Figure 1a , Expression levels of ISX , TWIST1 , Snail1 , Fibronectin ( FN1 ) , VEGF , and E - cadherin ( CDH1 ) mRNA were examined in a ISX - GFP inducible Tet - ON transformants at 8 h after addition of DOX . Data are presented as mean ± SD in graph ( p < 0 . 001 compared to time point 0h ; Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . b , Western blotting analysis of the protein levels of ISX , HIF1α , TWIST1 , Snail1 , Slug , ZEB1 , Bmi1 , E - cadherin ( E - Cad . ) , Fibronectin , N - cadherin ( N - Cad . ) , Vimentin , and VEGF in A549 and H1299 cells with DOX - inducible ISX expression system 8hs after DOX induction . ISX transcriptionally activates luciferase activity driven by TWIST1 ( c ) promoter regions in A549 cells . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . d , ISX transcriptionally activates luciferase activity driven by Snail1 ( d ) promoter regions in A549 cells . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . e , ChIP analysis of ISX binding to the TWIST1 promoter region in A549 and H1299 cells ( 10 % input of each groups were pull down and applied to qPCR ) . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . f , ISX transactivation activity analyzed by luciferase activity driven by the TWIST1 promoter . Data are presented as mean ± SD in graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . g , ChIP analysis of ISX binding to the endogenous promoters of TWIST1 ( - 180 / + 35 ) and Snail1 ( - 160 / + 40 ) in A549 and cells ( 10 % input of each groups were pull down and applied to qPCR ) . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . h , Effect of forced expression and knockdown of ISX on cell migration ( wound healing ) measured in A549 cells . ISXi , ISX - specific shRNAi Data are presented as mean ± SD in graph ( * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . i , Effect of forced expression and knockdown of ISX on cell invasion ( transwell ) activity measured in A549 cells . Data are presented as mean ± SD in bar graph ( * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm .

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

Figure 1a, Expression levels of ISX, TWIST1, Snail1, Fibronectin (FN1), VEGF, and E-cadherin (CDH1) mRNA were examined in a ISX-GFP inducible Tet-ON transformants at 8 h after addition of DOX. Data are presented as mean ± SD in graph (p< 0.001 compared to time point 0h; Student's t‐test) of 3 independent experiments, each performed in triplicate.b, Western blotting analysis of the protein levels of ISX, HIF1α, TWIST1, Snail1, Slug, ZEB1, Bmi1, E-cadherin (E-Cad.), Fibronectin, N-cadherin (N-Cad.), Vimentin, and VEGF in A549 and H1299 cells with DOX-inducible ISX expression system 8hs after DOX induction.ISX transcriptionally activates luciferase activity driven by TWIST1 (c) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.d, ISX transcriptionally activates luciferase activity driven by Snail1 (d) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.e, ChIP analysis of ISX binding to the TWIST1 promoter region in A549 and H1299 cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.f, ISX transactivation activity analyzed by luciferase activity driven by the TWIST1 promoter. Data are presented as mean ± SD in graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.g, ChIP analysis of ISX binding to the endogenous promoters of TWIST1 (-180/+35) and Snail1(-160/+40) in A549 and cells (10% input of each groups were pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate.h, Effect of forced expression and knockdown of ISX on cell migration (wound healing) measured in A549 cells. ISXi, ISX-specific shRNAi Data are presented as mean ± SD in graph (**, p < 0.01, ***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.i, Effect of forced expression and knockdown of ISX on cell invasion (transwell) activity measured in A549 cells. Data are presented as mean ± SD in bar graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm.


Figure 2a , BRD4 peptide ( 291 - 314 a . a ) obtained from anti - ISX immunoprecipitates of A549 cell lysates identified through liquid chromatography ‒ tandem mass spectrometry ( LC - tandem - MS ) . Data information : Each experiment was repeated at least three times . b , ISX association proteins ( PCAF , BRD4 , CBP , CREB , RNA Pol II , and Histone H3 ) determined by Western blot in immunoprecipitation of A549 cells . Data information : Each experiment was repeated at least three times . c , ISX association proteins ( PCAF , BRD4 , CBP , CREB , and RNA Pol II ) determined by Western blot in anti - ISX immunoprecipitates of tumor tissues from patients with lung cancer . Data information : Each experiment was repeated at least three times . d , Proximity ligation assay ( PLA ) of ISX and BRD4 ( PCAF ) interaction in A549 cells . Red foci indicate close proximity of the two proteins . NC : negative control . Blank arrows indicate interactions of ISX with BRD4 ( PCAF ) in the cells . Data information : Each experiment was repeated at least three times . e , Western blotting analysis of the protein levels of ISX , PCAF , and EMT markers in DOX - inducible ISX expression system A549 cells treated with TH1834 , Garcinol , C646 , MB - 3 for 8 h after DOX induction . Data information : Each experiment was repeated at least three times . f , Proximity ligation assay of ISX and BRD4 interaction in A549 cells treated with Garcinol . Red foci indicate close proximity of the two proteins . Blank arrows indicate interactions of ISX with BRD4 in the cells . NC : negative control . Data information : Each experiment was repeated at least three times . g , Western blotting analysis of the protein levels of ISX , PCAF , and EMT markers in A549 cells with PCAF knockdown and DOX - inducible ISX expression system at 8 h time point after DOX induction . Data information : Each experiment was repeated at least three times . Cell migration ( wound healing , h ) ssay of A549 lung cancer cells treated with Garcinol . V , vehicle . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times . i , Cell invasion ( transwell , i ) assay of A549 lung cancer cells treated with Garcinol . V , vehicle . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times .

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

Figure 2a, BRD4 peptide (291-314 a.a) obtained from anti-ISX immunoprecipitates of A549 cell lysates identified through liquid chromatography‒tandem mass spectrometry (LC-tandem -MS). Data information: Each experiment was repeated at least three times.b, ISX association proteins (PCAF, BRD4, CBP, CREB, RNA Pol II, and Histone H3) determined by Western blot in immunoprecipitation of A549 cells. Data information: Each experiment was repeated at least three times.c, ISX association proteins (PCAF, BRD4, CBP, CREB, and RNA Pol II) determined by Western blot in anti-ISX immunoprecipitates of tumor tissues from patients with lung cancer. Data information: Each experiment was repeated at least three times.d, Proximity ligation assay (PLA) of ISX and BRD4(PCAF) interaction in A549 cells. Red foci indicate close proximity of the two proteins. NC: negative control. Blank arrows indicate interactions of ISX with BRD4 (PCAF) in the cells. Data information: Each experiment was repeated at least three times.e, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in DOX-inducible ISX expression system A549 cells treated with TH1834, Garcinol, C646, MB-3 for 8 h after DOX induction. Data information: Each experiment was repeated at least three times.f, Proximity ligation assay of ISX and BRD4 interaction in A549 cells treated with Garcinol. Red foci indicate close proximity of the two proteins. Blank arrows indicate interactions of ISX with BRD4 in the cells. NC: negative control. Data information: Each experiment was repeated at least three times.g, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in A549 cells with PCAF knockdown and DOX-inducible ISX expression system at 8 h time point after DOX induction. Data information: Each experiment was repeated at least three times.Cell migration (wound healing, h) ssay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times. i, Cell invasion (transwell, i) assay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.


Figure 3c , Recombinant PCAF acetylates His6 - ISX at lysine residue 69 by in vitro acetylation assay . Acetylated ISX was detected by anti - acetyl Lysine antibody . Data information : Each experiment was repeated at least three times . d and e , The protein levels of GFP - tagged WT or mutant ISX , PCAF and BRD4 were determined in cytosol , nuclei , and anti - GFP immunoprecipites of A549 cells by Western blotting . Acetylated ISX was detected by anti - acetyl Lysine antibody . Data information : Each experiment was repeated at least three times . f , The protein levels of total and acetylated histone H3 were determined in anti - histone H3 immunoprecipites of A549 cells by Western blotting . Data information : Each experiment was repeated at least three times . The cell migration ( wound healing , g ) activity were determined in A549 cells with GFP - tagged wild or ISX mutants . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times . h , The cell invasion ( transwell , h ) activity were determined in A549 cells with GFP - tagged wild or ISX mutants . Data are presented as mean ± SD in graph ( * * * , p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Scale bar , 100μm . Data information : Each experiment was repeated at least three times . j . Tumor xenografts metastasis activity of constitutively expressing RFP A549 cells transfected with wild type or ISX AC3 mutant cDNA were imaged by IVIS imaging system at fifth weeks Data information : Each experiment was repeated at least three times . k , Kaplan - Meier survival curve analysis of nude mice xenograft injected with A549 cells carrying GFP , ISX - GFP and ISX Ac - GFP ( AC3 ) ( n = 10 ) . p = 0 . 0003 . p ‐ values were calculated by log ‐ rank ( Mantel - Cox ) test comparing the two Kaplan - Meier curves . Data information : Each experiment was repeated at least three times .

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

Figure 3c, Recombinant PCAF acetylates His6-ISX at lysine residue 69 by in vitro acetylation assay. Acetylated ISX was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.d and e, The protein levels of GFP-tagged WT or mutant ISX, PCAF and BRD4 were determined in cytosol, nuclei, and anti-GFP immunoprecipites of A549 cells by Western blotting. Acetylated ISX was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.f, The protein levels of total and acetylated histone H3 were determined in anti-histone H3 immunoprecipites of A549 cells by Western blotting. Data information: Each experiment was repeated at least three times.The cell migration (wound healing, g) activity were determined in A549 cells with GFP-tagged wild or ISX mutants. Data are presented as mean ± SD in graph (***, p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.h, The cell invasion (transwell, h) activity were determined in A549 cells with GFP-tagged wild or ISX mutants. Data are presented as mean ± SD in graph (***, p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.j. Tumor xenografts metastasis activity of constitutively expressing RFP A549 cells transfected with wild type or ISX AC3 mutant cDNA were imaged by IVIS imaging system at fifth weeks Data information: Each experiment was repeated at least three times.k, Kaplan-Meier survival curve analysis of nude mice xenograft injected with A549 cells carrying GFP, ISX-GFP and ISX Ac-GFP (AC3) (n = 10). p = 0.0003. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves. Data information: Each experiment was repeated at least three times.


Figure 4c , Recombinant PCAF acetylates His6 - BRD4 at lysine residue 332 . Acetylated BRD4 was detected by anti - acetyl Lysine antibody . Data information : Each experiment was repeated at least three times . d , The mCherry - tagged WT and BRD4 mutants were detected in anti - GFP immunoprecipitates by Western blotting in A549 cells . Data information : Each experiment was repeated at least three times . e and f , The mRNA levels of TWIST1 ( e ) and Snail1 ( f ) were verified in A549 cells co - expressing mCherry - tagged WT or mutant BRD4 and GFP - tagged ISX by RT - PCR . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times . g and h , The DNA binding activity of TWIST1 and Snail1 were evaluated in anti - GFP - mCherry ChIP - ChIP immunoprecipitates by RT - PCR in A549 cells . Red , Hprt1 promoter ( - 190 / + 40 bp , negative control ) ( 10 % input of each groups were pulled down and used for qPCR analysis ) . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times . i , The cell invasion ( transwell ) activity were determined in A549 cells co - transfected with cDNA coding for GFP - tagged ISX and mCherry - tagged BRD4 mutants . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times .

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

Figure 4c, Recombinant PCAF acetylates His6-BRD4 at lysine residue 332. Acetylated BRD4 was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.d, The mCherry-tagged WT and BRD4 mutants were detected in anti-GFP immunoprecipitates by Western blotting in A549 cells. Data information: Each experiment was repeated at least three times.e and f, The mRNA levels of TWIST1(e) and Snail1(f) were verified in A549 cells co-expressing mCherry-tagged WT or mutant BRD4 and GFP-tagged ISX by RT-PCR. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.g and h, The DNA binding activity of TWIST1 and Snail1 were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190/+40 bp, negative control) (10% input of each groups were pulled down and used for qPCR analysis). Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.i, The cell invasion (transwell) activity were determined in A549 cells co-transfected with cDNA coding for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.


Figure 5a , Schematic depiction of ISX and deletion constructs used . b GFP - tagged truncated ISX mutant proteins or HA - tagged BRD4 were detected in anti - HA or GFP immunoprecipitates by Western blotting analysis . Data information : Each experiment was repeated at least three times .

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Figure 5a, Schematic depiction of ISX and deletion constructs used. b GFP-tagged truncated ISX mutant proteins or HA-tagged BRD4 were detected in anti-HA or GFP immunoprecipitates by Western blotting analysis. Data information: Each experiment was repeated at least three times.


Figure 6a mCherry - tagged truncated BRD4 mutant proteins and GFP - tagged ISX were detected in anti - GFP immunoprecipitates by Western blot . Data information : Each experiment was repeated at least three times . The DNA binding activity of Snail ( b ) were evaluated in anti - GFP - mCherry ChIP - ChIP immunoprecipitates by RT - PCR in A549 cells . Red , Hprt1 promoter ( - 190 - + 40 bp , negative control ) . ( 10 % input of each groups were pull down and applied to qPCR ) Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times . c , The DNA binding activity of TWIST1 ( c ) were evaluated in anti - GFP - mCherry ChIP - ChIP immunoprecipitates by RT - PCR in A549 cells . Red , Hprt1 promoter ( - 190 - + 40 bp , negative control ) . ( 10 % input of each groups were pull down and applied to qPCR ) Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times . The cell migration ( wound healing , d ) activity were determined in A549 cells co - transfected with cDNAs for GFP - tagged ISX and mCherry - tagged BRD4 mutants . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times . e , The cell invasion ( transwell , e ) activity were determined in A549 cells co - transfected with cDNAs for GFP - tagged ISX and mCherry - tagged BRD4 mutants . Data are presented as mean ± SD in bar graph ( p < 0 . 001 , Student ' s t ‐ test ) of 3 independent experiments , each performed in triplicate . Data information : Each experiment was repeated at least three times .

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

Figure 6a mCherry-tagged truncated BRD4 mutant proteins and GFP-tagged ISX were detected in anti-GFP immunoprecipitates by Western blot. Data information: Each experiment was repeated at least three times.The DNA binding activity of Snail (b) were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190-+40 bp, negative control). (10% input of each groups were pull down and applied to qPCR) Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.c, The DNA binding activity of TWIST1 (c) were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190-+40 bp, negative control). (10% input of each groups were pull down and applied to qPCR) Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.The cell migration (wound healing, d) activity were determined in A549 cells co-transfected with cDNAs for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.e, The cell invasion (transwell, e) activity were determined in A549 cells co-transfected with cDNAs for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.


Figure 7a , IHC staining with ISX ( brown ) or PCAF ( brown ) antibody in lung tumors from patients with lung cancer . N , normal tissue ; T , tumor mass ; Nuclei , hematoxylin ( blue ) ; Blue and red arrow , cytosol and nuclear ISX ( PCAF ) . Scale bar , 500μm ( 400X ) ; Scale bar , 2mm ( 100X ) . b , Confocal immunofluorescence detection of ISX ( green ) and BRD4 ( red ) in lung tumors from patients with NSCLC . Cell nuclei were visualized by DAPI ( blue ) ; Yellow arrows indicate co - localization ; IgG , negative control . N , normal tissue ; T , tumor mass . c , Confocal immunofluorescence detection of ISX ( green ) and PCAF ( red ) in lung tumors from patients with NSCLC . Cell nuclei were visualized by DAPI ( blue ) ; Yellow arrows indicate co - localization ; IgG , negative control . N , normal tissue ; T , tumor mass . Correlation analysis of mRNA expression for , ( d ) ISX and BRD4 , in 157 lung cancer samples . e , Correlation analysis of mRNA expression for , ( e ) ISX and PCAF in 157 lung cancer samples . f , Protein expression of ISX , ISX ( 69Kac ) and Vimentin in tumor and normal parts from patients with NSCLC by western blotting . N , normal tissue ; T , tumor mass ; Arrow , target protein ; * , unspecific band . The Kaplan - Meier survival curve was used to analyze survival correlation between patients ( n = 157 ) with NSCLC and ISX ( g ) levels . On the basis of the cut - off values of fold differences , the study population was dichotomized into the " high " and " low " expression group . p ‐ values were calculated by log ‐ rank ( Mantel - Cox ) test comparing the two Kaplan - Meier curves . g and h , The Kaplan - Meier survival curve was used to analyze survival correlation between patients ( n = 157 ) with NSCLC and PCAF ( h ) levels . On the basis of the cut - off values of fold differences , the study population was dichotomized into the " high " and " low " expression group . p ‐ values were calculated by log ‐ rank ( Mantel - Cox ) test comparing the two Kaplan - Meier curves .

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

Figure 7a, IHC staining with ISX (brown) or PCAF (brown) antibody in lung tumors from patients with lung cancer. N, normal tissue; T, tumor mass; Nuclei, hematoxylin (blue); Blue and red arrow, cytosol and nuclear ISX (PCAF). Scale bar, 500μm (400X); Scale bar, 2mm (100X).b, Confocal immunofluorescence detection of ISX (green) and BRD4 (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass.c, Confocal immunofluorescence detection of ISX (green) and PCAF (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass.Correlation analysis of mRNA expression for, (d) ISX and BRD4, in 157 lung cancer samples.e, Correlation analysis of mRNA expression for, (e) ISX and PCAF in 157 lung cancer samples.f, Protein expression of ISX, ISX(69Kac) and Vimentin in tumor and normal parts from patients with NSCLC by western blotting. N, normal tissue; T, tumor mass; Arrow, target protein; *, unspecific band.The Kaplan-Meier survival curve was used to analyze survival correlation between patients (n=157) with NSCLC and ISX (g) levels. On the basis of the cut-off values of fold differences, the study population was dichotomized into the "high" and "low" expression group. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves.g and h, The Kaplan-Meier survival curve was used to analyze survival correlation between patients (n=157) with NSCLC and PCAF (h) levels. On the basis of the cut-off values of fold differences, the study population was dichotomized into the "high" and "low" expression group. p‐values were calculated by log‐rank (Mantel-Cox) test comparing the two Kaplan-Meier curves.


Figure 1B . Bright - field picture of a 0B K8 - eYFP embryo ( Left ) and time series of two - photon live imaging of ExE region ( Right ) . Time interval is 5 min ( n = 3 ) . C . Bright - field picture of EB K8 - eYFP embryo ( Left ) and time series of two - photon live imaging of ExE region ( Right ) . Time interval is 5 min ( n = 4 ) . D . Bright - field pictures of K8 - eYFP LB embryos ( n = 30 ) , and high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed white areas . K8 - eYFP and DAPI appear in yellow and cyan , respectively .

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Figure 1B. Bright-field picture of a 0B K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=3). C. Bright-field picture of EB K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=4). D. Bright-field pictures of K8-eYFP LB embryos (n=30), and high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed white areas. K8-eYFP and DAPI appear in yellow and cyan, respectively. Da


Figure 3A - H . SEM images of an opened embryo ( A ) , the ExE cavity wall ( B ) where blood islands are indicated by white arrow , the allantois surface ( C - C ' ) , an opened allantois ( D ) and its zooms ( E - E ' ) where filaments are indicated by white arrows , the epiblast - derived side of the amnion ( F ) , the mesoderm - derived side of the amnion ( G ) and its zoom ( G ' ) .

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Figure 3A-H. SEM images of an opened embryo (A), the ExE cavity wall (B) where blood islands are indicated by white arrow, the allantois surface (C-C'), an opened allantois (D) and its zooms (E-E') where filaments are indicated by white arrows, the epiblast-derived side of the amnion (F), the mesoderm-derived side of the amnion (G) and its zoom (G').


Figure 4B . Sagittal sections of C57BL / 6J wild - type and KtyII - / - E7 . 5 embryos immunostained for Krt8 ( n = 15 and n = 5 , respectively ) , F - actin ( n = 11 and 2 ) , Vimentin ( n = 5 and 1 ) , E - cadherin ( n = 1 and 1 ) , T ( n = 4 and 3 ) , Dsg2 ( n = 15 and 5 ) and Pecam - 1 ( n = 4 and 3 ) . Data information : White arrows point toward allantois and scale bars represent 25 µm . Quantification of embryo morphology at LB stage in C57BL / 6J wild - type ( white boxes / black dots , n = 22 including 16 in which allantois could be measured ) , and KtyII - / - embryos ( grey boxes / black triangles , n = 9 including 8 in which allantois could be measured ) . ( D ) Embryonic region length . Ratios of whole embryo and ExE cavity lengths ( E ) , over embryonic region length . I - I ' ' . SEM images of opened C57BL / 6J wild - type ( n = 3 ) and KtyII - / - ( n = 2 ) LB stage embryos with zooms on the allantois region ( I ' - I ' ' ) . Scale bars represent 200 ( I ) , 20 ( I ' ) and 10 ( I ' ' ) µm .

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

Figure 4B. Sagittal sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Krt8 (n= 15 and n=5, respectively), F-actin (n=11 and 2), Vimentin (n=5 and 1), E-cadherin (n=1 and 1), T (n=4 and 3), Dsg2 (n=15 and 5) and Pecam-1 (n=4 and 3). Data information: White arrows point toward allantois and scale bars represent 25 µm.Quantification of embryo morphology at LB stage in C57BL/6J wild-type (white boxes/ black dots, n=22 including 16 in which allantois could be measured), and KtyII-/- embryos (grey boxes/ black triangles, n=9 including 8 in which allantois could be measured). (D) Embryonic region length. Ratios of whole embryo and ExE cavity lengths (E) , over embryonic region length.I-I''. SEM images of opened C57BL/6J wild-type (n=3) and KtyII-/- (n=2) LB stage embryos with zooms on the allantois region (I'-I''). Scale bars represent 200 (I), 20 (I') and 10 (I'') µm.


Figure 5F . Dot plot of specific gene expression in amnion clusters . G . Immunostaining for Cdx2 ( n = 20 ) . H . Immunostaining for Nrp1 ( n = 8 ) and Col1a1 ( n = 7 ) , primarily expressed in Amnion Mesenchyme . I . Immunostaining for Epcam ( n = 12 ) , Cldn7 ( n = 7 ) , and Lrp2 ( n = 6 ) , primarily expressed in Amnion Ectoderm . J - K . Sections of C57BL / 6J wild - type and KtyII - / - E7 . 5 embryos immunostained for Nrp1 ( K ) ( n = 8 and 2 , respectively ) , and Lrp2 ( L ) ( n = 3 and 2 ) .

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Figure 5F. Dot plot of specific gene expression in amnion clusters.G. Immunostaining for Cdx2 (n=20). H. Immunostaining for Nrp1 (n=8) and Col1a1 (n=7), primarily expressed in Amnion Mesenchyme. I. Immunostaining for Epcam (n=12), Cldn7 (n=7), and Lrp2 (n=6), primarily expressed in Amnion Ectoderm.J-K. Sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Nrp1 (K) (n=8 and 2, respectively), and Lrp2 (L) (n=3 and 2).


Figure 6D . Dot plot of specific gene expression in chorion clusters . E . Immunostaining for Cdx2 ( n = 20 ) . F . Immunostaining for DAPI ( cyan , left ) , Sox2 ( middle ) , and Gata2 ( right ) . Sox2 is primarily expressed in Trophoblast Progenitors and Gata2 in Chorion Ectoderm . G . Immunostaining for Cebpb , detectable in most cells with variable intensity . H . Immunostaining for Fn1 ( n = 22 ) , Dlk1 ( n = 8 ) , and Acta2 ( n = 12 ) , primarily expressed in Chorion Mesenchyme . I . Sections from C57BL / 6J wild - type and KtyII - / - LB embryos immunostained for Dlk - 1 ( n = 8 and 4 , respectively ) and Acta2 ( n = 6 and 3 ) .

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Figure 6D. Dot plot of specific gene expression in chorion clusters.E. Immunostaining for Cdx2 (n=20). F. Immunostaining for DAPI (cyan, left), Sox2 (middle), and Gata2 (right). Sox2 is primarily expressed in Trophoblast Progenitors and Gata2 in Chorion Ectoderm. G. Immunostaining for Cebpb, detectable in most cells with variable intensity. H. Immunostaining for Fn1 (n=22), Dlk1 (n=8), and Acta2 (n=12), primarily expressed in Chorion Mesenchyme. I. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Dlk-1 (n=8 and 4, respectively) and Acta2 (n=6 and 3). Data


Figure 7D . Dot plot of specific gene expression in allantois clusters . F . Immunostaining for Ifitm3 ( n = 7 ) ( Allantois Base ) . G . Immunostaining for Cdh11 ( n = 9 ) , Stard8 ( n = 4 ) and Nrp1 ( n = 8 ) ( Allantois Cortex ) . H . Immunostaining for Pitx1 ( n = 4 ) ( Allantois Mesenchyme ) . I . Hhex - GFP ( n = 5 ) and immunostaining for Kdr ( n = 8 ) ( Endothelial Progenitors ) . J . Immunostaining for Runx1 ( n = 3 ) ( Erythroid Progenitors ) . K - L . K - L . Sections from C57BL / 6J wild - type and KtyII - / - LB embryos immunostained for Cdh11 ( Top ) and Krt8 ( Bottom ) ( K , n = 8 and 4 , respectively ) and Ifitm3 ( L , n = 2 and 2 ) . Data information : White arrows point toward signal in allantois . Sagittal sections with anterior to the left .

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

Figure 7D. Dot plot of specific gene expression in allantois clusters.F. Immunostaining for Ifitm3 (n=7) (Allantois Base). G. Immunostaining for Cdh11 (n=9), Stard8 (n=4) and Nrp1 (n=8) (Allantois Cortex). H. Immunostaining for Pitx1 (n=4) (Allantois Mesenchyme). I. Hhex-GFP (n=5) and immunostaining for Kdr (n=8) (Endothelial Progenitors). J. Immunostaining for Runx1 (n=3) (Erythroid Progenitors). K-L.K-L. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Cdh11 (Top) and Krt8 (Bottom) (K, n=8 and 4, respectively) and Ifitm3 (L, n=2 and 2). Data information: White arrows point toward signal in allantois. Sagittal sections with anterior to the left.


Figure 1A OT - I CD8 + CTLs were conjugated with 10 nM OVA257 - 264 - pulsed EL - 4 cells for 30 min , fixed , and stained with anti - SKAP55 ( red ) , anti - LFA - 1 ( green ) and Hoechst ( blue ) . B , C WT and SKAP55 − / − OT - ICD8 + CTLs were generated from WT or SKAP55 − / − OT - I Tg mice , then incubated with 10 nM OVA257 - 264 - pulsed EL4 targets for 4 h to assess the in vitro cytotoxicity at different Effector : Target ratios ( B ; mean of triplicates ± SD ) ; surface expression and the mRNA levels of PD - 1 ( C ) . Graphs are representative of at least three independent experiments . D WT and SKAP55 − / − OT - I CD8 + CTLs ( 3 × 106 ) were injected into C57BL / 6 mice , followed by injection of non - pulsed ( CFSEhi ) or 10 nM OVA257 - 264 - pulsed ( CFSElo ) splenocytes ( 5 × 106 ) to measure in vivo cytotoxicity ( mean ± SD , n = 3 mice per group ) . Representative data of three independent experiments . E WT and SKAP55 − / − OT - I CD8 + CTLs were pretreated with anti - PD - 1 antibody or IgG control , followed by incubation with OVA257 - 264 - pulsed EL4 cells to examine in vitro killing ability ( mean of triplicates ± SD ) . Graphs are representative of three independent experiments . F CD8 + CTLs were transfected with plasmids expressing SKAP55 - GFP or EGFP , then treated with non - pulsed or 10 nM OVA257 - 264 - pulsed EL4 cells to examine surface PD - 1 expression ( left panel ) or the in vitro killing assay ( mean of triplicates ± SD ) ( right panel ) .

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

Figure 1A OT-I CD8+ CTLs were conjugated with 10 nM OVA257-264-pulsed EL-4 cells for 30 min, fixed, and stained with anti-SKAP55 (red), anti-LFA-1 (green) and Hoechst (blue).B, C WT and SKAP55−/−OT-ICD8+CTLs were generated from WT or SKAP55−/−OT-I Tg mice, then incubated with 10 nM OVA257-264-pulsed EL4 targets for 4 h to assess the in vitro cytotoxicity at different Effector:Target ratios (B; mean of triplicates ± SD); surface expression and the mRNA levels of PD-1 (C). Graphs are representative of at least three independent experiments.D WT and SKAP55−/− OT-I CD8+ CTLs (3 × 106) were injected into C57BL/6 mice, followed by injection of non-pulsed (CFSEhi) or 10 nM OVA257-264-pulsed (CFSElo) splenocytes (5 × 106) to measure in vivo cytotoxicity (mean ± SD, n = 3 mice per group). Representative data of three independent experiments.E WT and SKAP55−/− OT-I CD8+ CTLs were pretreated with anti-PD-1 antibody or IgG control, followed by incubation with OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD). Graphs are representative of three independent experiments.F CD8+ CTLs were transfected with plasmids expressing SKAP55-GFP or EGFP, then treated with non-pulsed or 10 nM OVA257-264-pulsed EL4 cells to examine surface PD-1 expression (left panel) or the in vitro killing assay (mean of triplicates ± SD) (right panel).


Figure 2A OT - I CD8 + CTLs were conjugated with CFSE - labeled OVA257 - 264 - pulsed or non - pulsed EL - 4 cells for 30 min , fixed , and stained with anti - ADAP ( red ) . B OT - I CD8 + CTLs were transfected with plasmids expressing ADAP or GFP , then stimulated with 10 nM OVA257 - 264 - pulsed EL - 4 cells to detect surface PD - 1 expression . Representative of three independent experiments . C , D WT and ADAP − / − OT - I CD8 + CTLs were stimulated with 10 nM OVA257 - 264 - pulsed or unpulsed EL - 4 cells for 4 h to examine surface expression and the mRNA levels of PD - 1 ( C ) , in vitro cytotoxicity at different Effector : Target ratios ( D ; mean of triplicates ± SD ) . Graphs are representative of at least three independent experiments . E WT and ADAP − / − OT - I CD8 + CTLs were pretreated with 10 μg / ml anti - PD - 1 antibody or IgG control , followed by incubation with 10 nM OVA257 - 264 - pulsed EL4 cells to examine in vitro killing ability ( mean of triplicates ± SD ) . F Naïve CD8 + T cells were isolated from WT , SKAP55 − / − , ADAP − / − , and SKAP55 − / − ADAP − / − splenocytes , then stimulated with plate - bound anti - CD3 / CD28 for 12 h to assess PD - 1 mRNA levels , and for 48 h to check surface PD - 1 expression ( mean of triplicates ± SD ) .

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Figure 2A OT-I CD8+ CTLs were conjugated with CFSE-labeled OVA257-264-pulsed or non-pulsed EL-4 cells for 30 min, fixed, and stained with anti-ADAP (red).B OT-I CD8+ CTLs were transfected with plasmids expressing ADAP or GFP, then stimulated with 10 nM OVA257-264-pulsed EL-4 cells to detect surface PD-1 expression. Representative of three independent experiments.C, D WT and ADAP−/− OT-I CD8+ CTLs were stimulated with 10 nM OVA257-264-pulsed or unpulsed EL-4 cells for 4 h to examine surface expression and the mRNA levels of PD-1 (C), in vitro cytotoxicity at different Effector:Target ratios (D; mean of triplicates ± SD). Graphs are representative of at least three independent experiments.E WT and ADAP−/− OT-I CD8+ CTLs were pretreated with 10 μg/ml anti-PD-1 antibody or IgG control, followed by incubation with 10 nM OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD).F Naïve CD8+ T cells were isolated from WT, SKAP55−/−, ADAP−/−, and SKAP55−/−ADAP−/− splenocytes, then stimulated with plate-bound anti-CD3/CD28 for 12 h to assess PD-1 mRNA levels, and for 48 h to check surface PD-1 expression (mean of triplicates ± SD).


Figure 3A - C WT , ADAP − / − , or SKAP55 − / − OT - I CD8 + CTLs were stimulated with OVA257 - 264 - pulsed or unpulsed EL4 cells to detect the levels of NFATc1 expression , activation and nuclear entry by immunoblotting ( A ) or immunostaining ( B ) . Alternatively , nuclear extracts from these CTLs were incubated with the N1 DNA probes containing NFAT - binding sites for an EMSA . The unlabeled competitor probe ( Comp . N1 ) and the mutant probe that contains the same sequence except for carrying a mutated NFAT - binding site ( Comp . mutN1 ) were included as controls ( C ) . D WT , ADAP − / − , or SKAP55 − / − OT - I CD8 + CTLs were pretreated with DMSO or 5 μM CsA , then stimulated with OVA257 - 264 - pulsed EL - 4 cells for 4 h to examine surface PD - 1 expression ( mean of triplicates ± SD ) . E WT , ADAP − / − , or SKAP55 − / − OT - I CTLs were pretreated with the DMSO control , 10 μM PP2 or 75 μM 2APB , then stimulated with OVA257 - 264 - pulsed EL - 4 cells for 4 h to examine surface PD - 1 expression ( mean of triplicates ± SD ) . F GFP , ADAP , or SKAP55 was overexpressed with pGL3 - NFAT Luciferase reporter plasmid into Jurkat cells . Cells were stimulated with anti - CD3 and anti - CD28 in the presence or absence of PP2 or 2APB for 6 h , followed by measuring luciferase readings ( mean of triplicates ± SD ) .

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Figure 3A-C WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were stimulated with OVA257-264-pulsed or unpulsed EL4 cells to detect the levels of NFATc1 expression, activation and nuclear entry by immunoblotting (A) or immunostaining (B). Alternatively, nuclear extracts from these CTLs were incubated with the N1 DNA probes containing NFAT-binding sites for an EMSA. The unlabeled competitor probe (Comp. N1) and the mutant probe that contains the same sequence except for carrying a mutated NFAT-binding site (Comp. mutN1) were included as controls (C).D WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were pretreated with DMSO or 5 μM CsA, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD).E WT, ADAP−/−, or SKAP55−/− OT-I CTLs were pretreated with the DMSO control, 10 μM PP2 or 75 μM 2APB, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD).F GFP, ADAP, or SKAP55 was overexpressed with pGL3-NFAT Luciferase reporter plasmid into Jurkat cells. Cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of PP2 or 2APB for 6 h, followed by measuring luciferase readings (mean of triplicates ± SD).


Figure 4A WT and SKAP55 KO C57BL / 6 mice were s . c . immunized on day − 14 and day − 7 by DCs that were prepulsed with B16F10 tumor lysates . On day 0 , the immunized mice were i . v . inoculated with B16F10 cells . Number of lung tumors were counted 26 days after B16F10 challenge ( n ≥ 5 mice ) . B , C At day 0 ( B , before tumor injection ) or at day 26 ( C , after tumor injection ) , surface PD - 1 expression in CD44hi CD8 + or CD44hi CD4 + T cells , and the percentages of CD44hi CD8 + T cells or Foxp3 + CD4 + cells were analyzed ( mean ± SD , n ≥ 5 mice ) . D , E The mRNA levels of PD - 1 , NFATc1 , or the mRNA and protein levels of granzyme B in lung infiltrating CD8 + T cells were examined at day 26 ( mean of triplicates ± SD , and the mRNA samples were prepared from at least 3 mice per group ) . F CTLA - 4 levels in the lung - infiltrated CD44hiCD8 + T cells or the percentages of CD4 + Foxp3 + T cells in lungs , MLN , and spleen were measured at day 26 ( mean ± SD , n ≥ 5 mice ) .

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Figure 4A WT and SKAP55 KO C57BL/6 mice were s.c. immunized on day −14 and day −7 by DCs that were prepulsed with B16F10 tumor lysates. On day 0, the immunized mice were i.v. inoculated with B16F10 cells. Number of lung tumors were counted 26 days after B16F10 challenge (n ≥ 5 mice).B, C At day 0 (B, before tumor injection) or at day 26 (C, after tumor injection), surface PD-1 expression in CD44hi CD8+ or CD44hi CD4+T cells, and the percentages of CD44hi CD8+T cells or Foxp3+ CD4+ cells were analyzed (mean ± SD, n ≥ 5 mice).D, E The mRNA levels of PD-1, NFATc1, or the mRNA and protein levels of granzyme B in lung infiltrating CD8+ T cells were examined at day 26 (mean of triplicates ± SD, and the mRNA samples were prepared from at least 3 mice per group).F CTLA-4 levels in the lung-infiltrated CD44hiCD8+ T cells or the percentages of CD4+Foxp3+ T cells in lungs, MLN, and spleen were measured at day 26 (mean ± SD, n ≥ 5 mice).


Figure 5A The same procedures as in Fig4A were performed with WT and ADAP − / − C57BL / 6 mice . HE staining of lung tissue was examined on day 26 ( representative graph of 5 mice ) . B , C PD - 1 expression in CD44hiCD8 + or CD44hiCD4 + T cells ( mean ± SD , n ≥ 5 mice per group ) or granzyme B expression in CD44hiCD8 + T cells were examined from the lungs at day 26 ( representative data of 3 mice per group ) . D , E CTLA - 4 levels in lung infiltrating CD44hiCD8 + T cells and the percentages of CD4 + Foxp3 + T cells in the lungs or MLN were measured at day 26 ( mean ± SD , n ≥ 5 mice per group ) .

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Figure 5A The same procedures as in Fig4A were performed with WT and ADAP−/− C57BL/6 mice. HE staining of lung tissue was examined on day 26 (representative graph of 5 mice).B, C PD-1 expression in CD44hiCD8+ or CD44hiCD4+ T cells (mean ± SD, n ≥ 5 mice per group) or granzyme B expression in CD44hiCD8+ T cells were examined from the lungs at day 26 (representative data of 3 mice per group).D, E CTLA-4 levels in lung infiltrating CD44hiCD8+ T cells and the percentages of CD4+Foxp3+ T cells in the lungs or MLN were measured at day 26 (mean ± SD, n ≥ 5 mice per group).


Figure 6A , B WT and SKAP55 − / − mice were s . c . injected with MO5 melanoma cells followed by two injection at day 7 and day 14 of OVA257 - 264 - pulsed DCs or the PBS control respectively ( n ≥ 5 ) . The growth of tumors under skin was measured every three days according to tumor diameter ( mean ± SEM , n ≥ 5 mice per group ) ( A ) . Surface expression levels of PD - 1 were checked on OVA257 - 264 - specific Vα2high / Vβ5 + CD8 + effector cells at day 30 ( mean ± SD , n ≥ 5 mice ) ( B ) . C WT and SKAP55 − / − mice were s . c . injected with MO5 melanoma cells followed by s . c . two injection of OVA257 - 264 - pulsed DCs at day 7 and day 14 . anti - PD - 1 monoantibody or IgG controls were i . v . injected three times at day 7 , day 10 , and day 14 ( 50 μg / injection ) . The diameter and volume of tumors were measured every three days ( mean ± SEM , n ≥ 5 mice ) . D The WT recipient mice were s . c . injected with MO5 melanoma cells followed by injection of 10 nM OVA257 - 264 - stimulated WT or SKAP55 − / − CTLs at day 8 ( n ≥ 8 ) . The diameter and volume of tumors were measured every three days ( mean ± SEM , n ≥ 8 mice ) .

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Figure 6A, B WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by two injection at day 7 and day 14 of OVA257-264-pulsed DCs or the PBS control respectively (n ≥ 5). The growth of tumors under skin was measured every three days according to tumor diameter (mean ± SEM, n ≥ 5 mice per group) (A). Surface expression levels of PD-1 were checked on OVA257-264-specific Vα2high/Vβ5+ CD8+ effector cells at day 30 (mean ± SD, n ≥ 5 mice) (B).C WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by s.c. two injection of OVA257-264-pulsed DCs at day 7 and day 14. anti-PD-1 monoantibody or IgG controls were i.v. injected three times at day 7, day 10, and day 14 (50 μg/injection). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 5 mice).D The WT recipient mice were s.c. injected with MO5 melanoma cells followed by injection of 10 nM OVA257-264-stimulated WT or SKAP55−/− CTLs at day 8 (n ≥ 8). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 8 mice).


Figure 7A , B During generation of OVA257 - 264 peptide - specific CTLs , naïve OT - I splenocytes were stimulated with 10 nM OVA257 - 264 with or without CsA ( 10 nM ) for 3 days , then washed , and further cultured in the RPMI growth medium until day 6 . The CsA - treated or untreated OVA257 - 264 peptide - specific CTLs were incubated with OVA257 - 264 - pulsed EL - 4 cells for 4 h to assess the in vitro cytotoxicity ( A , mean of triplicates ± SD ) or i . v . injected into WT mice to measure in vivo cytotoxicity ( B , mean ± SD , n = 4 mice ) . C WT mice were s . c . injected with B16 - MO5 melanoma cells followed by injection of the CsA - treated or untreated OVA257 - 264 peptide - specific CTLs at day 9 ( n ≥ 12 ) . The diameter and volume of tumors were measured every 3 days ( mean ± SEM , n ≥ 12 mice ) .

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

Figure 7A, B During generation of OVA257-264 peptide-specific CTLs, naïve OT-I splenocytes were stimulated with 10 nM OVA257-264 with or without CsA (10 nM) for 3 days, then washed, and further cultured in the RPMI growth medium until day 6. The CsA-treated or untreated OVA257-264 peptide-specific CTLs were incubated with OVA257-264-pulsed EL-4 cells for 4 h to assess the in vitro cytotoxicity (A, mean of triplicates ± SD) or i.v. injected into WT mice to measure in vivo cytotoxicity (B, mean ± SD, n = 4 mice).C WT mice were s.c. injected with B16-MO5 melanoma cells followed by injection of the CsA-treated or untreated OVA257-264 peptide-specific CTLs at day 9 (n ≥ 12). The diameter and volume of tumors were measured every 3 days (mean ± SEM, n ≥ 12 mice).


Figure 1C : Time course of PIKfyve downregulation in brains of prion - infected mice ( 3 mice / time point ) . PIKfyve ( but not FIG4 , calnexin , NeuN and synaptophysin ) was suppressed between 60 and 120 dpi . T : terminal disease stage . E : Prion - infected Gt1 cells ( 75 dpi ) stained with Alexa - Fluor647 - succinimidyl ester which stains vacuoles negatively ( arrows ) . G : tga20 COCS were treated with pooled IgG or POM1 ( 72 hours ) . POM1 suppressed PIKfyve but not FIG4 and VAC14 . Right : Quantification of immunoblots . Each dot represents an independent experiment . * * : p < 0 . 01 ; * * * : p < 0 . 001 ; unpaired t - test Error bars represent s . e . m . H : Immunoblots of human CJD brains ( red : sampled region ) . Gt1Sc : prion - infected Gt1 cells . Loading controls : actin and calnexin ( Cnx )

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Figure 1C: Time course of PIKfyve downregulation in brains of prion-infected mice (3 mice/time point). PIKfyve (but not FIG4, calnexin, NeuN and synaptophysin) was suppressed between 60 and 120 dpi. T: terminal disease stage.E: Prion-infected Gt1 cells (75 dpi) stained with Alexa-Fluor647-succinimidyl ester which stains vacuoles negatively (arrows).G: tga20 COCS were treated with pooled IgG or POM1 (72 hours). POM1 suppressed PIKfyve but not FIG4 and VAC14. Right: Quantification of immunoblots. Each dot represents an independent experiment. **: p<0.01; ***: p<0.001; unpaired t-test Error bars represent s.e.m.H: Immunoblots of human CJD brains (red: sampled region). Gt1Sc: prion-infected Gt1 cells. Loading controls: actin and calnexin (Cnx)


Figure 2C : Treatment of COCS with POM1 reduces the levels of PIKfyve starting 48h post treatment . Each dot represents an individual experiment . Statistics : Unpaired t - test . * * : p < 0 . 01 . G : Left : partially restored PIKfyve levels in prion - infected tga20 mice treated with LIN5044 ( LCP ) or vehicle . Right : quantification of the western blot . Each dot represents an individual biological replicate . * : p0 . 05 ; unpaired t - test Error bars represent s . e . m . H : Left , tga20 COCS were inoculated with RML or NBH , optionally treated with GSK2606414 starting at 21 dpi , and lysed at 35 dpi . Depletion of PIKfyve in prion - infected samples was largely rescued by GSK2606414 ( GSK ) . Right , quantification of western blot . Each dot represents an individual biological replicate . Statistics : Unpaired t - test . * * : p < 0 . 01 ; unpaired t - test ; Error bars represent s . e . m .

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Figure 2C: Treatment of COCS with POM1 reduces the levels of PIKfyve starting 48h post treatment. Each dot represents an individual experiment. Statistics: Unpaired t-test. **: p<0.01.G: Left: partially restored PIKfyve levels in prion-infected tga20 mice treated with LIN5044 (LCP) or vehicle. Right: quantification of the western blot. Each dot represents an individual biological replicate. *: p0.05; unpaired t-test Error bars represent s.e.m.H: Left, tga20 COCS were inoculated with RML or NBH, optionally treated with GSK2606414 starting at 21 dpi, and lysed at 35 dpi. Depletion of PIKfyve in prion-infected samples was largely rescued by GSK2606414 (GSK). Right, quantification of western blot. Each dot represents an individual biological replicate. Statistics: Unpaired t-test. **: p<0.01; unpaired t-test; Error bars represent s.e.m.


Figure 3E , F : Acyl - rac of Gt1 cells subjected to siRNA against zDHHC9 , zDHHC21 or both ( 72 hours post transfection ) , showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 ( quantified in F ) . Each dot represents a separate experiment ( unpaired t - test ) . * * : p < 0 . 01 . Error bars represent s . e . m . H - I : Flag - tagged zDHHC9 or zDHHC21 were expressed in Gt1 cells were transfected with plasmids encoding . After 72 hours , zDHHC9 / 21 ( magenta ) were localized to the Golgi apparatus ( GM130 + , cyan ) in NBH - exposed cells but not in prion - infected cells . n = 3 individual biological replicates .

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Figure 3E, F: Acyl-rac of Gt1 cells subjected to siRNA against zDHHC9, zDHHC21 or both (72 hours post transfection), showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 (quantified in F). Each dot represents a separate experiment (unpaired t-test). **: p<0.01. Error bars represent s.e.m.H-I: Flag-tagged zDHHC9 or zDHHC21 were expressed in Gt1 cells were transfected with plasmids encoding. After 72 hours, zDHHC9/21 (magenta) were localized to the Golgi apparatus (GM130+, cyan) in NBH-exposed cells but not in prion-infected cells. n=3 individual biological replicates.


Figure 4A : Upper row : prion - infected Gt1 cells ( 75 dpi ) immunostained for LAMP1 showing prominent vacuoles . Control : NBH - inoculated cells . Middle row : Gt1 cells transfected with shRNA targeting PIKfyve or luciferase ( control ) for 5 days and immunostained for LAMP1 . Depletion of PIKfyve resulted in LAMP1 + vacuoles ( yellow ) . DAPI : nuclear stain . Lower row : prion - infected and NBH treated cells ( 75 dpi ) contained LAMP2 - vacuoles . n = 3 individual biological replicates . D : tga20 COCS treated with POM1 or control IgG ( 10 days ) and immunostained with NeuN and LAMP1 . Nuclei : DAPI . POM1 - treated COCS showed increased LAMP1 expression ( quantification : Appendix Fig . S4B ) . NeuN + cells exhibited vacuoles ( arrowheads ) . G : Lysosomal enzymes , but not β - actin , were elevated in brains of terminally scrapie - sick mice . Panels show independent triplicates . Statistics : ANOVA . . * * : p < 0 . 01 . Error bars represent s . e . m .

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Figure 4A: Upper row: prion-infected Gt1 cells (75 dpi) immunostained for LAMP1 showing prominent vacuoles. Control: NBH-inoculated cells. Middle row: Gt1 cells transfected with shRNA targeting PIKfyve or luciferase (control) for 5 days and immunostained for LAMP1. Depletion of PIKfyve resulted in LAMP1+ vacuoles (yellow). DAPI: nuclear stain. Lower row: prion-infected and NBH treated cells (75 dpi) contained LAMP2- vacuoles. n=3 individual biological replicates.D: tga20 COCS treated with POM1 or control IgG (10 days) and immunostained with NeuN and LAMP1. Nuclei: DAPI. POM1-treated COCS showed increased LAMP1 expression (quantification: Appendix Fig. S4B). NeuN+ cells exhibited vacuoles (arrowheads).G: Lysosomal enzymes, but not β-actin, were elevated in brains of terminally scrapie-sick mice. Panels show independent triplicates. Statistics: ANOVA.. **: p<0.01. Error bars represent s.e.m.


Figure 5A : Time course of TFEB phosphorylation in prion - infected C57BL / 6 mice . Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥ 90 days . Quantification : Each dot represents an individual biological replicate . Statistics : Unpaired t - test . * : p0 . 05 ; * * : p < 0 . 01 . Error bars represent s . e . m . B : TFEB - controlled transcripts in brains of terminally scrapie - sick C57BL / 6 mice were measured by qPCR . Prion infection led to upregulation of these genes , but not of STAT3 . Control : NBH - inoculated mice . ANOVA on independent triplicates . * * * : p < 0 . 001 . Error bars represent s . e . m . E : Gt1 cells ( as in D ) were fixed and stained with anti - TFEB antibody . Prion - infected cells showed nuclear translocation of TFEB . DAPI : Blue . Quantification : Each dot represents an individual biological replicate ( 30 images were quantified per replicate ) . Statistics : Unpaired t - test . * * : p < 0 . 01 . Error bars represent s . e . m . F : TFEB responsive genes were upregulated in Gt1 cells chronically infected with RML prions ( 75 dpi ) . Panels depict independent triplicates . Statistics : ANOVA . * : p0 . 05 . Error bars represent s . e . m . G : TFEB responsive genes were upregulated in cells transfected with shPIKfyve for 5 days compared to shCtrl ( shRNA against luciferase ) . Panels depict independent triplicates . Statistics : ANOVA . * : p0 . 05 . Error bars represent s . e . m .

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Figure 5A: Time course of TFEB phosphorylation in prion-infected C57BL/6 mice. Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥90 days. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. *: p0.05; **: p<0.01. Error bars represent s.e.m.B: TFEB-controlled transcripts in brains of terminally scrapie-sick C57BL/6 mice were measured by qPCR. Prion infection led to upregulation of these genes, but not of STAT3. Control: NBH-inoculated mice. ANOVA on independent triplicates. ***: p<0.001. Error bars represent s.e.m.E: Gt1 cells (as in D) were fixed and stained with anti-TFEB antibody. Prion-infected cells showed nuclear translocation of TFEB. DAPI: Blue. Quantification: Each dot represents an individual biological replicate (30 images were quantified per replicate). Statistics: Unpaired t-test. **: p<0.01. Error bars represent s.e.m.F: TFEB responsive genes were upregulated in Gt1 cells chronically infected with RML prions (75 dpi). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m. G: TFEB responsive genes were upregulated in cells transfected with shPIKfyve for 5 days compared to shCtrl (shRNA against luciferase). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m.


Figure 6C : Prion - infected Gt1 cells were lentivirally transduced with GADD34 at 75 dpi . Control : empty lentiviral vector . At 79 dpi , GADD34 overexpression had normalized eIF2α phosphorylation and restored PIKfyve levels . Right : Quantification of western blots ( n = 3 ; unpaired t - test ) . Each dot represents an individual experiment . * * : p < 0 . 01 . Error bars represent s . e . m . E : tga20 COCS were infected with RML and optionally treated with bPIP ( 5µg / ml ) . At 45 dpi , NeuN morphometry revealed ablation of cerebellar granule layer ( CGN ) in prion - infected slices and its rescue by bPIP . Control : NBH treated COCS . Each dot represents an individual slice ( Statistics : ANOVA ) . * : p < 0 . 05 . * * * * p < 0 . 0001 . F : tga20 COCS were treated with POM1 ( optionally pre - blocked with recPrP ) and treated with bPIP ( 5µg / ml ) . At 14 dpi NeuN morphometry revealed POM1 - induced ablation of cerebellar granule layer ( CGL ) and rescue by bPIP .

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

Figure 6C: Prion-infected Gt1 cells were lentivirally transduced with GADD34 at 75 dpi. Control: empty lentiviral vector. At 79 dpi, GADD34 overexpression had normalized eIF2α phosphorylation and restored PIKfyve levels. Right: Quantification of western blots (n=3; unpaired t-test). Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m.E: tga20 COCS were infected with RML and optionally treated with bPIP (5µg/ml). At 45 dpi, NeuN morphometry revealed ablation of cerebellar granule layer (CGN) in prion-infected slices and its rescue by bPIP. Control: NBH treated COCS. Each dot represents an individual slice (Statistics: ANOVA). *: p<0.05. **** p<0.0001.F: tga20 COCS were treated with POM1 (optionally pre-blocked with recPrP) and treated with bPIP (5µg/ml). At 14 dpi NeuN morphometry revealed POM1-induced ablation of cerebellar granule layer (CGL) and rescue by bPIP.


Figure 1B ) Binding affinity of CodB for cytosine as measured using the thermostability assay . Cytosine was titrated into detergent solubilised membranes from cells overexpressing CodB . The Kd was estimated to be 51 ± 9 µM . The measurements are the average of 4 independent titrations with error bars of the s . e . m .

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Figure 1B) Binding affinity of CodB for cytosine as measured using the thermostability assay. Cytosine was titrated into detergent solubilised membranes from cells overexpressing CodB. The Kd was estimated to be 51 ± 9 µM. The measurements are the average of 4 independent titrations with error bars of the s.e.m.


Figure 1A . Glioma tumour micro arrays ( grade II - IV gliomas ) were stained to visualise MDGI ( red ) . Samples from the epileptic brain served as a negative control . Scale bar 100 µm . B . Overall survival of grade II and III glioma patients ( n = 76 ) was significantly better when none / low MDGI ( blue line ) was detected compared to patients with moderate / high MDGI ( red line ) expression ( P = 0 . 007 ) . The cumulative survival rates were estimated by using the Kaplan - Meier method . C . Analysis of the association of MDGI mRNA expression with glioma patient survival using the GlioVis data portal . In the TCGA GBMLGG dataset ( n = 667 ) , patients with low MDGI expression ( green line ; n = 333 , events = 101 , median = 67 , 5 ) show significantly better survival than patients with high MDGI ( red line ; n = 334 , events = 138 , median = 41 . 5 ) expression ( P = 2e - 04 ) . HR = 0 . 62 ( 0 . 48 - 0 . 8 ) . The cumulative survival rates were estimated by using the Kaplan - Meier method . D . In glioblastoma samples , highest MDGI expression was observed in the mesenchymal subtype ( M ) compared to the classical ( Cl ) or pro - neural ( PN ) ones . However , it did not reach the statistical significance . GlioVis data portal , TCGA GBM dataset , n = 156 . Pairwise t - test with corrections for multiple testing , p - values with Bonferroni correction . E . MDGI expression was high in the leading edge of the tumour ( LE ) and in infiltrating tumour cells ( IT ) in the IVY _ Gap dataset ( n = 122 ) where the gene expression profile in different anatomical structures of glioblastomas was analysed . LE , leading edge ; IT , infiltrating tumour cells ; CT , cellular tumour ( cells within the tumour mass ) ; PC , pseudopalisading cells ; MP , microvascular proliferation . Numbers in the graph depict the P values : Pairwise t - test with corrections for multiple testing , p - values with Bonferroni correction . F . Western blot analysis shows MDGI expression in human glioma cells including patient - derived spheroids ( S24 , ZH161 , ZH305 , BT3 , BT11 , BT12 , BT13 ) , patient - derived adherent cells ( BT5 and BT5R ) , and long - term cell lines ( U87MG , LN229 , LN308 ) . Numbers above the lanes show the relative expression of MDGI compared to the levels of β - tubulin that served as loading control in each cell line . Representative Western blot image of 3 separate experiments is shown . G . High MDGI and HIF - 1α expression in the patient - derived glioblastoma spheroids decreased significantly when cells were cultured in medium containing 10 % of serum ( FBS ) for seven days . Numbers show the relative expression of MDGI and HIF - 1α compared to the levels of β - tubulin . Expression in the serum - free medium was set as 1 . Representative Western blot image of 2 separate experiments is shown . H . MDGI expression in adherent glioma cell lines and in glioma cell spheroids after 24h culture under hypoxia ( 1 % O2 ) or normoxia ( 21 % O2 ) . Numbers show the relative MDGI expression compared to the levels of β - tubulin . Expression under normoxia was set as 1 . Representative Western blot image of 3 separate experiments is shown .

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

Figure 1A. Glioma tumour micro arrays (grade II-IV gliomas) were stained to visualise MDGI (red). Samples from the epileptic brain served as a negative control. Scale bar 100 µm.B. Overall survival of grade II and III glioma patients (n = 76) was significantly better when none/low MDGI (blue line) was detected compared to patients with moderate/high MDGI (red line) expression (P = 0.007). The cumulative survival rates were estimated by using the Kaplan-Meier method.C. Analysis of the association of MDGI mRNA expression with glioma patient survival using the GlioVis data portal. In the TCGA GBMLGG dataset (n = 667), patients with low MDGI expression (green line; n = 333, events = 101, median = 67,5) show significantly better survival than patients with high MDGI (red line; n = 334, events = 138, median = 41.5) expression (P = 2e-04). HR = 0.62 (0.48 - 0.8). The cumulative survival rates were estimated by using the Kaplan-Meier method.D. In glioblastoma samples, highest MDGI expression was observed in the mesenchymal subtype (M) compared to the classical (Cl) or pro-neural (PN) ones. However, it did not reach the statistical significance. GlioVis data portal, TCGA GBM dataset, n = 156. Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction.E. MDGI expression was high in the leading edge of the tumour (LE) and in infiltrating tumour cells (IT) in the IVY_Gap dataset (n = 122) where the gene expression profile in different anatomical structures of glioblastomas was analysed. LE, leading edge; IT, infiltrating tumour cells; CT, cellular tumour (cells within the tumour mass); PC, pseudopalisading cells; MP, microvascular proliferation. Numbers in the graph depict the P values: Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction.F. Western blot analysis shows MDGI expression in human glioma cells including patient-derived spheroids (S24, ZH161, ZH305, BT3, BT11, BT12, BT13), patient-derived adherent cells (BT5 and BT5R), and long-term cell lines (U87MG, LN229, LN308). Numbers above the lanes show the relative expression of MDGI compared to the levels of β-tubulin that served as loading control in each cell line. Representative Western blot image of 3 separate experiments is shown.G. High MDGI and HIF-1α expression in the patient-derived glioblastoma spheroids decreased significantly when cells were cultured in medium containing 10 % of serum (FBS) for seven days. Numbers show the relative expression of MDGI and HIF-1α compared to the levels of β-tubulin. Expression in the serum-free medium was set as 1. Representative Western blot image of 2 separate experiments is shown.H. MDGI expression in adherent glioma cell lines and in glioma cell spheroids after 24h culture under hypoxia (1 % O2) or normoxia (21 % O2). Numbers show the relative MDGI expression compared to the levels of β-tubulin. Expression under normoxia was set as 1. Representative Western blot image of 3 separate experiments is shown.


Figure 2A , B Representative whole coronal section micrographs of murine brain injected with GFP ( A ) or MDGI - GFP ( B ) expressing U87MG glioma cells . Xenografted human cells were visualised by using anti - human vimentin ( hVim ) antibodies ( red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm . Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for human vimentin ( hVim red in C and D ) , Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . Arrows in D point to the angiotropic tumours . Scale bar : 50 µm . Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for MDGI ( green in E and F ) , and CD31 ( red in E and F ) . Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . Arrows in F point to the MDGI - expressing angiotropic tumour cells . Scale bar : 50 µm . G . Quantification of the number of U87MG secondary tumours ( diameter > 300 µm ) detected in the whole brain ( GFP : n = 5 , MDGI - GFP : n = 9 ) . Data are represented as mean ± SEM . * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test . H . Quantification of the number of invasive U87MG cells that grew next to brain blood vessels ( angiotropic co - opting tumour cells , diameter < 300 µm ) ( GFP : n = 5 , MDGI - GFP : n = 9 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . I , J Representative micrographs of whole murine brain coronal sections injected with GFP ( I ) or GFP - MDGI ( J ) expressing LN229 glioma cells . Xenografted human cells were visualised by using anti - human vimentin ( hVim ) antibodies ( red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm . Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for human vimentin ( hVim red in K and L ) Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . ) . Arrows in L point to the angiotropic tumours . Scale bar : 50 µm . Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for MDGI ( green in M and N ) , and CD31 ( red in M and N ) . Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . ) . Arrows in N point to the MDGI - expressing angiotropic tumour cells . Scale bar : 50 µm . O . Quantification of the number of invasive LN229 cells detected in the brain ( GFP : n = 10 , MDGI - GFP : n = 10 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . P . Quantification of the number of invasive LN229 cells that grew next to brain blood vessels ( angiotropic co - opting tumour cells , diameter < 300 µm ) detected in the brain ( GFP : n = 10 , MDGI - GFP : n = 10 ) . Data are represented as mean ± SEM . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

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

Figure 2A, B Representative whole coronal section micrographs of murine brain injected with GFP (A) or MDGI-GFP (B) expressing U87MG glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in C and D), Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in D point to the angiotropic tumours. Scale bar: 50 µm.Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in E and F), and CD31 (red in E and F). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in F point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.G. Quantification of the number of U87MG secondary tumours (diameter >300 µm) detected in the whole brain (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.H. Quantification of the number of invasive U87MG cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.I, J Representative micrographs of whole murine brain coronal sections injected with GFP (I) or GFP-MDGI (J) expressing LN229 glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in K and L) Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in L point to the angiotropic tumours. Scale bar: 50 µm.Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in M and N), and CD31 (red in M and N). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in N point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.O. Quantification of the number of invasive LN229 cells detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.P. Quantification of the number of invasive LN229 cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


Figure 3A . Transmitted light micrographs of MDGI - silenced ( shMDGI1 and shMDGI2 ) and control ( Scr ) patient - derived BT12 and BT13 spheroids 11 days after gene silencing . Representative images of at least 3 independent experiments are shown . Scale bar : 200 μm . B . Methylcellulose assay demonstrating the ability for self - renewal of control ( Scr ) and MDGI - silenced ( shMDGI1 and shMDGI2 ) patient - derived BT12 and BT13 cells after three weeks of culture . Representative images of at least 3 independent experiments are shown . Scale bar : 200 μm . C . Quantification of the colony size in methylcellulose of MDGI - silenced ( sh1 and sh2 ) cells relative to the controls ( Scr ) that is set as 100 ( n = 3 ) . Data are represented as mean ± SD . * * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test . D . MTT - proliferation assay of control ( Scr ) and MDGI - silenced ( shMDGI1 ) BT12 and BT13 cells . The values of each cell line were normalized to the values at day 1 marked with a dashed line ( n = 3 ) . Data are represented as mean ± SD . * * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test . E , F Representative whole coronal section micrographs of murine brain injected intracranially with control ( BT12 Scr , n = 5 ) and MDGI - silenced ( BT12 sh1 , n = 5 ) BT12 cells . After four weeks animals were sacrificed , the brains were excised and prepared for immunohistochemistry . Control ( Scr ) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells ( single cell invasion ) that eventually co - opted existing blood vessels ( angiotropism ) and formed secondary tumours ( upper panels ) . No tumour growth was observed when MDGI - silenced cells were implanted ( lower panels ) . Glioma cells were visualised by using anti - human vimentin ( hVim red ) and blood vessels using anti - podocalyxin antibodies ( green ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm ( E ) and 50 µm ( F ) .

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Figure 3A. Transmitted light micrographs of MDGI-silenced (shMDGI1 and shMDGI2) and control (Scr) patient-derived BT12 and BT13 spheroids 11 days after gene silencing. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.B. Methylcellulose assay demonstrating the ability for self-renewal of control (Scr) and MDGI-silenced (shMDGI1 and shMDGI2) patient-derived BT12 and BT13 cells after three weeks of culture. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.C. Quantification of the colony size in methylcellulose of MDGI-silenced (sh1 and sh2) cells relative to the controls (Scr) that is set as 100 (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.D. MTT-proliferation assay of control (Scr) and MDGI-silenced (shMDGI1) BT12 and BT13 cells. The values of each cell line were normalized to the values at day 1 marked with a dashed line (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.E, F Representative whole coronal section micrographs of murine brain injected intracranially with control (BT12 Scr, n = 5) and MDGI-silenced (BT12 sh1, n = 5) BT12 cells. After four weeks animals were sacrificed, the brains were excised and prepared for immunohistochemistry. Control (Scr) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells (single cell invasion) that eventually co-opted existing blood vessels (angiotropism) and formed secondary tumours (upper panels). No tumour growth was observed when MDGI-silenced cells were implanted (lower panels). Glioma cells were visualised by using anti-human vimentin (hVim red) and blood vessels using anti-podocalyxin antibodies (green). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm (E) and 50 µm (F).