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

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


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

O O O O O O B-MEASURED_VAR 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

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


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

B-MEASURED_VAR O 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 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 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 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 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.


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

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

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


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

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

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


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

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

O O O B-CONTROLLED_VAR O 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 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 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 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 - / - 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 .

O O O B-CONTROLLED_VAR O 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 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 B-MEASURED_VAR O 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 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 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 - / - 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 .

O O O B-CONTROLLED_VAR O 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 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 O B-MEASURED_VAR 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 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 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 ) and 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 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 images of HE staining of colon dissected from an Ifih1 - / - and a Cd4cre A1fl / fl Ifih1 - / - mouse . Scale bar , 200 μm .

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

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 ) 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 .

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

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

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

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 and immunoprecipitated with anti - C99

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


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 .

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 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

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 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 .

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

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 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 ) .

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

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 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 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 ) .

O O O O O O 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 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-MEASURED_VAR 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

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 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 .

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

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 ) 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 ) .

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

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 ) 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 .

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

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 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 .

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 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 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

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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


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

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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 .

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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 - 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


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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 .

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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 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 .

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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 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 % .

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

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 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 .

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

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

O O 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

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 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 .

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 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

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 ) 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 ) .

(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 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

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

(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 ) 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

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

(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 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

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 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

(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 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 (

O O O O O B-MEASURED_VAR 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

(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 staining , Sirius red staining , and KLF10 immunofluorescence staining of liver sections from NAFL and NASH patients . n = 3 .

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

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


( 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 ) .

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

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

(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 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

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 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

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 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 .

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

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 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

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 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

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 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

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 B-MEASURED_VAR 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 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 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

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

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 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

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

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 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

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 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 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 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

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 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

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 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 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 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

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 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

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 B-MEASURED_VAR 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 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 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

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 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

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 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

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 ) 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 ) .

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

(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 ) 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 (

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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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

(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 ) 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

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 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 ) 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

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

(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 ) 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 ) .

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


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 .

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


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 (

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


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

O 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 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


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 ,

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-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 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,


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 .

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


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

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

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


The liver tissues were collected from HFD - fed mice expressing KLF10 or control ( H ) 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

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 O O O O O

The liver tissues were collected from HFD-fed mice expressing KLF10 or control (H) 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


The liver tissues were collected from HFD - fed mice expressing KLF10 or control ( I ) Protein levels of palmitoylated CD36 in mouse livers . ( J ) 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 J ) .

O O O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (I) Protein levels of palmitoylated CD36 in mouse livers. (J) 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 J).


HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control ( Ctrl ) . n = 3 . Results represent three independent experiments . ( A ) Protein level of KLF10 , and its normalization to β - actin .

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

HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control (Ctrl). n=3. Results represent three independent experiments. (A) Protein level of KLF10, and its normalization to β-actin.


HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control ( Ctrl ) . n = 3 . Results represent three independent experiments . ( B ) Protein levels of palmitoylated CD36 . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . Student ' s t test (

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

HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control (Ctrl). n=3. Results represent three independent experiments. (B) Protein levels of palmitoylated CD36. Data information: ** P < 0.01. Results are shown as mean ± SD. Student's t test (B


HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control ( Ctrl ) . n = 3 . Results represent three independent experiments . ( C ) CD36 protein expression the plasma membrane fractions , and its normalization to ATP1a1 . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . Student ' s t test C ) .

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 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

HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control (Ctrl). n=3. Results represent three independent experiments. (C) CD36 protein expression the plasma membrane fractions, and its normalization to ATP1a1. Data information: ** P < 0.01. Results are shown as mean ± SD. Student's t test C).


HepG2 cells were treated with 2 - BP and / or lentivirus vectors expressing KLF10 . n = 3 . Results represent three independent experiments . ( D ) Protein levels of KLF10 , and its normalization to β - actin . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way

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

HepG2 cells were treated with 2-BP and/or lentivirus vectors expressing KLF10. n=3. Results represent three independent experiments. (D) Protein levels of KLF10, and its normalization to β-actin. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA


HepG2 cells were treated with 2 - BP and / or lentivirus vectors expressing KLF10 . n = 3 . Results represent three independent experiments . ( E ) Protein levels of palmitoylated CD36 . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way

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

HepG2 cells were treated with 2-BP and/or lentivirus vectors expressing KLF10. n=3. Results represent three independent experiments. (E) Protein levels of palmitoylated CD36. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA


HepG2 cells were treated with 2 - BP and / or lentivirus vectors expressing KLF10 . n = 3 . Results represent three independent experiments . ( F ) CD36 protein expression the plasma membrane fractions , and its normalization to ATP1a1 . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way

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

HepG2 cells were treated with 2-BP and/or lentivirus vectors expressing KLF10. n=3. Results represent three independent experiments. (F) CD36 protein expression the plasma membrane fractions, and its normalization to ATP1a1. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA


HepG2 cells were treated with 2 - BP and / or lentivirus vectors expressing KLF10 . n = 3 . Results represent three independent experiments . ( G ) BODIPY staining showing the lipid droplets .

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

HepG2 cells were treated with 2-BP and/or lentivirus vectors expressing KLF10. n=3. Results represent three independent experiments. (G) BODIPY staining showing the lipid droplets.


HepG2 cells were transfected with lentiviruses expressing KLF10 and / or shRNA targeting zDHHC7 . n = 3 independent experiments . ( H ) Protein levels of KLF10 and zDHHC7 , and its normalization to β - actin . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way

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

HepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (H) Protein levels of KLF10 and zDHHC7, and its normalization to β-actin. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA


HepG2 cells were transfected with lentiviruses expressing KLF10 and / or shRNA targeting zDHHC7 . n = 3 independent experiments . ( I ) Protein levels of palmitoylated CD36 . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way

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

HepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (I) Protein levels of palmitoylated CD36. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA


HepG2 cells were transfected with lentiviruses expressing KLF10 and / or shRNA targeting zDHHC7 . n = 3 independent experiments . ( J ) Immunofluorescence staining of CD36 and Na + / K + - ATPase ( left ) , and the quantitation of CD36 colocalization rate ( right ) . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way

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

HepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (J) Immunofluorescence staining of CD36 and Na+/K+-ATPase (left), and the quantitation of CD36 colocalization rate (right). Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA


HepG2 cells were transfected with lentiviruses expressing KLF10 and / or shRNA targeting zDHHC7 . n = 3 independent experiments . ( K ) BODIPY staining for lipid droplets .

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

HepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (K) BODIPY staining for lipid droplets.


HepG2 cells were transfected with lentiviruses expressing KLF10 and / or shRNA targeting zDHHC7 . n = 3 independent experiments . ( L ) Cellular TG contents . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way ANOVA , L ) .

O O O O O O O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O

HepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (L) Cellular TG contents. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA , L).


6 - week - old male CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( A ) Protein levels of KLF10 and CD36 in the liver ( left ) , and their normalization to β - actin ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA (

O O O O O O B-CONTROLLED_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 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 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 O O O O O

6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (A) Protein levels of KLF10 and CD36 in the liver (left), and their normalization to β-actin (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA (A


6 - week - old male CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( B ) Representative immunofluorescence staining of KLF10 and CD36 ( left ) , and the quantitative results ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

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

6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (B) Representative immunofluorescence staining of KLF10 and CD36 (left), and the quantitative results (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA


6 - week - old male CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( C ) H & E and Sirius red staining of liver sections . ( D ) Quantitative results of Sirius red staining . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

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

6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (C) H&E and Sirius red staining of liver sections. (D) Quantitative results of Sirius red staining. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA