( 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 .

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(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 .

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(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.


( A ) Representative immunoblot for GATA4 and actin from neonatal cardiomyocytes treated with control ( Ad . Con ) or GATA4 expressing adenovirus ( Ad . GATA4 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(I) Quantification of the capillary/cardiomyocyte ratio in mice 7 days after cryoinjury. ***p=0.0002


( 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 .

(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 .

(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 .

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(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 .

(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 .

(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 .

(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.


A . The production of Sox1 - GFP + neural - lineage cells during 7 days of neural differentiation , as shown by microscopy . Scale bars , 100 µm .

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A. 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 ) .

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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 ) .

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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 ) .

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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).


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 .

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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.


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 .

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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 .

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 .

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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 .

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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 .

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 .

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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 .

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 .

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 ) .

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N. Relative expression of Emx1, Brn2, Sox5, and Zeb2 mRNA in different differentiation stages of Ctrl and NDIME-knockout cells (compared with control cells).


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 .

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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 .

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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 .

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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

Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by microscopy


Overexpression of MEF2C in the NDIME - knockout cells rescued neural differentiation defects at day 5 of neural differentiation , as shown by


E . Overexpression of MEF2C in the NDIME - knockout cells rescued neural differentiation defects at day 5 of neural differentiation , as shown by FACS

E. Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by FACS


E . Overexpression of MEF2C in the NDIME - knockout cells rescued neural differentiation defects at day 5 of neural differentiation , as shown by

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F . NESTIN and GFP immunostaining of the cells in ( D ) at day 5 of neural differentiation . Scale bars , 100 µm .

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 .

G. Expression of neural-lineage genes in Ctrl, NDIME-/-, and NDIME-/-+Mef2c cells during 7 days of neural differentiation.


Enrichment of H3K27me3 at the Mef2c promoter in the NDIME - knockout cells at day 5 of neural differentiation compared with control cells .

Enrichment 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 .

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 ) .

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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 ) .

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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 ) .

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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 .

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 .

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 .

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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.


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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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 .

I. Western blot analysis of expression of MEF2C in the cells in (F) at day 5 of neural differentiation.


A . Summary of the DEGs in the NDIME - knockout cells compared with control cells at day 5 of neural differentiation .

A. 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 .

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 .

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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 .

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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 .

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.


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 .

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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 .

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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 ) .

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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 .

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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.


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 .

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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.


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

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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


G . Immunofluorescence images for GFP in the hippocampus of saline - treated mice , VPA - treated mice , and VPA - treated mice with viral - mediated expression of

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H . Immunofluorescence images for MEF2C of mice in ( G ) ; n = 9 per group . Scale bars , 50 µm .

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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 .

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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.


E . 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 .

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E. 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 ) .

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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 ) .

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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).


F . 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 ) .

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F. 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 ) .

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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 .

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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.


B . 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 .

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B. 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 .

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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.


A . 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 ) .

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A. 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 .

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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 .

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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.


B . Immunoblot analysis of extracts immunodepleted with indicated antibodies . Asterisks denote antibody cross - reactivity .

B. 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 .

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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 ) .

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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).


A - 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 .

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A-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 .

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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 293FT cells-transfected with FLT3/ITD mRNA (I)


FLT3 signaling was detected by Western blotting in zebrafish embryos - injected with FLT3 / ITD mRNA ( J ) .

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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 .

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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 .

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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 .

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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 .

Quantification of fst expression by WISH (N) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.


A - 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 .

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A-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 .

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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 ) .

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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 ) .

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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 .

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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 .

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.


B - 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 ) .

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

D 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 .

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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 ) .

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

F-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 ) .

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

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 .

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 .

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

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 .

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 ) .

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

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

P The rescue effect of CREB inhibitor 666-15 on FLT3/ITD-induced dorsalization and axis duplication in zebrafish embryos at 1 dpf.


A FST expression in different AML cell lines was detected by Western Blotting .

A 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 .

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

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 .

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

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 .

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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 .

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

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 .

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

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 .

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 .

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

Overall survival analysis of patients from TCGA-AML based on the differential expression of IL2RA


Overall survival analysis of patients from TCGA - AML based on the differential expression of CCL5 .

Overall 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 O O O O O O O O O O O O O O O O O O O O O O O O O

M Activation of MAPK/ERK pathway in ML-2 by FST344 overexpression were validated by Western Blotting. Scale bar = 1 mm.


A FST knockdown in MOLM - 13 by shRNA effectively reduced FST expression .

A 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 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 ) .

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

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 ) .

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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 .

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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 ) .

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

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

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.


The morphology of MOLM - 13 after FST knockout by CRISPR / Cas9 in vitro . Scale bar = 10 μm .

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

The 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 ) .

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

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.


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 ) .

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 .

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 ) .

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

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 ) .

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

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

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.


Flt3 / ITD knock - in mouse were genotyped ( A ) .

Flt3/ITD knock-in mouse were genotyped (A).


The spleen weight ( B and C , 4 mice each ) were measured in WT siblings and Flt3 / ITD - knock - in mice ( 8 mice each ) . data were presented in box plot . The whiskers , boxes , and central lines represented the minimum - to - maximum values , 25th - to - 75th percentile , and the 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - test ) .

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

The spleen weight (B and C, 4 mice each) were measured in WT siblings and Flt3/ITD-knock-in mice (8 mice each). data were presented in box plot. The whiskers, boxes, and central lines represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).


The serum Fst level ( D ) were measured in WT siblings and Flt3 / ITD - knock - in mice ( 8 mice each ) . data were presented in box plot . The whiskers , boxes , and central lines represented the minimum - to - maximum values , 25th - to - 75th percentile , and the 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - test ) .

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

The serum Fst level (D) were measured in WT siblings and Flt3/ITD-knock-in mice (8 mice each). data were presented in box plot. The whiskers, boxes, and central lines represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).


The engraftment of MOLM - 13 in NSG mice was confirmed at week 1 post injection ( E - F ) .

The engraftment of MOLM-13 in NSG mice was confirmed at week 1 post injection (E-F).


Serum FST level was measured in MOLM - 13 - engrafted NSG mice at pre - injection and week 2 post injection ( G , PBS group , 3 mice ; MOLM - 13 group , 4 mice ) . Data information : data were presented as scatter dot plot . n . s : not significant , * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) .

Serum FST level was measured in MOLM-13-engrafted NSG mice at pre-injection and week 2 post injection (G, PBS group, 3 mice; MOLM-13 group, 4 mice). Data information: data were presented as scatter dot plot. n.s: not significant, *P<0.05, **P<0.01 (Student's t-test). In


After FST knockdown , serum FST level was also measured in MOLM - 13 - engrafted NSG mice at week 2 post injection ( H , 3 mice for each group ) . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) .

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

After FST knockdown, serum FST level was also measured in MOLM-13-engrafted NSG mice at week 2 post injection (H, 3 mice for each group). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


Serum FST level was significantly increased in primary AML - derived xenografted mouse at week 6 post injection . Human leukemic engraftments were confirmed by flow cytometry of human CD45 and mouse CD45 . 1 cells in recipient mouse BM aspiration ( J ) .

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Serum FST level was significantly increased in primary AML-derived xenografted mouse at week 6 post injection. Human leukemic engraftments were confirmed by flow cytometry of human CD45 and mouse CD45.1 cells in recipient mouse BM aspiration (J).


Serum FST level was significantly increased in primary AML - derived xenografted mouse at week 6 post injection . Serum FST from pre - injection and post - engraftment mouse were measured ( K , one mouse for each primary AML sample ) . Data information : data were presented as scatter dot plot . n . s : not significant , * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) .

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Serum FST level was significantly increased in primary AML-derived xenografted mouse at week 6 post injection. Serum FST from pre-injection and post-engraftment mouse were measured (K, one mouse for each primary AML sample). Data information: data were presented as scatter dot plot. n.s: not significant, *P<0.05, **P<0.01 (Student's t-test).


L Correlation between serum FST levels and leukemia blast percentage from FLT3 / ITD - mutated AML at diagnosis . Correlation analysis ( Pearson correlation coefficient ) was performed by Graphpad Prism 6 .

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L Correlation between serum FST levels and leukemia blast percentage from FLT3/ITD-mutated AML at diagnosis. Correlation analysis (Pearson correlation coefficient) was performed by Graphpad Prism 6.


Serum FST decreased in CR and increased after relapse in 4 AML patients receiving Quizartinib monotherapy . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) .

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Serum FST decreased in CR and increased after relapse in 4 AML patients receiving Quizartinib monotherapy. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


N Serum FST continued to rise during disease progression from a patient who did not respond to Quizartinib . Patients in N were recruited in the QUANTUM - R and patient accrual has been completed . data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) .

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N Serum FST continued to rise during disease progression from a patient who did not respond to Quizartinib. Patients in N were recruited in the QUANTUM-R and patient accrual has been completed. data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


( a ) The ternary NDP52 - GAL8 - LC3C complex ( green line ) , binary NDP52 - GAL8 complex ( blue line ) and NDP52 complex alone ( red line ) by SEC - MALS . The dotted line represents measured MW and the vertical bars ( 1 and 2 ) indicate the gap in MW . The SEC - MALS result of NDP52 alone ( red line ) shows that the oligomeric state is ambiguous between dimer and trimer . However , the NDP52 - GAL8 complex shows a MW difference of 70 kDa compared with NDP52 alone , which confirmed that two molecules of monomeric GAL8 ( MW : ~ 33 . 5 kDa ; Supplementary Fig . S1b ) bound to homodimeric NDP52 . Furthermore , the ternary NDP52 - GAL8 - LC3C complex showed a MW difference of ~ 30 kDa compared with the binary NDP52 - GAL8 complex , which also confirmed that two additional molecules of monomeric LC3C bound to the binary complex .

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(a) The ternary NDP52-GAL8-LC3C complex (green line), binary NDP52-GAL8 complex (blue line) and NDP52 complex alone (red line) by SEC-MALS. The dotted line represents measured MW and the vertical bars (1 and 2) indicate the gap in MW. The SEC-MALS result of NDP52 alone (red line) shows that the oligomeric state is ambiguous between dimer and trimer. However, the NDP52-GAL8 complex shows a MW difference of 70 kDa compared with NDP52 alone, which confirmed that two molecules of monomeric GAL8 (MW: ~33.5 kDa; Supplementary Fig. S1b) bound to homodimeric NDP52. Furthermore, the ternary NDP52-GAL8-LC3C complex showed a MW difference of ~30 kDa compared with the binary NDP52-GAL8 complex, which also confirmed that two additional molecules of monomeric LC3C bound to the binary complex.


( b ) Size - exclusion chromatography data of NDP52 - GAL8 - LC3C complex . The first peak ( marked by an orange bar ) represents the ternary complex , as shown by the SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) results marked by an orange bar ( lane 1 , 2 and 3 ) . The second merged peak ( marked by green and yellow bar ) and corresponding SDS - PAGE results show the remaining free GAL8 ( lane 4 , 5 , 6 and 7 ) and LC3C ( lane 5 , 6 , 7 and 8 ) .

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(b) Size-exclusion chromatography data of NDP52-GAL8-LC3C complex. The first peak (marked by an orange bar) represents the ternary complex, as shown by the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results marked by an orange bar (lane 1, 2 and 3). The second merged peak (marked by green and yellow bar) and corresponding SDS-PAGE results show the remaining free GAL8 (lane 4, 5, 6 and 7) and LC3C (lane 5, 6, 7 and 8).


( a ) Overall structure of GAL8 with bound Galbi peptide ( cyan ) and NAD ( purple ) in the asymmetric unit . The other pair of crystallographic dimer is coloured grey . The first β - strand forming unique interaction is labelled as β1 . ( b ) A topology diagram of GAL8 . The β - strands in each N - CRD and C - CRD are numbered sequentially with prime ( ′ ) in the C - CRD . The β - strands with a solid line indicate a carbohydrate - binding side in each CRD and those of with a dashed line indicate an opposite side . The elongated purple and cyan ovals indicate NAD - and NDP52 - binding sites , respectively . ( c ) Overall view of Galbi peptide ( cyan ) bound to the C - CRD of GAL8 ( β - strands in yellow and residues in gray ) .

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(a) Overall structure of GAL8 with bound Galbi peptide (cyan) and NAD (purple) in the asymmetric unit. The other pair of crystallographic dimer is coloured grey. The first β-strand forming unique interaction is labelled as β1. (b) A topology diagram of GAL8. The β-strands in each N-CRD and C-CRD are numbered sequentially with prime (′) in the C-CRD. The β-strands with a solid line indicate a carbohydrate-binding side in each CRD and those of with a dashed line indicate an opposite side. The elongated purple and cyan ovals indicate NAD- and NDP52-binding sites, respectively. (c) Overall view of Galbi peptide (cyan) bound to the C-CRD of GAL8 (β-strands in yellow and residues in gray).


( a ) Magnified view showing the detailed interaction around Leu374 ( p ) . ( b ) Magnified view showing the detailed interaction around Tyr380 ( p ) . ( c ) Magnified view showing the importance of main - chain hydrogen bonds of the Galbi peptide . ( a - c ) Red dashed lines designate hydrogen bonds .

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(a) Magnified view showing the detailed interaction around Leu374(p). (b) Magnified view showing the detailed interaction around Tyr380(p). (c) Magnified view showing the importance of main-chain hydrogen bonds of the Galbi peptide. (a-c) Red dashed lines designate hydrogen bonds.


( d ) An FP assay using FITC - labelled Galbi peptides , including mutants ( L374A , Y380F and Y380A ) against an increasing concentration of GAL8 . ( e ) An FP assay using a FITC - labelled Galbi peptide against an increasing concentration of GAL8 mutants ( E268A , I270A , Y272A and Trp317 ) . ( d , e ) The KD values were calculated from at least three independent experiments . ND , not determined .

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(d) An FP assay using FITC-labelled Galbi peptides, including mutants (L374A, Y380F and Y380A) against an increasing concentration of GAL8. (e) An FP assay using a FITC-labelled Galbi peptide against an increasing concentration of GAL8 mutants (E268A, I270A, Y272A and Trp317). (d,e) The KD values were calculated from at least three independent experiments. ND, not determined.


( a ) Sequence alignment of N - CRD and C - CRD of GAL8 . The residues important for interaction with the Galbi peptide and NAD are marked by yellow and magenta circles , respectively . ( b ) Detailed view of the peptide - binding site of C - CRD superposed with N - CRD . The residues for the Galbi peptide ( cyan ) interaction are labelled and the side chains of the corresponding N - CRD residues ( magenta ) are shown . ( c ) A detailed view of the NAD - binding site of N - CRD superposed with C - CRD . The residues for NAD ( purple ) interaction are labelled and the side chains of corresponding C - CRD residues ( yellow ) are shown .

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(a) Sequence alignment of N-CRD and C-CRD of GAL8. The residues important for interaction with the Galbi peptide and NAD are marked by yellow and magenta circles, respectively. (b) Detailed view of the peptide-binding site of C-CRD superposed with N-CRD. The residues for the Galbi peptide (cyan) interaction are labelled and the side chains of the corresponding N-CRD residues (magenta) are shown. (c) A detailed view of the NAD-binding site of N-CRD superposed with C-CRD. The residues for NAD (purple) interaction are labelled and the side chains of corresponding C-CRD residues (yellow) are shown.


( a ) Three GAL8 structures , including both CRDs , are available from PDB ( 4FQZ - lactose bound in only N - CRD ; 3VKL - lactose bound in both N - CRD and N - C - CRD ; 3VKM - sialylated lactose in N - CRD and N - CRD in C - CRD ) . The C - CRDs of these Gal8 structures were superposed with that of our Gal8 . For clarity , only Cα - traces of each structure ( our GAL8 - yellow ; 4FQZ - red ; 3VKM - blue ; and 3VKL - cyan ) are shown . The large rotation of the N - CRD domain relative to GAL8 compared with the other NDP52 structures is observed in the GAL8 - NDP52 complex . A representative angle between CRD of our Gal8 and that of the 4FQZ structure is indicated in degrees . ( b ) A 90 ° rotation of panel ( a ) along the horizontal axis showing a top view . Arg59 , which is a unique residue of Gal8CRD that provides carbohydrate specificity , is used as a standard for measuring the distance of domain movement .

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

(a) Three GAL8 structures, including both CRDs, are available from PDB (4FQZ-lactose bound in only N-CRD; 3VKL-lactose bound in both N-CRD and N-C-CRD; 3VKM-sialylated lactose in N-CRD and N-CRD in C-CRD). The C-CRDs of these Gal8 structures were superposed with that of our Gal8. For clarity, only Cα-traces of each structure (our GAL8-yellow; 4FQZ-red; 3VKM-blue; and 3VKL-cyan) are shown. The large rotation of the N-CRD domain relative to GAL8 compared with the other NDP52 structures is observed in the GAL8-NDP52 complex. A representative angle between CRD of our Gal8 and that of the 4FQZ structure is indicated in degrees. (b) A 90° rotation of panel (a) along the horizontal axis showing a top view. Arg59, which is a unique residue of Gal8CRD that provides carbohydrate specificity, is used as a standard for measuring the distance of domain movement.


A αSMA mRNA levels in TGFβ - treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C . * P = 0 , 001 .

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A αSMA mRNA levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. *P = 0,001.


B Collagen1α2 mRNA levels in TGFβ - treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C . * P = 0 , 01 .

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B Collagen1α2 mRNA levels in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C. *P = 0,01.


C mRNA levels of ER - stress related genes in TGFβ - treated HFL1 fibroblasts . * P CHOP = 0 , 045 , * P Bip = 0 , 023 , * P spliced XBP - 1 P = 0 , 001 , compared to corresponding control .

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C mRNA levels of ER-stress related genes in TGFβ-treated HFL1 fibroblasts. *P CHOP = 0,045, *P Bip = 0,023, *P spliced XBP-1 P = 0,001, compared to corresponding control.


D Western blot of CHOP protein levels in TGFβ - treated HFL1 cells ± the IRE1α inhibitor 4μ8C .

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D Western blot of CHOP protein levels in TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C.


E Agarose gel showing the presence of spliced and unspliced XBP - 1 mRNA in TGFβ - treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C .

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E Agarose gel showing the presence of spliced and unspliced XBP-1 mRNA in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C.


F Immunohistochemistry staining of αSMA in TGFβ - treated HFL1fibroblasts ± the IRE1α inhibitor 4μ8C . Scale bars = 20μm .

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F Immunohistochemistry staining of αSMA in TGFβ-treated HFL1fibroblasts ± the IRE1α inhibitor 4μ8C. Scale bars = 20μm.


G Quantification of secreted soluble collagen proteins produced by TGFβ - treated HFL1 cells ± the IRE1α inhibitor 4μ8C . * P = 0 , 049 .

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G Quantification of secreted soluble collagen proteins produced by TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C. *P = 0,049.


H αSMA mRNA expression in TGFβ - treated HFL1 cells overexpressing two RNase dead mutants ( K599A and K907A ) of IRE1α . * P K599A = 0 . 013 , * P K907A = 0 . 0008 compared to WT .

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H αSMA mRNA expression in TGFβ-treated HFL1 cells overexpressing two RNase dead mutants (K599A and K907A) of IRE1α. *P K599A = 0.013, *P K907A = 0.0008 compared to WT.


I αSMA mRNA expression in TGFβ - treated IRE1α - / - or WT MEF cells . * P = 0 . 025 .

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I αSMA mRNA expression in TGFβ-treated IRE1α -/- or WT MEF cells. *P = 0.025.


A miR - 150 levels in TGFβ - treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C . * P = 0 . 046 compared to control .

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A miR-150 levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. * P = 0.046 compared to control.


B miR - 150 levels in HFL1 fibroblasts overexpressing two RNase dead mutants ( K599A and K907A ) of IRE1α . * P = 0 . 0043 ( K599A ) , P = 0 . 043 ( K907A ) , compared to control .

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B miR-150 levels in HFL1 fibroblasts overexpressing two RNase dead mutants (K599A and K907A) of IRE1α. * P = 0.0043 (K599A), P = 0.043 (K907A), compared to control.


C miR - 150 levels in TGFβ - treated WT ( IRE1α + / + ) and IRE1α - / - MEF cells . * P = 0 . 018 . Results are expressed as fold change compared to the Ctrl .

C miR-150 levels in TGFβ-treated WT (IRE1α +/+) and IRE1α -/- MEF cells. *P = 0.018. Results are expressed as fold change compared to the Ctrl.


D miR - 150 levels in IRE1α - / - MEF cells that were either untransfected ( UT ) or transfected with miRNA inhibitor control ( siCtrl ) or with a miR - 150 - 5p miRNA inhibitor . * P = 0 . 034 compared to UT .

D miR-150 levels in IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR-150-5p miRNA inhibitor. * P = 0.034 compared to UT.


E αSMA mRNA expression in TGFβ - treated IRE1α - / - MEF cells that were either untransfected ( UT ) or transfected with miRNA inhibitor control ( siCtrl ) or with a miR150 - 5p miRNA inhibitor . * P = 0 . 043 compared to UT .

E αSMA mRNA expression in TGFβ-treated IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR150-5p miRNA inhibitor. *P = 0.043 compared to UT.


F miR - 150 levels in HFL1 fibroblasts transfected with a control plasmid ( Ctrl ) or a miR - 150 expression plasmid ( miR150 ) . * P = 0 . 038 .

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F miR-150 levels in HFL1 fibroblasts transfected with a control plasmid (Ctrl) or a miR-150 expression plasmid (miR150). * P = 0.038.


G Western blot of c - Myb in TGFβ - treated HFL1 fibroblasts that were either untransfected ( UT ) , transfected with a control plasmid ( pCtrl ) or a miR150 expression plasmid ( miR150 ) . * P = 0 . 0206 in UT Ctrl vs . UT TGFβ and P = 0 . 0126 in pCtrl Ctrl vs . pCtrl TGFβ .

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G Western blot of c-Myb in TGFβ-treated HFL1 fibroblasts that were either untransfected (UT), transfected with a control plasmid (pCtrl) or a miR150 expression plasmid (miR150). *P = 0.0206 in UT Ctrl vs. UT TGFβ and P = 0.0126 in pCtrl Ctrl vs. pCtrl TGFβ .


H Western blot of αSMA in the same experimental setup as in G . * P = 0 . 0405 in UT Ctrl vs . UT TGFβ , and P = 0 . 0284 in pCtrl Ctrl vs . pCtrl TGFβ .

H Western blot of αSMA in the same experimental setup as in G. * P = 0.0405 in UT Ctrl vs. UT TGFβ, and P = 0.0284 in pCtrl Ctrl vs. pCtrl TGFβ.


I c - Myb mRNA levels in untransfected HFL1 cells ( UT ) and in cells transfected with a control siRNA ( siCtrl ) or an siRNA targeting c - Myb ( sicMyb ) . * P = 0 . 00001 compared to UT .

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I c-Myb mRNA levels in untransfected HFL1 cells (UT) and in cells transfected with a control siRNA (siCtrl) or an siRNA targeting c-Myb (sicMyb). * P = 0.00001 compared to UT.


J αSMA mRNA expression in untransfected HFL1 cells ( UT ) and in cells transfected with a control siRNA ( siCtrl ) or with a siRNA targeting c - Myb ( sicMyb ) . * P = 0 . 0042 in UT Ctrl vs . UT TGFβ .

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J αSMA mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with a control siRNA (siCtrl) or with a siRNA targeting c-Myb (sicMyb). * P = 0.0042 in UT Ctrl vs. UT TGFβ.


L In vitro cleavage assay with recombinant IRE1α and miR - 150 ( left ) or XBP - 1 ( right ) .

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L In vitro cleavage assay with recombinant IRE1α and miR-150 (left) or XBP-1 (right).


M miR - 150 expression in HFL - 1 cells that were pre - treated with TGFβ in the absence of presence of 4μ8C and then treated with Actinomycin D ( ActD ) for 4 and 24 hours . * P = 0 . 027 compared to TGFβ .

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M miR-150 expression in HFL-1 cells that were pre-treated with TGFβ in the absence of presence of 4μ8C and then treated with Actinomycin D (ActD) for 4 and 24 hours. * P = 0.027 compared to TGFβ.


A Analysis of ER size in HFL1 fibroblasts treated with TGFβ in the absence ( Ctrl ) or presence of the IRE1α inhibitor 4μ8C , or in HFL1 cells transfected with a siRNA control siRNA ( siCtrl ) or with siRNA targeting XBP - 1 ( siXBP - 1 ) . The ER was visualized with ER tracker Red and nuclei stained with Hoechst ( left , scale bars = 20 μm ) or by electron microscopy ( right , scale bars = 200 nm ) where red lines mark the ER . B Quantification of ER size relative to the nucleus from the experimental setup in A . * P = 0 . 0001 in Ctrl , and * P = 0 . 0223 in siCtrl .

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A Analysis of ER size in HFL1 fibroblasts treated with TGFβ in the absence (Ctrl) or presence of the IRE1α inhibitor 4μ8C, or in HFL1 cells transfected with a siRNA control siRNA (siCtrl) or with siRNA targeting XBP-1 (siXBP-1). The ER was visualized with ER tracker Red and nuclei stained with Hoechst (left, scale bars = 20 μm) or by electron microscopy (right, scale bars = 200 nm) where red lines mark the ER.B Quantification of ER size relative to the nucleus from the experimental setup in A. * P = 0.0001 in Ctrl, and * P = 0.0223 in siCtrl.