Figure 5C 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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Figure 5C 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.


Figure 6D 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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Figure 6D 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.


Figure 1D 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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Figure 1D 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.


Figure 2G 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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Figure 2G 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.


Figure 3D 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 . 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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Figure 3D 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.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.


Figure 4Progestin 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 . 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 .

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

Figure 4Progestin 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.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.


Figure 5E 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5E 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. 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%.


Figure 6A - 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 .

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Figure 6A-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.


Figure 7A - 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 . 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 .

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Figure 7A-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.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.


Figure 1 ( 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 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 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 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 ( E ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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 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 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 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 (E(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).(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).


Figure 28 - 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 test8 - 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 test8 - 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 test8 - 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 test8 - 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 test8 - 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 test8 - 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 test8 - 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 test8 - 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 test8 - 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 test8 - 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 test8 - 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

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

Figure 28-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 test8-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 test8-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 test8-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 test8-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 test8-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 test8-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 test8-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 test8-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 test8-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 test8-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 test8-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


Figure 3 ( 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 ( 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 ) . ( 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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 (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).(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 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 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).


Figure 4The 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 ( BThe 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 testThe 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 . 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 testThe 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 testThe 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4The 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 (BThe 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 testThe 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.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 testThe 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 testThe 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).


Figure 5HepG2 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 ( BHepG2 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 ANOVAHepG2 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 ANOVAHepG2 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 ANOVAHepG2 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 ANOVAHepG2 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 ANOVAHepG2 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 ANOVAHepG2 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5HepG2 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 (BHepG2 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 ANOVAHepG2 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 ANOVAHepG2 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 ANOVAHepG2 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 ANOVAHepG2 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 ANOVAHepG2 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 ANOVAHepG2 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).


Figure 66 - 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 ( A6 - 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 ANOVA6 - 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 ANOVA6 - 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 . ( E ) Body weights and liver / body weight ratio . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - 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 . ( F ) Hepatic TG contents . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - 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 . ( G ) Serum AST and ALT levels . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

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

Figure 66-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 (A6-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 ANOVA6-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 ANOVA6-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. (E) Body weights and liver/body weight ratio. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-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. (F) Hepatic TG contents. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-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. (G) Serum AST and ALT levels. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA


Figure 76 - week - old male CD36 knockout mice were administered with AAV - TBG - wildtype - CD36 , wildtype - CD36 plus KLF10 , palmitoylation site mutant CD36 , mutant - CD36 plus KLF10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( A ) Serum AST and ALT levels . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - week - old male CD36 knockout mice were administered with AAV - TBG - wildtype - CD36 , wildtype - CD36 plus KLF10 , palmitoylation site mutant CD36 , mutant - CD36 plus KLF10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( B ) Hepatic TG contents . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - week - old male CD36 knockout mice were administered with AAV - TBG - wildtype - CD36 , wildtype - CD36 plus KLF10 , palmitoylation site mutant CD36 , mutant - CD36 plus KLF10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( C ) Body weights and liver / body weight ratio . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - week - old male CD36 knockout mice were administered with AAV - TBG - wildtype - CD36 , wildtype - CD36 plus KLF10 , palmitoylation site mutant CD36 , mutant - CD36 plus KLF10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( D ) H & E and Sirius red staining of liver sections ( left ) , and quantitation of Sirius red staining ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - week - old male CD36 knockout mice were administered with AAV - TBG - wildtype - CD36 , wildtype - CD36 plus KLF10 , palmitoylation site mutant CD36 , mutant - CD36 plus KLF10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( E ) Immunofluorescence staining of CD36 and β - catenin . ( F ) Quantitation of CD36 positive area . ( G ) Co - localization rate of CD36 and β - catenin . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - week - old male CD36 knockout mice were administered with AAV - TBG - wildtype - CD36 , wildtype - CD36 plus KLF10 , palmitoylation site mutant CD36 , mutant - CD36 plus KLF10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( H ) Protein levels of palmitoylated CD36 in mouse livers ( left ) , and its quantitative results ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - week - old male CD36 knockout mice were administered with AAV - TBG - wildtype - CD36 , wildtype - CD36 plus KLF10 , palmitoylation site mutant CD36 , mutant - CD36 plus KLF10 through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( I ) CD36 protein expression in the plasma membrane fractions , and its normalization to ATP1a1 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

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

Figure 76-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (A) Serum AST and ALT levels. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (B) Hepatic TG contents. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (C) Body weights and liver/body weight ratio. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (D) H&E and Sirius red staining of liver sections (left), and quantitation of Sirius red staining (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (E) Immunofluorescence staining of CD36 and β-catenin. (F) Quantitation of CD36 positive area. (G) Co-localization rate of CD36 and β-catenin. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (H) Protein levels of palmitoylated CD36 in mouse livers (left), and its quantitative results (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (I) CD36 protein expression in the plasma membrane fractions, and its normalization to ATP1a1. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA


Figure 8Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( A - B ) Protein levels of KLF10 , zDHHC7 and TNF - α ( A ) , and their normalization to β - actin ( B ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testAlbumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( C ) Body weights . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testAlbumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( D ) Liver weights and liver / body weight ratio . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testAlbumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( E ) Hepatic TG contents . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testAlbumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( F ) Serum ALT and AST levels . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testAlbumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( G ) H & E staining and Sirius red staining of liver sections . Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( H ) Quantitative results of Sirius red staining . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testAlbumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( I ) Immunofluorescence staining of CD36 and β - catenin . The arrows represent the colocalization of CD36 and β - catenin . Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( J ) Quantitative results of co - localization rate of CD36 and β - catenin . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testAlbumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( K ) Protein levels of palmitoylated CD36 in mouse livers . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testAlbumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( L ) CD36 protein expression in plasma membrane fractions from mouse livers , and its normalization to ATP1a1 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t testAlbumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old male KLF10hep - / - mice and KLF10flox / flox were treated with WD / CCl4 for 12 weeks . KLF10flox / flox mice were used as the control ( Ctrl ) . n = 5 per group . ( M ) Relative mRNA level of genes involved in the liver inflammation and fibrosis . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

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

Figure 8Albumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (A-B) Protein levels of KLF10, zDHHC7 and TNF-α (A), and their normalization to β-actin (B). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t testAlbumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (C) Body weights. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t testAlbumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (D) Liver weights and liver/body weight ratio. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t testAlbumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (E) Hepatic TG contents. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t testAlbumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (F) Serum ALT and AST levels. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t testAlbumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (G) H&E staining and Sirius red staining of liver sections.Albumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (H) Quantitative results of Sirius red staining. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t testAlbumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (I) Immunofluorescence staining of CD36 and β-catenin. The arrows represent the colocalization of CD36 and β-catenin.Albumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (J) Quantitative results of co-localization rate of CD36 and β-catenin. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t testAlbumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (K) Protein levels of palmitoylated CD36 in mouse livers. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t testAlbumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (L) CD36 protein expression in plasma membrane fractions from mouse livers, and its normalization to ATP1a1. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t testAlbumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (M) Relative mRNA level of genes involved in the liver inflammation and fibrosis. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test


Figure 1Representative microscopic views of Ndrg4 + / + and Ndrg4 - / - murine intestinal sections ( n = 4 , 12 months of age ) reveal an even distribution of alkaline phosphatase along the enterocyte brush border , and a similar number and distribution of Paneth cells ( Lysozyme ) in the small intestine ; and neuroendocrine ( Chromogranin A ) , Goblet ( PAS + ) , and proliferating ( Ki67 ) cells in the colon . Scale bars , 50 µm . Representative microscopic views of the myenteric plexus of Ndrg4 + / + and Ndrg4 - / - murine colonic sections labeled with either TuJ1 and S100 ( B , n = 3 ) or HuC / D ( C , n = 6 ) did not reveal structural or organizational differences in the ganglionic network of Ndrg4 + / + and Ndrg4 - / - mice . Scale bars , 100 µm . Quantification of the enteric neuronal cell number ( HuC / D ) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4 + / + and Ndrg4 - / - mice ( n = 6 ) .

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

Figure 1Representative microscopic views of Ndrg4+/+ and Ndrg4-/- murine intestinal sections (n=4, 12 months of age) reveal an even distribution of alkaline phosphatase along the enterocyte brush border, and a similar number and distribution of Paneth cells (Lysozyme) in the small intestine; and neuroendocrine (Chromogranin A), Goblet (PAS+), and proliferating (Ki67) cells in the colon. Scale bars, 50 µm.Representative microscopic views of the myenteric plexus of Ndrg4+/+ and Ndrg4-/- murine colonic sections labeled with either TuJ1 and S100 (B, n=3) or HuC/D (C, n=6) did not reveal structural or organizational differences in the ganglionic network of Ndrg4+/+ and Ndrg4-/- mice. Scale bars, 100 µm. Quantification of the enteric neuronal cell number (HuC/D) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4+/+ and Ndrg4-/- mice (n=6).


Figure 2In contrast , Ndrg4fl / fl and Ndrg4fl / fl - VillinCre mice have a similar bodyweight ( I ) and develop an equal number of adenomas ( J ) with an equivalent diameter ( K , L ) . N = 13 vs . 12 in I , n = 13 vs . 11 in J and n = 10 vs . 8 in K .

Figure 2In contrast, Ndrg4fl/fl and Ndrg4fl/fl-VillinCre mice have a similar bodyweight (I) and develop an equal number of adenomas (J) with an equivalent diameter (K,L). N=13 vs. 12 in I, n=13 vs. 11 in J and n=10 vs. 8 in K.


Figure 3Compared to Ndrg4 + / + ENS - derived medium , addition of medium derived from Ndrg4 - / - ENS cultures enhances the relative growth of intestinal epithelial organoids ( E , IEOs ) and accelerates the formation of new crypt buds per IEO in the course of five days ( F ) .

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Figure 3Compared to Ndrg4+/+ ENS-derived medium, addition of medium derived from Ndrg4-/- ENS cultures enhances the relative growth of intestinal epithelial organoids (E, IEOs) and accelerates the formation of new crypt buds per IEO in the course of five days (F).


Figure 4Box plot showing the quantitative protein expression results of nanoLC - MS / MS analysis reveals the significantly higher presence of Nid1 and Fbln2 in the Ndrg4 - / - compared to the Ndrg4 + / + ENS cell secretome ( n = 4 ; NSAF , normalized spectral abundance factor ) . Data are analyzed with R version 3 . 5 . 2 and the ibb R package . Each dot within the box plot represents the NSAF of an individual sample ; the inside band reflects the median , and the bottom and top of the box the first and third quartile , respectively . The whiskers reflect the minimum and maximal values within 1 . 5 × the interquartile range . NSAF values were compared using the Mann - Whitney U test . Representative immunohistochemistry ( C ) and more detailed immunofluorescence ( D , E ) labeling indicate that Nid1 ( C , D ) and Fbln2 ( C , E ) are expressed within the myenteric plexus and by primary ENS - cells ( ( C ) , black arrowheads , scale bars , 50 µm ; ( D , E ) , n = 3 , scale bars , 10 µm ) .

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

Figure 4Box plot showing the quantitative protein expression results of nanoLC-MS/MS analysis reveals the significantly higher presence of Nid1 and Fbln2 in the Ndrg4-/- compared to the Ndrg4+/+ ENS cell secretome (n=4; NSAF, normalized spectral abundance factor). Data are analyzed with R version 3.5.2 and the ibb R package. Each dot within the box plot represents the NSAF of an individual sample; the inside band reflects the median, and the bottom and top of the box the first and third quartile, respectively. The whiskers reflect the minimum and maximal values within 1.5× the interquartile range. NSAF values were compared using the Mann-Whitney U test.Representative immunohistochemistry (C) and more detailed immunofluorescence (D, E) labeling indicate that Nid1 (C, D) and Fbln2 (C, E) are expressed within the myenteric plexus and by primary ENS-cells ((C), black arrowheads, scale bars, 50 µm; (D, E), n=3, scale bars, 10 µm).


Figure 5Representative microscopic views show that NID1 and FBLN2 are highly expressed in the human colonic ganglia ( Black arrowheads ) and interconnecting nerve fibers , and in a limited level throughout the epithelium ( n = 3 ) . Scale bar , 50 µm . Addition of NID1 & FBLN2 to the Matrigel ® dome significantly enhances the relative growth rate of HIOs after five days of culture as compared to the PBS control condition . Box plot reflecting the quantitative nanoLC - MS / MS analysis results shows the up - regulation of NID1 and FBLN2 in the colorectal cancer tissue secretome ( n = 17 ) compared to the normal colon tissue secretome ( n = 17 ) . Data are analyzed with R version 3 . 5 . 2 and the ibb R package . Within the box plots , each dot represents the log10 transformed raw count of an individual sample ; the inside band is the median value ; the bottom and top of the box the first and third quartile , respectively ; and the whiskers reflect the minimum and maximal values within 1 . 5 × the interquartile range . Representative images of human colonic cancer epithelium ( n = 3 ) shows a high scattered expression of NID1 and FBLN2 within the cancerous epithelium . Scale bar , 50 μm .

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

Figure 5Representative microscopic views show that NID1 and FBLN2 are highly expressed in the human colonic ganglia (Black arrowheads) and interconnecting nerve fibers, and in a limited level throughout the epithelium (n=3). Scale bar, 50 µm.Addition of NID1&FBLN2 to the Matrigel® dome significantly enhances the relative growth rate of HIOs after five days of culture as compared to the PBS control condition.Box plot reflecting the quantitative nanoLC-MS/MS analysis results shows the up-regulation of NID1 and FBLN2 in the colorectal cancer tissue secretome (n=17) compared to the normal colon tissue secretome (n=17). Data are analyzed with R version 3.5.2 and the ibb R package. Within the box plots, each dot represents the log10 transformed raw count of an individual sample; the inside band is the median value; the bottom and top of the box the first and third quartile, respectively; and the whiskers reflect the minimum and maximal values within 1.5× the interquartile range.Representative images of human colonic cancer epithelium (n=3) shows a high scattered expression of NID1 and FBLN2 within the cancerous epithelium. Scale bar, 50 μm.


Figure 1B . Kaplan - Meier curve represents the survival of BALB / c mice over time . Four BALB / c mice in each experimental group were infected i . v with 104 L . monocytogenes wild - type ( EGD - e ) or ΔinlK mutant . C . The L . monocytogenes EGD - e wild - type strain ( WT ) , the ΔinlK mutant ( ΔinlK ) and the complemented strain ( ΔinlK + pPL2 inlK ) 104 CFU were inoculated i . v into BALB / c mice . Animals were euthanized 24 h , 48 h , 72 h or 96 h after infection and organs were recovered , homogenized , and homogenates serially plated on BHI . The number of bacteria able to colonize liver ( left panel ) and spleen ( right panel ) is expressed as log10 CFU . Four animals per bacterial strain , per time points and per experiment were used . Statistical analyses were performed on the results of 3 independent experiments using the Student t test . P values of < 0 . 05 were considered statistically different and are labeled here as * .

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Figure 1B. Kaplan-Meier curve represents the survival of BALB/c mice over time. Four BALB/c mice in each experimental group were infected i.v with 104 L. monocytogenes wild-type (EGD-e) or ΔinlK mutant.C. The L. monocytogenes EGD-e wild-type strain (WT), the ΔinlK mutant (ΔinlK) and the complemented strain (ΔinlK+pPL2 inlK) 104 CFU were inoculated i.v into BALB/c mice. Animals were euthanized 24 h, 48 h, 72 h or 96 h after infection and organs were recovered, homogenized, and homogenates serially plated on BHI. The number of bacteria able to colonize liver (left panel) and spleen (right panel) is expressed as log10 CFU. Four animals per bacterial strain, per time points and per experiment were used. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.