Figure 4Ai - x . Lateral view of the adult fly head . Scale bar = 100 μm . i . longGMR , UAS - Rac1W / TM6B , Hu , Tb causes a mild rough eye phenotype with fused ( arrows ) and disorganized but still separated ommatidia ( arrowheads ) . ii / iii . Rac1 overexpression with KD of dlrrk ( UAS - dlrrk - RNAi ( v22139 or v22140 ) / longGMR - GAL4 , UAS - Rac1W ) , results in smaller eyes where most ommatidia are fused . iv / v . Rac1 overexpression with KD of Clc ( UAS - Clc - RNAi ( v22318 or v106632 ) / + ; longGMR - GAL4 , UAS - Rac1W / + ) , results in smaller eyes with some ommatidia fused . vi . Rac1 overexpression with KD of the inwardly rectifying potassium channel 3 ( irk3 ) , a non - relevant RNAi control ( UAS - irk3 - RNAi ( v101174 ) / + ; longGMR - GAL4 , UAS - Rac1W / + ) , has no effect . vii / viii . KD of dlrrk with GMR - GAL4 / + ; UAS - dlrrk - RNAi ( v22139 or 22140 ) / + , respectively as control have no effect on eye morphology . ix / x . KD of Clc with GMR - GAL4 / UAS - Clc - RNAi ( v22318 or v106632 ) , respectively as control have no effect on eye morphology . Bi - ii . Lateral view of the adult fly head . Scale bar : 100 μm . i . Rac1 overexpression with removing one copy of dlrrk ( dlrrke03680 / longGMR - GAL4 , UAS - Rac1W ) , results in smaller eyes where most ommatidia are fused together . ii . Removing one copy of dlrrke03680 in GMR - GAL4 background , as control has no effect on eye morphology ( GMR - GAL4 / + ; dlrrke03680 / + ) . Ci - viii . Lateral view of the adult fly head . Scale bar : 100 μm . i . longGMR , UAS - Rac1W / TM6B , Hu , Tb causes a mild rough eye phenotype with fused and disorganized but still separated ommatidia ( as explained in Ai ) . ii . Rac1 co - expression with dlrrk - WT using UAS - dlrrk - WT / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye morphology . iii . Rac1 co - expression with Clc using UAS - GFP - Clc / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye morphology . iv . Rac1 co - expression with dlrrk - WT - 3KD using UAS - dlrrk - 3KD / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye morphology . v . A wild - type eye of the longGMR - GAL4 promoter as a control . vi - viii . Overexpression of dlrrk - WT , dlrrk - 3KD , or Clc alone with GMR - GAL4 as control , have no effect on eye morphology .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Ai-x. Lateral view of the adult fly head. Scale bar = 100 μm.i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused (arrows) and disorganized but still separated ommatidia (arrowheads).ii/iii. Rac1 overexpression with KD of dlrrk (UAS-dlrrk-RNAi (v22139 or v22140)/longGMR-GAL4, UAS-Rac1W), results in smaller eyes where most ommatidia are fused.iv/v. Rac1 overexpression with KD of Clc (UAS-Clc-RNAi (v22318 or v106632)/+;longGMR-GAL4,UAS-Rac1W/+), results in smaller eyes with some ommatidia fused.vi. Rac1 overexpression with KD of the inwardly rectifying potassium channel 3 (irk3), a non-relevant RNAi control (UAS-irk3-RNAi(v101174)/+; longGMR-GAL4,UAS-Rac1W/+), has no effect.vii/viii. KD of dlrrk with GMR-GAL4/+; UAS-dlrrk-RNAi(v22139 or 22140)/+, respectively as control have no effect on eye morphology.ix/x. KD of Clc with GMR-GAL4/UAS-Clc-RNAi (v22318 or v106632), respectively as control have no effect on eye morphology.Bi-ii. Lateral view of the adult fly head. Scale bar: 100 μm.i. Rac1 overexpression with removing one copy of dlrrk (dlrrke03680/longGMR-GAL4,UAS-Rac1W), results in smaller eyes where most ommatidia are fused together.ii. Removing one copy of dlrrke03680 in GMR-GAL4 background, as control has no effect on eye morphology (GMR-GAL4/+;dlrrke03680/+).Ci-viii. Lateral view of the adult fly head. Scale bar: 100 μm.i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused and disorganized but still separated ommatidia (as explained in Ai).ii. Rac1 co-expression with dlrrk-WT using UAS-dlrrk-WT/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.iii. Rac1 co-expression with Clc using UAS-GFP-Clc/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.iv. Rac1 co-expression with dlrrk-WT-3KD using UAS-dlrrk-3KD/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.v. A wild-type eye of the longGMR-GAL4 promoter as a control.vi-viii. Overexpression of dlrrk-WT, dlrrk-3KD, or Clc alone with GMR-GAL4 as control, have no effect on eye morphology.


Figure 1 ( A ) Cardiac GATA4 protein abundance analyzed by immunoblotting . GAPDH was the loading control . + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4 overexpressing adenovirus . ( B ) Densitometric quantification of the immunoblot shown under ( A ) ; * * * p = 0 . 0003 for P1 vs . P7 and P1 vs . P21 ; p < 0 . 0001 for P1 vs . P60 . The number within bars indicates the number of mice analyzed in that particular group . ( C ) Immunoblot of cardiac GATA4 abundance in control ( Con ) and cardiomyocyte specific Gata4 knock - out mice ( CM - G4 - KO ) at postnatal day ( P ) 1 . ( D ) Quantification of the immunoblot shown in ( C ) ; * p = 0 . 018 . ( E ) Cardiac immunofluorescence staining and its quantification from Con and CM - G4 - KO mice at P1 . Scale bar : 25 µm ; * * * * p < 0 . 0001 . ( F ) Transversal sections of mouse hearts 7 days after cryoinjury as indicated , stained with Sirius - Red . Scale bar : 500 µm . The quantification of the left ventricular ( LV ) scar area ( red , arrows ) 7 days after injury is shown on the right ; * * p = 0 . 0027 . ( G ) The quantification of the LV scar area of the indicated mice 21 days after cryoinjury ; * p = 0 . 0261 . ( H ) The quantification of the LV scar area of the indicated mice 60 days after cryoinjury ; * * * * p < 0 . 0001 . ( I ) Echocardiography analysis of left ventricular systolic function ( measured as fractional area change ) in the indicated mice 7 days after cryoinjury ; * p = 0 . 033 between Con mice after sham or cryoinjury and p = 0 . 04 between Con and CM - G4 - KO mice after cryoinjury .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Cardiac GATA4 protein abundance analyzed by immunoblotting. GAPDH was the loading control. + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4 overexpressing adenovirus.(B) Densitometric quantification of the immunoblot shown under (A); ***p=0.0003 for P1 vs. P7 and P1 vs. P21; p<0.0001 for P1 vs. P60. The number within bars indicates the number of mice analyzed in that particular group.(C) Immunoblot of cardiac GATA4 abundance in control (Con) and cardiomyocyte specific Gata4 knock-out mice (CM-G4-KO) at postnatal day (P) 1.(D) Quantification of the immunoblot shown in (C); *p=0.018.(E) Cardiac immunofluorescence staining and its quantification from Con and CM-G4-KO mice at P1. Scale bar: 25 µm; ****p<0.0001.(F) Transversal sections of mouse hearts 7 days after cryoinjury as indicated, stained with Sirius-Red . Scale bar: 500 µm. The quantification of the left ventricular (LV) scar area (red, arrows) 7 days after injury is shown on the right; **p=0.0027.(G) The quantification of the LV scar area of the indicated mice 21 days after cryoinjury; *p=0.0261.(H) The quantification of the LV scar area of the indicated mice 60 days after cryoinjury; ****p<0.0001.(I) Echocardiography analysis of left ventricular systolic function (measured as fractional area change) in the indicated mice 7 days after cryoinjury; *p=0.033 between Con mice after sham or cryoinjury and p=0.04 between Con and CM-G4-KO mice after cryoinjury.


Figure 2 ( A ) Representative pictures of TUNEL , WGA ( wheat germ agglutinin to mark cardiomyocyte membranes ) and DAPI immunofluorescence staining of mouse hearts as indicated . The white dashed line encircles the infarcted area ( arrows ) . Scale bar : 200µm . The quantification of the area of TUNEL positive cells 3 hours after cryoinjury is shown on the right ( A ´ ) . The number within bars indicates the number of mice analyzed in that particular group . ( B ) Representative merged and single channel pictures of cardiac CD31 , DAPI and WGA immunostaining in mouse hearts as indicated . The quantification of the myocardial capillary density as capillaries / cardiomyocyte ( CM ) ratio , 7 days ( d ) after cryoinjury or sham surgery is shown as bar graph ( B ´ ) . Scale bar : 20µm . * * p = 0 . 0039 . ( C ) Representative pictures of myocardial immunofluorescence staining for the mitosis marker phospho - histone H3 ( pH3 ) , troponin T and DAPI in mice as indicated . Enlarged pH3 positive cardiomyocyte nuclei are shown separately . The arrows indicate pH3 positive cardiomyocyte nuclei . Scale bar : 25µm . The quantification of pH3 positive ( + ) cardiomyocytes per microscopic field is shown as bar graph ( C ´ ) ; * p = 0 . 046 . ( D ) Representative myocardial immunostaining for aurora B , DAPI and troponin T ( TropT ) . The arrow points towards a cardiomyocyte cell division , in which aurora B localizes at the midbody in cytokinesis . Scale bar : 25µm . The quantification of dividing cardiomyocytes per microscopic field is shown as bar graph ( D ´ ) ; * p = 0 . 0104 . ( E ) Determination of cardiac gene - expression by qPCR 7 days after sham or cryoinjury for the indicated genes ; * p = 0 . 039 for Il13 and p = 0 . 049 for Vegfa ; * * p = 0 . 0043 for Ccne1 , p = 0 . 0027 for Cdk4 , p = 0 . 0096 for Cenpa , p = 0 . 0015 for Zfp191 ; * * * p = 0 . 0009 for Ccna2 , p = 0 . 0002 for Cenpa .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative pictures of TUNEL, WGA (wheat germ agglutinin to mark cardiomyocyte membranes) and DAPI immunofluorescence staining of mouse hearts as indicated. The white dashed line encircles the infarcted area (arrows). Scale bar: 200µm. The quantification of the area of TUNEL positive cells 3 hours after cryoinjury is shown on the right (A´). The number within bars indicates the number of mice analyzed in that particular group.(B) Representative merged and single channel pictures of cardiac CD31, DAPI and WGA immunostaining in mouse hearts as indicated. The quantification of the myocardial capillary density as capillaries/cardiomyocyte (CM) ratio, 7 days (d) after cryoinjury or sham surgery is shown as bar graph (B´). Scale bar: 20µm. **p=0.0039.(C) Representative pictures of myocardial immunofluorescence staining for the mitosis marker phospho-histone H3 (pH3), troponin T and DAPI in mice as indicated. Enlarged pH3 positive cardiomyocyte nuclei are shown separately. The arrows indicate pH3 positive cardiomyocyte nuclei. Scale bar: 25µm. The quantification of pH3 positive (+) cardiomyocytes per microscopic field is shown as bar graph (C´); *p=0.046.(D) Representative myocardial immunostaining for aurora B, DAPI and troponin T (TropT). The arrow points towards a cardiomyocyte cell division, in which aurora B localizes at the midbody in cytokinesis. Scale bar: 25µm. The quantification of dividing cardiomyocytes per microscopic field is shown as bar graph (D´); *p=0.0104.(E) Determination of cardiac gene-expression by qPCR 7 days after sham or cryoinjury for the indicated genes; *p=0.039 for Il13 and p=0.049 for Vegfa; **p=0.0043 for Ccne1, p=0.0027 for Cdk4, p=0.0096 for Cenpa, p=0.0015 for Zfp191; ***p=0.0009 for Ccna2, p=0.0002 for Cenpa.


Figure 3 ( A ) Representative immunoblots for GATA4 and GAPDH ( as loading control ) of fetal cardiomyocytes treated with adenoviruses ( Ad ) expressing control shRNA ( shCon ) or shRNA against Gata4 ( shGATA4 ) for 48 hours . ( B ) Number of cardiomyocytes ( CM ) in a defined low magnification ( 50x ) microscopic field before the adenoviral infection and 48h later . The number in the bars indicates the number of cell culture plates analyzed . * * * * p < 0 . 0001 . ( C ) Representative pictures and quantification of BrdU incorporation in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for BrdU , troponin I ( Trop I ) and DAPI . Scale bar : 50µm ; p = 0 . 0169 . ( D ) Representative pictures and quantification of cardiomyocytes positive for phospho - histone H3 ( pH3 ) in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for pH3 , troponin I and DAPI . Scale bar : 50µm ; * p = 0 . 0145 . ( E ) Representative picture and quantification of cardiomyocyte cell division in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for aurora B ( Auro B ) , troponin I and DAPI . Scale bar : 50µm . ( F ) Representative pictures , single channel enlargements and quantification of cardiomyocyte apoptosis in fetal cardiomyocytes 48 hours after application of the adenoviruses and after immunostaining for cleaved caspase 3 , troponin I and DAPI . Scale bar : 100µm . ( G ) Representative pictures of cardiac explants of the indicated mice embedded in Matrigel shortly after cryoinjury and immunostained for troponin I 7 days later . Quantification of outgrowing cardiomyocytes is shown on the right . The number within bars indicates the number of mice analyzed in that particular group . Scale bar : 50µm . * * p = 0 . 0037 . ( H ) Representative pictures of cardiac explants like in ( G ) , stained for pH3 in addition . The quantification is shown on the right . Scale bar : 50µm . * p = 0 . 0215 .

O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative immunoblots for GATA4 and GAPDH (as loading control) of fetal cardiomyocytes treated with adenoviruses (Ad) expressing control shRNA (shCon) or shRNA against Gata4 (shGATA4) for 48 hours.(B) Number of cardiomyocytes (CM) in a defined low magnification (50x) microscopic field before the adenoviral infection and 48h later. The number in the bars indicates the number of cell culture plates analyzed. ****p<0.0001.(C) Representative pictures and quantification of BrdU incorporation in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for BrdU, troponin I (Trop I) and DAPI. Scale bar: 50µm; p=0.0169.(D) Representative pictures and quantification of cardiomyocytes positive for phospho-histone H3 (pH3) in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for pH3, troponin I and DAPI. Scale bar: 50µm; *p=0.0145.(E) Representative picture and quantification of cardiomyocyte cell division in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for aurora B (Auro B), troponin I and DAPI. Scale bar: 50µm.(F) Representative pictures, single channel enlargements and quantification of cardiomyocyte apoptosis in fetal cardiomyocytes 48 hours after application of the adenoviruses and after immunostaining for cleaved caspase 3, troponin I and DAPI. Scale bar: 100µm.(G) Representative pictures of cardiac explants of the indicated mice embedded in Matrigel shortly after cryoinjury and immunostained for troponin I 7 days later. Quantification of outgrowing cardiomyocytes is shown on the right. The number within bars indicates the number of mice analyzed in that particular group. Scale bar: 50µm. **p=0.0037.(H) Representative pictures of cardiac explants like in (G), stained for pH3 in addition. The quantification is shown on the right. Scale bar: 50µm. *p=0.0215.


Figure 4 ( A ) Representative immunoblot for GATA4 and actin from neonatal cardiomyocytes treated with control ( Ad . Con ) or GATA4 expressing adenovirus ( Ad . GATA4 ) . ( B ) Quantification of BrdU incorporation , pH3 labelling and cytokinesis in neonatal cardiomyocytes treated as shown . * p = 0 . 0272 for BrdU incorporation , p = 0 . 0329 for pH3 labelling , p = 0 . 0217 for cytokinesis . ( C ) Immunoblot for GATA4 and GAPDH of mouse hearts treated as shown in the experimental time scale above . ( D ) A quantification of the immunoblot from ( C ) is shown ; * * p = 0 . 0062 . ( E ) Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad . Con or Ad . GATA4 . The quantification of cardiomyocytes ( CM ) staining positive for GATA4 vs . total cardiomyocytes is shown as bar graph ( E ´ ) . The number within bars indicates the number of mice analyzed in that particular group . Scale bar : 20µm . * * * * p < 0 . 0001 . ( E ) Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad . Con or Ad . GATA4 . The quantification of cardiomyocytes ( CM ) staining positive for GATA4 vs . total cardiomyocytes is shown as bar graph ( E ´ ) . The number within bars indicates the number of mice analyzed in that particular group . Scale bar : 20µm . * * * * p < 0 . 0001 . ( F ) Representative Sirius - Red stained heart sections of mice 7 days after cryoinjury ( performed at P7 ) and treated with adenoviruses as indicated . The scar is displayed in red ( arrows ) . Scale bar : 500 µm . The quantification of left ventricular ( LV ) scar area of mice 7 days after cryoinjury is shown as bar graph ( F ´ ) . * p = 0 . 0135 . ( G ) Representative picture of a myocardial section immunostained for the indicated proteins 7 days after cryoinjury . Scale bar : 10 µm . Quantification of Ki67 positive cardiomyocytes in these mice ( G ´ ) . * * p = 0 . 0023 . ( G ) Representative picture of a myocardial section immunostained for the indicated proteins 7 days after cryoinjury . Scale bar : 10 µm . Quantification of Ki67 positive cardiomyocytes in these mice ( G ´ ) . * * p = 0 . 0023 . ( H ) Quantification of pH3 positive cardiomyocytes and cardiomyocyte ( CM ) cytokinesis in mice 7 days after cryoinjury ; * p = 0 . 0264 , * * * p = 0 . 0006 . ( I ) Quantification of the capillary / cardiomyocyte ratio in mice 7 days after cryoinjury . * * * p = 0 .

O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Representative immunoblot for GATA4 and actin from neonatal cardiomyocytes treated with control (Ad.Con) or GATA4 expressing adenovirus (Ad.GATA4).(B) Quantification of BrdU incorporation, pH3 labelling and cytokinesis in neonatal cardiomyocytes treated as shown. *p=0.0272 for BrdU incorporation, p=0.0329 for pH3 labelling, p=0.0217 for cytokinesis.(C) Immunoblot for GATA4 and GAPDH of mouse hearts treated as shown in the experimental time scale above.(D) A quantification of the immunoblot from (C) is shown; **p=0.0062.(E) Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad.Con or Ad.GATA4. The quantification of cardiomyocytes (CM) staining positive for GATA4 vs. total cardiomyocytes is shown as bar graph (E´). The number within bars indicates the number of mice analyzed in that particular group. Scale bar: 20µm. ****p<0.0001.(E) Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad.Con or Ad.GATA4. The quantification of cardiomyocytes (CM) staining positive for GATA4 vs. total cardiomyocytes is shown as bar graph (E´). The number within bars indicates the number of mice analyzed in that particular group. Scale bar: 20µm. ****p<0.0001.(F) Representative Sirius-Red stained heart sections of mice 7 days after cryoinjury (performed at P7) and treated with adenoviruses as indicated. The scar is displayed in red (arrows). Scale bar: 500 µm. The quantification of left ventricular (LV) scar area of mice 7 days after cryoinjury is shown as bar graph (F´). *p=0.0135.(G) Representative picture of a myocardial section immunostained for the indicated proteins 7 days after cryoinjury. Scale bar: 10 µm. Quantification of Ki67 positive cardiomyocytes in these mice (G´). **p=0.0023.(G) Representative picture of a myocardial section immunostained for the indicated proteins 7 days after cryoinjury. Scale bar: 10 µm. Quantification of Ki67 positive cardiomyocytes in these mice (G´). **p=0.0023.(H) Quantification of pH3 positive cardiomyocytes and cardiomyocyte (CM) cytokinesis in mice 7 days after cryoinjury; *p=0.0264, ***p=0.0006.(I) Quantification of the capillary/cardiomyocyte ratio in mice 7 days after cryoinjury. ***p=0.0002


Figure 5 ( B ) Quantification of left ventricular ( LV ) scar area of mice 7 days after cryoinjury and treated as indicated . The number within bars indicates the number of mice analyzed in that particular group . * * p = 0 . 0017 between Con and CM - G4 - KO after PBS treatment and p = 0 . 0078 between CM - G4 - KO mice with PBS or IL - 13 treatment . ( C ) Echocardiography analysis of left ventricular systolic function in the indicated mice 7 days after cryoinjury ; * p = 0 . 0218 ( D ) Quantification of Ki67 or pH3 positive cardiomyocytes in mice treated as described in ( A ) ; * p = 0 . 0106 and * * p = 0 . 0052 between Con and CM - G4 - KO after PBS treatment and * p = 0 . 0439 or * * p = 0 . 0023 between CM - G4 - KO mice with PBS or IL - 13 treatment . ( E ) Quantification of capillary density as capillary / cardiomyocyte ( CM ) ratio in mice treated as described in ( A ) . * p = 0 . 0365 , * * p = 0 . 0031 . ( F ) Quantification of α - SMA positive small conductance vessel ( ~ 20 - 50µM in diameter ) in myocardial sections of the mice treated as indicated . ( G ) Representative immunoblots for STAT6 and GAPDH from neonatal mouse hearts ( KO = CM - G4 - KO ) treated as indicated . The quantification of the blots is shown on the right . * p = 0 . 0114 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Quantification of left ventricular (LV) scar area of mice 7 days after cryoinjury and treated as indicated. The number within bars indicates the number of mice analyzed in that particular group. **p=0.0017 between Con and CM-G4-KO after PBS treatment and p=0.0078 between CM-G4-KO mice with PBS or IL-13 treatment.(C) Echocardiography analysis of left ventricular systolic function in the indicated mice 7 days after cryoinjury; *p=0.0218(D) Quantification of Ki67 or pH3 positive cardiomyocytes in mice treated as described in (A); *p=0.0106 and **p=0.0052 between Con and CM-G4-KO after PBS treatment and *p=0.0439 or **p=0.0023 between CM-G4-KO mice with PBS or IL-13 treatment.(E) Quantification of capillary density as capillary/cardiomyocyte (CM) ratio in mice treated as described in (A). *p=0.0365, **p=0.0031.(F) Quantification of α-SMA positive small conductance vessel (~20-50µM in diameter) in myocardial sections of the mice treated as indicated.(G) Representative immunoblots for STAT6 and GAPDH from neonatal mouse hearts (KO = CM-G4-KO) treated as indicated. The quantification of the blots is shown on the right. *p=0.0114.


Figure 1A . The production of Sox1 - GFP + neural - lineage cells during 7 days of neural differentiation , as shown by microscopy . Scale bars , 100 µm . B . qRT - PCR analysis of NDIME expression during 7 days of neural differentiation ( D3 , P = 0 . 068 ; D5 , P = 0 . 0049 ; D7 , P = 0 . 0003 ) . C . Relative expression level of NDIME mRNA in the Sox1 - GFP - population and Sox1 - GFP + population at day 7 of neural differentiation ( P = 0 . 0118 ) . D . mESCs were cultured in differentiation medium alone , or with 10 % FBS , at day 5 of neural differentiation as shown by microscopy . Scale bars , 100 µm . E . Temporal expression profile of NDIME by qRT - PCR . mESCs were cultured in differentiation medium alone or with 10 % FBS ( D3 , P = 0 . 074 ; D5 , P = 0 . 0048 ; D7 , P = 0 . 0034 ) . F . Distribution of NDIME in the cytoplasm and nucleus at day 7 of neural differentiation . The XIST RNA and GAPDH mRNA were considered as the positive controls for the nuclear and cytosolic fractions , respectively . Data are represented as mean , and error bars represent SEM in all panels . n = 3 . G . Verification of the efficiency of knockout of exon 3 of NDIME in mESCs via qRT - PCR . Knockout of NDIME strongly inhibited neural differentiation , as shown by microscopy analyses during 7 days of neural differentiation . Scale bars , 100 µm . I . Knockout of NDIME strongly inhibited neural differentiation , as shown by FACS analyses during 7 days of neural differentiation . Scale bars , 100 µm . J . Knockout of NDIME inhibits the expression of neural - lineage genes at day 5 of neural differentiation . K . NESTIN and GFP immunostaining of the cells in ( H ) at day 5 of neural differentiation . Nuclei were stained with Hoechst 33342 ( blue ) . Scale bars , 100 µm . L . NESTIN and GFP immunostaining of Ctrl , NDIME - / - , and NDIME - / - + Rs26 - NDIME cells at day 5 of neural differentiation . Scale bars , 100 µm . M . Expression of neural - lineage genes of Ctrl , NDIME - / - , and NDIME - / - + Rs26 - NDIME cells at day 5 of neural differentiation . N . Relative expression of Emx1 , Brn2 , Sox5 , and Zeb2 mRNA in different differentiation stages of Ctrl and NDIME - knockout cells ( compared with control cells ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. The production of Sox1-GFP+ neural-lineage cells during 7 days of neural differentiation, as shown by microscopy. Scale bars, 100 µm. B. qRT-PCR analysis of NDIME expression during 7 days of neural differentiation (D3, P = 0.068; D5, P = 0.0049; D7, P = 0.0003).  C. Relative expression level of NDIME mRNA in the Sox1-GFP- population and Sox1-GFP+ population at day 7 of neural differentiation (P = 0.0118).  D. mESCs were cultured in differentiation medium alone, or with 10% FBS, at day 5 of neural differentiation as shown by microscopy. Scale bars, 100 µm. E. Temporal expression profile of NDIME by qRT-PCR. mESCs were cultured in differentiation medium alone or with 10% FBS (D3, P = 0.074; D5, P = 0.0048; D7, P = 0.0034).  F. Distribution of NDIME in the cytoplasm and nucleus at day 7 of neural differentiation. The XIST RNA and GAPDH mRNA were considered as the positive controls for the nuclear and cytosolic fractions, respectively. Data are represented as mean, and error bars represent SEM in all panels. n= 3.  G. Verification of the efficiency of knockout of exon 3 of NDIME in mESCs via qRT-PCR.  Knockout of NDIME strongly inhibited neural differentiation, as shown by microscopy analyses during 7 days of neural differentiation. Scale bars, 100 µm.  I. Knockout of NDIME strongly inhibited neural differentiation, as shown by FACS analyses during 7 days of neural differentiation. Scale bars, 100 µm.  J. Knockout of NDIME inhibits the expression of neural-lineage genes at day 5 of neural differentiation.  K. NESTIN and GFP immunostaining of the cells in (H) at day 5 of neural differentiation. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100 µm.  L. NESTIN and GFP immunostaining of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation. Scale bars, 100 µm. M. Expression of neural-lineage genes of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation. N. Relative expression of Emx1, Brn2, Sox5, and Zeb2 mRNA in different differentiation stages of Ctrl and NDIME-knockout cells (compared with control cells).


Figure 2 A , Expression levels of MEF2C ( D0 , P = 0 . 0020 ; D3 , P = 0 . 0025 ; D5 , P = 0 . 0003 ; D7 , P = 0 . 0142 ) of Ctrl and NDIME - / - cells during 7 days of neural differentiation , following knockout of NDIME at day 5 of neural differentiation . B . ( D0 , P = 0 . 0020 ; D3 , P = 0 . 0025 ; D5 , P = 0 . 0003 ; D7 , P = 0 . 0142 ) of Ctrl and NDIME - / - cells during 7 days of neural differentiation , and protein level of MEF2C following knockout of NDIME at day 5 of neural differentiation . C . Western blot analysis of the effects of MEF2C overexpression in NDIME - knockout cells at day 5 of neural differentiation . Overexpression of MEF2C in the NDIME - knockout cells rescued neural differentiation defects at day 5 of neural differentiation , as shown by microscopy E . Overexpression of MEF2C in the NDIME - knockout cells rescued neural differentiation defects at day 5 of neural differentiation , as shown by FACS F . NESTIN and GFP immunostaining of the cells in ( D ) at day 5 of neural differentiation . Scale bars , 100 µm . G . Expression of neural - lineage genes in Ctrl , NDIME - / - , and NDIME - / - + Mef2c cells during 7 days of neural differentiation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 A, Expression levels of MEF2C (D0, P = 0.0020; D3, P = 0.0025; D5, P = 0.0003; D7, P = 0.0142) of Ctrl and NDIME-/- cells during 7 days of neural differentiation, following knockout of NDIME at day 5 of neural differentiation.  B. (D0, P = 0.0020; D3, P = 0.0025; D5, P = 0.0003; D7, P = 0.0142) of Ctrl and NDIME-/- cells during 7 days of neural differentiation, and protein level of MEF2C following knockout of NDIME at day 5 of neural differentiation. C. Western blot analysis of the effects of MEF2C overexpression in NDIME-knockout cells at day 5 of neural differentiation. Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by microscopy  E. Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by FACS  F. NESTIN and GFP immunostaining of the cells in (D) at day 5 of neural differentiation. Scale bars, 100 µm.  G. Expression of neural-lineage genes in Ctrl, NDIME-/-, and NDIME-/-+Mef2c cells during 7 days of neural differentiation.


Figure 3Enrichment of H3K27me3 at the Mef2c promoter in the NDIME - knockout cells at day 5 of neural differentiation compared with control cells . B . Enrichment of RNA polymerase II at the Mef2c promoter in the NDIME - knockout cells at day 5 of neural differentiation compared with control cells . C . NDIME interacts with EZH2 ( P = 0 . 0043 ) and SUZ12 ( P = 0 . 0003 ) , but not EED ( P = 0 . 6720 ) and EZH1 ( P = 0 . 0951 ) compared with IgG , as determined by RIP using EZH2 , SUZ12 , EED , and EZH1 antibodies at day 5 of neural differentiation . Nuclear RNA U1 as a negative control . The presence of NDIME and U1 was measured by qRT - PCR with or without reverse transcriptase ( RT ) . NDIME levels following EZH2 - RIP ( P = 0 . 0028 ) in HEK293T cells compared with pcDNA3 . 1 . Nuclear RNA U1 was used as a negative control . The presence of NDIME and U1 was measured by qRT - PCR with or without reverse transcriptase ( RT ) . E . NDIME levels following SUZ12 - RIP ( P = 0 . 0015 ) in HEK293T cells compared with pcDNA3 . 1 . Nuclear RNA U1 was used as a negative control . The presence of NDIME and U1 was measured by qRT - PCR with or without reverse transcriptase ( RT ) . F , Enrichment of EZH2 at the Mef2c promoter in NDIME - knockout cells at day 5 of neural differentiation compared with control cells . G . Enrichment of SUZ12 at the Mef2c promoter in NDIME - knockout cells at day 5 of neural differentiation compared with control cells . H . Expression of neural - lineage genes at day 5 of neural differentiation in the NDIME - knockout cells after adding the EZH2 inhibitor GSK126 ( 7 . 5 µM ) partially rescued neural differentiation defects , as shown by qRT - PCR ; the wild - type and NDIME - knockout cells treated with DMSO .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Enrichment of H3K27me3 at the Mef2c promoter in the NDIME-knockout cells at day 5 of neural differentiation compared with control cells.B. Enrichment of RNA polymerase II at the Mef2c promoter in the NDIME-knockout cells at day 5 of neural differentiation compared with control cells. C. NDIME interacts with EZH2 (P = 0.0043) and SUZ12 (P = 0.0003), but not EED (P = 0.6720) and EZH1 (P = 0.0951) compared with IgG, as determined by RIP using EZH2, SUZ12, EED, and EZH1 antibodies at day 5 of neural differentiation. Nuclear RNA U1 as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).  NDIME levels following EZH2-RIP (P = 0.0028) in HEK293T cells compared with pcDNA3.1. Nuclear RNA U1 was used as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).  E. NDIME levels following SUZ12-RIP (P = 0.0015) in HEK293T cells compared with pcDNA3.1. Nuclear RNA U1 was used as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).  F, Enrichment of EZH2 at the Mef2c promoter in NDIME-knockout cells at day 5 of neural differentiation compared with control cells.  G. Enrichment of SUZ12 at the Mef2c promoter in NDIME-knockout cells at day 5 of neural differentiation compared with control cells.  H. Expression of neural-lineage genes at day 5 of neural differentiation in the NDIME-knockout cells after adding the EZH2 inhibitor GSK126 (7.5 µM) partially rescued neural differentiation defects, as shown by qRT-PCR; the wild-type and NDIME-knockout cells treated with DMSO.


Figure 4 B . RIP analysis using EZH2 antibody for the co - transfection of Flag - EZH2 and NDIME exons 1 - 3 ( P = 0 . 0177 ) or exon 4 ( P = 0 . 4335 ) in HEK293T extracts compared with IgG . C . Verification of the efficiency of overexpression of exons 1 - 3 or exon 4 of NDIME in NDIME - knockout cells at day 5 of neural differentiation via qRT - PCR . Three different primers P1 , P2 , and P3 were used to analyze NDIME or exons of NDIME expression in Ctrl , NDIME - / - , NDIME - / - + Rs26 - Exon123 , and NDIME - / - + Rs26 - Exon4 cells ( compared with control cells ) . The position of primers P1 , P2 , and P3 are shown in the schematic diagram . FACS , in Ctrl , NDIME - / - , NDIME - / - + Rs26 - Exon123 , NDIME - / - + Rs26 - Exon4 cells at day 5 of neural differentiation . E . , expression of neural - lineage genes , in Ctrl , NDIME - / - , NDIME - / - + Rs26 - Exon123 , NDIME - / - + Rs26 - Exon4 cells at day 5 of neural differentiation . F . Microscopy of the control , NDIME - / - , NDIME - / - + Rs26 - Exon123 , and NDIME - / - + Rs26 - Exon4 cells at day 5 of neural differentiation . Scale bars , 100 µm . G . NESTIN and GFP immunostaining of the cells in ( F ) at day 5 of neural differentiation . Scale bars , 100 µm . H . Enrichment of H3K27me3 at the Mef2c promoter of the cells in ( F ) at day 5 of neural differentiation . I . Western blot analysis of expression of MEF2C in the cells in ( F ) at day 5 of neural differentiation .

O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 4 B. RIP analysis using EZH2 antibody for the co-transfection of Flag-EZH2 and NDIME exons 1-3 (P = 0.0177) or exon 4 (P = 0.4335) in HEK293T extracts compared with IgG. C. Verification of the efficiency of overexpression of exons 1-3 or exon 4 of NDIME in NDIME-knockout cells at day 5 of neural differentiation via qRT-PCR. Three different primers P1, P2, and P3 were used to analyze NDIME or exons of NDIME expression in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells (compared with control cells). The position of primers P1, P2, and P3 are shown in the schematic diagram. FACS , in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation.  E. , expression of neural-lineage genes , in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation.  F. Microscopy of the control, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation. Scale bars, 100 µm.  G. NESTIN and GFP immunostaining of the cells in (F) at day 5 of neural differentiation. Scale bars, 100 µm. H. Enrichment of H3K27me3 at the Mef2c promoter of the cells in (F) at day 5 of neural differentiation.I. Western blot analysis of expression of MEF2C in the cells in (F) at day 5 of neural differentiation.


Figure 5A . Summary of the DEGs in the NDIME - knockout cells compared with control cells at day 5 of neural differentiation . C . Summary of the DEGs in the MEF2C - knockdown cells compared with control cells at day 5 of neural differentiation . I . Heatmap illustrating the expression of selected neurectoderm genes and mESCs - specific genes that were shown as log2 FPKM in Ctrl , NDIME - / - , shCtrl and shMef2c mESCs at day 5 of neural differentiation . Each lane corresponds to an independent biological sample . J . qRT - PCR analysis of representative co - upregulated and co - downregulated genes in ( G ) at day 5 of neural differentiation . L . Relative expression of selectively down - regulated ASD risk genes from the DEGs of the NDIME - knockout cells compared with control cells at day 5 and from the DEGs of the MEF2C - knockdown cells compared with control cells at day 5 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Summary of the DEGs in the NDIME-knockout cells compared with control cells at day 5 of neural differentiation.C. Summary of the DEGs in the MEF2C-knockdown cells compared with control cells at day 5 of neural differentiation.I. Heatmap illustrating the expression of selected neurectoderm genes and mESCs-specific genes that were shown as log2 FPKM in Ctrl, NDIME-/- , shCtrl and shMef2c mESCs at day 5 of neural differentiation. Each lane corresponds to an independent biological sample. J. qRT-PCR analysis of representative co-upregulated and co-downregulated genes in (G) at day 5 of neural differentiation.  L. Relative expression of selectively down-regulated ASD risk genes from the DEGs of the NDIME-knockout cells compared with control cells at day 5 and from the DEGs of the MEF2C-knockdown cells compared with control cells at day 5.


Figure 6 A . qRT - PCR analysis of NDIME ( P = 0 . 0051 ) and MEF2C ( P = 0 . 0062 ) in VPA - treated mice and saline - treated mice ; n = 9 per group . B . Enrichment of EZH2 at the Mef2c promoter in VPA - treated mice and saline - treated mice ( compared with saline - treated mice ) ; n = 9 per group . C . Western blot analysis of the protein levels of MEF2C and GAPDH in the hippocampus of VPA - treated mice ( n = 9 ) and saline - treated mice ( n = 10 ) . D . Immunofluorescence images for MEF2C in the hippocampus of VPA - treated mice and saline - treated mice . Nuclei were stained with Hoechst 33342 ( blue ) ; n = 9 per group . Scale bars , 50 µm . E . Statistical analysis of the grayscale levels of MEF2C in ( C ) by ImageJ ( P = 0 . 0074 ) ; saline - treated mice ( n = 10 ) , VPA - treated mice ( n = 9 ) . F . Statistical analysis of the Mean MEF2C staining intensity / unit area in ( D ) by ImageJ ( P = 0 . 0096 ) ; n = 9 per group . G . Immunofluorescence images for GFP in the hippocampus of saline - treated mice , VPA - treated mice , and VPA - treated mice with viral - mediated expression of NDIME H . Immunofluorescence images for MEF2C of mice in ( G ) ; n = 9 per group . Scale bars , 50 µm . I - K . Adeno - associated virus overexpression of NDIME in the dentate gyrus ( DG ) of the dorsal hippocampus of VPA - treated mice rescued autism - like social deficits . Open field test ( I ) , three - chamber test ( J ) , and elevated - plus maze ( EPM ) test ( K ) . Data in ( I - K ) are represented as mean ± SEM ; n = 9 per group . Asterisks indicate a difference between VPA + AAV - GFP and Saline + AAV - GFP , whereas hash tags indicate a difference between VPA + AAV - NDIME - GFP and VPA + AAV - GFP . # P < 0 . 05 , * * / # # P < 0 . 01 , * * * P < 0 . 001 ( one - way ANOVA with Bonferroni post - hoc test ) . NS - non ‐ significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 A. qRT-PCR analysis of NDIME (P = 0.0051) and MEF2C (P = 0.0062) in VPA-treated mice and saline-treated mice; n = 9 per group.  B. Enrichment of EZH2 at the Mef2c promoter in VPA-treated mice and saline-treated mice (compared with saline-treated mice); n = 9 per group. C. Western blot analysis of the protein levels of MEF2C and GAPDH in the hippocampus of VPA-treated mice (n = 9) and saline-treated mice (n = 10). D. Immunofluorescence images for MEF2C in the hippocampus of VPA-treated mice and saline-treated mice. Nuclei were stained with Hoechst 33342 (blue); n = 9 per group. Scale bars, 50 µm. E. Statistical analysis of the grayscale levels of MEF2C in (C) by ImageJ (P = 0.0074); saline-treated mice (n = 10), VPA-treated mice (n = 9). F. Statistical analysis of the Mean MEF2C staining intensity/unit area in (D) by ImageJ (P = 0.0096); n = 9 per group.  G. Immunofluorescence images for GFP in the hippocampus of saline-treated mice, VPA-treated mice, and VPA-treated mice with viral-mediated expression of NDIME H. Immunofluorescence images for MEF2C of mice in (G); n = 9 per group. Scale bars, 50 µm. I-K. Adeno-associated virus overexpression of NDIME in the dentate gyrus (DG) of the dorsal hippocampus of VPA-treated mice rescued autism-like social deficits. Open field test (I), three-chamber test (J), and elevated-plus maze (EPM) test (K). Data in (I-K) are represented as mean ± SEM; n = 9 per group. Asterisks indicate a difference between VPA+AAV-GFP and Saline+AAV-GFP, whereas hash tags indicate a difference between VPA+AAV-NDIME-GFP and VPA+AAV-GFP. #P < 0.05, **/##P < 0.01, ***P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant.


Figure 1E . As in ( D ) , except that cells were treated with Cisplatin for 24 h ( mean ± SEM ; n = 3 independent experiments ) . F . U2OS WT , U2OS / SCAI KO and U2OS / SCAI KO / Strep - HA - SCAI cells were treated or not with MMC ( 9 nM ) for 48 h , fixed and co - stained with PCNA antibody and DAPI . Cell cycle distribution was analyzed by quantitative image - based cytometry ( QIBC ) ( ≥ 2000 cells analyzed per condition ) . Data from a representative experiment are shown . G . U2OS WT , U2OS / SCAI KO and U2OS / SCAI KO / Strep - HA - SCAI cells were treated or not with MMC ( 90 nM ) for 1 h , fixed 24 h later and co - stained with RPA2 antibody and DAPI . RPA2 foci were quantified by QIBC ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * , p < 0 . 05 ; ns , not significant , two - tailed paired t - test ) . H . As in ( G ) , except that cells were co - stained with γH2AX antibody and DAPI ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ; ns , not significant , two - tailed paired t - test ) . I . Experimental workflow for metaphase chromosome morphology analysis ( top ) and representative images of metaphase spreads from indicated cell lines treated or not with MMC ( bottom ) . DNA was stained with DAPI . Scale bars , 10 µm . J . Quantification of radial chromosomes in ( I ) ( mean ± SD ; 180 cells analyzed per condition ; n = 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 , ns , not significant , two - tailed t - test ) . K . Quantification of chromosomal breaks / gaps in ( I ) ( mean ± SEM ; 99 metaphase cells analyzed for each condition pooled from 3 independent experiments ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 , ns , not significant , Mann - Whitney U test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1E. As in (D), except that cells were treated with Cisplatin for 24 h (mean±SEM; n=3 independent experiments). F. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (9 nM) for 48 h, fixed and co-stained with PCNA antibody and DAPI. Cell cycle distribution was analyzed by quantitative image-based cytometry (QIBC) (≥2000 cells analyzed per condition). Data from a representative experiment are shown.G. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test). H. As in (G), except that cells were co-stained with γH2AX antibody and DAPI (≥3000 cells analyzed per condition; mean± SD; n=3 independent experiments; *, p<0.05; **, p<0.01; ns, not significant, two-tailed paired t-test).I. Experimental workflow for metaphase chromosome morphology analysis (top) and representative images of metaphase spreads from indicated cell lines treated or not with MMC (bottom). DNA was stained with DAPI. Scale bars, 10 µm. J. Quantification of radial chromosomes in (I) (mean±SD; 180 cells analyzed per condition; n=3 independent experiments; *, p<0.05; **, p<0.01, ns, not significant, two-tailed t-test). K. Quantification of chromosomal breaks/gaps in (I) (mean±SEM; 99 metaphase cells analyzed for each condition pooled from 3 independent experiments; **, p<0.01; ****, p<0.0001, ns, not significant, Mann-Whitney U test).


Figure 2F . U2OS and U2OS / SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC ( 90 nM ) for 1 h , fixed 24 h later and co - stained with RPA2 antibody and DAPI . RPA2 foci were quantified by QIBC ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * , p < 0 . 05 ; ns , not significant , two - tailed paired t - test ) . G . As in ( F ) , except that cells were stained with γH2AX antibody ( ≥ 3000 cells analyzed per condition ; mean ± SD ; n = 3 independent experiments ; * * , p < 0 . 01 ; ns , not significant , two - tailed paired t - test ) . I . Clonogenic survival of U2OS and U2OS / FANCA KO cells subjected to indicated doses of MMC for 24 h ( mean ± SEM ; n = 3 independent experiments ) . J . Clonogenic survival of U2OS , U2OS / SCAI KO , U2OS / FANCA KO , and U2OS / FANCA + SCAI DKO cells subjected to indicated doses of MMC for 24 h ( mean ± SEM ; n = 3 independent experiments ) . K . Immunoblot analysis of U2OS , U2OS / SCAI KO , U2OS / FANCA KO , and U2OS / FANCA SCAI DKO cell lines exposed or not to MMC as indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2F. U2OS and U2OS/SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test). G. As in (F), except that cells were stained with γH2AX antibody (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; **, p<0.01; ns, not significant, two-tailed paired t-test).I. Clonogenic survival of U2OS and U2OS/FANCA KO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments). J. Clonogenic survival of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA+SCAI DKO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).K. Immunoblot analysis of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA SCAI DKO cell lines exposed or not to MMC as indicated.


Figure 3B . pICLpt was isolated via plasmid pulldown at the indicated times after incubation in Xenopus egg extract in the presence or absence of CDC7i ( 100 µM ) and associated proteins were analyzed by immunoblotting . F . pICLpt was replicated in mock - or SCAI - depleted egg extract in the presence of [ α - 32P ] dATP for the indicated times , and reactions were analyzed by native agarose gel electrophoresis . ∆ SCAI - C and ∆ SCAI - N denote SCAI immunodepletion with an antibody raised against the C - or N - terminus of SCAI , respectively ; RI , replication intermediates ; OC , open circular ; SC , supercoiled .

O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. pICLpt was isolated via plasmid pulldown at the indicated times after incubation in Xenopus egg extract in the presence or absence of CDC7i (100 µM) and associated proteins were analyzed by immunoblotting.F. pICLpt was replicated in mock- or SCAI-depleted egg extract in the presence of [α-32P]dATP for the indicated times, and reactions were analyzed by native agarose gel electrophoresis. ∆SCAI-C and ∆SCAI-N denote SCAI immunodepletion with an antibody raised against the C- or N-terminus of SCAI, respectively; RI, replication intermediates; OC, open circular; SC, supercoiled.


Figure 4A . Analysis of SCAI - interacting proteins in Xenopus egg extracts . Volcano plot shows enrichment of proteins in both SCAI - C and SCAI - N immunoprecipitates ( IPs ) compared to an IgG control , plotted against the P - value ( n = 4 independent experiments ; permutation - based FDR < 0 . 01 , s0 = 2 ) . D . GFP IPs from U2OS or U2OS / GFP - SCAI cells transfected with indicated siRNAs were immunoblotted with indicated antibodies . Note that a small fraction of REV7 is still immunoprecipitated with GFP - SCAI in REV3 knockdown cells . This could be due to incomplete knockdown , and / or additional interactions of SCAI with the REV1 - Polζ complex that are independent of REV3 . J . pICLpt was replicated in egg extracts immunodepleted with indicated antibodies , isolated at various times via plasmid pulldown and associated proteins were analyzed by immunoblotting .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Analysis of SCAI-interacting proteins in Xenopus egg extracts. Volcano plot shows enrichment of proteins in both SCAI-C and SCAI-N immunoprecipitates (IPs) compared to an IgG control, plotted against the P-value (n=4 independent experiments; permutation-based FDR<0.01, s0=2).D. GFP IPs from U2OS or U2OS/GFP-SCAI cells transfected with indicated siRNAs were immunoblotted with indicated antibodies. Note that a small fraction of REV7 is still immunoprecipitated with GFP-SCAI in REV3 knockdown cells. This could be due to incomplete knockdown, and/or additional interactions of SCAI with the REV1-Polζ complex that are independent of REV3.J. pICLpt was replicated in egg extracts immunodepleted with indicated antibodies, isolated at various times via plasmid pulldown and associated proteins were analyzed by immunoblotting.


Figure 5B . Immunoblot analysis of extracts immunodepleted with indicated antibodies . Asterisks denote antibody cross - reactivity . E . Representative images of metaphase spreads from U2OS / SCAI KO cells transfected with indicated siRNAs and processed as in Figure 1I . Scale bars , 10 µm . H . Clonogenic survival of U2OS and U2OS / SCAI KO cells transfected with non - targeting control ( CTRL ) or Polθ siRNAs and subjected to indicated doses of MMC for 24 h ( mean ± SEM ; n = 3 independent experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B. Immunoblot analysis of extracts immunodepleted with indicated antibodies. Asterisks denote antibody cross-reactivity.E. Representative images of metaphase spreads from U2OS/SCAI KO cells transfected with indicated siRNAs and processed as in Figure 1I. Scale bars, 10 µm.H. Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non-targeting control (CTRL) or Polθ siRNAs and subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).


Figure 1A - D The morphology of uninjected , FLT3 / WT mRNA and FLT3 / ITD mRNA injected ( 150 ng per embryo ) embryos on day 2 post fertilization ( dpf ) . Data information : ov : otic vesicles Scale bar = 500 μm . E - H Whole mount in situ hybridization ( WISH ) of notochord - specific marker col9a2 in uninjected , FLT3 / WT mRNA and FLT3 / ITD mRNA injected embryos on 2 dpf . Data information : ov : otic vesicles ; cm : cephalic mesoderm ; nc : notochord . Scale bar = 500 μm . FLT3 signaling was detected by Western blotting in 293FT cells - transfected with FLT3 / ITD mRNA ( I ) FLT3 signaling was detected by Western blotting in zebrafish embryos - injected with FLT3 / ITD mRNA ( J ) . K The effect of FLT3 inhibitor quizartinib ( Qui ) on the dorsalization and axis - duplication phenotype - induced by FLT3 / ITD mRNA injection in zebrafish . Data information : the experiments were performed in triplicates and the data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . NS , not significant . Quantification of fst expression by RT - qPCR ( L ) after FLT3 / ITD overexpression in zebrafish embryos at 6 hpf . Data information : the experiments were performed in triplicates and the data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . NS , not significant . Quantification of fst expression by Western Blotting ( M ) , after FLT3 / ITD overexpression in zebrafish embryos at 6 hpf . Quantification of fst expression by WISH ( N ) after FLT3 / ITD overexpression in zebrafish embryos at 6 hpf .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 1A-D The morphology of uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected (150 ng per embryo) embryos on day 2 post fertilization (dpf). Data information: ov: otic vesicles Scale bar = 500 μm.E-H Whole mount in situ hybridization (WISH) of notochord-specific marker col9a2 in uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected embryos on 2 dpf. Data information: ov: otic vesicles; cm: cephalic mesoderm; nc: notochord. Scale bar = 500 μm.FLT3 signaling was detected by Western blotting in 293FT cells-transfected with FLT3/ITD mRNA (I)FLT3 signaling was detected by Western blotting in zebrafish embryos-injected with FLT3/ITD mRNA (J).K The effect of FLT3 inhibitor quizartinib (Qui) on the dorsalization and axis-duplication phenotype-induced by FLT3/ITD mRNA injection in zebrafish. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant.Quantification of fst expression by RT-qPCR (L) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant.Quantification of fst expression by Western Blotting (M), after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.Quantification of fst expression by WISH (N) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.


Figure 2A - C WISH of fst in FLT3 / WT - ( A ) , FLT3 / ITD - plasmid DNA injected zebrafish embryos without ( B ) or with ( C ) quizartinib treatment ( 2 . 5 μM ) from 6 to 36 hpf . fst expression was expanded by FLT3 / ITD DNA in 86 % of embryos ( B , arrow , 32 / 37 ) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83 % of embryos ( C , 29 / 35 ) . Data information : Scale bar = 500 μm . Generation and characterization of FLT3 / ITD - transgenic zebrafish . GFP expression was detected by florescent microscopy ( F - H ) and in blood circulation and thymus by WISH ( I and J , blue arrow ) in WT sibling and Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) embryos at 4 dpf . FLT3 / ITD - positive zebrafish ( F1 ) were confirmed by PCR genotyping of GFP and FLT3 / ITD using genomic DNA from fin clip of WT siblings and Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at two months old . Fish 4 , 5 and 6 showed germline transmission of FLT3 / ITD transgene ( K ) . Data information : Scale bar = 500 μm . Kidney marrow ( KM ) was collected from Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at 18 months old . The morphology and hematopoietic composition of KM from WT siblings ( n = 6 ) and Runx1 - FLT3 / ITD transgenic ( n = 6 ) zebrafish were examined by Giemsa staining ( L ) and flow cytometry ( abbreviation for panel M : M , Myeloid cells ; P , Progenitor cells ; L , Lymphoid cells ; E , Erythroid cells ) . Data are presented in box plot . * * P < 0 . 01 ( Student ' s t - test ) . Kidney marrow ( KM ) was collected from Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at 18 months old . The morphology and hematopoietic composition of KM from WT siblings ( n = 6 ) and Runx1 - FLT3 / ITD transgenic ( n = 6 ) zebrafish were examined by flow cytometry Data are presented in box plot . The whiskers , boxes , and central lines in panel N represented the minimum - to - maximum values , 25th - to - 75th percentile , and the 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - test ) . O Expression of fst was detected by RT - qPCR in KM from WT sibling and Runx1 - FLT3 / ITD transgenic zebrafish at 18 months old . The RT - qPCR experiments were performed in triplicates and data were presented as mean ± SEM . * * P < 0 . 01 . P Detection of FST expression , p - ERK1 / 2 , and p - CREB in mononuclear cells from normal peripheral blood stem cell ( PBSC ) and FLT3 / ITD AML patients ( diagnostic samples with leukemia blasts > 80 % ) by Western blotting . ^ : non - specific staining of p - ATF1 protein due to the conserved motif .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-C WISH of fst in FLT3/WT- (A), FLT3/ITD- plasmid DNA injected zebrafish embryos without (B) or with (C) quizartinib treatment (2.5 μM) from 6 to 36 hpf. fst expression was expanded by FLT3/ITD DNA in 86% of embryos (B, arrow, 32/37) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83% of embryos (C, 29/35). Data information: Scale bar = 500 μm.Generation and characterization of FLT3/ITD-transgenic zebrafish. GFP expression was detected by florescent microscopy (F-H) and in blood circulation and thymus by WISH (I and J, blue arrow) in WT sibling and Runx1-FLT3/ITD transgenic zebrafish (F1) embryos at 4 dpf. FLT3/ITD-positive zebrafish (F1) were confirmed by PCR genotyping of GFP and FLT3/ITD using genomic DNA from fin clip of WT siblings and Runx1-FLT3/ITD transgenic zebrafish (F1) at two months old. Fish 4, 5 and 6 showed germline transmission of FLT3/ITD transgene (K). Data information: Scale bar = 500 μm.Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by Giemsa staining (L) and flow cytometry (abbreviation for panel M: M, Myeloid cells; P, Progenitor cells; L, Lymphoid cells; E, Erythroid cells). Data are presented in box plot. **P<0.01 (Student's t-test).Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by flow cytometry Data are presented in box plot. The whiskers, boxes, and central lines in panel N represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).O Expression of fst was detected by RT-qPCR in KM from WT sibling and Runx1-FLT3/ITD transgenic zebrafish at 18 months old. The RT-qPCR experiments were performed in triplicates and data were presented as mean ± SEM. **P<0.01.P Detection of FST expression, p-ERK1/2, and p-CREB in mononuclear cells from normal peripheral blood stem cell (PBSC) and FLT3/ITD AML patients (diagnostic samples with leukemia blasts > 80%) by Western blotting. ^: non-specific staining of p-ATF1 protein due to the conserved motif.


Figure 3B - C The direct binding of p - CREB to human FST promoter was detected by ChIP - PCR ( B ) and ChIP - qPCR ( C ) . c - Fos was used as positive control of p - CREB target gene . Normal IgG was used as negative control of ChIP . Data information the experiments were performed in triplicates , and the data were presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . D Dual luciferase assay demonstrating the direct binding of pCREB on human FST promoter . pRL - CMV , Renilla luciferase vector ; pGL - CRE - and pGL - CRE + , firefly luciferase expression driven by human FST promoter with deleted CRE site ( CRE - ) or wildtype ( CRE + ) ; p - GFPSpark , GFP expressing vector ; p - CREBY134F , CREBY134F - GFP expressing vector . Data information : the experiments were performed in triplicates , and the data were presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . E FST expression and FLT3 / ITD signaling were detected by Western blotting in Ba / F3 - parental ( P in short ) and Ba / F3 - FLT3 / ITD ( ITD in short ) cells . F - H Phosphor - flow analysis of p - CREB in Ba / F3 - parental , Ba / F3 - FLT3 / ITD , and Ba / F3 - FLT3 / ITD cells - treated with FLT3 inhibitor Quizartinib ( Qui in short ) . Isotype antibody were used as control to calculate the mean fluorescence intensity ( MFI ) ratio ( F - G ) . The transcription and expression of Fst was detected by RT - qPCR after Quizartinib treatment ( 10 nM ) in Ba / F3 - FLT3 / ITD cells for one day ( H ) . Data information : the experiments were performed in triplicates , and the data were presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . I - K The expression of FST and phosphorylation of CREB were detected by Western Blotting ( I and K ) and phospho - flow analysis ( J ) in MOLM - 13 ( I ) and Ba / F3 - FLT3 / ITD ( K ) cells treated with Quizartinib and BRD7389 for one day , respectively . Data information : the experiments were performed in triplicates , and the data were presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . L RSK and FST expression were detected by Western Blotting after p90RSK knockout by CRISPR / Cas9 in MOLM - 13 cells . M The phosphorylation of CREB and FST expression were detected by Western Blotting in Ba / F3 - FLT3 / ITD cells treated with CREB inhibitor 666 - 15 for one day . ^ : non - specific staining of p - ATF1 protein due to the conserved motif . N CREB and FST expression were detected by Western Blotting after CREB knockout by CRISPR / Cas9 in MOLM - 13 cells . O The growth of Ba / F3 - parental ( with IL - 3 ) , Ba / F3 - FLT3 / ITD ( without IL - 3 ) and Ba / F3 - FLT3 / ITD ( with IL - 3 ) cells was measured after three days treatment of CREB inhibitor 666 - 15 in vitro . Data information : the experiments were performed in triplicates , and the data were presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . P The rescue effect of CREB inhibitor 666 - 15 on FLT3 / ITD - induced dorsalization and axis duplication in zebrafish embryos at 1 dpf .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B-C The direct binding of p-CREB to human FST promoter was detected by ChIP-PCR (B) and ChIP-qPCR (C). c-Fos was used as positive control of p-CREB target gene. Normal IgG was used as negative control of ChIP. Data information the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).D Dual luciferase assay demonstrating the direct binding of pCREB on human FST promoter. pRL-CMV, Renilla luciferase vector; pGL-CRE- and pGL-CRE+, firefly luciferase expression driven by human FST promoter with deleted CRE site (CRE-) or wildtype (CRE+); p-GFPSpark, GFP expressing vector; p-CREBY134F, CREBY134F-GFP expressing vector. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).E FST expression and FLT3/ITD signaling were detected by Western blotting in Ba/F3-parental (P in short) and Ba/F3-FLT3/ITD (ITD in short) cells.F-H Phosphor-flow analysis of p-CREB in Ba/F3-parental, Ba/F3-FLT3/ITD, and Ba/F3-FLT3/ITD cells-treated with FLT3 inhibitor Quizartinib (Qui in short). Isotype antibody were used as control to calculate the mean fluorescence intensity (MFI) ratio (F-G). The transcription and expression of Fst was detected by RT-qPCR after Quizartinib treatment (10 nM) in Ba/F3-FLT3/ITD cells for one day (H). Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).I-K The expression of FST and phosphorylation of CREB were detected by Western Blotting (I and K) and phospho-flow analysis (J) in MOLM-13 (I) and Ba/F3-FLT3/ITD (K) cells treated with Quizartinib and BRD7389 for one day, respectively. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).L RSK and FST expression were detected by Western Blotting after p90RSK knockout by CRISPR/Cas9 in MOLM-13 cells.M The phosphorylation of CREB and FST expression were detected by Western Blotting in Ba/F3-FLT3/ITD cells treated with CREB inhibitor 666-15 for one day. ^: non-specific staining of p-ATF1 protein due to the conserved motif.N CREB and FST expression were detected by Western Blotting after CREB knockout by CRISPR/Cas9 in MOLM-13 cells.O The growth of Ba/F3-parental (with IL-3), Ba/F3-FLT3/ITD (without IL-3) and Ba/F3-FLT3/ITD (with IL-3) cells was measured after three days treatment of CREB inhibitor 666-15 in vitro. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).P The rescue effect of CREB inhibitor 666-15 on FLT3/ITD-induced dorsalization and axis duplication in zebrafish embryos at 1 dpf.


Figure 4A FST expression in different AML cell lines was detected by Western Blotting . FST317 and FST344 overexpression resulted in significant increases of FST transcription by RT - qPCR and protein by Western Blot ( B ) Green , ML - 2 - GFP ; Blue , ML - 2 - FST317 ; Red , ML - 2 - FST344 . The RT - qPCR experiments were performed in triplicates ( B ) . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ns : Not significant . FST317 and FST344 overexpression promoted ML - 2 cell growth in vitro ( C ) . Green , ML - 2 - GFP ; Blue , ML - 2 - FST317 ; Red , ML - 2 - FST344 . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ns : Not significant . D - E The clonogenicity of ML - 2 overexpressing GFP , FST317 , and FST344 in vitro for 14 days . The CFU experiments were performed in triplicates ( E ) . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ns : Not significant . The engraftment of ML - 2 ( with luciferase gene ) overexpressing GFP , FST317 , and FST344 was quantified by bioluminescence imaging ( F and G ) Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ns : Not significant . The engraftment of ML - 2 ( with luciferase gene ) overexpressing GFP , FST317 , and FST344 and the survival of ML - 2 - engrafted NSG mice in vivo was recorded ( H ) . Survival curve in panel H was analyzed by Log - rank test . * P < 0 . 05 , * * P < 0 . 01 . RNA - seq of upregulation of RET , IL2RA and CCL5 after FST344 overexpression in ML - 2 cells . RT - qPCR validation of upregulation of RET , IL2RA and CCL5 after FST344 overexpression in ML - 2 cells . RT - qPCR experiments were performed in triplicates ( J ) . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ns : Not significant . Overall survival analysis of patients from TCGA - AML based on the differential expression of IL2RAOverall survival analysis of patients from TCGA - AML based on the differential expression of CCL5 . M Activation of MAPK / ERK pathway in ML - 2 by FST344 overexpression were validated by Western Blotting . Scale bar = 1 mm .

O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A FST expression in different AML cell lines was detected by Western Blotting.FST317 and FST344 overexpression resulted in significant increases of FST transcription by RT-qPCR and protein by Western Blot (B) Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. The RT-qPCR experiments were performed in triplicates (B). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.FST317 and FST344 overexpression promoted ML-2 cell growth in vitro (C). Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.D-E The clonogenicity of ML-2 overexpressing GFP, FST317, and FST344 in vitro for 14 days. The CFU experiments were performed in triplicates (E). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.The engraftment of ML-2 (with luciferase gene) overexpressing GFP, FST317, and FST344 was quantified by bioluminescence imaging (F and G) Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.The engraftment of ML-2 (with luciferase gene) overexpressing GFP, FST317, and FST344 and the survival of ML-2-engrafted NSG mice in vivo was recorded (H). Survival curve in panel H was analyzed by Log-rank test. *P<0.05, **P<0.01.RNA-seq of upregulation of RET, IL2RA and CCL5 after FST344 overexpression in ML-2 cells.RT-qPCR validation of upregulation of RET, IL2RA and CCL5 after FST344 overexpression in ML-2 cells. RT-qPCR experiments were performed in triplicates (J). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.Overall survival analysis of patients from TCGA-AML based on the differential expression of IL2RAOverall survival analysis of patients from TCGA-AML based on the differential expression of CCL5.M Activation of MAPK/ERK pathway in ML-2 by FST344 overexpression were validated by Western Blotting. Scale bar = 1 mm.


Figure 5A FST knockdown in MOLM - 13 by shRNA effectively reduced FST expression . The morphology were measured after FST knockdown in vitro . Scale bar = 10 μm . The apoptosis ( C ) measured after FST knockdown in vitro . Scale bar = 10 μm . The apoptosis assays ( C ) were performed in triplicates . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . The clonogenicity of MOLM - 13 ( D and E ) were measured after FST knockdown in vitro . Scale bar = 10 μm . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . The engraftment of MOLM - 13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45 . 1 positive cells in recipient mouse BM aspiration at week 2 post transplantation . The engraftment of MOLM - 13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45 . 1 positive cells in recipient mouse BM aspiration at week 2 post transplantation . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . The effect of FST knockdown on the survival of NSG mice - engrafted with MOLM - 13 . scr : scrambled sequence control ( 7 mice ) ; sh : short hairpin RNA ( 8 mice for sh1 and sh2 respectively ) . The survival curve was analyzed by Log - rank test . * * P < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A FST knockdown in MOLM-13 by shRNA effectively reduced FST expression.The morphology were measured after FST knockdown in vitro. Scale bar = 10 μm.The apoptosis (C) measured after FST knockdown in vitro. Scale bar = 10 μm. The apoptosis assays (C) were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).The clonogenicity of MOLM-13 (D and E) were measured after FST knockdown in vitro. Scale bar = 10 μm. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).The engraftment of MOLM-13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation.The engraftment of MOLM-13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).The effect of FST knockdown on the survival of NSG mice-engrafted with MOLM-13. scr: scrambled sequence control (7 mice); sh: short hairpin RNA (8 mice for sh1 and sh2 respectively). The survival curve was analyzed by Log-rank test. **P<0.01.


Figure 6The morphology of MOLM - 13 after FST knockout by CRISPR / Cas9 in vitro . Scale bar = 10 μm . The clonogenicity of MOLM - 13 after FST knockout by CRISPR / Cas9 in vitro . Scale bar = 10 μm . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . The engraftment of MOLM - 13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45 . 1 positive cells in recipient mouse BM aspiration at week 2 post transplantation . The engraftment of MOLM - 13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45 . 1 positive cells in recipient mouse BM aspiration at week 2 post transplantation . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . E The effect of FST knockout on the survival of NSG mice - engrafted with MOLM - 13 cells . Cas 9 : Cas 9 only ( 10 mice ) ; sgRNA # 3 : Cas 9 + sgRNA # 3 ( 10 mice ) ; sgRNA # 4 : Cas 9 + sgRNA # 4 ( 8 mice ) . Data information : survival curves were analyzed by Log - rank test . * P < 0 . 05 . F The knockdown efficiency of different FST - specific antisense oligoes ( ASOs ) in MOLM - 13 cells was detected by RT - qPCR after three days treatment in vitro . The knockdown and RT - qPCR experiments were performed in triplicates . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . G MOLM - 13 cell growth was measured after 3 days treatment of FST - ASO in vitro . The ASO treatment experiments were performed in triplicates . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . H Intraperitoneal injection of FST - ASO ( 10mg / kg weekly , 6 mice ) significantly prolonged the survival of MOLM - 13 - engrafted NSG mice . The random sequence was used for negative control ( Neg - ASO , 6 mice ) . Data information : survival curves were analyzed by Log - rank test . * P < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6The morphology of MOLM-13 after FST knockout by CRISPR/Cas9 in vitro. Scale bar = 10 μm.The clonogenicity of MOLM-13 after FST knockout by CRISPR/Cas9 in vitro. Scale bar = 10 μm. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).The engraftment of MOLM-13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation.The engraftment of MOLM-13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).E The effect of FST knockout on the survival of NSG mice-engrafted with MOLM-13 cells. Cas 9: Cas 9 only (10 mice); sgRNA#3: Cas 9 + sgRNA#3 (10 mice); sgRNA#4: Cas 9 + sgRNA#4 (8 mice). Data information: survival curves were analyzed by Log-rank test. *P<0.05.F The knockdown efficiency of different FST-specific antisense oligoes (ASOs) in MOLM-13 cells was detected by RT-qPCR after three days treatment in vitro. The knockdown and RT-qPCR experiments were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).G MOLM-13 cell growth was measured after 3 days treatment of FST-ASO in vitro. The ASO treatment experiments were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).H Intraperitoneal injection of FST-ASO (10mg/kg weekly, 6 mice) significantly prolonged the survival of MOLM-13-engrafted NSG mice. The random sequence was used for negative control (Neg-ASO, 6 mice). Data information: survival curves were analyzed by Log-rank test. *P<0.05.