E . Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout . HEK293 wild type or HEK293 NLK knockout were transfected with indicated plasmids ( " EV " stands for " empty vector " ) , wounded areas after 0 24 hr were measured by using TScratch program ( left panel ) . Graphs show of unfilled areas at 24 hr compared to 0 hr , average values from a representative of multiple experiments performed in triplicate ( right - top panel ) .

O O O O O B-CONTROLLED_VAR 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 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

E. Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout. HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ("EV" stands for "empty vector"), and the wounded areas after 0 and 24 hr were measured by using the TScratch program (left panel). Graphs show percentage of unfilled areas at 24 hr compared to 0 hr, and average values from a representative of multiple experiments performed in triplicate (right-top panel).


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

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

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.


relative expression of ADAR1 p150 mRNA ( B ) total ADAR1 mRNA ( C ) in sorted DN , DP , 4SP 8SP thymocytes isolated from WT mice . relative expression of ADAR1 p150 mRNA ( B ) total ADAR1 mRNA ( C ) were normalized to level of expression in DN thymocytes ( DN values set to 1 ) . Values represent relative gene expression normalized to GAPDH mRNA are displayed as mean ± ( n = 5 ; Mann - Whitney U - , * p < 0 . 05 , * * p < 0 . 01 ) .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


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

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 ) CD4 + - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice .

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

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


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

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR 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 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (B).


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

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

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


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

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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).


percentages of DN , DP , 4SP , 8SP thymocytes were compared between WT 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 Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD69 anti - MHC - I antibodies were gated on 4SP thymocytes . Representative are shown ( upper panel ) . percentages of CD69 + MHC - I - ( SM ) , CD69 + MHC - I + ( M1 ) , CD69 - MHC - I - ( M2 ) thymocytes were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 Cd4cre A1fl / fl mice were stained with antibodies against CD5 analyzed by flow cytometry . Representative flow cytometric for DP 4SP thymocytes are shown ( upper panel ) . percentages of CD5 - DP 4SP thymocytes were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD25 anti - Foxp3 antibodies were gated on 4SP thymocytes . Representative are shown ( upper panel ) . percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg ) were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

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

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

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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).


relative expression of anti - apoptotic genes in sorted 4SP thymocytes isolated from WT Cd4cre A1fl / fl mice . Values represent relative gene expression normalized to Actinβ mRNA are displayed as mean ± ( n = 3 ; Mann - Whitney U - , * p < 0 . 05 , N . S . ; not significant ) . mean value for 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of total phosphorylated ZAP70 PLCγ1 expression in anti - CD3 - stimulated thymocytes isolated from WT Cd4cre A1fl / fl mice . expression of GAPDH is shown as a reference . Representative are shown ( left panel ) . band of p - ZAP70 p - PLCγ1 was normalized to that of total ZAP70 PLCγ1 ,

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 ) CD4 + - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice ( n = 11 for each group ;

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

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 of HE of colon dissected from a WT Cd4cre A1fl / fl mouse . Scale bar , 200 μm .

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

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


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

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 Cd4cre A1fl / fl mice were stained with anti - IFNγ anti - IL17 together with anti - CD4 antibodies were gated on CD4 + . Representative flow cytometry are shown ( upper panel ) . percentages of IFNγ - IL17 - secreting CD4 + T were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 Cd4cre A1fl / fl mice were stained with anti - Foxp3 together with anti - CD4 antibodies were gated on CD4 + . Representative flow cytometry are shown ( upper panel ) . percentages of Foxp3 - expressing CD4 + T were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

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

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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%.


editing in 3 ′ UTR ( C ) intronic region ( D ) of each gene indicated in figures with their chromosomal location was validated by direct Sanger sequencing frequency of editing was compared among wild - type ( WT ) DP , WT 4SP thymocytes , 4SP thymocytes isolated from CD4 + - specific Adar1 knockout ( 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O

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.


relative expression of each interferon - stimulated gene ( ISG ) indicated in figure in sorted DP 4SP thymocytes isolated from WT Cd4cre A1fl / fl mice . Values represent relative gene expression normalized to GAPDH mRNA are displayed as mean ± ( n = 3 ; Mann - Whitney U - , * p < 0 . 05 ) . mean value for 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of total phosphorylated STAT1 expression in thymocytes isolated from WT Cd4cre A1fl / fl mice ( n = 2 for each group ) . expression of GAPDH is shown as a reference .

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

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 + - 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 - / - Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 anti - TCRβ antibodies analyzed by flow cytometry . Representative are shown ( left panel ) . of DN , DP , 4SP 8SP thymocytes were compared among Ifih1 - / - ( n = 4 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 5 ) Cd4cre A1fl / fl ( n = 7 ) mice ( right panel ) . same data shown in Fig 2C was used for Cd4cre A1fl / fl mice .

B-MEASURED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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.


relative expression of each interferon - stimulated gene ( ISG ) indicated in figure in sorted 4SP thymocytes isolated from Ifih1 - / - Cd4cre A1fl / fl Ifih1 - / - mice . Values represent relative gene expression normalized to GAPDH mRNA are displayed as mean ± ( n = 3 ; Mann - Whitney U - , * p < 0 . 05 ) . mean value for 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of total phosphorylated ZAP70 PLCγ1 expression in anti - CD3 - stimulated thymocytes isolated from Ifih1 - / - Cd4cre A1fl / fl Ifih1 - / - mice . expression of GAPDH is shown as a reference . Representative are shown ( left panel ) . band of p - ZAP70 p - PLCγ1 was normalized to that of total ZAP70 PLCγ1 , respectively displayed as mean ± ( n = 3 ; Mann - Whitney U - , N . S . ; not significant ) ( right panel ) by setting mean value of stimulated Ifih1 - / - samples at 60 sec as 1 .

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

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

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

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

O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 - / - Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 , anti - CD25 anti - Foxp3 antibodies were gated on 4SP thymocytes . Representative are shown ( upper panel ) . percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg ) were compared among Ifih1 - / - , Cd4cre A1fl / fl Ifih1 - / - Cd4cre A1fl / fl mice ( lower panel ) . same data shown in Fig 2H was used for Cd4cre A1fl / fl mice .

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

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

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

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 ) Cd4cre A1fl / fl Ifih1 - / - ( n = 11 ) mice . For side - by - side comparison with Cd4cre A1fl / fl

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

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

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

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


A Indicated tail - anchored ( TA ) were in vitro translated in rabbit reticulocyte lysate ( RRL ) using RNAs containing a stop codon . Translations were carried out in presence of 2 μM HisTrx or HisTrx - SGTA ( HT - SGTA ) , followed by incubation with HisPur Cobalt resin Bound were eluted at high imidazole , samples resolved by SDS - PAGE results visualised by phosphorimaging . Sec61β 3R carries three Arg residues within Sec61β transmembrane domain ( TMD ) which abolish its hydrophobic character was used as a negative control for SGTA binding . A schematic representation of a TA - is also shown . Open circles indicate TA - selectively bound by HisTrx - SGTA . RAMP4 - stress - associated endoplasmic reticulum 1 ; Syb2 - synaptobrevin 2 ; Synt5 - syntaxin 5 . B As for ( A ) but total translation products ( input ) were resolved by SDS - PAGE results visualised by phosphorimaging . When total products are analysed high amounts of haemoglobin in RRL distort migration of Sec61β its variant .

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

A Indicated tail-anchored (TA) proteins were in vitro translated in rabbit reticulocyte lysate (RRL) using RNAs containing a stop codon. Translations were carried out in the presence of 2 μM HisTrx or HisTrx-SGTA (HT-SGTA), followed by incubation with HisPur Cobalt resin Bound proteins were eluted at high imidazole concentration, samples resolved by SDS-PAGE and results visualised by phosphorimaging. Sec61β 3R carries three Arg residues within Sec61β transmembrane domain (TMD) which abolish its hydrophobic character and was used as a negative control for SGTA binding. A schematic representation of a TA-protein is also shown. Open circles indicate TA-proteins selectively bound by HisTrx-SGTA. RAMP4 - stress-associated endoplasmic reticulum protein 1; Syb2 - synaptobrevin 2; Synt5 - syntaxin 5. B As for (A) but total translation products (input) were resolved by SDS-PAGE and results visualised by phosphorimaging. When total products are analysed high amounts of haemoglobin in the RRL distort the migration of Sec61β and its variant.


RNAs coding for C99 variants containing a stop codon introduced either after complete coding or before predicted TMD were used . E As for ( D ) but total translation products were subjected to immunoprecipation with either anti - C99 antibody ( for full - variants ) or with anti - β - amyloid antibody ( for products lacking TMD ) . A filled square indicates inefficiently translated APP - C99 - TMD immunoprecipitated with anti - β - amyloid antibody .

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

RNAs coding for C99 variants and containing a stop codon introduced either after the complete coding sequence or before the predicted TMD were used. E As for (D) but total translation products were subjected to immunoprecipation with either anti-C99 antibody (for the full-length variants) or with anti-β-amyloid antibody (for products lacking the TMD). A filled square indicates inefficiently translated APP-C99-TMD immunoprecipitated with anti-β-amyloid antibody.


A Full - polypeptides were translated in presence of 2 μM HisTrx or HisTrx - SGTA using RNAs lacking a stop codon either stabilised at ribosome with cycloheximide ( CHX ) or released with puromycin ( puro ) . Reactions were incubated with HisPur Cobalt resin , bound eluted at high imidazole , resolved by SDS - PAGE visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX - treated samples .

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

A Full-length polypeptides were translated in the presence of 2 μM HisTrx or HisTrx-SGTA using RNAs lacking a stop codon and either stabilised at the ribosome with cycloheximide (CHX) or released with puromycin (puro). Reactions were incubated with HisPur Cobalt resin, bound proteins eluted at high imidazole concentration, resolved by SDS-PAGE and visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX-treated samples.


total translation reactions were treated with RNaseA immunoprecipitated with anti - C99

total translation reactions were treated with RNaseA and immunoprecipitated with anti-C99 antibody


Following in vitro translation of indicated in presence of 2 μM HisTrx - SGTA recovery on HisPur Cobalt resin eluted fractions were subjected to RNaseA treatment ( C ) - tRNA " " + tRNA " indicate tRNA - lacking tRNA - bound species , respectively .

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

Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx-SGTA and recovery on HisPur Cobalt resin the eluted fractions were subjected to RNaseA treatment (C) -tRNA" and "+ tRNA" indicate tRNA-lacking and tRNA-bound protein species, respectively.


D Following in vitro translation of indicated in presence of 2 μM HisTrx - SGTA recovery on HisPur Cobalt resin precipitation with hexadecyltrimethylammonium bromide ( CTAB ) ( D ) ; - tRNA " " + tRNA " indicate tRNA - lacking tRNA - bound species , respectively .

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

D Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx-SGTA and recovery on HisPur Cobalt resin precipitation with hexadecyltrimethylammonium bromide (CTAB) (D) ;-tRNA" and "+ tRNA" indicate tRNA-lacking and tRNA-bound protein species, respectively.


B Variants of PPLssKO - C99 were translated in presence of 2 μM HisTrx - SGTA using templates lacking a stop codon terminating either before ( " - TMD " ) or at indicated acid after TMD . Pull - down with HisPur Cobalt resin was carried out as described in Materials Methods , eluted material resolved by SDS - PAGE results visualised by phosphorimaging . tRNA - bound species were verified by treating PPLssKO - C99 variant terminating immediately after TMD ( TMD + 0 ) with RNaseA ( lane 9 ) .

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

B Variants of PPLssKO-C99 were translated in the presence of 2 μM HisTrx-SGTA using templates lacking a stop codon and terminating either before ("-TMD") or at indicated amino acid distance after the TMD. Pull-down with HisPur Cobalt resin was carried out as described in Materials and Methods, eluted material resolved by SDS-PAGE and results visualised by phosphorimaging. tRNA-bound species were verified by treating PPLssKO-C99 variant terminating immediately after the TMD (TMD + 0) with RNaseA (lane 9).


translation reactions were carried out using RNAs coding for indicated TA - lacking a stop codon . Sec61β 3R was used as a control without a functional TMD . Filled circles indicate tRNA - bound TA - species recovered with HisTrx - SGTA .

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 B-CONTROLLED_VAR O

translation reactions were carried out using RNAs coding for the indicated TA-proteins and lacking a stop codon. Sec61β 3R was used as a control protein without a functional TMD. Filled circles indicate tRNA-bound TA-protein species recovered with HisTrx-SGTA.


D Rabbit reticulocyte lysate was left untreated ( " untreat . " ) , incubated with control immobilised antibodies ( " contr . depl . " ) or immunodepleted of Hbs1L , a factor that together with Pelota ABCE1 [ 4 ] , mediates splitting of ribosomes stalled on truncated mRNAs ( see diagram ) . These lysates were used to translate FLAG - tagged Sec61β from an mRNA lacking a stop codon in presence of 2 μM HisTrx or HisTrx - SGTA samples were processed as described for ( B ) .

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

D Rabbit reticulocyte lysate was left untreated ("untreat."), incubated with control immobilised antibodies ("contr. depl.") or immunodepleted of Hbs1L, a factor that together with Pelota and ABCE1 [4], mediates splitting of ribosomes stalled on truncated mRNAs (see diagram). These lysates were used to translate FLAG-tagged Sec61β from an mRNA lacking a stop codon in the presence of 2 μM HisTrx or HisTrx-SGTA and samples were processed as described for (B).


E tRNA - bound species of FLAG - tagged Sec61β from experiment shown in panel ( D ) were quantified expressed relative to value obtained for control - depleted RRL . Shown is mean with standard error of mean for n = 4 biological replicates . Statistical significance was calculated using unpaired t with Welch ' s correction . ns - not significant .

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

E tRNA-bound species of FLAG-tagged Sec61β from experiment shown in panel (D) were quantified and expressed relative to the value obtained for the control-depleted RRL. Shown is the mean with standard error of mean for n=4 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction. ns - not significant.


Sec61β wild - type ( WT ) 3R variant tagged at their N - terminus with FLAG epitope were translated in RRL from RNAs lacking a stop codon . Ribosome - nascent chain complexes ( RNCs ) were recovered using anti - FLAG affinity resin , beads were washed , bound eluted with 3xFLAG peptide RNCs isolated by pelleting through a sucrose cushion Samples were resolved by SDS - PAGE immunoblotted for endogenous rabbit SGTA , FLAG - tagged translation products , of 40S ( Rps3 ) 60S ( Rpl17 ) ribosomal subunits or analysed by phosphorimaging ( 35S ) . 3xFLAG peptide eluted material ( lanes 4 5 ) corresponds to ~ 10 % of sedimented RNCs ( lanes 7 8 ) .

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

Sec61β wild-type (WT) and the 3R variant tagged at their N-terminus with the FLAG epitope were translated in RRL from RNAs lacking a stop codon. Ribosome-nascent chain complexes (RNCs) were recovered using anti-FLAG affinity resin, beads were washed, bound proteins eluted with 3xFLAG peptide and RNCs isolated by pelleting through a sucrose cushion Samples were resolved by SDS-PAGE and immunoblotted for endogenous rabbit SGTA, FLAG-tagged translation products, proteins of the 40S (Rps3) and 60S (Rpl17) ribosomal subunits or analysed by phosphorimaging (35S). The 3xFLAG peptide eluted material (lanes 4 and 5) corresponds to ~10% of the sedimented RNCs (lanes 7 and 8).


A , B PPL - C99 - TMD ( A ) PPL - C99FL ( B ) were in vitro translated in RRL from mRNAs lacking a stop codon in presence of 2 μM HisTrx or HisTrx - SGTA ε - TDBA - Lys - tRNA analogue . Reactions were irradiated with UV to induce photo - cross - linking , RNCs isolated adducts immunoprecipitated with mouse anti - SRP54 , mouse anti - SGTA or a control antibody as described in Materials Methods . Samples were resolved by SDS - PAGE results visualised by phosphorimaging . C - terminal 40 acid residues located in ribosomal exit tunnel are indicated . " x SRP54 " " x HisTrx - SGTA " indicate cross - linking adducts between translated nascent chain endogenous SRP54 or recombinant HisTrx - SGTA , respectively . Arrows indicate unmodified translation products .

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

A, B PPL-C99-TMD (A) and PPL-C99FL (B) were in vitro translated in RRL from mRNAs lacking a stop codon in the presence of 2 μM HisTrx or HisTrx-SGTA and ε-TDBA-Lys-tRNA analogue. Reactions were irradiated with the UV light to induce photo-cross-linking, RNCs isolated and adducts immunoprecipitated with mouse anti-SRP54, mouse anti-SGTA or a control antibody as described in Materials and Methods. Samples were resolved by SDS-PAGE and results visualised by phosphorimaging. C-terminal 40 amino acid residues located in the ribosomal exit tunnel are indicated. "x SRP54" and "x HisTrx-SGTA" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively. Arrows indicate unmodified translation products.


C PPL - C99FL variants lacking a stop codon carrying indicated Lys to Arg substitutions were in vitro translated in presence of 2 μM HisTrx - SGTA . Reactions were processed using chicken anti - SGTA antibody for immunoprecipitation of SGTA adducts . D Cross - linking adducts between ribosome - stalled PPL - C99FL lysine mutants recombinant HisTrx - SGTA shown in panel ( C ) were quantified , any differences in translation efficiency accounted for cross - linking efficiency expressed relative to wild - type ( WT ) . Shown is mean with standard error of mean for n = 3 biological replicates . Statistical significance was calculated using unpaired t with Welch ' s correction .

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

C PPL-C99FL variants lacking a stop codon and carrying the indicated Lys to Arg substitutions were in vitro translated in the presence of 2 μM HisTrx-SGTA. Reactions were processed using chicken anti-SGTA antibody for immunoprecipitation of SGTA adducts. D Cross-linking adducts between ribosome-stalled PPL-C99FL lysine mutants and recombinant HisTrx-SGTA shown in panel (C) were quantified, any differences in translation efficiency accounted for and cross-linking efficiency expressed relative to the wild-type (WT) protein. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.


A , B PPL - C99FL lacking a stop codon was in vitro translated in RRL in presence of canine pancreatic microsomes or buffer control in reactions supplemented with ε - TDBA - Lys - tRNA analogue . Reactions were carried out in presence ( A ) or absence ( B ) of 2 μM HisTrx - SGTA . Samples were subjected to UV - induced photo - cross - linking processed as described for Fig 5 . " x SRP54 " " x HisTrx - SGTA " indicate cross - linking adducts between translated nascent chain endogenous SRP54 or recombinant HisTrx - SGTA , respectively , whilst " endo . SGTA " indicates cross - linking adducts with endogenous SGTA . Cross - linking products between stalled PPL - C99FL SGTA are also indicated with arrowheads , whilst arrows indicate unmodified translation products .

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

A, B PPL-C99FL lacking a stop codon was in vitro translated in RRL in the presence of canine pancreatic microsomes or buffer control in reactions supplemented with ε-TDBA-Lys-tRNA analogue. Reactions were carried out in the presence (A) or absence (B) of 2 μM HisTrx-SGTA. Samples were subjected to UV light-induced photo-cross-linking and processed as described for Fig 5. "x SRP54" and "x HisTrx-SGTA" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively, whilst "endo. SGTA" indicates cross-linking adducts with endogenous SGTA. Cross-linking products between stalled PPL-C99FL and SGTA are also indicated with arrowheads, whilst arrows indicate unmodified translation products.


C , D chemical cross - linking using DSS reagent was carried out . E DSS - mediated HisTrx - SGTA cross - linking efficiency to PPL - C99FL in absence or presence of ER - derived microsomes was quantified . For each repeat , of cross - linked SGTA adduct in absence of microsomes was set as 100 % . Shown is mean with standard error of mean for n = 3 biological replicates . Statistical significance was calculated using unpaired t with Welch ' s correction .

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

C, D chemical cross-linking using DSS reagent was carried out. E DSS-mediated HisTrx-SGTA cross-linking efficiency to PPL-C99FL in the absence or presence of ER-derived microsomes was quantified. For each repeat, intensity of the cross-linked SGTA adduct in the absence of microsomes was set as 100%. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.


A Indicated precursor were translated in vitro using RRL supplemented with 20 μM HA - tagged ubiquitin 2 μM of either HisTrx or HisTrx - SGTA . RNCs were isolated , ubiquitinated nascent chains recovered using anti - HA agarose , samples resolved by SDS - PAGE analysed by phosphorimaging . Ubiquitinated precursor species were assigned on SDS - PAGE electrophoretic mobility are indicated in red . A schematic diagram of used is shown ~ 40 residues of nascent chain located in ribosomal exit tunnel are indicated . " ss " - signal , " TMD " - transmembrane domain .

O 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

A Indicated precursor proteins were translated in vitro using RRL supplemented with 20 μM HA-tagged ubiquitin and 2 μM of either HisTrx or HisTrx-SGTA. RNCs were isolated, ubiquitinated nascent chains recovered using anti-HA agarose, samples resolved by SDS-PAGE and analysed by phosphorimaging. Ubiquitinated precursor protein species were assigned based on SDS-PAGE electrophoretic mobility and are indicated in red. A schematic diagram of the proteins used is shown and the ~40 residues of the nascent chain located in the ribosomal exit tunnel are indicated. "ss" - signal sequence, "TMD" - transmembrane domain.


B , clearly resolved ubiquitinated species of indicated ( labelled 1 to 4 ) were quantified their in HisTrx - SGTA containing samples shown relative to samples supplemented with HisTrx control . Shown is mean with standard error of mean for n = 3 biological replicates . Statistical significance was calculated using unpaired t with Welch ' s correction . ns - not significant .

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

B Individual, clearly resolved ubiquitinated species of the indicated proteins (labelled 1 to 4) were quantified and their intensity in HisTrx-SGTA containing samples shown relative to samples supplemented with HisTrx control protein. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction. ns - not significant.


G Verify mobility of GFP ( control ) mRNA GFP - MdCAX3 mRNA by RT - PCR in Mx leaf , , root . recombinant vectors ( F ) were transformed into A . tumefaciens GV3101 introduced into Mx leaves by vacuum infiltration .

O O O O O O O O O O O O O O O O O O O O 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 Verify the mobility of GFP (control) mRNA and GFP-MdCAX3 mRNA by RT-PCR in Mx leaf, stem, and root. The recombinant vectors (F) were transformed into A. tumefaciens GV3101 and introduced into Mx leaves by vacuum infiltration.


H GFP image of Mx roots under different treatment . Nuclei were labeled using DAPI . Scale Bar : 100 μm .

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

H GFP fluorescence image of the Mx roots under different treatment. Nuclei were labeled using DAPI. Scale Bar: 100 μm.


H ( 488 / 440 ) image of BCECF in roots of MdCAX3 - suppressed in leaves Md / Mx . Scale bars : 100 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

H Ratio (488/440) image of BCECF fluorescence in the roots of MdCAX3-suppressed in leaves Md/Mx. Scale bars: 100 μm.


I Rhizosphere acidification of MdCAX3 - suppressed in leaves Md / Mx . Bromocresol purple was used as a pH indicator for visualization . Scale bars : 1 cm .

O O O O O O 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 Rhizosphere acidification of MdCAX3-suppressed in leaves Md/Mx. Bromocresol purple was used as a pH indicator for visualization. Scale bars: 1 cm.


F GUS in N . benthamiana leaves after transient co - expression of pro35S : : GFP with proMxCXIP1 : GUS or proMbCXIP1 : GUS , co - expression with pBI101 : GUS as a control .

O O O O O O O O O O O O O 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 GUS staining in N. benthamiana leaves after transient co-expression of pro35S::GFP with proMxCXIP1:GUS or proMbCXIP1:GUS, and co-expression with pBI101:GUS as a control.


G Interaction between MdCXIP1 MdCAX3 tested by yeast two - hybrid ( Y2H ) assays . A dilution series of NMY51 yeast co - expressing MdCXIP1 MdCAX3 cultured on SD / II SDIV screening media . pPBT3 - N ( empty vector ) co - expressed with MdCXIP1 was used as a negative control . X - gal was used in SD / - Trp / - Leu / - His / - Ade screening medium to further for possible interactions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Interaction between MdCXIP1 and MdCAX3 tested by yeast two-hybrid (Y2H) assays. A dilution series of NMY51 yeast cells co-expressing MdCXIP1 and MdCAX3 cultured on SD/II and SDIV screening media. pPBT3-N (empty vector) co-expressed with MdCXIP1 was used as a negative control. X-gal was used in SD/-Trp/-Leu/-His/-Ade screening medium to further test for possible interactions.


I Interaction between MdCXIP1 MdCAX3 N - terminal autoinhibitory domain in an in vitro pull - down . His - MdCXIP1 , GST GST - N - MdCAX3 fusion were expressed in Escherichia coli BL21 , GST alone was used as a negative control . ' + ' indicates presence " − " indicates absence . IB , immunoblot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Interaction between MdCXIP1 and the MdCAX3 N-terminal autoinhibitory domain in an in vitro pull-down assay. The His-MdCXIP1, GST and GST-N-MdCAX3 fusion proteins were expressed in the Escherichia coli BL21 strain, and the GST protein alone was used as a negative control. '+' indicates presence and "−" indicates absence. IB, immunoblot.


C ( 488 / 440 nm excitations ) of BCECF in roots of MdCAX3 MdCXIP1 co - overexpressing Mb . central band represents median box ranges showing interquartile range covers central 50 % of data . whiskers showing minimum maximum of data . Three biological replicates ; 3 roots were quantified for each replicate . Asterisks indicate statistically significant differences ( * * * , P < 0 . 001 ; ANOVA , Tukey correction ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescence ratio (488/440 nm excitations) of BCECF fluorescence in the roots of MdCAX3 and MdCXIP1 co-overexpressing Mb. The central band represents the median and the box ranges showing the interquartile range and covers the central 50% of the data. The whiskers showing the minimum and maximum of the data. Three biological replicates; 3 roots were quantified for each replicate. Asterisks indicate statistically significant differences (***, P < 0.001; ANOVA, Tukey correction).


D ( 488 / 440 ) image of BCECF in MdCAX3 MdCXIP1 co - overexpressing Mb . Scale Bar : 100 μm . E Rhizosphere acidification in MdCAX3 MdCXIP1 co - overexpressing Mb . Bromocresol purple was used as a pH indicator for visualization . Scale Bar : 1 cm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Ratio (488/440) image of BCECF fluorescence in MdCAX3 and MdCXIP1 co-overexpressing Mb. Scale Bar: 100 μm. E Rhizosphere acidification in MdCAX3 and MdCXIP1 co-overexpressing Mb. Bromocresol purple was used as a pH indicator for visualization. Scale Bar: 1 cm.


D localization of Zn2 + ( blue ) in Mx MdCAX3 - suppressed Mx roots under Fe deficient conditions . Arrowhead indicates cytoplasm . Scale Bars : 20 μm . E detection at position indicated by long narrow arrow in Fig 6D .

O O O O O O O O O O O O O O O O O O O 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 Cellular localization of Zn2+ (blue) in Mx and MdCAX3-suppressed Mx roots under Fe deficient conditions. Arrowhead indicates the cytoplasm. Scale Bars: 20 μm. E Fluorescence intensity detection at the position indicated by the long and narrow arrow in the Fig 6D.


D Volcano plot showing differentially expressed genes ( adj . P . Val < 0 . 05 ) between ethanol - R5020 - treated mouse mammary organoids , n = 3 . Genes with log2 ( FC ) > 2 are highlighted in red , genes with log2FC < - 2 in blue . Names of genes with - log10 ( adj . P . Val ) > 10 abs ( log2 ( FC ) ) > 2 are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Volcano plot showing differentially expressed genes (adj. P.Val < 0.05) between ethanol- and R5020-treated mouse mammary organoids, n=3. Genes with log2(FC) > 2 are highlighted in red, genes with log2FC < -2 in blue. Names of genes with -log10 (adj.P.Val) > 10 and abs(log2(FC)) > 2 are indicated.


H , I Bar graphs showing relative Rankl Wnt4 transcript normalized to 36B4 expression in C57Bl6 - derived mammary organoids treated for 6 hours with R5020 ( H ) or LNG ( I ) either 10 μM or 100 μM enzalutamide , n = 3 independent experiments .

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

H, I Bar graphs showing relative Rankl and Wnt4 transcript levels normalized to 36B4 expression in C57Bl6-derived mammary organoids treated for 6 hours with R5020 (H) or LNG (I) and either 10 μM or 100 μM enzalutamide, n=3 independent experiments.


G Scatter plot showing genes which are commonly altered upon R5020 stimulation ( adj . P - value < 0 . 05 ) in both mouse human organoids relative to their CTRL samples . Color scale as a function of averaged human mouse logFC values . red dashed lines represent an adj . P - value threshold of 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Scatter plot showing the genes which are commonly altered upon R5020 stimulation (adj. P-value < 0.05) in both mouse and human organoids relative to their CTRL samples. Color scale as a function of the averaged human and mouse logFC values. The red dashed lines represent an adj. P-value threshold of 0.001.


D Stereo of whole mounted mammary 12 after injection of HBECs . Scale bar , 2 mm . Inset , arrows point to mouse distended by presence of HBECs . Scale bar , 0 . 8 mm .

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

D Stereo micrographs of whole mounted mammary glands 12 weeks after injection of HBECs. Scale bar, 2 mm. Inset, arrows point to mouse ducts distended by the presence of HBECs. Scale bar, 0.8 mm.


I Representative co - IF of a cross - sectioned " humanized " mouse milk 5 months after intraductal injection for AR , ERα PR , in red , human E - CAD , in green . Scale bar , 50 μm .

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

I Representative co-IF micrographs of a cross-sectioned "humanized" mouse milk duct 5 months after intraductal injection for AR, ERα and PR, in red, and human E-CAD, in green. Scale bar, 50 μm.


K Bar graphs showing threshold cycles of semi quantitative RT - PCR with human - specific primers on from virgin ( n = 4 ) pregnant ( n = 3 ) mice xenografted with human .

O O O O O O O O O O O O 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 Bar graphs showing the threshold cycles of semi quantitative RT-PCR with human-specific primers on RNA from virgin (n=4) and pregnant (n=3) mice xenografted with human cells.


Progestin plasma in treated mice ( n = 10 , 14 , 18 , 7 , 6 , 10 ) in women ( n = 20 , 11 , 3 , 4 , 6 , 8 ) on hormonal contraceptives measured by LC - MS , shown as means ± SD , as well as values reported in literature ( Cmax ) . active form of DSG , 3 - ketodesogestrel was measured .

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

Progestin plasma levels in treated mice (n=10, 14, 18, 7, 6, 10) and in women (n=20, 11, 3, 4, 6, 8) on hormonal contraceptives measured by LC-MS, shown as means ± SD, as well as values reported in the literature (Cmax). The active form of DSG, 3-ketodesogestrel was measured.


E Representative showing co - IF with anti - KI67 ( red ) , anti - hECAD ( green ) of xenografted milk after 21 days treatment with vehicle or LNG . Scale bar , 50 μm . F Violin plot showing of KI67 hECAD double + of total hECAD + , dots represent sectors counted in 3 different , control ( n = 30 ) or LNG ( n = 28 ) , median ( red ) , statistical significance was assessed by non - parametric Mann - Whitney . G Bar plot showing relative transcript of MKI67 in xenografted from control ( n = 6 ) LNG - treated ( n = 3 ) mice , Student ' s t - .

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

E Representative micrographs showing co-IF with anti-KI67 (red), and anti-hECAD (green) of xenografted milk ducts after 21 days treatment with vehicle or LNG. Scale bar, 50 μm. F Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ cells, dots represent individual sectors counted in 3 different glands, control (n=30) or LNG (n=28), median (red), statistical significance was assessed by non-parametric Mann-Whitney test. G Bar plot showing relative transcript levels of MKI67 in xenografted glands from control (n=6) and LNG-treated (n=3) mice, Student's t-test.


Representative showing co - IF with anti - KI67 ( red ) anti - hECAD ( green ) of histological sections from xenografted with HBECs from mammoplasty M310 exposed to vehicle or progestins . Scale bar , 50 μm . J Violin plot showing of KI67 hECAD double + of total hECAD + in M310 derived HBECs , dots represent sectors counted in different , median ( red ) , CTRL ( n = 180 ) , P4 ( n = 134 ) , DSG ( n = 52 ) , GSN ( n = 161 ) , LNG ( n = 32 ) , CMA ( n = 56 ) , CPA ( n = 42 ) , DSP ( n = 100 ) . Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions using CTRL group as reference .

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

Representative micrographs showing co-IF with anti-KI67 (red) and anti-hECAD (green) of histological sections from glands xenografted with HBECs from mammoplasty M310 exposed to vehicle or progestins. Scale bar, 50 μm. J Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ cells in M310 derived HBECs, dots represent individual sectors counted in different glands, median (red), CTRL (n=180), P4 (n=134), DSG (n=52), GSN (n=161), LNG (n=32), CMA (n=56), CPA (n=42), DSP (n=100). Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions using the CTRL group as reference.


K Violin plot showing of KI67 hECAD double + of total hECAD + HBECs derived from 11 different upon 21 days of CTRL ( n = 360 ) , P4 ( n = 180 ) , DSG ( n = 147 ) , GSN ( n = 298 ) , LNG ( n = 284 ) , CMA ( n = 115 ) , CPA ( n = 156 ) , DSP ( n = 123 ) treatment . Red lines show medians . L Violin plot showing of AR + intraductally - engrafted HBECs derived from 11 different upon 21 days of CTRL ( n = 258 ) , P4 ( n = 97 ) , DSG ( n = 173 ) , GSN ( n = 159 ) , LNG ( n = 168 ) , CMA ( n = 68 ) , CPA ( n = 76 ) , DSP ( n = 135 ) pellets . Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions , with batches as random variables CTRL as reference , median in red . J - L dashed shows 0 % .

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

K Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ HBECs derived from 11 different patients upon 21 days of CTRL (n=360), P4 (n=180), DSG (n=147), GSN (n=298), LNG (n=284), CMA (n=115), CPA (n=156), DSP (n=123) treatment. Red lines show medians. L Violin plot showing percentage of AR+ intraductally-engrafted HBECs derived from 11 different patients upon 21 days of CTRL (n=258), P4 (n=97), DSG (n=173), GSN (n=159), LNG (n=168), CMA (n=68), CPA (n=76), DSP (n=135) pellets. Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions, with batches and patients as random variables and CTRL as reference, median in red. J-L dashed line shows 0%.


A - F of H & E stained tissue sections from mouse mammary engrafted with HBECS exposed to LNG or vehicle for 6 months . In control mice ( A , B ) most have a narrow lumen a thin wall due to limited growth of human ( A ) ; a few mildly dilated exhibit a proliferation made out of multiple small acini burgeoning at external side of their wall ( B , arrows ) . In LNG - treated mice ( C - F ) peripheral acinar proliferation is diffuse , marked in some foci ( C ) , associated with a variable degree of dilatation from mild ( C , arrow ) up to true cysts formation ( D ) . Proliferating human epithelial showing apocrine changes ( e . g . abundant eosinophilic cytoplasm central round nuclei ) partly fill lumen with a architecture of micropapillae ( E ) bridges ( F , arrows ) . Scalebars , 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Micrographs of H&E stained tissue sections from mouse mammary glands engrafted with HBECS and exposed to LNG or vehicle for 6 months. In control mice (A, B) most ducts have a narrow lumen and a thin wall due to limited growth of human cells (A); a few mildly dilated ducts exhibit a cellular proliferation made out of multiple small acini burgeoning at the external side of their wall (B, arrows). In LNG-treated mice (C-F) the peripheral acinar proliferation is diffuse, marked in some foci (C), and associated with a variable degree of duct dilatation from mild (C, arrow) up to true cysts formation (D). Proliferating human epithelial cells showing apocrine changes (e.g. abundant eosinophilic cytoplasm and central round nuclei) partly fill the ductal lumen with a complex architecture of micropapillae (E) and bridges (F, arrows). Scalebars, 50 μm.


A - C Graphs showing in vivo growth of HBECs from different donors as measured by radiance after treatment with LNG , ENZ , or LNG ENZ , means of radiance in ± , n = 8 - 10 per treatment , Wilcoxon matched - pairs . Right : Dot plot showing plasma of progesterone ( P ) , testosterone ( T ) LNG in xenografted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Graphs showing in vivo growth of HBECs from different donors as measured by radiance after treatment with LNG, ENZ, or LNG and ENZ, means of radiance in individual glands ± SEM, n= 8-10 per treatment, Wilcoxon matched-pairs test. Right: Dot plot showing plasma levels of progesterone (P), testosterone (T) and LNG in individual xenografted animals.


D Graph showing in vivo growth of HBECs from a 20 - year - old transduced with either sh scramble or shAR treated with LNG . Points show means of radiance in ± ; n = 8 - 10 per treatment . Wilcoxon matched - pairs .

O O O O O O O O O O O O O O O O O O O O O O O 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 Graph showing in vivo growth of HBECs from a 20-year-old patient transduced with either sh scramble or shAR and treated with LNG. Points show means of radiance in individual glands ± SEM; n= 8-10 per treatment. Wilcoxon matched-pairs test.


E Representative showing co - IF with anti - GFP ( red ) , anti - AR ( green ) antibodies on histological sections from xenografted with HBECs transduced either with sh scramble or shAR - expressing lentivirus . Scale bar , 50 μm . F Violin plot showing of AR - GFP - double + of total GFP + in sh scramble ( n = 6 ) shAR ( n = 12 ) conditions , dots represent sectors counted , median ( red ) . Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions using CTRL as reference . Red shows median .

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

E Representative micrographs showing co-IF with anti-GFP (red), and anti-AR (green) antibodies on histological sections from glands xenografted with HBECs transduced either with sh scramble or shAR-expressing lentivirus. Scale bar, 50 μm. F Violin plot showing the percentage of AR- and GFP-double+ cells of total GFP+ cells in sh scramble (n=6) and shAR (n=12) conditions, dots represent individual sectors counted, median (red). Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions using CTRL as reference. Red line shows median.


( A ) of genes in livers of HFD - feeding induced NAFL mice WD / CCl4 - treatment induced NASH mice . n = 5 . color gradient represents log2 ( Fold change ) .

(A) Heatmap of genes in the livers of HFD-feeding induced NAFL mice and WD/CCl4-treatment induced NASH mice. n=5. The color gradient represents log2(Fold change).


( B ) Relative mRNA of KLF10 in livers from NAFL NASH mice . n = 5 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Student ' s t

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

(B) Relative mRNA levels of KLF10 in the livers from NAFL and NASH mice. n=5. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Student's t test


( C ) of KLF10 examined by western blots ( left ) its expression was normalized to β - actin ( right ) . n = 3 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Student ' s t

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

(C) Protein levels of KLF10 examined by western blots (left) and its expression was normalized to the β-actin (right). n=3. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Student's t test


( D ) Representative immunohistochemistry of KLF10 expression in liver sections from NAFL NASH mice . bar chart showed fold change of KLF10 positive per field ( NASH vs . NAFL ) . n = 5 . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Student ' s t

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

(D) Representative immunohistochemistry of KLF10 protein expression in the liver sections from NAFL and NASH mice. The bar chart showed the fold change of KLF10 positive cells per field (NASH vs. NAFL). n=5. Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Student's t test


( E ) Correlations between KLF10 NAS , inflammation score , fibrosis scores , hepatic triglyceride contents ( TG ) from NAFL mice ( n = 7 ) NASH mice ( n = 8 ) . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Spearman ' s correlation (

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

(E) Correlations between KLF10 and NAS, inflammation score, fibrosis scores, and hepatic triglyceride contents (TG) from NAFL mice (n=7) and NASH mice (n=8). Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Spearman's correlation (E


( F ) H & E , Sirius red , KLF10 immunofluorescence of liver sections from NAFL NASH . n = 3 .

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

(F) H&E staining, Sirius red staining, and KLF10 immunofluorescence staining of liver sections from NAFL and NASH patients. n=3.


( G ) Gene expression in liver tissues from NAFL NASH using GEO datasets ( GSE48452 GSE61260 ) from NCBI database . In dataset GSE48452 , n = 17 for NAFL n = 15 for NASH . In dataset GSE61260 , n = 21 for NAFL n = 24 for NASH . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Student ' s t 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

(G) Gene expression analysis in liver tissues from NAFL and NASH patients using GEO datasets (GSE48452 and GSE61260) from NCBI database. In the dataset GSE48452, n=17 for NAFL patients and n=15 for NASH patients. In the dataset GSE61260, n=21 for NAFL patients and n=24 for NASH patients. Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Student's t test G).


( H ) Spearman correlations between KLF10 expression NAS in liver tissues from NAFL NASH using GEO datasets ( GSE48452 ) . Left panel , n = 32 ( including 17 NAFL 15 NASH ) . Right panel , n = 21 ( including 6 NAFL 15 NASH ) . Data information : * P < 0 . 05 , * * P < 0 . 01 - . Results are shown as mean ± SD . Spearman ' s correlation H ) .

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

(H) Spearman correlations between KLF10 expression and NAS in liver tissues from NAFL and NASH patients using GEO datasets (GSE48452). Left panel, n=32 (including 17 NAFL patients and 15 NASH patients). Right panel, n=21 (including 6 NAFL and 15 NASH patients). Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Spearman's correlation H).


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on WD / CCl4 for 12 . n = 5 per group . ( B ) of KLF10 in livers ( left ) its expression was normalized to β - actin ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (B) Protein levels of KLF10 in the livers (left) and its expression was normalized to the β-actin (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on WD / CCl4 for 12 . n = 5 per group . ( C ) H & E of liver sections .

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

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (C) H&E staining of liver sections.


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on WD / CCl4 for 12 . n = 5 per group . ( D ) CD68 immunofluorescence of liver sections ( left ) its quantitation ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (D) CD68 immunofluorescence staining of liver sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on WD / CCl4 for 12 . n = 5 per group . ( E ) ( BW ) , liver / , serum AST ALT , hepatic TG contents of two groups of mice . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (E) Body weight (BW), liver/body weight ratio, serum AST and ALT levels, and hepatic TG contents of two groups of mice. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on WD / CCl4 for 12 . n = 5 per group . ( F ) Sirius red of liver sections ( left ) its quantitation ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (F) Sirius red staining of liver sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on WD / CCl4 for 12 . n = 5 per group . ( G ) Relative mRNA of genes involved in hepatic inflammation fibrosis . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (G) Relative mRNA levels of genes involved in the hepatic inflammation and fibrosis. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on WD / CCl4 for 12 . n = 5 per group . ( H ) of TNF - α in livers ( left ) its expression was normalized to β - actin ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (H) Protein levels of TNF-α in the livers (left) and its expression was normalized to the β-actin (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on HFD - feeding for 12 . n = 5 per group . ( J ) of KLF10 in livers ( left ) its expression was normalized to β - actin ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (J) Protein levels of KLF10 in the livers (left) and its expression was normalized to β-actin (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on HFD - feeding for 12 . n = 5 per group . ( K ) H & E of liver sections .

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (K) H&E staining of liver sections.


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on HFD - feeding for 12 . n = 5 per group . ( L ) CD68 immunofluorescence of liver sections ( left ) its quantitation ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (L) CD68 immunofluorescence staining of liver sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on HFD - feeding for 12 . n = 5 per group . ( M ) ( BW ) , liver / , serum AST ALT , hepatic TG contents . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (M) Body weight (BW), liver/body weight ratio, serum AST and ALT levels, and hepatic TG contents. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on HFD - feeding for 12 . n = 5 per group . ( N ) Sirius red of liver sections ( left ) its quantitation ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (N) Sirius red staining of liver sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on HFD - feeding for 12 . n = 5 per group . ( O ) Relative mRNA of genes involved in hepatic inflammation fibrosis . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (O) Relative mRNA levels of genes involved in the hepatic inflammation and fibrosis. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


8 - week - old C57BL / 6J mice were administered with AAV - GFP or AAV - Klf10 through tail vein injection , then kept on HFD - feeding for 12 . n = 5 per group . ( P ) of TNF - α in livers ( left ) its expression was normalized to β - actin ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

8-week-old male C57BL/6J mice were administered with AAV-GFP or AAV -Klf10 through tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (P) Protein levels of TNF-α in the livers (left) and its expression was normalized to the β-actin (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


( A ) of genes in mouse hepatocytes transfected with lentivirus vectors expressing KLF10 ( KLF10OE ) or empty vector ( Ctrl ) , P ＜ 0 . 001 . n = 5 per group . color gradient represents log2 ( Fold change ) .

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

(A) Heatmap of genes in mouse primary hepatocytes transfected with lentivirus vectors expressing KLF10 (KLF10OE) or empty vector (Ctrl), P＜0.001. n=5 per group. The color gradient represents log2(Fold change).


( B ) Relative mRNA ( n = 5 ) ( n = 3 ) of zDHHC7 in MPHs expressing KLF10 or empty vector ( Ctrl ) . level of KLF10 or zDHHC7 was normalized to β - actin . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t (

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

(B) Relative mRNA (n=5) and protein (n=3) levels of zDHHC7 in MPHs expressing KLF10 or empty vector (Ctrl). The protein level of KLF10 or zDHHC7 was normalized to β-actin. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test (B


( C - D ) Spearman correlations between KLF10 zDHHC7 in liver tissues from NASH NAFL using GEO datasets ( GSE48452 GSE61260 ) . In dataset GSE61260 ( C ) , n = 83 ( including 38 healthy controls , 21 NAFL 24 NASH ) . In dataset GSE48452 ( D ) , n = 73 ( including 41 healthy controls , 17 NAFL 15 NASH ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Spearman ' s correlation ( C , D ) .

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

(C-D) Spearman correlations between KLF10 and zDHHC7 in liver tissues from NASH and NAFL patients using GEO datasets (GSE48452 and GSE61260). In the dataset GSE61260 (C), n=83 (including 38 healthy controls, 21 NAFL patients and 24 NASH patients). In the dataset GSE48452 (D), n=73 (including 41 healthy controls, 17 NAFL patients and 15 NASH patients). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Spearman's correlation (C, D).


( E ) Relative mRNA of DHHCs family in HepG2 overexpressing KLF10 or empty vector ( ctrl ) . n = 5 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

(E) Relative mRNA levels of DHHCs family in HepG2 cells overexpressing KLF10 or empty vector (ctrl). n=5. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test


( F ) of zDHHC7 in livers of HFD mice or WD / CCl4 mice level of zDHHC7 was normalized to β - actin . n = 5 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

(F) Protein levels of zDHHC7 in the livers of HFD mice or WD/CCl4 mice The protein level of zDHHC7 was normalized to the β-actin. n=5. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test


( H ) transcriptional activity of wild - type or mutant zDHHC7 promoter in HepG2 . n = 3 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

(H) The transcriptional activity of wild-type or mutant zDHHC7 promoter in HepG2 cells. n=3. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test


( I ) immunoprecipitation of KLF10 binding activity at zDHHC7 promoter . Soluble was prepared from livers of WD / CCl4 treatment - induced NASH mice immunoprecipitated with antibodies against KLF10 or against IgG . final extractions were amplified using pairs of primers that cover regions of zDHHC7 GAPDH gene promoters . ( J ) Quantitative real - time RCR results of ChIP assays . IgG , immunoglobulin G . n = 3 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t J ) .

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

(I) Chromatin immunoprecipitation analysis of KLF10 binding activity at the zDHHC7 promoter. Soluble chromatin was prepared from the livers of WD/CCl4 treatment-induced NASH mice and immunoprecipitated with antibodies against KLF10 or against IgG. The final DNA extractions were amplified using pairs of primers that cover the regions of zDHHC7 and GAPDH gene promoters. (J) Quantitative real-time RCR results of ChIP assays. IgG, immunoglobulin G. n=3. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test J).


liver tissues were collected from WD / CCl4 - treated mice expressing KLF10 or control ( A ) Representative immunofluorescence of CD36 β - catenin . arrows represent colocalization of CD36 β - catenin .

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

The liver tissues were collected from WD/CCl4-treated mice expressing KLF10 or control (A) Representative immunofluorescence staining of CD36 and β-catenin. The arrows represent the colocalization of CD36 and β-catenin.


liver tissues were collected from WD / CCl4 - treated mice expressing KLF10 or control ( B ) Quantitative results of CD36 colocalization rate . n = 5 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t (

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

The liver tissues were collected from WD/CCl4-treated mice expressing KLF10 or control (B) Quantitative results of CD36 colocalization rate. n=5. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test (B


liver tissues were collected from WD / CCl4 - treated mice expressing KLF10 or control ( C ) Expression of CD36 in whole livers plasma fractions . were normalized to β - actin or ATP1a1 , respectively . n = 5 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

The liver tissues were collected from WD/CCl4-treated mice expressing KLF10 or control (C) Expression of CD36 in whole livers and plasma membrane fractions. Protein levels were normalized to the β-actin or ATP1a1, respectively. n=5. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test


liver tissues were collected from WD / CCl4 - treated mice expressing KLF10 or control ( D ) of palmitoylated CD36 in mouse livers . ( E ) Quantitative results of CD36 colocalization rate . n = 3 . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t E ,

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

The liver tissues were collected from WD/CCl4-treated mice expressing KLF10 or control (D) Protein levels of palmitoylated CD36 in mouse livers. (E) Quantitative results of CD36 colocalization rate. n=3. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test E,


liver tissues were collected from HFD - fed mice expressing KLF10 or control ( F ) Representative immunofluorescence of CD36 β - catenin . arrows represent colocalization of CD36 β - catenin .

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

The liver tissues were collected from HFD-fed mice expressing KLF10 or control (F) Representative immunofluorescence staining of CD36 and β-catenin. The arrows represent the colocalization of CD36 and β-catenin.


liver tissues were collected from HFD - fed mice expressing KLF10 or control ( G ) Quantitative results of CD36 colocalization rate . Results represent three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

The liver tissues were collected from HFD-fed mice expressing KLF10 or control (G) Quantitative results of CD36 colocalization rate. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test