H Scatter plots showing the number of cleaved caspase 3 - positive cells / mm2 in TN BC , categorized according to major - or minor - H2AX protein level decrease after chemotherapy .

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H Scatter plots showing the number of cleaved caspase 3-positive cells / mm2 in TN BC, categorized according to major- or minor-H2AX protein level decrease after chemotherapy.


I Scatter plots showing variation of NRF2 histological score in patients with major - H2AX decrease before and after chemotherapy .

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I Scatter plots showing variation of NRF2 histological score in patients with major-H2AX decrease before and after chemotherapy.


J Box plots showing expression levels of representative NRF2 - target genes in TN tumours with minor - or major - H2AX decrease , as indicated . Lines indicate Means and whiskers show 10 - 90 percentiles . ACOT7 : Acyl - CoA Thioesterase 7 ; PGD : Phosphogluconate Dehydrogenase . AHR : Aryl Hydrocarbon Receptor ; JAG1 : Jagged1 . P - values are from 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

J Box plots showing expression levels of representative NRF2-target genes in TN tumours with minor- or major-H2AX decrease, as indicated. Lines indicate Means and whiskers show 10-90 percentiles. ACOT7: Acyl-CoA Thioesterase 7; PGD: Phosphogluconate Dehydrogenase. AHR: Aryl Hydrocarbon Receptor; JAG1: Jagged1. P-values are from t-test.


Immunoblot analysis of ADAR1 p110 , ADAR1 p150 and ADAR2 expression in various organs isolated from wild - type ( WT ) mice . The expression of GAPDH is shown as a reference .

Immunoblot analysis of ADAR1 p110, ADAR1 p150 and ADAR2 expression in various organs isolated from wild-type (WT) mice. The expression of GAPDH is shown as a reference.


The relative expression of ADAR1 p150 mRNA ( B ) and total ADAR1 mRNA ( C ) in sorted DN , DP , 4SP and 8SP thymocytes isolated from WT mice . The relative expression of ADAR1 p150 mRNA ( B ) and total ADAR1 mRNA ( C ) were normalized to the level of expression in DN thymocytes ( DN values set to 1 ) . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 5 ; Mann - Whitney U - 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) were normalized to the level of expression in DN thymocytes (DN values set to 1). Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 5; Mann-Whitney U-test, *p < 0.05, **p < 0.01).


The relative expression of type I interferon - stimulated genes ( ISGs ) in sorted DN , DP , 4SP and 8SP thymocytes isolated from WT mice . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 ) . The relative expression of ISGs was normalized against the level of expression in DN thymocytes ( DN values set to 1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 relative expression of type I interferon-stimulated genes (ISGs) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The relative expression of ISGs was normalized against the level of expression in DN thymocytes (DN values set to 1).


Total number of thymocytes in wild - type ( WT ) and CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice .

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Total number of thymocytes in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice.


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 and anti - CD8 antibodies and analyzed by flow cytometry . Representative images are shown ( B ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (B).


The percentages of DN , DP , 4SP , and 8SP thymocytes were compared between WT and Cd4cre A1fl / fl mice ( C ) .

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The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (C).


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 and anti - TCRβ antibodies and were gated by the high expression of TCRβ ( TCRβhigh ) . Representative images are shown ( D ) .

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

Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and were gated by the high expression of TCRβ (TCRβhigh). Representative images are shown (D).


The percentages of DN , DP , 4SP , and 8SP thymocytes were compared between WT and Cd4cre A1fl / fl mice ( E ) .

The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (E).


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD69 and anti - MHC - I antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD69 + MHC - I - ( SM ) , CD69 + MHC - I + ( M1 ) , and CD69 - MHC - I - ( M2 ) thymocytes were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

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

Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with antibodies against CD5 and analyzed by flow cytometry . Representative flow cytometric images for DP and 4SP thymocytes are shown ( upper panel ) . The percentages of CD5 - DP and 4SP thymocytes were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

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

Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with antibodies against CD5 and analyzed by flow cytometry. Representative flow cytometric images for DP and 4SP thymocytes are shown (upper panel). The percentages of CD5- DP and 4SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD25 and anti - Foxp3 antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg cells ) were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

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

Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared between WT and Cd4cre A1fl/fl mice (lower panel).


CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice were crossed with HY - TCR + mice to generate Cd4cre A1fl / fl HY - TCR + mice . Thymocytes isolated from male ( A ) and female ( B ) HY - TCR + or Cd4cre A1fl / fl HY - TCR + mice were stained with anti - CD4 and anti - CD8 antibodies and analyzed by flow cytometry . Representative images are shown ( left panels ) . The absolute number of DP and 8SP thymocytes were compared between HY - TCR + and Cd4cre A1fl / fl HY - TCR + mice ( right panels ) .

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

CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with HY-TCR+ mice to generate Cd4cre A1fl/fl HY-TCR+ mice. Thymocytes isolated from male (A) and female (B) HY-TCR+ or Cd4cre A1fl/fl HY-TCR+ mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (left panels). The absolute number of DP and 8SP thymocytes were compared between HY-TCR+ and Cd4cre A1fl/fl HY-TCR+ mice (right panels).


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were labeled with Cell Proliferation Dye and then stimulated with plate - bound anti - CD3 and anti - CD28 antibodies for 4 days . Apoptotic ( Annexin V + ) and proliferating thymocytes ( reduced intensity of Proliferation Dye ) were determined by flow cytometry . Representative flow cytometric images are shown ( left panel ) . The percentages of either Annexin V + or Annexin V - proliferating thymocytes were compared between WT ( n = 5 ) and Cd4cre A1fl / fl ( n = 4 ) mice ( right panel ) .

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

Thymocytes isolated from WT and Cd4cre A1fl/fl mice were labeled with Cell Proliferation Dye and then stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 4 days. Apoptotic (Annexin V+) and proliferating thymocytes (reduced intensity of Proliferation Dye) were determined by flow cytometry. Representative flow cytometric images are shown (left panel). The percentages of either Annexin V+ or Annexin V- proliferating thymocytes were compared between WT (n = 5) and Cd4cre A1fl/fl (n = 4) mice (right panel).


The relative expression of anti - apoptotic genes in sorted 4SP thymocytes isolated from WT and Cd4cre A1fl / fl mice . Values represent the relative gene expression normalized to Actinβ mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 , N . S . ; not significant ) . The mean value for the relative expression of each anti - apoptotic gene in WT 4SP thymocytes was set as one .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 relative expression of anti-apoptotic genes in sorted 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to Actinβ mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05, N.S.; not significant). The mean value for the relative expression of each anti-apoptotic gene in WT 4SP thymocytes was set as one.


Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti - CD3 - stimulated thymocytes isolated from WT and Cd4cre A1fl / fl mice . The expression of GAPDH is shown as a reference . Representative images are shown ( left panel ) . The band intensity of p - ZAP70 and p - PLCγ1 was normalized to that of total ZAP70 and PLCγ1 ,

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

Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from WT and Cd4cre A1fl/fl mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively


Incidence of spontaneous colitis observed in wild - type ( WT ) and CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice ( n = 11 for each group ;

Incidence of spontaneous colitis observed in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice (n = 11 for each group;


Representative images of HE staining of the colon dissected from a WT and Cd4cre A1fl / fl mouse . Scale bar , 200 μm .

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Representative images of HE staining of the colon dissected from a WT and Cd4cre A1fl/fl mouse. Scale bar, 200 μm.


Lymphocytes isolated from the colonic lamina propria of WT and Cd4cre A1fl / fl mice were stained with anti - IFNγ , anti - IL17 or anti - Foxp3 antibodies together with anti - CD4 antibodies and analyzed by flow cytometry . Representative images are shown ( left panel ) . The absolute number of IFNγ - , IL17 - , or Foxp3 - expressing CD4 + T cells were compared between WT and Cd4cre A1fl / fl mice ( right panel ) .

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

Lymphocytes isolated from the colonic lamina propria of WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ, anti-IL17 or anti-Foxp3 antibodies together with anti-CD4 antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The absolute number of IFNγ-, IL17-, or Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (right panel).


Splenocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - IFNγ and anti - IL17 together with anti - CD4 antibodies and were gated on CD4 + cells . Representative flow cytometry images are shown ( upper panel ) . The percentages of IFNγ - and IL17 - secreting CD4 + T cells were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

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

Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Splenocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - Foxp3 together with anti - CD4 antibodies and were gated on CD4 + cells . Representative flow cytometry images are shown ( upper panel ) . The percentages of Foxp3 - expressing CD4 + T cells were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

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

Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-Foxp3 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Effector T ( Teff ) cells isolated from the spleen of a WT mouse were stimulated with soluble anti - CD3 ( 5 μg / ml ) in the presence of mitomycin C - treated splenocytes and were cultured with regulatory T ( Treg ) cells isolated from WT and Cd4cre A1fl / fl mice at the indicated ratios . After 72 h , the proliferative activity was measured . Values are displayed as the mean ± SEM ( n = 3 for each group ; Mann - Whitney U - test , N . S . ; not significant ) . The mean value for the suppression of proliferative activity of Teff cells in a condition without Treg cells was set as 0 % .

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

Effector T (Teff) cells isolated from the spleen of a WT mouse were stimulated with soluble anti-CD3 (5 μg/ml) in the presence of mitomycin C-treated splenocytes and were cultured with regulatory T (Treg) cells isolated from WT and Cd4cre A1fl/fl mice at the indicated ratios. After 72 h, the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, N.S.; not significant). The mean value for the suppression of proliferative activity of Teff cells in a condition without Treg cells was set as 0%.


RNA editing in the 3 ′ UTR ( C ) and intronic region ( D ) of each gene indicated in the figures with their chromosomal location was validated by direct Sanger sequencing and the frequency of editing was compared among wild - type ( WT ) DP , WT 4SP thymocytes , and 4SP thymocytes isolated from CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice .

RNA editing in the 3′UTR (C) and intronic region (D) of each gene indicated in the figures with their chromosomal location was validated by direct Sanger sequencing and the frequency of editing was compared among wild-type (WT) DP, WT 4SP thymocytes, and 4SP thymocytes isolated from CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice.


The relative expression of each interferon - stimulated gene ( ISG ) indicated in the figure in sorted DP and 4SP thymocytes isolated from WT and Cd4cre A1fl / fl mice . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 ) . The mean value for the relative expression of each ISG in WT DP thymocytes was set as one .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted DP and 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in WT DP thymocytes was set as one.


Immunoblot analysis of total and phosphorylated STAT1 expression in thymocytes isolated from WT and Cd4cre A1fl / fl mice ( n = 2 for each group ) . The expression of GAPDH is shown as a reference .

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

Immunoblot analysis of total and phosphorylated STAT1 expression in thymocytes isolated from WT and Cd4cre A1fl/fl mice (n = 2 for each group). The expression of GAPDH is shown as a reference.


CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice were crossed with MDA5 knockout ( Ifih1 - / - ) mice to generate Cd4cre A1fl / fl Ifih1 - / - mice . Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 and anti - TCRβ antibodies and analyzed by flow cytometry . Representative images are shown ( left panel ) . The percentage of DN , DP , 4SP and 8SP thymocytes were compared among Ifih1 - / - ( n = 4 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 5 ) and Cd4cre A1fl / fl ( n = 7 ) mice ( right panel ) . The same data shown in Fig 2C was used for Cd4cre A1fl / fl mice .

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

CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with MDA5 knockout (Ifih1-/-) mice to generate Cd4cre A1fl/fl Ifih1-/- mice. Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The percentage of DN, DP, 4SP and 8SP thymocytes were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl (n = 7) mice (right panel). The same data shown in Fig 2C was used for Cd4cre A1fl/fl mice.


The relative expression of each interferon - stimulated gene ( ISG ) indicated in the figure in sorted 4SP thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 ) . The mean value for the relative expression of each ISG in Ifih1 - / - 4SP thymocytes was set as one .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted 4SP thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in Ifih1-/- 4SP thymocytes was set as one.


Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti - CD3 - stimulated thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice . The expression of GAPDH is shown as a reference . Representative images are shown ( left panel ) . The band intensity of p - ZAP70 and p - PLCγ1 was normalized to that of total ZAP70 and PLCγ1 , respectively and displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , N . S . ; not significant ) ( right panel ) by setting the mean value of stimulated Ifih1 - / - samples at 60 sec as 1 .

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

Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively and displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, N.S.; not significant) (right panel) by setting the mean value of stimulated Ifih1-/- samples at 60 sec as 1.


Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stimulated with plate - bound anti - CD3 ( 5 μg / ml ) and anti - CD28 ( 1 μg / ml ) antibodies for 72 h and the proliferative activity was measured . Values are displayed as the mean ± SEM ( n = 3 for each group ; Mann - Whitney U - test , * p < 0 . 05 ) . The mean value for the activity of Ifih1 - / - thymocytes in an unstimulated condition was set as one . For comparative analysis , the same data shown in Appendix Fig S1B was used for Cd4cre A1fl / fl mice .

Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stimulated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 72 h and the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05). The mean value for the activity of Ifih1-/- thymocytes in an unstimulated condition was set as one. For comparative analysis, the same data shown in Appendix Fig S1B was used for Cd4cre A1fl/fl mice.


Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 , anti - CD69 and anti - MHC - I antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD69 + MHC - I - ( SM ) , CD69 + MHC - I + ( M1 ) , and CD69 - MHC - I - ( M2 ) thymocytes were compared among Ifih1 - / - ( n = 5 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 3 ) and Cd4cre A1fl / fl ( n = 3 ) mice ( lower panel ) . The same data shown in Fig 2F was used for Cd4cre A1fl / fl mice .

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

Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared among Ifih1-/- (n = 5), Cd4cre A1fl/fl Ifih1-/- (n = 3) and Cd4cre A1fl/fl (n = 3) mice (lower panel). The same data shown in Fig 2F was used for Cd4cre A1fl/fl mice.


Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 , anti - CD25 and anti - Foxp3 antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg cells ) were compared among Ifih1 - / - , Cd4cre A1fl / fl Ifih1 - / - and Cd4cre A1fl / fl mice ( lower panel ) . The same data shown in Fig 2H was used for Cd4cre A1fl / fl mice .

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Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared among Ifih1-/-, Cd4cre A1fl/fl Ifih1-/- and Cd4cre A1fl/fl mice (lower panel). The same data shown in Fig 2H was used for Cd4cre A1fl/fl mice.


Splenocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - IFNγ and anti - IL17 together with anti - CD4 antibodies and were gated on CD4 + cells . Representative flow cytometry images are shown ( left panel ) . The percentages of IFNγ - and IL17 - secreting CD4 + T cells were compared among Ifih1 - / - ( n = 4 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 5 ) and Cd4cre A1fl / fl mice ( n = 6 ) ( right panel ) . The same data shown in Fig 4E was used for Cd4cre A1fl / fl mice .

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Splenocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (left panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl mice (n = 6) (right panel). The same data shown in Fig 4E was used for Cd4cre A1fl/fl mice.


No incidence of spontaneous colitis observed in Ifih1 - / - ( n = 5 ) and Cd4cre A1fl / fl Ifih1 - / - ( n = 11 ) mice . For side - by - side comparison with Cd4cre A1fl / fl

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No incidence of spontaneous colitis observed in Ifih1-/- (n = 5) and Cd4cre A1fl/fl Ifih1-/- (n = 11) mice. For side-by-side comparison with Cd4cre A1fl/fl mice


Representative images of HE staining of colon dissected from an Ifih1 - / - and a Cd4cre A1fl / fl Ifih1 - / - mouse . Scale bar , 200 μm .

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Representative images of HE staining of colon dissected from an Ifih1-/- and a Cd4cre A1fl/fl Ifih1-/- mouse. Scale bar, 200 μm.


( B ) Whole mount low magnification images show marking in interscale ( K10 + ) vs scale regions ,

(B) Whole mount low magnification images show marking in interscale (K10+) vs scale regions,


C ) interscale ( K10 + ) vs scale regions , quantified in ( C ) . Error bars are SDs . The data shown is average of 4 different timepoints ( 2 week , 3month , 6month and 1 year ) each consisting of 2 mice and 5 images per mice . p - value is calculated for difference between scale vs interscale using Student ' s t - test ( mixed Anova ) .

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C) interscale (K10+) vs scale regions, quantified in (C). Error bars are SDs. The data shown is average of 4 different timepoints (2 week, 3month, 6month and 1 year) each consisting of 2 mice and 5 images per mice. p-value is calculated for difference between scale vs interscale using Student's t-test (mixed Anova).


( D ) Tail skin sections immunofluorescence stained for interscale ( K10 ) or scale ( K31 ) supra - basal markers , and basal markers . Scale bar = 20 µm or 50 µm as indicated on the panels . Asterisks indicate that photoshop was used to erase the autoflourescent cornified layer . Dashed lines indicate ROI in basement membrane .

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(D) Tail skin sections immunofluorescence stained for interscale (K10) or scale (K31) supra-basal markers, and basal markers. Scale bar =20 µm or 50 µm as indicated on the panels. Asterisks indicate that photoshop was used to erase the autoflourescent cornified layer. Dashed lines indicate ROI in basement membrane.


( E ) Quantification of images like those in ( D ) with n = 3 - 4 mice and 6 - 7 images per mouse showing preferred - gene enrichment . Error bars are SDs . P - values calculated by Student ' s t - test ( mixed effect model )

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(E) Quantification of images like those in (D) with n=3-4 mice and 6-7 images per mouse showing preferred-gene enrichment. Error bars are SDs. P-values calculated by Student's t-test (mixed effect model)


( A - F ) Human skin immunofluorescence images of mouse basal non - LRC / LRC preferred - factor staining in rete ridges ( RR ) versus inter - ridges ( IR ) . Representative images from 30 samples of different body regions and ages Scale bars are indicated on the

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(A-F) Human skin immunofluorescence images of mouse basal non-LRC/LRC preferred-factor staining in rete ridges (RR) versus inter-ridges (IR). Representative images from 30 samples of different body regions and ages Scale bars are indicated on the panels


( G ) LRC / non - LRC preferred gene expression in RR and IR samples from two 30 - year - old breast samples , quantified using background subtraction P - values from paired Student ' s t - test . Error bars are SDs . 8 - 10 images were quantified each from two females .

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(G) LRC/non-LRC preferred gene expression in RR and IR samples from two 30-year-old breast samples, quantified using background subtraction P-values from paired Student's t-test. Error bars are SDs. 8-10 images were quantified each from two females.


( A ) Schematic showing the tamoxifen injection paradigm for tdTomato - labelling of all IFE lineages in tail and back . Co - expression of markers with the lineage - traced cells at 1 - month post - tamoxifen . Dlx1 - CrER is shown as example in tail ; Scale bar = 50 µm ( B ) Quantification of images like those in ( A ) showing % tdTomato + cells expressing indicated markers . Sox6 ( black bar ) was not analyzed in K14 - Cre / EYFP lineage . Error bars are SDs . P - values calculated by Student ' s t - test ( mixed effects model ) from n = 2 - 3 mice and 5 - 6 images per replicate .

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(A) Schematic showing the tamoxifen injection paradigm for tdTomato-labelling of all IFE lineages in tail and back. Co-expression of markers with the lineage-traced cells at 1-month post-tamoxifen. Dlx1-CrER is shown as example in tail; Scale bar =50 µm (B) Quantification of images like those in (A) showing % tdTomato+ cells expressing indicated markers. Sox6 (black bar) was not analyzed in K14-Cre/EYFP lineage. Error bars are SDs. P-values calculated by Student's t-test (mixed effects model) from n=2-3 mice and 5-6 images per replicate.


( G ) Immunofluorescence images of Sox6 staining in noUVB ( 0hr ) or UVB - exposed ( 6hrs - or 24hrs - post ) mouse back skin . ( H ) Boxplot shows % Sox6 expressing basal cells quantified from images like those in ( G ) . Central band is the mean value , the top of box indicates 25th percentile while the bottom indicates 75th percentile . Whiskers indicate minimum and maximum values in the data , with dots indicating potential outliers . Linear mixed model statistical test was used with data from 18 images from 3 biological replicates .

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(G) Immunofluorescence images of Sox6 staining in noUVB (0hr) or UVB-exposed (6hrs- or 24hrs-post) mouse back skin. (H) Boxplot shows % Sox6 expressing basal cells quantified from images like those in (G). Central band is the mean value, the top of box indicates 25th percentile while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 images from 3 biological replicates.


( B - G ) ( B , D , F ) Images of tail skin immune - stained for markers indicated and ( C , E , G ) corresponding box plots . Central band is the mean value , the top of box indicates 25th percentile , while the bottom indicates 75th percentile . Whiskers indicate minimum and maximum values in the data , with dots indicating potential outliers . Linear mixed model statistical test was used with data from 18 total images from 3 biological replicates .

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(B-G) (B,D,F) Images of tail skin immune-stained for markers indicated and (C,E,G) corresponding box plots. Central band is the mean value, the top of box indicates 25th percentile, while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 total images from 3 biological replicates.


( I - N ) ( I , K , M ) Images of skin immune - stained for markers indicated and ( J , L , N ) corresponding box plots . Central band is the mean value , the top of box indicates 25th percentile , while the bottom indicates 75th percentile . Whiskers indicate minimum and maximum values in the data , with dots indicating potential outliers . Linear mixed model statistical test was used with data from 18 images from 3 biological replicates .

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(I-N) (I,K,M) Images of skin immune-stained for markers indicated and (J,L,N) corresponding box plots. Central band is the mean value, the top of box indicates 25th percentile, while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 images from 3 biological replicates.


BN - PAGE of 20 µg solubilized BHM with several of the distinct complexes annotated . The dashed box indicates the monomeric ATP synthase ( complex V ) band subjected to IGX - MS .

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BN-PAGE of 20 µg solubilized BHM with several of the distinct complexes annotated. The dashed box indicates the monomeric ATP synthase (complex V) band subjected to IGX-MS.


D . ( Top ) Relative enrichment of the three microRNAs across different cell types . Quantitative expression of microRNAs in primary hippocampal neurons , primary astrocytes and primary microglia . miR - 146a - 5p is significantly enriched in microglia , while miR - 148a - 3p and miR - 181a - 5p are significantly enriched in neurons . N = 5 / group , Two - way ANOVA , Tukey ' s multiple comparisons test , * P < 0 . 05 , * * P < 0 . 01 . Bars and error bar indicates mean ± sem . ( Bottom ) miRNA expression in different cell types of mouse brain stem . The data was retrieved from Hoye et al . 2017 .

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D. (Top) Relative enrichment of the three microRNAs across different cell types. Quantitative expression of microRNAs in primary hippocampal neurons, primary astrocytes and primary microglia. miR-146a-5p is significantly enriched in microglia, while miR-148a-3p and miR-181a-5p are significantly enriched in neurons. N = 5/group, Two-way ANOVA, Tukey's multiple comparisons test, *P<0.05, **P<0.01. Bars and error bar indicates mean ± sem. (Bottom) miRNA expression in different cell types of mouse brain stem. The data was retrieved from Hoye et al. 2017.


E . Overexpression of microRNAs in relevant cell types for 48 hours followed by genome wide RNA - seq analysis . miR - 181a - 5p was overexpressed in primary hippocampal neurons , while miR - 146a - 5p was overexpressed in microglia culture . Immortalized microglial cell line was used for this purpose . Given that miR - 148a - 3p was highly enriched in neurons ( D ) , primary hippocampal neurons were treated with the corresponding miR - 148a - 3p mimic . PCA plot shows that the mimic and control treated samples cluster distinguishingly separate from one another . Volcano plot displays the genes significantly deregulated in mimic treated samples compared to control samples ( FDR < 0 . 05 ) . Red color indicates the up - regulated genes while the blue color represents the genes those were down - regulated .

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E. Overexpression of microRNAs in relevant cell types for 48 hours followed by genome wide RNA-seq analysis. miR-181a-5p was overexpressed in primary hippocampal neurons, while miR-146a-5p was overexpressed in microglia culture. Immortalized microglial cell line was used for this purpose. Given that miR-148a-3p was highly enriched in neurons (D), primary hippocampal neurons were treated with the corresponding miR-148a-3p mimic. PCA plot shows that the mimic and control treated samples cluster distinguishingly separate from one another. Volcano plot displays the genes significantly deregulated in mimic treated samples compared to control samples (FDR <0.05). Red color indicates the up-regulated genes while the blue color represents the genes those were down-regulated.


H . qPCR analysis confirms the over - expression of pro - inflammation related genes ( IL - 1ß , IL - 6 , TNF - alpha ) due to overexpression of miR - 146a - 5p . Expression of anti - inflammatory gene , IL - 10 was downregulated in mimic treated cells compared to the controls . Unpaired t - tests , two - tailed , * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 . Bars and error bar indicates mean ± sem . Number of biological replicates : 5 - 6 / group .

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H. qPCR analysis confirms the over-expression of pro-inflammation related genes (IL-1ß, IL-6, TNF-alpha) due to overexpression of miR-146a-5p. Expression of anti-inflammatory gene, IL-10 was downregulated in mimic treated cells compared to the controls. Unpaired t-tests, two-tailed, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Bars and error bar indicates mean ± sem. Number of biological replicates: 5-6/group.


B . Functional mature synapses were quantified via co - localizations of pre - ( Synatophysin 1 ) and the post - synaptic ( PSD - 95 ) markers and compared between 3 - miR - mix and control groups . Scale bar : 10 μm . Two independent methods ( SynQuant and Colocalization ) were used for quantification . 3 - miR - mix reduced the number of functional synapses compared to controls ( n = 24 - 30 images )

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B. Functional mature synapses were quantified via co-localizations of pre- (Synatophysin 1) and the post-synaptic (PSD-95) markers and compared between 3-miR-mix and control groups. Scale bar: 10 μm. Two independent methods (SynQuant and Colocalization) were used for quantification. 3-miR-mix reduced the number of functional synapses compared to controls (n = 24-30 images)


C . Dendrite labeling and quantification . Dendritic spines were stained with Dil . Scale bar : 10 μm . Spine density and total spine length are substantially reduced in 3 - miR mix treated primary neurons compared to those treated with scramble RNA ( n = 49 - 97 images )

C. Dendrite labeling and quantification. Dendritic spines were stained with Dil. Scale bar: 10 μm. Spine density and total spine length are substantially reduced in 3-miR mix treated primary neurons compared to those treated with scramble RNA (n = 49-97 images)


All data have been adjusted for age , gender and other latent covariates for downstream eigenvalue calculation . A . Eigen value showing the increased expression of the 3 - microRNA signature in blood plasma samples of age matched MCI ( n = 23 ) patients compared to controls ( n = 27 ) . B . Expression of 3 - microRNA signature is increased in PAXgene blood samples of MCI patients ( n = 71 ) compared to controls ( n = 65 ) from the DELCODE cohort .

All data have been adjusted for age, gender and other latent covariates for downstream eigenvalue calculation. A. Eigen value showing the increased expression of the 3-microRNA signature in blood plasma samples of age matched MCI (n = 23) patients compared to controls (n = 27). B. Expression of 3-microRNA signature is increased in PAXgene blood samples of MCI patients (n=71) compared to controls (n=65) from the DELCODE cohort.


E . Eigen value showing the expression the 3 - microRNA signature in MCI patients for those the follow up diagnostics data assessed 2 years after were available . 15 % of these MCI patients developed Alzheimer ´ s disease ( AD ) , while the rest 85 % patients remained with MCI ( stable MCI ) . The boxplot depicts the increased expression levels of 3 - miR signature in patients who converted to AD ( n = 8 ) compared to those that had stable MCI ( n = 47 ) . F . Increased expression of 3 - microRNA signature in cerebrospinal fluid ( CSF ) of MCI patients ( n = 9 ) compared to controls ( n = 26 ) . Wilcoxon - rank test , P value is given on the corresponding panel . In the boxplots in ( A , B , E , F ) , the center line indicates the median , while the upper and lower lines represent the 75th and 25th percentiles , respectively . The whiskers represent the smallest and largest values in the 1 . 5 × interquartile range .

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E. Eigen value showing the expression the 3-microRNA signature in MCI patients for those the follow up diagnostics data assessed 2 years after were available. 15% of these MCI patients developed Alzheimer´s disease (AD), while the rest 85% patients remained with MCI (stable MCI). The boxplot depicts the increased expression levels of 3-miR signature in patients who converted to AD (n=8) compared to those that had stable MCI (n=47). F. Increased expression of 3-microRNA signature in cerebrospinal fluid (CSF) of MCI patients (n = 9) compared to controls (n = 26). Wilcoxon-rank test, P value is given on the corresponding panel. In the boxplots in (A, B, E, F), the center line indicates the median, while the upper and lower lines represent the 75th and 25th percentiles, respectively. The whiskers represent the smallest and largest values in the 1.5× interquartile range.


H . Escape latency during the water maze training comparing 7 months old wild type mice ( WT - control , n = 17 , male : 9 , female : 8 ) and APPPS - 21 mice ( APP - control , n = 8 , male : 6 , female : 2 ) injected with scramble control oligonucleotides and APPPS1 - 21 mice injected with microRNA inhibitors ( APP miR - inhibtor mix , n = 12 , male : 8 , female : 4 ) . Bars and error bars indicate mean ± sem . .

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H. Escape latency during the water maze training comparing 7 months old wild type mice (WT-control, n = 17, male: 9, female: 8) and APPPS-21 mice (APP-control, n = 8, male: 6, female: 2) injected with scramble control oligonucleotides and APPPS1-21 mice injected with microRNA inhibitors (APP miR-inhibtor mix, n = 12, male: 8, female: 4). Bars and error bars indicate mean ± sem..


D . qPCR assay for several synaptic genes ( LRKK2 , Cadm3 and Slc6a11 ) confirms reinstatement of gene expression with anti - miR - mix ( n = 5 - 7 , Kruskal - wallis test ) .

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D. qPCR assay for several synaptic genes (LRKK2, Cadm3 and Slc6a11) confirms reinstatement of gene expression with anti-miR-mix (n = 5-7, Kruskal-wallis test).


G . qPCR data shows rescue of AFF2 / FMR2 and Hivep3 expression in APP / PS1 mice treated with inhibitor cocktail ( n = 6 - 7 , Kruskal - wallis test ) .

G. qPCR data shows rescue of AFF2/FMR2 and Hivep3 expression in APP/PS1 mice treated with inhibitor cocktail (n = 6-7, Kruskal-wallis test).


a , 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 .

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a, 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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.


a , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

O O O O O O O O O O O O O O O O O O O O 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, 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 .

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

c, 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

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

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 .

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

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 .

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

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 .

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

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 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

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.


c , 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 .

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

c, 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 .

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

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.


c , 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 .

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

c, 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 .

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 .

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

e 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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.


a , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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.


a 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 .

a 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

c, 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 .

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

e, 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.


a , 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

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 .

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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


B . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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


A - 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 ' ) .

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


B . 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 .

B. 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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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.


F . Dot plot of specific gene expression in amnion clusters .

F. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) .

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).


D . Dot plot of specific gene expression in chorion clusters .

D. 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 ) .

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

E. 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


D . Dot plot of specific gene expression in allantois clusters .

D. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O 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

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 .

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

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 .

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

E 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 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

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).


A 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 ) .

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

A 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 .

O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 ) .

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

E 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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).


A - 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 ) .

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

A-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 ) .

O O 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 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

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 ) .

O O 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 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

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 ) .

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

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).


A 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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).