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

B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O

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


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

B-CELL_LINE O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O

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


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

B-CELL_LINE O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (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 ANOVA


6 - week - old CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , then kept on WD / CCl4 for 12 . 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

O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


6 - week - old CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , then kept on WD / CCl4 for 12 . n = 5 per group . ( G ) Serum AST ALT . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (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


6 - week - old 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 , then kept on WD / CCl4 for 12 . n = 5 per group . ( A ) Serum AST ALT . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

O O O O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

6-week-old male CD36 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 ANOVA


6 - week - old 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 , then kept on WD / CCl4 for 12 . 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

O O O O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

6-week-old male CD36 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 ANOVA


6 - week - old 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 , then kept on WD / CCl4 for 12 . n = 5 per group . ( C ) weights liver / . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

O O O O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

6-week-old male CD36 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 ANOVA


6 - week - old 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 , then kept on WD / CCl4 for 12 . n = 5 per group . ( D ) H & E Sirius red of liver sections ( left ) , quantitation of Sirius red ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

O O O O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

6-week-old male CD36 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 ANOVA


6 - week - old 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 , then kept on WD / CCl4 for 12 . n = 5 per group . ( E ) Immunofluorescence of CD36 β - catenin . ( F ) Quantitation of CD36 positive . ( G ) Co - localization rate of CD36 β - catenin . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

O O O O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

6-week-old male CD36 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 ANOVA


6 - week - old 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 , then kept on WD / CCl4 for 12 . n = 5 per group . ( H ) of palmitoylated CD36 in mouse livers ( left ) , its quantitative results ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

O O O O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

6-week-old male CD36 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 ANOVA


6 - week - old 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 , then kept on WD / CCl4 for 12 . n = 5 per group . ( I ) CD36 expression in plasma fractions , its normalization to ATP1a1 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

O O O O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

6-week-old male CD36 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


Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( A - B ) of KLF10 , zDHHC7 TNF - α ( A ) , 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

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. (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 test


Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( C ) weights . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. (C) Body weights. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test


Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( D ) Liver weights liver / . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. (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 test


Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as 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

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. (E) Hepatic TG contents. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test


Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( F ) Serum ALT AST . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O B-TISSUE B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. (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 test


Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( G ) H & E Sirius red of liver sections .

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE O O

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. (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 KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( H ) Quantitative results of Sirius red . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 test


Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( I ) Immunofluorescence of CD36 β - catenin . arrows represent colocalization of CD36 β - catenin .

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

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. (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 KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( J ) Quantitative results of co - localization rate of CD36 β - catenin . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 test


Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( K ) 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

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. (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 test


Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( L ) CD36 expression in plasma fractions from mouse livers , its normalization to ATP1a1 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O B-ORGANISM B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. (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 test


Albumin - cre mice were crossed with KLF10flox / flox to generate hepatocyte - specific KLF10 knockout mice ( KLF10hep - / - ) . Then , 8 - week - old KLF10hep - / - mice KLF10flox / flox were treated with WD / CCl4 for 12 . KLF10flox / flox mice were used as control ( Ctrl ) . n = 5 per group . ( M ) Relative mRNA level of genes involved in liver inflammation fibrosis . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . Student ' s t

B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM 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-TISSUE B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. (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


Representative microscopic views of Ndrg4 + / + Ndrg4 - / - murine intestinal sections ( n = 4 , 12 months of ) reveal an even distribution of alkaline phosphatase along enterocyte brush border , a similar number distribution of Paneth ( Lysozyme ) in small intestine ; neuroendocrine ( Chromogranin A ) , Goblet ( PAS + ) , proliferating ( Ki67 ) in colon . Scale bars , 50 µm .

O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-CELL_TYPE B-TISSUE I-TISSUE O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O B-TISSUE I-TISSUE O B-TISSUE O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O O O O B-GENEPROD O O B-TISSUE O O O O O O O

Representative 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 myenteric plexus of Ndrg4 + / + Ndrg4 - / - murine colonic sections labeled with either TuJ1 S100 ( B , n = 3 ) or HuC / D ( C , n = 6 ) did not reveal structural or organizational differences in ganglionic network of Ndrg4 + / + Ndrg4 - / - mice . Scale bars , 100 µm . of enteric neuronal number ( HuC / D ) shows a similar number of enteric neurons in proximal distal colon of Ndrg4 + / + Ndrg4 - / - mice ( n = 6 ) .

O B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O

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).


In contrast , Ndrg4fl / fl Ndrg4fl / fl - VillinCre mice have a similar bodyweight ( I ) 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 n = 10 vs . 8 in K .

O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O B-DISEASE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In 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.


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

O O B-GENEPROD O O O B-TISSUE O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O

Compared 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).


Box plot showing quantitative expression results of nanoLC - MS / MS reveals significantly higher presence of Nid1 Fbln2 in Ndrg4 - / - compared to Ndrg4 + / + ENS secretome ( n = 4 ; NSAF , normalized spectral abundance factor ) . Data are analyzed with R version 3 . 5 . 2 ibb R package . Each dot within box plot represents NSAF of an sample ; inside band reflects median , bottom top of box first third quartile , respectively . whiskers reflect minimum maximal values within 1 . 5 × interquartile range . NSAF values were compared using Mann - Whitney U .

O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Box 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 ) more detailed immunofluorescence ( D , E ) labeling indicate that Nid1 ( C , D ) Fbln2 ( C , E ) are expressed within myenteric plexus by ENS - ( ( C ) , black arrowheads , scale bars , 50 µm ; ( D , E ) , n = 3 , scale bars , 10 µm ) .

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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).


Representative microscopic views show that NID1 FBLN2 are highly expressed in human colonic ganglia ( Black arrowheads ) interconnecting nerve , in a limited level throughout epithelium ( n = 3 ) . Scale bar , 50 µm .

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Representative 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 Matrigel ® dome significantly enhances relative growth rate of HIOs after five days of culture as compared to PBS control condition .

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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 quantitative nanoLC - MS / MS results shows up - regulation of NID1 FBLN2 in colorectal cancer tissue secretome ( n = 17 ) compared to normal colon tissue secretome ( n = 17 ) . Data are analyzed with R version 3 . 5 . 2 ibb R package . Within box plots , each dot represents log10 transformed raw of an sample ; inside band is median value ; bottom top of box first third quartile , respectively ; whiskers reflect minimum maximal values within 1 . 5 × interquartile range .

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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 of human colonic cancer epithelium ( n = 3 ) shows a high scattered expression of NID1 FBLN2 within cancerous epithelium . Scale bar , 50 μm .

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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.


B . Kaplan - Meier curve represents 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 .

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B. 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 . L . monocytogenes EGD - e wild - type ( WT ) , ΔinlK mutant ( ΔinlK ) complemented ( ΔinlK + pPL2 inlK ) 104 CFU were inoculated i . v into BALB / c mice . were euthanized 24 h , 48 h , 72 h or 96 h after infection organs were recovered , homogenized , homogenates serially plated on BHI . number of able to colonize liver ( left panel ) spleen ( right panel ) is expressed as log10 CFU . Four per , per time points per experiment were used . Statistical analyses were performed on results of 3 independent experiments using Student t . P values of < 0 . 05 were considered statistically different are labeled here as * .

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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 *.


A . InlK acid . signal is underlined different regions of leucine rich repeats ( LRRs ) are outlined . consensus pentapeptide LPXTG at C - terminal end is boxed .

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A. InlK amino acid sequence. The signal sequence is underlined and the different regions of leucine rich repeats (LRRs) are outlined. The consensus pentapeptide LPXTG at the C-terminal end is boxed.


B . Detection by immunofluorescence microscopy of InlK over - expressing in L . monocytogenes EGD - e ( WT ) , ΔinlK , WT + pADc - inlK , ΔinlK + pPRT - inlK ΔsrtA mutant over - expressing inlK ( ΔsrtA + pPRT - inlK ) grown in BHI medium using rabbit polyclonal anti - InlK antibody . InlK was detected at surface of InlK over - expressing ( WT + pADc - inlK ΔinlK + pPRT - inlK ) , whereas it was undetectable at surface WT or at surface of ΔstrA mutant over - expressing inlK .

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B. Detection by immunofluorescence microscopy of InlK over-expressing in L. monocytogenes EGD-e (WT), ΔinlK, WT+pADc-inlK, ΔinlK+pPRT-inlK and the ΔsrtA mutant over-expressing inlK (ΔsrtA+pPRT-inlK) grown in BHI medium using the rabbit polyclonal anti-InlK antibody. InlK was detected at the surface of InlK over-expressing bacteria (WT+pADc-inlK and ΔinlK+pPRT-inlK), whereas it was undetectable at the surface WT bacteria or at the surface of the ΔstrA mutant over-expressing inlK.


C . Detection of InlK by Western blot on total lysates of L . monocytogenes EGD - e ( WT ) , ΔinlK ΔinlK + pPRT - inlK grown in BHI using rabbit polyclonal anti - InlK antibody . Decreased concentrations of recombinant purified InlK were used as a positive control .

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C. Detection of InlK by Western blot on total lysates of L. monocytogenes EGD-e (WT), ΔinlK and ΔinlK+pPRT-inlK grown in BHI using the rabbit polyclonal anti-InlK antibody. Decreased concentrations of recombinant purified InlK were used as a positive control.


D . Detection of secreted InlK in supernatant of ΔsrtA mutants over - expressing InlK . Western blotting was carried out on trichloroacetic acid precipitates of ΔsrtA ΔsrtA + pPRT - inlK culture ( OD600 = 1 ) supernatants using rabbit polyclonal anti - InlK antibody .

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D. Detection of secreted InlK in the supernatant of ΔsrtA mutants over-expressing InlK. Western blotting was carried out on trichloroacetic acid precipitates of ΔsrtA and ΔsrtA+pPRT-inlK culture (OD600 = 1) supernatants using the rabbit polyclonal anti-InlK antibody.


E . Detection of purified recombinant InlK with rabbit polyclonal anti - live Listeria antibody , rabbit polyclonal anti - killed Listeria antibody , rabbit polyclonal anti - InlK a rabbit pre - immune serum . InlK was detected only with rabbit polyclonal anti - live Listeria antibody indicating that it is expressed during in vivo infectious process . BSA was used as control . Two different amounts of were tested ( 500 ng 200 ng ) to access signal specificity .

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E. Detection of purified recombinant InlK protein with rabbit polyclonal anti-live Listeria antibody, rabbit polyclonal anti-killed Listeria antibody, rabbit polyclonal anti-InlK and a rabbit pre-immune serum. InlK was detected only with the rabbit polyclonal anti-live Listeria antibody indicating that it is expressed during the in vivo infectious process. BSA was used as control protein. Two different amounts of proteins were tested (500 ng and 200 ng) to access signal specificity.


A . pull - down of purified GST - MVP with L . monocytogenes strains WT + pPRT - empty , ΔinlK + pPRT - empty , ΔinlK + pPRT - inlK WT + pPRT - inlJ . GST - MVP bound to InlK over - expressing ( ΔinlK + pPRT - inlK ) but not to other . GST - ScarA was used as control of specificity of MVP precipitation by InlK over - expressing .

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A. Bacterial pull-down of purified GST-MVP with the L. monocytogenes strains WT+pPRT-empty, ΔinlK+pPRT-empty, ΔinlK+pPRT-inlK and WT+pPRT-inlJ. GST-MVP bound to InlK over-expressing bacteria (ΔinlK+pPRT-inlK) but not to other bacteria. GST-ScarA was used as control of the specificity of the MVP precipitation by InlK over-expressing bacteria.


B . pull - down of MVP - GFP from transfected HeLa lysates with L . monocytogenes strains WT + pPRT - empty , ΔinlK + pPRT - empty , ΔinlK + pPRT - inlK WT + pPRT - inlJ . MVP - GFP bound to InlK over - expressing ( ΔinlK + pPRT - inlK ) but not to other .

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B. Bacterial pull-down of MVP-GFP from transfected HeLa cell lysates with the L. monocytogenes strains WT+pPRT-empty, ΔinlK+pPRT-empty, ΔinlK+pPRT-inlK and WT+pPRT-inlJ. MVP-GFP bound to InlK over-expressing bacteria (ΔinlK+pPRT-inlK) but not to other bacteria.


C . Co - immunoprecipitation ( Co - IP ) of purified InlK ( 20 µg ) with endogenous MVP of HT29 . right panel shows anti - MVP co - IP performed on HT29 lysates . left panel shows control anti - MVP co - IP performed on lysis buffer .

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C. Co-immunoprecipitation (Co-IP) of purified InlK (20 µg) with endogenous MVP of HT29 cells. The right panel shows anti-MVP co-IP performed on HT29 cell lysates. The left panel shows the control anti-MVP co-IP performed on lysis buffer.


D . Detection of MVP recruitment at surface of InlK over - expressing . HeLa were transfected with MVP - GFP ( green ) , infected with L . monocytogenes EGD - e wild - type ( WT ) , ΔinlK or ΔinlK + pPRT - inlK for 4 h , fixed for microscopy , stained with anti - Listeria antibodies ( red ) . Inset regions are magnified . scale bar represents 1 µm .

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D. Detection of MVP recruitment at the surface of InlK over-expressing bacteria. HeLa cells were transfected with MVP-GFP (green), infected with L. monocytogenes EGD-e wild-type (WT), ΔinlK or ΔinlK+pPRT-inlK for 4 h, fixed for fluorescence light microscopy, and stained with anti-Listeria antibodies (red). Inset regions are magnified. The scale bar represents 1 µm.


A . Detection of MVP recruitment at surface of intracellular InlK over - expressing . HeLa were transfected with MVP - GFP ( red ) , infected with InlK expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for microscopy . Intra - ( only green ) extracellular ( cyan = green + blue ) were differentially stained with anti - Listeria antibody ( cf Material Methods ) . Inset regions are magnified . Arrows indicate another intracellular bacterium which recruit MVP - GFP . scale bar represents 1 µm . right panel represents of intracellular that recruit MVP ( mean % ± % ) shown in left panel . Statistical analyses were performed on results of 3 independent experiments using Student ' s t . No significant difference was found between 4 time points .

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A. Detection of MVP recruitment at the surface of intracellular InlK over-expressing bacteria. HeLa cells were transfected with MVP-GFP (red), infected with InlK expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy. Intra- (only green) and extracellular (cyan = green+blue) bacteria were differentially stained with anti- Listeria antibody (cf Material and Methods). Inset regions are magnified. Arrows indicate another intracellular bacterium which recruit MVP-GFP. The scale bar represents 1 µm. The right panel represents the quantification of the intracellular bacteria that recruit MVP (mean%±SEM%) shown in the left panel. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. No significant difference was found between the 4 time points.


B . Detection of MVP recruitment at surface of intracytosolic InlK over - expressing . HeLa were transfected with MVP - tomato ( red ) YFP - CBD ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for microscopy stained with phalloidin ( blue ) . MVP positive were also labeled with YFP - CBD revealing that MVP was recruited by intracytosolic after lysis of internalization vacuole . Inset regions are magnified . scale bar represents 1 µm .

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B. Detection of MVP recruitment at the surface of intracytosolic InlK over-expressing bacteria. HeLa cells were transfected with MVP-tomato (red) and YFP-CBD (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy and stained with phalloidin (blue). MVP positive bacteria were also labeled with YFP-CBD revealing that MVP was recruited by intracytosolic bacteria after the lysis of the internalization vacuole. Inset regions are magnified. The scale bar represents 1 µm.


C . Kinetics of MVP actin recruitment at surface of InlK over - expressing . HeLa were transfected with MVP - tomato ( red ) actin - GFP ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , prepared for real - time video microscopy . Image series were collected every 15 min for 2 h . left part shows an MVP positive bacterium that never recruits actin . right part shows MVP replacement by actin around bacterium . No colocalization of MVP - Tomato actin - GFP was detected . Time is indicated along Y axis . entire image can be viewed as Video S1 .

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C. Kinetics of MVP and actin recruitment at the surface of InlK over-expressing bacteria. HeLa cells were transfected with MVP-tomato (red) and actin-GFP (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, and prepared for real-time video microscopy. Image series were collected every 15 min for 2 h. The left part shows an MVP positive bacterium that never recruits actin. The right part shows MVP replacement by actin around the bacterium. No colocalization of MVP-Tomato and actin-GFP was detected. Time is indicated along the Y axis. The entire image sequence can be viewed as Video S1.


A . Impaired recruitment of p62 to MVP positive Listeria . HeLa were transfected with MVP - GFP ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for microscopy , stained with phalloidin ( blue ) anti - p62 antibody ( red ) . Inset regions are magnified . Arrows indicate independent scale bar represents 1 µm . vast majority of MVP - positive were completely devoid of anti - p62 labeling ( 95 . 1 ± 2 . 0 % ; mean ± from n = 3 experiments ) but 4 . 9 ± 2 . 0 % ( mean ± from n = 3 experiments ) were stained at one pole with MVP at other pole with p62 .

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A. Impaired recruitment of p62 to MVP positive Listeria. HeLa cells were transfected with MVP-GFP (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with phalloidin (blue) and anti-p62 antibody (red). Inset regions are magnified. Arrows indicate independent bacteria The scale bar represents 1 µm. The vast majority of MVP-positive bacteria were completely devoid of anti-p62 labeling (95.1±2.0%; mean ± SEM from n = 3 experiments) but 4.9±2.0% (mean ± SEM from n = 3 experiments) were stained at one pole with MVP and at the other pole with p62.


B . Impaired recruitment of GFP - LC3 on MVP positive Listeria . HeLa were transfected with MVP - tomato ( red ) GFP - LC3 ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , fixed for microscopy , stained with phalloidin ( blue ) . Inset regions are magnified . scale bar represents 1 µm . MVP / or actin positive were never recognized by GFP - LC3 . Arrows point to at different steps of infection process : 1 ) InlK over - expressing bacterium is totally covered by MVP ; 2 ) bacterium is partially labeled with MVP ( at poles ) actin ( at center ) ; 3 ) bacterium is completely covered by actin ; 4 ) bacterium is enclosed in an GFP - LC3 positive autophagosome .

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B. Impaired recruitment of GFP-LC3 on MVP positive Listeria. HeLa cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with phalloidin (blue). Inset regions are magnified. The scale bar represents 1 µm. MVP and/or actin positive bacteria were never recognized by GFP-LC3. Arrows point to bacteria at different steps of the infection process: 1) InlK over-expressing bacterium is totally covered by MVP; 2) bacterium is partially labeled with MVP (at the poles) and actin (at the center); 3) bacterium is completely covered by actin; 4) bacterium is enclosed in an GFP-LC3 positive autophagosome.


C . Kinetics of autophagy escape for MVP positive Listeria . Jeg3 were transfected with MVP - tomato ( red ) GFP - LC3 ( green ) , infected with InlK over - expressing Listeria ( ΔinlK + pPRT - inlK ) for 4 h , prepared for real - time video microscopy . Image series were collected every 5 min for 2 h . Time is indicated along Y axis . left panel shows that GFP - LC3 membranous aggregate detaches from MVP positive Listeria . entire image can be viewed as Video S2 . right panel shows that GFP - LC3 membranous aggregate on MVP positive does not lead to an autophagosome formation , whereas those efficiently divided . entire image can be viewed as Video S3 .

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C. Kinetics of autophagy escape for MVP positive Listeria. Jeg3 cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing Listeria (ΔinlK+pPRT-inlK) for 4 h, and prepared for real-time video microscopy. Image series were collected every 5 min for 2 h. Time is indicated along the Y axis. The left panel shows that the GFP-LC3 membranous aggregate detaches from the MVP positive Listeria. The entire image sequence can be viewed as Video S2. The right panel shows that the GFP-LC3 membranous aggregate on MVP positive bacteria does not lead to an autophagosome formation, whereas those bacteria efficiently divided. The entire image sequence can be viewed as Video S3.


D . Impaired recruitment of GFP - LC3 ubiquitin to MVP positive ΔactA Listeria . HeLa were transfected with MVP - tomato ( red ) GFP - LC3 ( green ) , infected with InlK over - expressing ΔactA ( ΔactA + pADc - inlK ) for 4 h , fixed for microscopy , stained with anti - ubiquitin antibody ( blue ) DAPI ( ) . Inset regions are magnified . scale bar represents 1 µm .

O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-ORGANISM O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

D. Impaired recruitment of GFP-LC3 and ubiquitin to MVP positive ΔactA Listeria. HeLa cells were transfected with MVP-tomato (red) and GFP-LC3 (green), infected with InlK over-expressing ΔactA (ΔactA+pADc-inlK) for 4 h, fixed for fluorescence light microscopy, and stained with anti-ubiquitin antibody (blue) and DAPI (white). Inset regions are magnified. The scale bar represents 1 µm.


E . LC3 in infected RAW 267 . 4 macrophages . Left panel : RAW 267 . 4 macrophages were infected with L . monocytogenes EGD ( WT ) , ΔactA or ΔactA + InlK for 6 h . total lysates were immunobloted for LC3 actin . Western blot is representative from 3 independent experiments . Right panel : of relative LC3 - II level ( mean ± ) shown in left panel . Statistical analyses were performed on results of 3 independent experiments using Student ' s t . P values of < 0 . 05 were considered statistically different .

O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. LC3 levels in infected RAW 267.4 macrophages. Left panel: RAW 267.4 macrophages were infected with L. monocytogenes EGD (WT), ΔactA or ΔactA+InlK for 6 h. Cell total lysates were immunobloted for LC3 and actin. Western blot is representative from 3 independent experiments. Right panel: Quantification of the relative LC3-II level (mean ± SEM) shown in the left panel. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different.


A . InlK ActA expression in Listeria strains used for survival assays . Total lysates of L . monocytogenes EGD - ( pADc - GFP ) , EGD - ( pADc - inlK ) , ΔactA - ( pADc - GFP ) ΔactA - ( pADc - inlK ) grown in BHI were immunoblotted using anti - ActA anti - InlK antibodies .

O O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O

A. InlK and ActA expression in Listeria strains used for survival assays. Total lysates of L. monocytogenes EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) grown in BHI were immunoblotted using anti-ActA and anti-InlK antibodies.


B . Intracellular survival of EGD - ( pADc - GFP ) , EGD - ( pADc - inlK ) , ΔactA - ( pADc - GFP ) ΔactA - ( pADc - inlK ) in RAW 267 . 4 macrophages . Statistical analyses were performed on results of 3 independent experiments using Student t . P values of < 0 . 05 were considered statistically different are labeled here as * .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O 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. Intracellular survival of EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in RAW 267.4 macrophages. 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 *.


C . Intracellular survival of ΔactA - ( pADc - GFP ) ΔactA - ( pADc - inlK ) in MVP - GFP transfected Jeg3 . Statistical analyses were performed on results of 3 independent experiments using Student ' s t . P values of < 0 . 05 were considered statistically different are labeled here as * .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP-GFP transfected Jeg3 cells. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different and are labeled here as *.


D . Intracellular survival of ΔactA - ( pADc - GFP ) ΔactA - ( pADc - inlK ) in MVP - GFP transfected HeLa . Statistical analyses were performed on results of 3 independent experiments using Student ' s t . P values of < 0 . 05 were considered statistically different are labeled here as * .

D. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP-GFP transfected HeLa cells. Statistical analyses were performed on the results of 3 independent experiments using the Student's t test. P values of <0.05 were considered statistically different and are labeled here as *.


E . MVP in RAW 267 . 4 macrophages treated with MVP - siRNA . Western blot is representative from 3 independent experiments .

O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

E. MVP levels in RAW 267.4 macrophages treated with MVP-siRNA. Western blot is representative from 3 independent experiments.


F . Intracellular survival of ΔactA - ( pADc - GFP ) ΔactA - ( pADc - inlK ) in MVP knock - down RAW 267 . 4 macrophages . Statistical analyses were performed on results of 3 independent experiments using Student t . P values of < 0 . 05 were considered statistically different are labeled here as * .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Intracellular survival of ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in MVP knock-down RAW 267.4 macrophages. 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 *.


A Double - plotted representative actograms of indicated genotypes . Flies are monitored in LD for 4 days then DD for 7 days . Dicer2 ( dcr2 ) is co - expressed to enhance effects of RNAi . boxes indicate phase black boxes indicate phase . Gray shades indicate DD . G4 , GAL4 ; U , UAS .

O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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-GENEPROD O B-GENEPROD O O O O O

A Double-plotted representative actograms of the indicated genotypes. Flies are monitored in LD for 4 days and then DD for 7 days. Dicer2 (dcr2) is co-expressed to enhance the effects of RNAi. White boxes indicate the light phase and black boxes indicate the dark phase. Gray shades indicate DD. G4, GAL4; U, UAS.


B - D period of DD locomotor rhythms of flies with Nipped - A knocked down controls . Data information : ( B - H ) Error bars represent standard error of mean ( ) . Digits on bars are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using One - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # P < 0 . 001 , * compared with GAL4 controls ; # compared with UAS controls . bar indicates UAS or GAL4 controls . Gray bar indicates flies with Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O

B-D The period of DD locomotor rhythms of flies with Nipped-A knocked down and controls. Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.


E , F Using - sensitive GAL80 system to control Nipped - A RNAi expression . ( E ) period of DD locomotor rhythms of flies raised at 18ºC tested at 29ºC . ( F ) period of DD locomotor rhythms of flies raised at 29ºC tested at 18ºC . Data information : ( B - H ) Error bars represent standard error of mean ( ) . Digits on bars are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using One - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # P < 0 . 001 , * compared with GAL4 controls ; # compared with UAS controls . bar indicates UAS or GAL4 controls . Gray bar indicates flies with Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O 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-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O

E, F Using temperature-sensitive GAL80 system to control Nipped-A RNAi expression. (E) The period of DD locomotor rhythms of flies raised at 18ºC and tested at 29ºC. (F) The period of DD locomotor rhythms of flies raised at 29ºC and tested at 18ºC. Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.


G , H period of DD locomotor rhythms of flies treated with RU486 to activate pdfG4 - geneswitch ( pdf - GS ) driver ( G ) or vehicle control ( H ) . Data information : ( B - H ) Error bars represent standard error of mean ( ) . Digits on bars are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using One - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # P < 0 . 001 , * compared with GAL4 controls ; # compared with UAS controls . bar indicates UAS or GAL4 controls . Gray bar indicates flies with Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O 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-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O

G, H The period of DD locomotor rhythms of flies treated with RU486 to activate the pdfG4-geneswitch (pdf-GS) driver (G) or vehicle control (H). Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.


A Plots of relative mRNA abundance vs circadian time ( CT ) for clock genes determined by qRT - PCR in whole head extracts of Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 / + ) control ( timG4 / + ; Udcr2 / + ) collected on DD1 ( tim / cry , n = 5 ; Pdp1ε / per / clk / cyc , n = 3 ; vri , n = 6 ) . For each time series , value of lowest time point was set to 1 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

A Plots of relative mRNA abundance vs circadian time (CT) for clock genes determined by qRT-PCR in whole head extracts of Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1/+) and control (timG4/+;Udcr2/+) collected on DD1 (tim/cry, n=5; Pdp1ε/per/clk/cyc, n=3; vri, n=6). For each time series, the value of the lowest time point was set to 1.


B Western blots of from whole head extracts of Nipped - A RNAi control flies collected on DD1 probed with TIM , PDP1ε PER antibodies . C of TIM ( n = 5 ) , PDP1ε ( n = 3 ) PER ( n = 3 ) of blots in ( B ) . TIM , PDP1ε PER were normalized to that of HSP70 . For each time series , value of control at peak time point was set to 1 .

O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

B Western blots of proteins from whole head extracts of Nipped-A RNAi and control flies collected on DD1 and probed with TIM, PDP1ε and PER antibodies. C Quantification of TIM (n=5), PDP1ε (n=3) and PER (n=3) protein levels of blots in (B). TIM, PDP1ε and PER protein levels were normalized to that of HSP70. For each time series, the value of the control at the peak time point was set to 1.


D Brains from Udcr2 / + ; cryG4 - 16 / + Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi flies collected at CT0 , 4 , 8 , 12 , 16 , 20 on DD1 were immunostained with TIM ( green ) , PDP1ε ( red ) , PER ( green ) PDF ( red or green ) antisera . scale bar represents 10µm . E of TIM , PDP1ε PER in s - LNvs of in ( D ) ( TIM : CT8 : Udcr2 / + ; cryG4 - 16 / + , n = 89 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 58 ; CT20 : Udcr2 / + ; cryG4 - 16 / + , n = 130 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 80 ; PDP1ε : CT4 : Udcr2 / + ; cryG4 - 16 / + , n = 46 ; Udcr2 / + ; cryG4 - 16 / UNipped - RNAi , n = 26 ; CT16 : Udcr2 / + ; cryG4 - 16 / + , n = 65 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 41 ; PER : CT0 : Udcr2 / + ; cryG4 - 16 / + , n = 90 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 58 ; CT12 : Udcr2 / + ; cryG4 - 16 / + , n = 86 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 64 ) .

O B-TISSUE O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O

D Brains from Udcr2/+;cryG4-16/+ and Udcr2/+;cryG4-16/UNipped-ARNAi flies collected at CT0, 4, 8, 12, 16, 20 on DD1 were immunostained with TIM (green) , PDP1ε (red), PER (green) and PDF (red or green) antisera. The scale bar represents 10µm. E Quantification of TIM, PDP1ε and PER protein levels in the s-LNvs of images in (D) (TIM: CT8: Udcr2/+;cryG4-16/+, n=89; Udcr2/+;cryG4-16/UNipped-ARNAi, n=58; CT20: Udcr2/+;cryG4-16/+, n=130; Udcr2/+;cryG4-16/UNipped-ARNAi, n=80; PDP1ε: CT4: Udcr2/+;cryG4-16/+, n=46; Udcr2/+;cryG4-16/UNipped- RNAi, n=26; CT16: Udcr2/+;cryG4-16/+, n=65; Udcr2/+;cryG4-16/UNipped-ARNAi, n=41; PER: CT0: Udcr2/+;cryG4-16/+, n=90; Udcr2/+;cryG4-16/UNipped-ARNAi, n=58; CT12: Udcr2/+;cryG4-16/+, n=86; Udcr2/+;cryG4-16/UNipped-ARNAi, n=64).


A , B period of DD locomotor rhythm of Nipped - A RNAi flies over - expressing tim ( A ) or carrying heterozygous tim01 mutation ( B ) . Ptim is a tim cDNA construct driven by tim promoter . Data information : Error bars represent . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with over - expression or mutant flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated clock gene . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated clock gene Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O

A, B The period of DD locomotor rhythm of Nipped-A RNAi flies over-expressing tim (A) or carrying heterozygous tim01 mutation (B). Ptim is a tim cDNA construct driven by tim promoter. Data information: Error bars represent SEM. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the over-expression or mutant flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated core clock gene. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated core clock gene and Nipped-A knocked down. G4, GAL4; U, UAS.


C , D period of DD locomotor rhythm of Nipped - A RNAi flies over - expressing Pdp1ε ( C ) or carrying heterozygous Pdp1ε3135 mutation ( D ) . Data information : Error bars represent . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with over - expression or mutant flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated clock gene . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated clock gene Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O

C, D The period of DD locomotor rhythm of Nipped-A RNAi flies over-expressing Pdp1ε (C) or carrying heterozygous Pdp1ε3135 mutation (D). Data information: Error bars represent SEM. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the over-expression or mutant flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated core clock gene. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated core clock gene and Nipped-A knocked down. G4, GAL4; U, UAS.


E , F period of DD locomotor rhythm of Nipped - A RNAi flies over - expressing per ( E ) or carrying perL mutation ( F ) . Data information : Error bars represent . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with over - expression or mutant flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated clock gene . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated clock gene Nipped - A knocked down . G4 , GAL4 ; U , UAS .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O

E, F The period of DD locomotor rhythm of Nipped-A RNAi flies over-expressing per (E) or carrying perL mutation (F). Data information: Error bars represent SEM. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the over-expression or mutant flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated core clock gene. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated core clock gene and Nipped-A knocked down. G4, GAL4; U, UAS.


A Plots of relative mRNA abundance of Nipped - A in whole head extracts of w1118 flies in LD DD1 determined by qRT - PCR ( n = 3 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

A Plots of relative mRNA abundance of Nipped-A in whole head extracts of w1118 flies in LD and DD1 determined by qRT-PCR (n=3).


B Left panel : Western blots of from whole heads of w1118 Nipped - A RNAi flies collected during LD or DD1 probed with NIPPED - A antibody . Right panel : of NIPPED - A of blots in left panel ( LD , n = 3 ; DD1 , n = 5 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

B Left panel: Western blots of proteins from whole heads of w1118 and Nipped-A RNAi flies collected during LD or DD1 and probed with NIPPED-A antibody. Right panel: quantification of NIPPED-A protein levels of blots in the left panel (LD, n=3; DD1, n=5).


C immunoprecipitation ( ChIP ) assays to detect NIPPED - A binding at E - box , transcription start site ( TSS ) gene of tim , Pdp1ε per using w1118 flies ( n = 3 ) . Data information : Error bars represent . Two - way ANOVA , significant effect between NIPPED - A IgG were found for tim E - box ( P < 0 . 001 ) , Pdp1ε E - box ( P < 0 . 001 ) , per E - box ( P < 0 . 001 ) , tim TSS ( P < 0 . 001 ) , Pdp1ε TSS ( P < 0 . 001 ) , per TSS ( P < 0 . 001 ) , tim gene ( P < 0 . 001 ) , Pdp1ε gene per gene ( P < 0 . 001 ) . Student ' s t - , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . G4 , GAL4 ; U , UAS .

O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O

C Chromatin immunoprecipitation (ChIP) assays to detect NIPPED-A binding at E-box, transcription start site (TSS) and gene body of tim, Pdp1ε and per using w1118 flies (n=3). Data information: Error bars represent SEM. Two-way ANOVA, significant effect between NIPPED-A and IgG were found for tim E-box (P<0.001), Pdp1ε E-box (P<0.001), per E-box (P<0.001), tim TSS (P<0.001), Pdp1ε TSS (P<0.001), per TSS (P<0.001), tim gene body (P<0.001), Pdp1ε gene body and per gene body (P<0.001). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. G4, GAL4; U, UAS.


A Plots of relative pre - mRNA abundance of tim ( n = 5 ) Pdp1ε ( n = 3 ) determined by qRT - PCR in whole head extracts of Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 ) control ( timG4 / + ; Udcr2 / + ) flies during DD1 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O

A Plots of relative pre-mRNA abundance of tim (n=5) and Pdp1ε (n=3) determined by qRT-PCR in the whole head extracts of Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) flies during DD1.


B ChIP assays to detect ub - H2B binding at E - box , TSS gene of tim , Pdp1ε , per clk in Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 ) control ( timG4 / + ; Udcr2 / + ) ( n ≥ 3 ) . Data information : Student ' s t - , * P < 0 . 05 . Two - way ANOVA , significant effect between ub - H2B IgG were found for genomic regions tested ( P < 0 . 001 ) . Significant effect of genotypes were found for tim E - box ( P < 0 . 05 ) , Pdp1ε E - box ( P < 0 . 01 ) , tim TSS ( P < 0 . 01 ) Pdp1ε TSS ( P < 0 . 001 ) .

O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O

B ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim, Pdp1ε, per and clk in Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) (n≥3). Data information: Student's t-test, *P < 0.05. Two-way ANOVA, significant effect between ub-H2B and IgG were found for all genomic regions tested (P<0.001). Significant effect of genotypes were found for tim E-box (P<0.05), Pdp1ε E-box (P<0.01), tim TSS (P<0.01) and Pdp1ε TSS (P<0.001).


A , B period ( A ) power ( B ) of DD locomotor rhythms of flies with not knocked down controls . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with not overexpression flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not Nipped - A knocked down .

O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O B-GENEPROD O B-ORGANISM O O O O O B-GENEPROD O O O O O B-ORGANISM O O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O

A, B The period (A) and power (B) of DD locomotor rhythms of flies with not knocked down and controls. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


C Plots of relative mRNA abundance vs CT for clock genes determined by qRT - PCR in whole head extracts of not RNAi ( timG4 / + ; Udcr2 / UnotRNAi ) control ( timG4 / + ; Udcr2 / + ) collected on DD1 ( n = 5 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

C Plots of relative mRNA abundance vs CT for clock genes determined by qRT-PCR in whole head extracts of not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) collected on DD1 (n=5).