing percent survival between mice treated with either IgG or AB-Tie1-39 (nIgG = 10, nAB-Tie1-39 = 9 mice). **, P<0.01 (Log-rank (Mantel-Cox) test).


( B ) Representative images of the whole lungs are shown on the left . Scale bars = 5 mm . Quantitation of the number of lung metastatic foci is plotted on the right ( mean ± SD , nIgG = 9 , nAB - Tie1 - 39 = 8 mice ) . * , P < 0 . 05 ( two - tailed Mann - Whitney U test ) .

(B) Representative images of the whole lungs are shown on the left. Scale bars = 5 mm. Quantitation of the number of lung metastatic foci is plotted on the right (mean ± SD, nIgG = 9, nAB-Tie1-39 = 8 mice). *, P<0.05 (two-tailed Mann-Whitney U test).


( C ) EC were seeded on gelatin - coated Transwell inserts . RFP - labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer . Thereafter , transmigrated tumor cells were counted . The dot plot presents the normalized data ( mean ± SD , n = 9 inserts from three independent experiments ) . * * * , P < 0 . 001 ( two - tailed Mann - Whitney U test ) .

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(C) EC were seeded on gelatin-coated Transwell inserts. RFP-labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer. Thereafter, transmigrated tumor cells were counted. The dot plot presents the normalized data (mean ± SD, n = 9 inserts from three independent experiments). ***, P<0.001 (two-tailed Mann-Whitney U test).


( D ) LLC tumor - bearing mice were pretreated with IgG or AB - Tie1 - 39 . On day 14 , lung tissue lysates were immunoblotted with anti - phospho - TIE2 , total - TIE2 and ACTB ( upper panel ; n = 6 mice ) . Densitometric quantitation of the blots presented in the upper panel is shown ( mean ± SD , n = 6 mice ) . * * , P < 0 . 01 ( two - tailed Mann - Whitney U test ) .

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(D) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung tissue lysates were immunoblotted with anti-phospho-TIE2, total-TIE2 and ACTB (upper panel; n = 6 mice). Densitometric quantitation of the blots presented in the upper panel is shown (mean ± SD, n = 6 mice). **, P<0.01 (two-tailed Mann-Whitney U test).


( E ) LLC tumor - bearing mice were pretreated with IgG or AB - Tie1 - 39 . On day 14 , lung EC were FACS - sorted and used for global gene expression ( n = 4 mice ) . On the left , a heatmap shows significantly - regulated genes ( red outline = AB - Tie1 - 39 - treated ; black outline = IgG - treated ) . On the right , top significantly regulated bio - functions in AB - Tie1 - 39 - treated as compared to IgG - treated mice in Ingenuity Pathway Analysis .

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(E) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung EC were FACS-sorted and used for global gene expression (n = 4 mice). On the left, a heatmap shows significantly- regulated genes (red outline = AB-Tie1-39-treated; black outline = IgG-treated). On the right, top significantly regulated bio-functions in AB-Tie1-39-treated as compared to IgG-treated mice in Ingenuity Pathway Analysis.


Formalin - fixed paraffin - embedded ( FFPE ) sections from control or Tgif1KO mice were immunostained with anti - pSmad2 antibody and revealed by immunofluorescence ( IF ) and DAPI . Representative pictures at 40x are shown ( n = 30 ) . Scale bars , 100 μM .

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Formalin-fixed paraffin-embedded (FFPE) sections from control or Tgif1KO mice were immunostained with anti-pSmad2 antibody and revealed by immunofluorescence (IF) and DAPI. Representative pictures at 40x are shown (n=30). Scale bars, 100 μM.


FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin and eosin ( H & E ) or immunostained with antibodies to CK19 or Amylase and revealed by IHC . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin and eosin (H&E) or immunostained with antibodies to CK19 or Amylase and revealed by IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Kaplan - Meyer survival analysis of control , Tgif1KO , KC and KTC mice . A regular mosaic two - colors line was used to discriminate between control and Tgif1KO mice .

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Kaplan-Meyer survival analysis of control, Tgif1KO, KC and KTC mice. A regular mosaic two-colors line was used to discriminate between control and Tgif1KO mice.


Pictures of whole pancreas and spleen tissues from control , Tgif1KO , KC or KTC mice ( left ) . Weight of pancreas was measured and presented as dot plot ( n = 30 ) .

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Pictures of whole pancreas and spleen tissues from control, Tgif1KO, KC or KTC mice (left). Weight of pancreas was measured and presented as dot plot (n=30).


FFPE pancreatic sections from control , Tgif1KO , KC or KTC were stained with H & E or subjected to IHC analysis using antibodies to CK19 or α - SMA . Yellow arrows indicate PanINs in KC mice . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

FFPE pancreatic sections from control, Tgif1KO, KC or KTC were stained with H&E or subjected to IHC analysis using antibodies to CK19 or α-SMA. Yellow arrows indicate PanINs in KC mice. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Representative pictures of dissected abdominal region of KC and KTC mice . Yellow arrows indicate metastatic nodules in liver . Yellow dotted lines show the pancreas .

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Representative pictures of dissected abdominal region of KC and KTC mice. Yellow arrows indicate metastatic nodules in liver. Yellow dotted lines show the pancreas.


FFPE sections of pancreas , liver , lungs or lymph - nodes from control , KC or KTC mice were stained with H & E or immunostained with anti - Vimentin antibody and revealed by IF . Representative pictures at 4x ( Lymph node , H & E ) , 10x ( Liver , H & E ) and 20x ( Pancreas , H & E ; Lung , H & E ; Lymph node , IF - Vimentin ) are shown ( n = 18 to 30 ) . Scale bars , 1 , 000 μM ( Lymph node , H & E ) , 400 μM ( Liver , H & E ) and 200 μM ( Pancreas , H & E ; Lung , H & E ; Lymph node , IF - Vimentin ) .

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FFPE sections of pancreas, liver, lungs or lymph-nodes from control, KC or KTC mice were stained with H&E or immunostained with anti-Vimentin antibody and revealed by IF. Representative pictures at 4x (Lymph node, H&E), 10x (Liver, H&E) and 20x (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin) are shown (n=18 to 30). Scale bars, 1,000 μM (Lymph node, H&E), 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin).


Tumor volume and metastasis in live KCLuc and KTCLuc mice were analyzed by IVIS bioluminescence . Following imaging live animals , pancreas , brain , liver and lung were dissected and analyzed again by IVIS bioluminescence ( n = 6 , 5 out of 6 mice developed metastasis to liver and lung ) .

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Tumor volume and metastasis in live KCLuc and KTCLuc mice were analyzed by IVIS bioluminescence. Following imaging live animals, pancreas, brain, liver and lung were dissected and analyzed again by IVIS bioluminescence (n= 6, 5 out of 6 mice developed metastasis to liver and lung).


Total cell lysates from pancreas of control , KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to pSmad2 , Smad2 or JunB ( n = 6 ) .

Total cell lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to pSmad2, Smad2 or JunB (n=6).


Expression of JunB in pancreas from control , KC or KTC mice ( same as in D ) was analyzed by qRT - PCR ( n = 6 ) . Data are expressed as mean ± SEM . * * p < 0 . 01 ; based on a two - tailed Student ' s test .

Expression of JunB in pancreas from control, KC or KTC mice (same as in D) was analyzed by qRT-PCR (n=6). Data are expressed as mean ± SEM. **p< 0.01; based on a two-tailed Student's test.


Protein extracts from MIAPaCa - 2 cells ( left ) and control or Tgif1KO mice ( right ) were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 ( A - C ) .

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Protein extracts from MIAPaCa-2 cells (left) and control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 (A-C).


Protein extracts from control or Tgif1KO mice ( right ) were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading , total cell lysates were analyzed by immunoblotting using antibodies to Twist1 , TGIF1 or β - Actin .

Protein extracts from control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading, total cell lysates were analyzed by immunoblotting using antibodies to Twist1, TGIF1 or β-Actin.


Protein extracts from MIAPaCa - 2 cells ( left ) and control were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C , cell lysates were treated with DNase I before coimmunopreciptation .

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Protein extracts from MIAPaCa-2 cells (left) and control were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C, cell lysates were treated with DNase I before coimmunopreciptation.


Panc - 1 cells were treated with TGF - β for 30 min , and protein extracts were immunoprecipitated with anti - TGIF1 antibody and analyzed by immunoblotting using anti - Twist1 antibody . To control for activation of TGF - β signaling , total cell lysates were analyzed by immunoblotting using anti - pSmad2 antibody .

Panc-1 cells were treated with TGF-β for 30 min, and protein extracts were immunoprecipitated with anti-TGIF1 antibody and analyzed by immunoblotting using anti-Twist1 antibody. To control for activation of TGF-β signaling, total cell lysates were analyzed by immunoblotting using anti-pSmad2 antibody.


PL45 stably expressing Dox - inducible control or sh - RNA targeting TGIF were treated with vehicle or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT - PCR ( n = 6 ) . In left panel , the intensity of the bands was assessed by densitometry , and the result was presented as a ratio of Twist1 / TGIF1 .

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PL45 stably expressing Dox-inducible control or sh-RNA targeting TGIF were treated with vehicle or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6). In left panel, the intensity of the bands was assessed by densitometry, and the result was presented as a ratio of Twist1/TGIF1.


PL45 stably expressing Dox - inducible TGIF1 were treated with vehicle or Dox for 48 hr and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel .

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PL45 stably expressing Dox-inducible TGIF1 were treated with vehicle or Dox for 48 hr and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.


Pancreatic chromatin from control or Tgif1KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel .

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Pancreatic chromatin from control or Tgif1KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.


PL45 stably expressing both Dox - inducible TGIF1 and Tam - inducible Twist1 ( Twist1ER ) were treated with Tam and / or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT - PCR ( n = 6 ) .

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PL45 stably expressing both Dox-inducible TGIF1 and Tam-inducible Twist1 (Twist1ER) were treated with Tam and/or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6).


MIAPaCa - 2 cells were transfected with wild - type or mutated ( Twist1 binding site ) Twist1 luciferase reporters ( TGTLuc ) together with increasing amounts of Twist1 expression vector in the presence of empty vector ( EV ) or TGIF1 expression vector . Luciferase activity was measured 48 hr following transfection and normalized on the basis of co - transfected Renilla luciferase ( n = 6 ) .

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MIAPaCa-2 cells were transfected with wild-type or mutated (Twist1 binding site) Twist1 luciferase reporters (TGTLuc) together with increasing amounts of Twist1 expression vector in the presence of empty vector (EV) or TGIF1 expression vector. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6).


Total lysates from pancreas of control , KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to E - Cadherin , Vimentin , p16Ink4A and β - Actin ( n = 6 ) .

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Total lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to E-Cadherin, Vimentin, p16Ink4A and β-Actin (n=6).


Expression of Cdh1 , Vimentin , and p16Ink4A in pancreas from control , KC or KTC mice ( same as in C ) was analyzed by qRT - PCR ( n = 6 ) .

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Expression of Cdh1, Vimentin, and p16Ink4A in pancreas from control, KC or KTC mice (same as in C) was analyzed by qRT-PCR (n=6).


FFPE pancreatic sections from control , KC or KTC mice were stained with H & E or immunostained with antibodies to E - Cadherin , Vimentin or p16Ink4a and revealed by IF or IHC . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

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FFPE pancreatic sections from control, KC or KTC mice were stained with H&E or immunostained with antibodies to E-Cadherin, Vimentin or p16Ink4a and revealed by IF or IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Kaplan - Meyer survival analysis of control , KTC and KTWC mice .

Kaplan-Meyer survival analysis of control, KTC and KTWC mice.


FFPE sections of pancreas or liver from control , KTC or KTWC mice were stained with H & E or immunostained with anti - CK19 antibody and revealed by IHC . Representative pictures at 10x ( Liver , H & E ) or 20x ( Pancreas , H & E ; Pancreas , IHC - CK19 ) are shown ( n = 30 ) . Scale bars , 400 μM ( Liver , H & E ) and 200 μM ( Pancreas , H & E ; Pancreas , IHC - CK19 ) .

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FFPE sections of pancreas or liver from control, KTC or KTWC mice were stained with H&E or immunostained with anti-CK19 antibody and revealed by IHC. Representative pictures at 10x (Liver, H&E) or 20x (Pancreas, H&E; Pancreas, IHC-CK19) are shown (n=30). Scale bars, 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Pancreas, IHC-CK19).


Expression of Cdh1 , Vimentin and p16Ink4A in pancreas from control , KTC or KTWC mice was analyzed by qRT - PCR ( n = 6 ) .

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Expression of Cdh1, Vimentin and p16Ink4A in pancreas from control, KTC or KTWC mice was analyzed by qRT-PCR (n=6).


Specificity of anti - phospho - TGIF1 ( pTGIF1 ) antibody . Cell extracts from Tgif1 - / - MEFs cells reconstituted with empty vector ( EV ) , wild - type ( TGIF1 ) or non - phosphorylated form ( TGIF1 . 2TA ) of Flag - TGIF1 were immunoblotted with anti - phospho - TGIF1 or β - Actin as a loading control .

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Specificity of anti-phospho-TGIF1 (pTGIF1) antibody. Cell extracts from Tgif1-/- MEFs cells reconstituted with empty vector (EV), wild-type (TGIF1) or non-phosphorylated form (TGIF1.2TA) of Flag-TGIF1 were immunoblotted with anti-phospho-TGIF1 or β-Actin as a loading control.


Expression of pTGIF1 and Twist1 in human tissue microarrays of human PDAC samples was analyzed by immunohistochemistry . Representative pictures at different stages ( 40x ) are shown .

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Expression of pTGIF1 and Twist1 in human tissue microarrays of human PDAC samples was analyzed by immunohistochemistry. Representative pictures at different stages (40x) are shown.


The percentages of samples with high versus low expression of pTGIF1 and Twist1 in normal tissues versus PDAC tissues are shown . Scale bars , 100 μM .

The percentages of samples with high versus low expression of pTGIF1 and Twist1 in normal tissues versus PDAC tissues are shown. Scale bars, 100 μM.


MIAPaCa - 2 cells were transfected with TGIF1 or TGIF1 . 2TD expression vectors , selected with G418 and resistance colonies were pooled ( n = 6 ) . Expression of Twist1 was examined by qRT - PCR ( D ) .

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MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Expression of Twist1 was examined by qRT-PCR (D).


MIAPaCa - 2 cells were transfected with TGIF1 or TGIF1 . 2TD expression vectors , selected with G418 and resistance colonies were pooled ( n = 6 ) . Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel .

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MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel.


BxPC3 cells were transfected with TGTLuc together with TGIF1 or TGIF1 . 2TA in the presence or absence of KrasG12D . Luciferase activity was measured 48 hr following transfection and normalized on the basis of co - transfected Renilla luciferase ( n = 6 ) .

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BxPC3 cells were transfected with TGTLuc together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6).


BxPC3 cells were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1 . 2TA in the presence or absence of KrasG12D and luciferase activity was measured 48 hr following transfection as described in ( E ) ( n = 6 ) .

BxPC3 cells were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D and luciferase activity was measured 48 hr following transfection as described in (E) (n=6).


Representative microscope pictures ( 20x ) of MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells . Scale bars , 200 μM .

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Representative microscope pictures (20x) of MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells. Scale bars, 200 μM.


Expression of TGIF1 and Twist1 in MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells was analyzed by immunoblotting .

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Expression of TGIF1 and Twist1 in MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells was analyzed by immunoblotting.


MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells were transfected with empty vector ( EV ) , wild - type ( TGIF1 ) or phosphorylation mimic mutant ( TGIF1 . 2TD ) of TGIF1 . Cell extracts were analyzed 48 hr following transfection by immunoblotting using anti - Flag antibody ( C ) .

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MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells were transfected with empty vector (EV), wild-type (TGIF1) or phosphorylation mimic mutant (TGIF1.2TD) of TGIF1. Cell extracts were analyzed 48 hr following transfection by immunoblotting using anti-Flag antibody (C).


MIAPaCa - 2 , Suit - 2 , Capan - 2 and Panc - 1 cells were transfected with empty vector ( EV ) , wild - type ( TGIF1 ) or phosphorylation mimic mutant ( TGIF1 . 2TD ) of TGIF1 . Cells were selected with neomycin for 2 - 3 weeks , and counted using an automatic cell counter ( D ) ( n = 6 ) .

MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells were transfected with empty vector (EV), wild-type (TGIF1) or phosphorylation mimic mutant (TGIF1.2TD) of TGIF1. Cells were selected with neomycin for 2-3 weeks, and counted using an automatic cell counter (D) (n=6).


A . Structure of Brca1ex2 allele . * Note that exon 4 was annotated in error in original descriptions of the gene structure and is not drawn . RT - PCR shows a novel product from the Brca1ex2 allele , corresponding to splicing of exon 1 directly to exon 3 .

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A. Structure of Brca1ex2 allele. * Note that exon 4 was annotated in error in original descriptions of the gene structure and is not drawn. RT-PCR shows a novel product from the Brca1ex2 allele, corresponding to splicing of exon 1 directly to exon 3.


B . Western blot to detect BRCA1 and BARD1 in MEFs expressing WT and mutant Brca1 .

B. Western blot to detect BRCA1 and BARD1 in MEFs expressing WT and mutant Brca1.


C . Immunofluorescent ( IF ) detection of BRCA1 and RAD51 at IR - induced nuclear foci in cells expressing WT and mutant forms of Brca1 . Scale bar : 10 ingD . Quantification of IF , showing proportion of cells with RAD51 foci that also had > 5 BRCA1 foci . N = 2 . E . Quantification of IF , showing proportion of RAD51 foci that colocalized with BRCA1 foci . N = 2 .

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C. Immunofluorescent (IF) detection of BRCA1 and RAD51 at IR-induced nuclear foci in cells expressing WT and mutant forms of Brca1. Scale bar: 10 ingD. Quantification of IF, showing proportion of cells with RAD51 foci that also had >5 BRCA1 foci. N=2.E. Quantification of IF, showing proportion of RAD51 foci that colocalized with BRCA1 foci. N=2.


F . Immunofluorescent detection of BARD1 at IR - induced nuclear foci in MEFs expressing WT and mutant forms of Brca1 . Scale bar : 10 μ

F. Immunofluorescent detection of BARD1 at IR-induced nuclear foci in MEFs expressing WT and mutant forms of Brca1. Scale bar: 10 μ S


A . Western blot showing abundance of BRCA1 and BARD1 protein in Brca1Δ2 / Δ2 ; Trp53bp1 - / - mouseB cells .

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A. Western blot showing abundance of BRCA1 and BARD1 protein in Brca1Δ2/Δ2;Trp53bp1-/- mouseB cells.


B . Analysis of chromosome aberrations in mice with targeted deletion of Brca1 exon 2 and Trp53bp1 . Left , metaphase spreads were prepared from B cells treated with olaparib . Arrows show examples of chromosome aberrations . Right , quantification of chromosome breaks ( CSB ) , chromatid - type breaks ( CTD ) , radial chromosomes , and other abnormalities in mouseB cells . Error bars indicate SD , N =

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B. Analysis of chromosome aberrations in mice with targeted deletion of Brca1 exon 2 and Trp53bp1. Left, metaphase spreads were prepared from B cells treated with olaparib. Arrows show examples of chromosome aberrations. Right, quantification of chromosome breaks (CSB), chromatid-type breaks (CTD), radial chromosomes, and other abnormalities in mouseB cells. Error bars indicate SD, N=3


C . RAD51 foci in B cells after 10 Gy IR exposure . Chart shows mean percentage of cells with > 5 foci , N = 3 . Error bars indicate SD , statistical analysis by two - tailed Student t test .

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C. RAD51 foci in B cells after 10 Gy IR exposure. Chart shows mean percentage of cells with >5 foci, N=3. Error bars indicate SD, statistical analysis by two-tailed Student t test.


D . Percentage of cells showing > 5 RAD51 foci after 4hrs treatment with 10 μM olaparib ( OLA ) or 10 μM camptothecin ( CPT ) . N = 2 .

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D. Percentage of cells showing >5 RAD51 foci after 4hrs treatment with 10 μM olaparib (OLA) or 10 μM camptothecin (CPT). N=2.


E . Average number of γ - H2AX foci per cell in cells that were not treated ( NT ) or exposed to 2 Gy ionizing radiation . N = 2 .

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E. Average number of γ-H2AX foci per cell in cells that were not treated (NT) or exposed to 2 Gy ionizing radiation. N=2.


F . Sister chromatid exchanges in cells that were not treated ( NT ) or exposed to 2 μM olaparib ( OLA ) for 16 hrs . Black arrows in images show SCEs in olaparib - treated cells . Scale bar : 5 μm . N = 2 .

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F. Sister chromatid exchanges in cells that were not treated (NT) or exposed to 2 μM olaparib (OLA) for 16 hrs. Black arrows in images show SCEs in olaparib-treated cells. Scale bar: 5 μm. N=2.


A . Experimental scheme and representative images of DNA fibers . Scale bar : 5 μm . B . Analysis of fibers showing proportions of replication forks that showed restart after HU treatment or remained stalled . N = 2 , > 500 fibers scored per experiment . C . Proportion of total forks showing de novo initiation after HU treatment . N = 2 , > 500 fibers scored per experiment . D . Length of initial replication fork tracts ( CldU tracts ) . N = 2 , > 200 fibers measured per experiment .

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A. Experimental scheme and representative images of DNA fibers. Scale bar: 5 μm.B. Analysis of fibers showing proportions of replication forks that showed restart after HU treatment or remained stalled. N=2, >500 fibers scored per experiment.C. Proportion of total forks showing de novo initiation after HU treatment. N=2, >500 fibers scored per experiment.D. Length of initial replication fork tracts (CldU tracts). N=2, >200 fibers measured per experiment.


A . Kaplan - Meier survival curve for Brca1ex2 / + ; Trp53bp1 - / - and Brca1ex2 / ex2 ; Trp53bp1 - / - mice . N = 20 animals in each group . Statistical analysis by Mantel - Cox log - rank test .

A. Kaplan-Meier survival curve for Brca1ex2/+;Trp53bp1-/- and Brca1ex2/ex2;Trp53bp1-/- mice. N=20 animals in each group. Statistical analysis by Mantel-Cox log-rank test.


B . Kaplan - Meier survival curve for Brca1ex2 / + ; Trp53bp1 - / - ; Trp53 + / - and Brca1ex2 / ex2 ; Trp53bp1 - / - ; Trp53 + / - mice . N = 20 animals in each group . Statistical analysis by Mantel - Cox log - rank test .

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B. Kaplan-Meier survival curve for Brca1ex2/+;Trp53bp1-/-;Trp53+/- and Brca1ex2/ex2;Trp53bp1-/-;Trp53+/- mice. N=20 animals in each group. Statistical analysis by Mantel-Cox log-rank test.


C . Percentage of Brca1ex2 / + ; Trp53bp1 - / - and Brca1ex2 / ex2 ; Trp53bp1 - / - mice showing signs of abnormal tissue morphology at death . N = 46 animals inspected for Brca1ex2 / + ; Trp53bp1 + / + , N = 54 animals inspected for Brca1ex2 / ex2 / Trp53bp1 - / - .

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C. Percentage of Brca1ex2/+;Trp53bp1-/- and Brca1ex2/ex2;Trp53bp1-/- mice showing signs of abnormal tissue morphology at death. N=46 animals inspected for Brca1ex2/+;Trp53bp1+/+, N=54 animals inspected for Brca1ex2/ex2/Trp53bp1-/-.


D . Tissues affected by abnormal growth from N = 46 Brca1ex2 / + / Trp53bp1 - / - and N = 54 Brca1ex2 / ex2 / Trp53bp1 - / - mice .

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D. Tissues affected by abnormal growth from N=46 Brca1ex2/+/Trp53bp1-/- and N=54 Brca1ex2/ex2/Trp53bp1-/- mice.


A . H & amp ; amp ; E - stained sections of seminiferous tubules from testes of WT and Brca1ex2 / ex2 / Trp53bp1 - / - male mice . Scale bar : 100 μm .

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A. H&amp;E-stained sections of seminiferous tubules from testes of WT and Brca1ex2/ex2/Trp53bp1-/- male mice. Scale bar: 100 μm.


B . Immunofluorescence on meiotic chromosomes from WT and Brca1ex2 / ex2 ; Trp53bp1 - / - spermatocytes . Spreads were stained for SYCP3 to indicate chromosome axes and extent of synapsis and were stained for SYCP3 to indicate chromosome axex body . Scale bar : 5

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B. Immunofluorescence on meiotic chromosomes from WT and Brca1ex2/ex2;Trp53bp1-/-spermatocytes. Spreads were stained for SYCP3 to indicate chromosome axes and extent of synapsis and were stained for SYCP3 to indicate chromosome axex body. Scale bar: 5 apsi


C . Quantification of the number of γ - H2AX foci observed at the pachytene stage in the indicated genotypes . P value is Mann - Whitney , two - tailed .

C. Quantification of the number of γ-H2AX foci observed at the pachytene stage in the indicated genotypes. P value is Mann-Whitney, two-tailed.


D . G2M checkpoint analysis in mouse B cells after IR treatment . Left , mitotic cells were identified by flow cytometry as having 4c DNA content ( based on propidium iodide staining ) and pSer10 - H3 + . Right , quantification of flow cytometry data , showing mitotic cells after IR as percentage of that seen in untreated cells . N ≥ 2 .

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D. G2M checkpoint analysis in mouse B cells after IR treatment. Left, mitotic cells were identified by flow cytometry as having 4c DNA content (based on propidium iodide staining) and pSer10-H3+. Right, quantification of flow cytometry data, showing mitotic cells after IR as percentage of that seen in untreated cells. N≥2.


Normalised Wnt / STOP signalling activity by quantification of the protein expression of two Wnt / STOP target proteins , β - catenin and c - Myc , in mitotic ( M ) and asynchronised cells ( A ) after silencing 47 Wnt signalling regulators .

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Normalised Wnt/STOP signalling activity by quantification of the protein expression of two Wnt/STOP target proteins, β-catenin and c-Myc, in mitotic (M) and asynchronised cells (A) after silencing 47 Wnt signalling regulators.


ELISA validation of selected Wnt / STOP signalling regulators ( n = 3 ) .

ELISA validation of selected Wnt/STOP signalling regulators (n=3).


Immunoblotting and ( D ) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX ( n = 3 ) .

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Immunoblotting and (D) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX (n=3).


Representative pictures ( left panel ) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3 - GFP biosensor following different treatments before and after metaphase ( n = 10 ) . Scale bars represent 10 μm .

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Representative pictures (left panel) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3-GFP biosensor following different treatments before and after metaphase (n=10). Scale bars represent 10 μm.


Myc and β - catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9 .

Myc and β-catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9.


The representative xenograft tumours ( top panel ) and tumour growth analysis ( bottom panel ) of scramble and BCL9 stable knockdown cells . LF : left flank of mouse , RF : right flank . n = 6 for shS1 vs . shB1 , n = 5 for shS1 vs . shB2 .

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The representative xenograft tumours (top panel) and tumour growth analysis (bottom panel) of scramble and BCL9 stable knockdown cells. LF: left flank of mouse, RF: right flank. n=6 for shS1 vs. shB1, n=5 for shS1 vs. shB2.


Immunohistochemical staining of the xenograft ; the red arrows indicate the representative mitotic cells . Scale bars represent 200 μm .

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Immunohistochemical staining of the xenograft; the red arrows indicate the representative mitotic cells. Scale bars represent 200 μm.


Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells ; the black arrow indicates the delayed band of mitotic BCL9 .

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Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells; the black arrow indicates the delayed band of mitotic BCL9.


Immunofluorescence confocal analysis of BCL9 localization on the spindle by antibody staining . Yellow arrows indicate the spindle pole . Scale bars represent : 2 μm for interphase and anaphase ; 5 μm for prophase , metaphase and telophase .

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Immunofluorescence confocal analysis of BCL9 localization on the spindle by antibody staining. Yellow arrows indicate the spindle pole. Scale bars represent: 2 μm for interphase and anaphase; 5 μm for prophase, metaphase and telophase.


Immunofluorescence confocal analysis of BCL9 localization on the centrosome . Yellow arrows indicate the centrosome . Scale bars represent : 5 μm for control treatment , 10 μm for BCL9 peptide treatment .

Immunofluorescence confocal analysis of BCL9 localization on the centrosome. Yellow arrows indicate the centrosome. Scale bars represent: 5 μm for control treatment, 10 μm for BCL9 peptide treatment.


Confocal microscope 3D - analysis and radial histograms of the spindle angle in the cells with siRNA treatment ; yellow arrows indicate the centrosome . Scale bars represent 5 μm .

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Confocal microscope 3D-analysis and radial histograms of the spindle angle in the cells with siRNA treatment; yellow arrows indicate the centrosome. Scale bars represent 5 μm.


Confocal ( left panel ) and live cell imaging ( right panel ) analysis of chromosome segregation in H2BGFP - cells following siRNA treatment ; yellow arrows indicate the typical abnormal chromosomes by BCL9 knockdown . 23 H2BGFP - mitotic cells were monitored for each time experiment ; data are shown from three independent experiments . Scale bars represent 5 μm . Quantification of aberrant chromosome segregation rate by live cell imaging of H2BGFP cells upon Wnt3a or DKK1 treatment . 23 H2BGFP - mitotic cells were monitored for each time experiment ; data are shown from three independent experiments .

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Confocal (left panel) and live cell imaging (right panel) analysis of chromosome segregation in H2BGFP-cells following siRNA treatment; yellow arrows indicate the typical abnormal chromosomes by BCL9 knockdown. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments. Scale bars represent 5 μm. Quantification of aberrant chromosome segregation rate by live cell imaging of H2BGFP cells upon Wnt3a or DKK1 treatment. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments.


Quantification of the duration of NEB to ANA ( NEB : nuclear envelope breakdown , ANA : anaphase ) , NEB to cell death in interphase after cell division ( blue colour bar ) or in mitosis ( dark red bar ) , or growth well in 30 hours ( pink bar ) by live cell imaging analysis in monitored H2BGFP cells with different treatments .

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Quantification of the duration of NEB to ANA (NEB: nuclear envelope breakdown, ANA: anaphase), NEB to cell death in interphase after cell division (blue colour bar) or in mitosis (dark red bar), or growth well in 30 hours (pink bar) by live cell imaging analysis in monitored H2BGFP cells with different treatments.


Volcano plot of the BCL9 mitotic interactome by the SILAC proteomics approach . Red dashed line indicates a strict cut off by a normalised SILAC H / L ratio > 2 .

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Volcano plot of the BCL9 mitotic interactome by the SILAC proteomics approach. Red dashed line indicates a strict cut off by a normalised SILAC H/L ratio > 2.


An independent immunoprecipitation assay for validation of potential BCL9 interactors in both asynchronised and mitotic cells . The relative binding of CLTC with BCL9 is quantified and labelled on top of the CLTC blot .

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An independent immunoprecipitation assay for validation of potential BCL9 interactors in both asynchronised and mitotic cells. The relative binding of CLTC with BCL9 is quantified and labelled on top of the CLTC blot.


Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells , from G1S to G2M . P - H3S10 , a mitotic marker indicating mitosis .

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Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells, from G1S to G2M. P-H3S10, a mitotic marker indicating mitosis.