Figure 5HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control ( Ctrl ) . n = 3 . Results represent three independent experiments . ( A ) Protein level of KLF10 , and its normalization to β - actin . HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control ( Ctrl ) . n = 3 . Results represent three independent experiments . ( B ) Protein levels of palmitoylated CD36 . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . Student ' s t test ( BHepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control ( Ctrl ) . n = 3 . Results represent three independent experiments . ( C ) CD36 protein expression the plasma membrane fractions , and its normalization to ATP1a1 . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . Student ' s t test C ) . HepG2 cells were treated with 2 - BP and / or lentivirus vectors expressing KLF10 . n = 3 . Results represent three independent experiments . ( D ) Protein levels of KLF10 , and its normalization to β - actin . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way ANOVAHepG2 cells were treated with 2 - BP and / or lentivirus vectors expressing KLF10 . n = 3 . Results represent three independent experiments . ( E ) Protein levels of palmitoylated CD36 . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way ANOVAHepG2 cells were treated with 2 - BP and / or lentivirus vectors expressing KLF10 . n = 3 . Results represent three independent experiments . ( F ) CD36 protein expression the plasma membrane fractions , and its normalization to ATP1a1 . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way ANOVAHepG2 cells were treated with 2 - BP and / or lentivirus vectors expressing KLF10 . n = 3 . Results represent three independent experiments . ( G ) BODIPY staining showing the lipid droplets . HepG2 cells were transfected with lentiviruses expressing KLF10 and / or shRNA targeting zDHHC7 . n = 3 independent experiments . ( H ) Protein levels of KLF10 and zDHHC7 , and its normalization to β - actin . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way ANOVAHepG2 cells were transfected with lentiviruses expressing KLF10 and / or shRNA targeting zDHHC7 . n = 3 independent experiments . ( I ) Protein levels of palmitoylated CD36 . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way ANOVAHepG2 cells were transfected with lentiviruses expressing KLF10 and / or shRNA targeting zDHHC7 . n = 3 independent experiments . ( J ) Immunofluorescence staining of CD36 and Na + / K + - ATPase ( left ) , and the quantitation of CD36 colocalization rate ( right ) . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way ANOVAHepG2 cells were transfected with lentiviruses expressing KLF10 and / or shRNA targeting zDHHC7 . n = 3 independent experiments . ( K ) BODIPY staining for lipid droplets . HepG2 cells were transfected with lentiviruses expressing KLF10 and / or shRNA targeting zDHHC7 . n = 3 independent experiments . ( L ) Cellular TG contents . Data information : * * P < 0 . 01 . Results are shown as mean ± SD . 1 - way ANOVA , L ) .

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

Figure 5HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control (Ctrl). n=3. Results represent three independent experiments. (A) Protein level of KLF10, and its normalization to β-actin.HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control (Ctrl). n=3. Results represent three independent experiments. (B) Protein levels of palmitoylated CD36. Data information: ** P < 0.01. Results are shown as mean ± SD. Student's t test (BHepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control (Ctrl). n=3. Results represent three independent experiments. (C) CD36 protein expression the plasma membrane fractions, and its normalization to ATP1a1. Data information: ** P < 0.01. Results are shown as mean ± SD. Student's t test C).HepG2 cells were treated with 2-BP and/or lentivirus vectors expressing KLF10. n=3. Results represent three independent experiments. (D) Protein levels of KLF10, and its normalization to β-actin. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVAHepG2 cells were treated with 2-BP and/or lentivirus vectors expressing KLF10. n=3. Results represent three independent experiments. (E) Protein levels of palmitoylated CD36. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVAHepG2 cells were treated with 2-BP and/or lentivirus vectors expressing KLF10. n=3. Results represent three independent experiments. (F) CD36 protein expression the plasma membrane fractions, and its normalization to ATP1a1. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVAHepG2 cells were treated with 2-BP and/or lentivirus vectors expressing KLF10. n=3. Results represent three independent experiments. (G) BODIPY staining showing the lipid droplets.HepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (H) Protein levels of KLF10 and zDHHC7, and its normalization to β-actin. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVAHepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (I) Protein levels of palmitoylated CD36. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVAHepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (J) Immunofluorescence staining of CD36 and Na+/K+-ATPase (left), and the quantitation of CD36 colocalization rate (right). Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVAHepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (K) BODIPY staining for lipid droplets.HepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (L) Cellular TG contents. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA , L).


Figure 66 - week - old male CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( A ) Protein levels of KLF10 and CD36 in the liver ( left ) , and their normalization to β - actin ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA ( A6 - week - old male CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( B ) Representative immunofluorescence staining of KLF10 and CD36 ( left ) , and the quantitative results ( right ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - week - old male CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( C ) H & E and Sirius red staining of liver sections . ( D ) Quantitative results of Sirius red staining . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - week - old male CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( E ) Body weights and liver / body weight ratio . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - week - old male CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( F ) Hepatic TG contents . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way ANOVA6 - week - old male CD36 wild - type ( WT ) or knockout mice ( CD36KO ) were administered with AAV - GFP ( Ctrl ) or AAV - KLF10 ( KLF10OE ) through tail vein injection , and then kept on WD / CCl4 for 12 weeks . n = 5 per group . ( G ) Serum AST and ALT levels . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Results are shown as mean ± SD . 1 - way

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

Figure 66-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (A) Protein levels of KLF10 and CD36 in the liver (left), and their normalization to β-actin (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA (A6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (B) Representative immunofluorescence staining of KLF10 and CD36 (left), and the quantitative results (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (C) H&E and Sirius red staining of liver sections. (D) Quantitative results of Sirius red staining. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (E) Body weights and liver/body weight ratio. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (F) Hepatic TG contents. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (G) Serum AST and ALT levels. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA


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

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

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


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

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

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


Figure 1 ( A m6A dot blot of the P493 - 6 cells treated with Tet for 0h or 72h ( A ) equal mRNA loading was verified by methylene blue staining . The shown data are representative of at least three independent experiments . B ) m6A dot blot of Raji cells that expressed NTC or MYC shRNAs ( B ) ; equal mRNA loading was verified by methylene blue staining . The shown data are representative of at least three independent experiments . ( C ) Quantification of m6A abundance by HPLC - MS . * * * P < 0 . 001 as compared to corresponding high MYC group ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) . ( D ) Metagene profiles of m6A peak distribution along a normalized transcript composed of three rescaled non - overlapping segments : 5 ′ UTR , CDS , and 3 ′ UTR in P493 - 6 cells treated with Tet for 0h or 72h . ( F ) IGV graph showing location of m6A peaks on representative genes . ( G ) m6A - RIP assay in P493 - 6 cells treated with Tet for 0h or 72h . HPRT1 serves as negative control . * * * P < 0 . 001 as compared to corresponding high MYC group , ns , not significant ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) . ( H Western blot analysis for protein levels in P493 - 6 cells treated with Tet for 0h , 24h , 48h and 72h ( H ) HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments . I ) Western blot analysis for protein levels in Raji cells that expressed NTC or MYC shRNAs ( I ) . HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments .

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

Figure 1(A m6A dot blot of the P493-6 cells treated with Tet for 0h or 72h (A) equal mRNA loading was verified by methylene blue staining. The shown data are representative of at least three independent experiments.B) m6A dot blot of Raji cells that expressed NTC or MYC shRNAs (B); equal mRNA loading was verified by methylene blue staining. The shown data are representative of at least three independent experiments.(C) Quantification of m6A abundance by HPLC-MS. *** P<0.001 as compared to corresponding high MYC group (mean ± SD, n = 3 biological replicates, Student's t‐test).(D) Metagene profiles of m6A peak distribution along a normalized transcript composed of three rescaled non-overlapping segments: 5′UTR, CDS, and 3′UTR in P493-6 cells treated with Tet for 0h or 72h.(F) IGV graph showing location of m6A peaks on representative genes.(G) m6A-RIP assay in P493-6 cells treated with Tet for 0h or 72h. HPRT1 serves as negative control. *** P<0.001 as compared to corresponding high MYC group, ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).(H Western blot analysis for protein levels in P493-6 cells treated with Tet for 0h, 24h, 48h and 72h (H) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.I) Western blot analysis for protein levels in Raji cells that expressed NTC or MYC shRNAs (I). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.


Figure 2 ( A Western blot analysis for protein levels of m6A methyltransferases ( METTL3 and METTL14 ) and demethylases ( ALKBH5 and FTO ) in P493 - 6 cells treated with Tet for 0h , 24h , 48h and 72h ( A ) β - actin serves as loading controls . Data are representative of at least three independent experiments . B ) Western blot analysis for protein levels of m6A methyltransferases ( METTL3 and METTL14 ) and demethylases ( ALKBH5 and FTO ) in Raji cells that expressed NTC or MYC shRNAs ( B ) . β - actin serves as loading controls . Data are representative of at least three independent experiments . ( C ) RT - qPCR analysis of the mRNA levels of methyltransferases and demethylases in P493 - 6 cells treated with Tet for 0h or 24h . Data were presented as mean ( ± SD ) , n = 3 biological replicates . * P < 0 . 05 relative to corresponding - Tet group ( Student ' s t ‐ test ) . ( D ) IGV graph showing location of MYC binding peaks on ALKBH5 and FTO from published ChIP - seq datasets ( Walz et al , 2014 ; Data ref : Eilers M , 2014 ) . In this dataset , high levels of MYC were induced with 1µg / ml doxycycline for 30h and ChIPed by MYC antibody . Red triangle indicates the E - box . ( E ) RIP assay , using ALKBH5 , FTO or IgG antibody to detect the binding to MRGs ( SPI1 and PHF12 ) in P493 - 6 cells treated with Tet or not . HPRT1 serves as negative control . * * * P < 0 . 001 as compared to corresponding IgG group ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) . ( F Western blot analysis for protein levels in P493 - 6 cells that overexpressed empty vector ( EV ) , ALKBH5 , or FTO and were then treated with Tet or not ( F ) HPRT1 and β - actin served as negative and loading controls , respectively . Data are representative of at least three independent experiments . G ) Western blot analysis for protein levels in Raji cells expressed NTC or MYC shRNAs ( G ) . HPRT1 and β - actin served as negative and loading controls , respectively . Data are representative of at least three independent experiments . ( H ) m6A RIP assay in P493 - 6 cells that overexpressed EV , ALKBH5 , or FTO and were then treated with Tet or not . HPRT1 serves as negative control . * * * P < 0 . 001 as compared between indicated groups . ns , not significant ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) . ( I , Western blot analysis for protein levels in P493 - 6 cells that expressed NTC or ALKBH5 shRNAs and were then treated with Tet or not ( I ) HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments . J ) Western blot analysis for protein levels in Raji cells expressed NTC or ALKBH5 shRNAs and knocked down MYC or not ( J ) . HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments . ( K ) m6A RIP assay HPRT1 serves as negative control . * * * P < 0 . 001 as compared between indicated groups ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) .

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

Figure 2(A Western blot analysis for protein levels of m6A methyltransferases (METTL3 and METTL14) and demethylases (ALKBH5 and FTO) in P493-6 cells treated with Tet for 0h, 24h, 48h and 72h (A) β-actin serves as loading controls. Data are representative of at least three independent experiments.B) Western blot analysis for protein levels of m6A methyltransferases (METTL3 and METTL14) and demethylases (ALKBH5 and FTO) in Raji cells that expressed NTC or MYC shRNAs (B). β-actin serves as loading controls. Data are representative of at least three independent experiments.(C) RT-qPCR analysis of the mRNA levels of methyltransferases and demethylases in P493-6 cells treated with Tet for 0h or 24h. Data were presented as mean (±SD), n = 3 biological replicates. * P<0.05 relative to corresponding -Tet group (Student's t‐test).(D) IGV graph showing location of MYC binding peaks on ALKBH5 and FTO from published ChIP-seq datasets (Walz et al, 2014; Data ref: Eilers M, 2014). In this dataset, high levels of MYC were induced with 1µg/ml doxycycline for 30h and ChIPed by MYC antibody. Red triangle indicates the E-box.(E) RIP assay, using ALKBH5, FTO or IgG antibody to detect the binding to MRGs (SPI1 and PHF12) in P493-6 cells treated with Tet or not. HPRT1 serves as negative control. *** P<0.001 as compared to corresponding IgG group (mean ± SD, n = 3 biological replicates, Student's t‐test).(F Western blot analysis for protein levels in P493-6 cells that overexpressed empty vector (EV), ALKBH5, or FTO and were then treated with Tet or not (F) HPRT1 and β-actin served as negative and loading controls, respectively. Data are representative of at least three independent experiments.G) Western blot analysis for protein levels in Raji cells expressed NTC or MYC shRNAs (G). HPRT1 and β-actin served as negative and loading controls, respectively. Data are representative of at least three independent experiments.(H) m6A RIP assay in P493-6 cells that overexpressed EV, ALKBH5, or FTO and were then treated with Tet or not. HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups. ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).(I, Western blot analysis for protein levels in P493-6 cells that expressed NTC or ALKBH5 shRNAs and were then treated with Tet or not (I) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.J) Western blot analysis for protein levels in Raji cells expressed NTC or ALKBH5 shRNAs and knocked down MYC or not (J). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.(K) m6A RIP assay HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups (mean ± SD, n = 3 biological replicates, Student's t‐test).


Figure 3 ( A ) RIP assay in P493 - 6 cells treated with Tet for 0h or 72h . HPRT1 serves as negative control . * * * P < 0 . 001 relative to indicating groups ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) . ( B , Western blot analysis for protein levels in P493 - 6 cells that expressed NTC or YTHDF3 shRNAs and were then treated with Tet or not ( B ) HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments . C ) Western blot analysis for protein levels in Raji cells that expressed NTC or YTHDF3 shRNAs and knocked down MYC or not ( C ) . HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments . ( D ) Polysomes profiling assay in P493 - 6 cells that expressed NTC or YTHDF3 shRNA . ( E ) RT - qPCR assay for mRNA levels of MRGs ( SPI1 and PHF12 ) in different fractions in ( D ) . HPRT1 serves as negative control . Data were presented as mean ( ± SD ) , n = 3 biological replicates . * * P < 0 . 01 and * * * P < 0 . 001 as compared to NTC group ( Student ' s t ‐ test ) . ( F ) Click - iT AHA ( L - azidohomoalaine ) experiments were performed using IgG , anti - SPI1 or anti - PHF12 antibody . P493 - 6 cells expressing NTC or YTHDF3 shRNAs were incubated for 1 hour in medium containing 100 ug / mL AHA . The translated proteins were detected by Western blot . Arrow indicates translated MRGs . ( G Western blot analysis for protein levels in P493 - 6 cells ( G ) β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments . H ) Western blot analysis for protein levels in Raji cells ( H ) that expressed NTC , or ALKBH5 shRNA , or YTHDF3 shRNA . β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments . ( I ) m6A RIP assay HPRT1 serves as negative control . * * * P < 0 . 001 as compared between indicated groups , ns , not significant ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) .

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

Figure 3(A) RIP assay in P493-6 cells treated with Tet for 0h or 72h. HPRT1 serves as negative control. *** P<0.001 relative to indicating groups (mean ± SD, n = 3 biological replicates, Student's t‐test).(B, Western blot analysis for protein levels in P493-6 cells that expressed NTC or YTHDF3 shRNAs and were then treated with Tet or not (B) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.C) Western blot analysis for protein levels in Raji cells that expressed NTC or YTHDF3 shRNAs and knocked down MYC or not (C). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.(D) Polysomes profiling assay in P493-6 cells that expressed NTC or YTHDF3 shRNA.(E) RT-qPCR assay for mRNA levels of MRGs (SPI1 and PHF12) in different fractions in (D). HPRT1 serves as negative control. Data were presented as mean (±SD), n = 3 biological replicates. ** P<0.01 and *** P<0.001 as compared to NTC group (Student's t‐test).(F) Click-iT AHA (L-azidohomoalaine) experiments were performed using IgG, anti-SPI1 or anti-PHF12 antibody. P493-6 cells expressing NTC or YTHDF3 shRNAs were incubated for 1 hour in medium containing 100 ug/mL AHA. The translated proteins were detected by Western blot. Arrow indicates translated MRGs.(G Western blot analysis for protein levels in P493-6 cells (G) β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.H) Western blot analysis for protein levels in Raji cells (H) that expressed NTC, or ALKBH5 shRNA, or YTHDF3 shRNA. β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.(I) m6A RIP assay HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups, ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).


Figure 4 ( A ) Trypan blue counting was used to analyze growth curves for P493 - 6 cells that expressed NTC or SPI1 shRNA or PHF12 shRNA . * * P < 0 . 01 as compared between indicated groups . ( mean ± SD , n = 4 biological replicates , Student ' s t ‐ test ) ( B , C ) Trypan blue counting was used to analyze growth curves for P493 - 6 cells overexpressing ALKBH5 and further infected with viruses expressing SPI1 ( B ) or PHF12 ( C ) . * * P < 0 . 01 as compared between indicated groups ( mean ± SD , n = 4 biological replicates , Student ' s t ‐ test ) . ( D , E ) Raji cells stably expressing EV or MYC were infected with viruses expressing shALKBH5 . Cells were injected subcutaneously into nude mice ( n = 5 for each group ) . Tumor growth curves were measured starting from 12 days post injection ( D ) . Photo of tumors collected at the end of the experiment ( day 27 ) ( E ) . Data are presented as mean ( ± SEM ) . * * P < 0 . 01 or * * * P < 0 . 001 as compared between indicated groups ( Student ' s t ‐ test ) . ( F P493 - 6 cells stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice ( n = 5 for each group ) . Tumor growth curves were measured starting from 12 days post injection ( F ) . Data are presented as mean ( ± SEM ) . * * P < 0 . 01 as compared between indicated groups ( Student ' s t ‐ test ) . G ) P493 - 6 cells stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice ( n = 5 for each group ) . Photo ( G ) of tumors collected at the end of the experiment ( day 25 ) . ( H , I ) P493 - 6 cells stably expressing EV or ALKBH5 were infected with viruses expressing SPI1 or PHF12 . Cells were injected subcutaneously into nude mice ( n = 5 for each group ) . Tumor growth curves were measured starting from 17 days post injection ( H ) . Photo ( I ) of tumors collected at the end of the experiment ( day 32 ) . Data are presented as mean ( ± SEM ) . * P < 0 . 05 as compared between indicated groups ( Student ' s t ‐ test ) . ( J , K ) Representative IHC images ( J ) , and analysis ( K ) of MYC , ALKBH5 , SPI1 and PHF12 expressions in normal lymphocyte tissue ( normal ) and lymphoma specimens . Scale bar : 50 um . Data are presented as mean ( ± SEM ) , clinical samples n = 30 for normal group , n = 28 for low MYC expression group , n = 27 for high MYC expression group , P value was presented between indicated groups ( Student ' s t ‐ test ) .

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

Figure 4(A) Trypan blue counting was used to analyze growth curves for P493-6 cells that expressed NTC or SPI1 shRNA or PHF12 shRNA. ** P<0.01 as compared between indicated groups. (mean ± SD, n = 4 biological replicates, Student's t‐test)(B, C) Trypan blue counting was used to analyze growth curves for P493-6 cells overexpressing ALKBH5 and further infected with viruses expressing SPI1 (B) or PHF12 (C). ** P<0.01 as compared between indicated groups (mean ± SD, n = 4 biological replicates, Student's t‐test).(D, E) Raji cells stably expressing EV or MYC were infected with viruses expressing shALKBH5. Cells were injected subcutaneously into nude mice (n=5 for each group). Tumor growth curves were measured starting from 12 days post injection (D). Photo of tumors collected at the end of the experiment (day 27) (E). Data are presented as mean (±SEM). ** P<0.01 or *** P<0.001 as compared between indicated groups (Student's t‐test).(F P493-6 cells stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice (n=5 for each group). Tumor growth curves were measured starting from 12 days post injection (F). Data are presented as mean (±SEM). ** P<0.01 as compared between indicated groups (Student's t‐test).G) P493-6 cells stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice (n=5 for each group). Photo (G) of tumors collected at the end of the experiment (day 25).(H, I) P493-6 cells stably expressing EV or ALKBH5 were infected with viruses expressing SPI1 or PHF12. Cells were injected subcutaneously into nude mice (n=5 for each group). Tumor growth curves were measured starting from 17 days post injection (H). Photo (I) of tumors collected at the end of the experiment (day 32). Data are presented as mean (±SEM). * P<0.05 as compared between indicated groups (Student's t‐test).(J, K) Representative IHC images (J), and analysis (K) of MYC, ALKBH5, SPI1 and PHF12 expressions in normal lymphocyte tissue (normal) and lymphoma specimens. Scale bar: 50 um. Data are presented as mean (±SEM), clinical samples n = 30 for normal group, n = 28 for low MYC expression group, n = 27 for high MYC expression group, P value was presented between indicated groups (Student's t‐test).


Figure 1B PCR performed on genomic DNA with indicated primers as shown : o1 : oPAP8 _ rtpF , o2 : oPAP8 _ E3R , o3 : oPAP8 _ rtpR , oLB : oLBb1 . 3 , EF1α : ELONGATION FACTOR 1α , WT : wild type , pap8 - 1 : Homozygous albino plant , Ht : Heterozygous green plant ; T : T - DNA , arrowhead : 670 - bp contaminant amplification product used as loading control . C RT - PCR on wild type and pap8 - 1 homozygous plants grown in the dark for 3 days followed with 72 - h growth under white light to allow greening of the wild type ; EF1α used as control . E Half - open siliques of a heterozygous plant showing the embryo greening ; scale bar equals 250 µm . The given number is the position rank of the silique from top to bottom of the inflorescence presenting the segregation of homozygous and heterozygous seeds based on their ability to transiently develop chloroplasts . F Mutant Rescue : WT and two representative pap8 - 1 plants were grown in vitro using sucrose and low white light intensity ( of 10 µmol . m - 2 . s - 1 ) ; scale bar equals 20 mm .

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Figure 1B PCR performed on genomic DNA with indicated primers as shown: o1: oPAP8_rtpF, o2: oPAP8_E3R, o3: oPAP8_rtpR, oLB: oLBb1.3, EF1α: ELONGATION FACTOR 1α, WT: wild type, pap8-1: Homozygous albino plant, Ht: Heterozygous green plant; T: T-DNA, arrowhead: 670-bp contaminant amplification product used as loading control.C RT-PCR on wild type and pap8-1 homozygous plants grown in the dark for 3 days followed with 72-h growth under white light to allow greening of the wild type; EF1α used as control.E Half-open siliques of a heterozygous plant showing the embryo greening; scale bar equals 250 µm. The given number is the position rank of the silique from top to bottom of the inflorescence presenting the segregation of homozygous and heterozygous seeds based on their ability to transiently develop chloroplasts.F Mutant Rescue: WT and two representative pap8-1 plants were grown in vitro using sucrose and low white light intensity (of 10 µmol.m-2.s-1); scale bar equals 20 mm.


Figure 2B Two or six ( - 97m3 ) representative primary transformants expressing GUS under the given PAP8 promoter version ; FC + , the corresponding promoters were tested positive in functional complementation of the mutant pap8 - 1 . Scale bar equals 3 mm . D Dual luciferase reporter assay ; Renilla luciferase ( Rluc ) used as internal control and GFPer used as control for the transfected area ; the promoters driving Firefly luciferase ( Fluc ) were transfected in onion epidermis cells without or with constitutively expressed HY5 . The Fluc / Rluc activity was set to 1 for the minus - HY5 control ; mean ± standard error corresponding to 3 replicates ; photon counts are given in source data . * , ε - test = 3 , 43 > 1 . 96 corresponding to p - value < 0 , 001 . E Electro - mobility shift assay of a probe corresponding to the near palindromic PAP8 element ( GAcGCTC ) with recombinant HY5 protein ; a probe containing a canonical G - box element ( CACGTG ) recognized by HY5 was used as cold competitor . F Integrative genomics viewer ( IGV ) images of the Chromatin immunoprecipitation ( ChIP ) sequencing data30 at the PAP8 locus ; TAIR10 , annotation according to the Arabidopsis thaliana information resource orange box indicates the PAP8 locus . ChIP on hy5 - ks50 ; 35S : HY5 - YFP exposed to blue light or red light using GFP antibody and compared to mock corresponding to ChIP control experiment done without antibody . Each treatment is presented as track overlay of triplicates : the read count is given within the " group autoscale " range in brackets . Close up on the 5 ' - UTR region centred on the - 95 - promoter element in yellow .

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Figure 2B Two or six (-97m3) representative primary transformants expressing GUS under the given PAP8 promoter version; FC+, the corresponding promoters were tested positive in functional complementation of the mutant pap8-1. Scale bar equals 3 mm.D Dual luciferase reporter assay; Renilla luciferase (Rluc) used as internal control and GFPer used as control for the transfected area; the promoters driving Firefly luciferase (Fluc) were transfected in onion epidermis cells without or with constitutively expressed HY5. The Fluc/Rluc activity was set to 1 for the minus-HY5 control; mean ± standard error corresponding to 3 replicates; photon counts are given in source data. *, ε-test = 3,43 > 1.96 corresponding to p-value < 0,001.E Electro-mobility shift assay of a probe corresponding to the near palindromic PAP8 element (GAcGCTC) with recombinant HY5 protein; a probe containing a canonical G-box element (CACGTG) recognized by HY5 was used as cold competitor.F Integrative genomics viewer (IGV) images of the Chromatin immunoprecipitation (ChIP) sequencing data30 at the PAP8 locus; TAIR10, annotation according to the Arabidopsis thaliana information resource orange box indicates the PAP8 locus. ChIP on hy5-ks50; 35S:HY5-YFP exposed to blue light or red light using GFP antibody and compared to mock corresponding to ChIP control experiment done without antibody. Each treatment is presented as track overlay of triplicates: the read count is given within the "group autoscale" range in brackets. Close up on the 5'-UTR region centred on the -95-promoter element in yellow.


Figure 3B Transiently expressed PAP8FL - GFP ( full - length coding sequence of PAP8 fused to GFP ) in onion epidermal cells displays a dual localization in the nucleus ( white arrowhead ) and in plastids ( yellow arrowheads ) . C Immuno - blots for the detection of PAP8 in total protein extracts ( Col - 0 Tot ) the nuclear fraction ( N ) and the plastidic fractions ( EP , etioplast ; CP , chloroplast ) of etiolated ( Dark ) or photomorphogenic ( Light ) Arabidopsis seedlings by immuno - Western blotting using a PAP8 antiserum ( PAP8 ) . As control , a mixture of antisera raised against Histone 3 ( H3 ) and the large subunit of ribulose 1 , 5 - bisphosphate carboxylase / oxygenase ( RbcL ) was used to evaluate reciprocal contaminations . The lanes are extracted from the same blots provided in the Source Data file . D - G Co - expression analysis of PAP8FL - GFP ( D ) with PAP8ΔcTP - RFP ( E ) in onion epidermal cells ; ( F ) green and red channels merged ; yellow arrowheads show plastids ; white arrowheads show nuclei as observed with DIC , differential interference contrast in ( G ) ; FL , PAP8 full length ORF ; ΔcTP , deletion of the cTP ; scale bars equal 20 µm . H Confocal imaging on Arabidopsis cotyledons expressing PAP8ΔcTP - GFP , details of a few palisade cells ( see Fig . S7B for a view in a cross section between the two cotyledons ) ; magenta , auto - fluorescence of the chloroplast ; the white arrowhead shows a nucleus ; scale bar equals 5 µm . I , J Confocal imaging on Arabidopsis cotyledons stably expressing pP8 : : PAP8ΔNLS - GFP ; ΔNLS , deletion of the NLS ( I ) during skotomorphogenesis and ( J ) after 24h light ; Yellow arrowheads show the GFP signal ; the picture is a merge of different channels : GFP in green , Pchl , : protochlorophyllide , or Chl : chlorophyll in magenta marked with arrowheads , and propidium iodide , showing the waxy cuticle in red , the empty space correspond to the layer of highly vacuolated epidermal cells .

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

Figure 3B Transiently expressed PAP8FL-GFP (full-length coding sequence of PAP8 fused to GFP) in onion epidermal cells displays a dual localization in the nucleus (white arrowhead) and in plastids (yellow arrowheads).C Immuno-blots for the detection of PAP8 in total protein extracts (Col-0 Tot) the nuclear fraction (N) and the plastidic fractions (EP, etioplast; CP, chloroplast) of etiolated (Dark) or photomorphogenic (Light) Arabidopsis seedlings by immuno-Western blotting using a PAP8 antiserum (PAP8). As control, a mixture of antisera raised against Histone 3 (H3) and the large subunit of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RbcL) was used to evaluate reciprocal contaminations. The lanes are extracted from the same blots provided in the Source Data file.D-G Co-expression analysis of PAP8FL-GFP (D) with PAP8ΔcTP-RFP (E) in onion epidermal cells; (F) green and red channels merged; yellow arrowheads show plastids; white arrowheads show nuclei as observed with DIC, differential interference contrast in (G); FL, PAP8 full length ORF; ΔcTP, deletion of the cTP; scale bars equal 20 µm.H Confocal imaging on Arabidopsis cotyledons expressing PAP8ΔcTP-GFP, details of a few palisade cells (see Fig. S7B for a view in a cross section between the two cotyledons); magenta, auto-fluorescence of the chloroplast; the white arrowhead shows a nucleus; scale bar equals 5 µm.I,J Confocal imaging on Arabidopsis cotyledons stably expressing pP8::PAP8ΔNLS-GFP; ΔNLS, deletion of the NLS (I) during skotomorphogenesis and (J) after 24h light; Yellow arrowheads show the GFP signal; the picture is a merge of different channels: GFP in green, Pchl,: protochlorophyllide, or Chl: chlorophyll in magenta marked with arrowheads, and propidium iodide, showing the waxy cuticle in red, the empty space correspond to the layer of highly vacuolated epidermal cells.


Figure 4 ( A , B ) ( A ) Transient expression of PAP8NLSm5 - GFP mutated in the NLS as described in Fig . EV4A . The circle marks the position of the nucleus ( N ) as observed with DIC in ( B ) ; yellow arrowheads show stromules ( str ) . Scale bars equal 20 µm . C - G Pictures of representative genotypes obtained in the functional complementation test of pap8 - 1 using pP8 : : PAP8 - NLSm5 ( C , F ) or pP8 : : PAP8ΔcTP ( G ) both constructions without GFP tags ; ⌀ indicates pap8 - 1 without any PAP8 transgene . ( D - G ) Pictures using Keyence technology of plants with genotypes as labelled ; transgenes expressed using the 1 . 1 - kb PAP8 promoter . Scale bars equal 1 mm .

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

Figure 4(A,B) (A) Transient expression of PAP8NLSm5-GFP mutated in the NLS as described in Fig. EV4A. The circle marks the position of the nucleus (N) as observed with DIC in (B); yellow arrowheads show stromules (str). Scale bars equal 20 µm.C-G Pictures of representative genotypes obtained in the functional complementation test of pap8-1 using pP8::PAP8-NLSm5 (C, F) or pP8::PAP8ΔcTP (G) both constructions without GFP tags; ⌀ indicates pap8-1 without any PAP8 transgene. (D-G) Pictures using Keyence technology of plants with genotypes as labelled; transgenes expressed using the 1.1-kb PAP8 promoter. Scale bars equal 1 mm.


Figure 5A Phenotypes of given genotypes subjected to 5 days of illumination at 8 µmol . m - 2 . s - 1 660 - nm red light . PBG , pCaMV35S : : PHYB - GFP transformed in pap8 - 1 / + ( among 25 lines selected for GFP expression see data source ; 2 doubly heterozygous pap8 - 1 / + ; PBG / - lines # 6 and # 7 segregated the photobodies alteration with the albinism ) . B Hypocotyl length , given as the mean ± SD , of plants grown as in ( A ) ( R8 , grey bars ) or at 30 µmol . m - 2 . s - 1 660 - nm red light ( R30 , pink bars ) showing partial insensitivity of pap8 - 1 to the PBG overexpression . Measurements are given in source data ; in the order of the graph n equals ( 50 , 133 , 58 , 43 , 36 , 112 , 25 , 60 ) δ - test ( comparison of the mean ) R8 : δPBG / PBGp8 - 1 = 20 . 38 corresponding to p - value < 10 - 31 δwt / p8 - 1 = 0 . 5 < 1 . 96 not significant at α set to 0 . 05 ) . Nuclear accumulation of PBG observed under GFP excitation in the given genotypes . ( C , D ) Epi - fluorescence microscopy . ( E , F ) Confocal microscopy showing the size of the nuclear bodies . Scale bars equal 5 µm . ( G ) Box plot ( Min , 1st quartile , median as the central band , 3rd quartile , max ) on the diameter of the nuclear bodies ( NBs ) ; n equals the number of records . H Immuno - blots using a GFP antibody or a PAP8 antibody showing respectively the levels of PHYB - GFP and PAP8 in the given genotypes grown in the dark for 3 days or in light ; n . a . , not applicable as the pap8 - 1 mutant can only be visually distinguished from wild type after light exposure ; 2 lines ( L # 06 and L # 07 ) for PBG / pap8 - 1 were tested . Coomassie blue staining presented as loading ; signals were quantified using ImageJ . I RT - qPCR analysis on wild type , pap8 - 1 , PBG and PBG pap8 - 1 . Seedlings were grown in the dark ( D ) or under white light ( L , 30 µmol . m - 2 . s - 1 ) ; levels of transcripts are given relative to EF1α ; error bars correspond to standard errors on technical triplicates and the dark sample is the wild - type PBG line . δ - test ( comparison of the mean ) * * * , P < 10 - 72 . J Immuno - blots showing the levels of PIF1 , PIF3 , HY5 in given genotypes grown in the Dark or Light condition as noted : p5 / + , mix of an heterozygous pap5 - 2 siblings progeny undistinguishable from wild type ; p5 - 2 , pap5 - 2 and pifq , quadruple pif1 - 1 pif3 - 3 pif4 - 2 pif5 - 3 mutant ; Histone H3 ( H3 ) , RbcL and PAP8 were used as controls ; n . a . , not applicable .

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

Figure 5A Phenotypes of given genotypes subjected to 5 days of illumination at 8 µmol.m-2.s-1 660-nm red light. PBG, pCaMV35S::PHYB-GFP transformed in pap8-1/+ (among 25 lines selected for GFP expression see data source; 2 doubly heterozygous pap8-1/+; PBG/- lines #6 and #7 segregated the photobodies alteration with the albinism).B Hypocotyl length, given as the mean ± SD, of plants grown as in (A) (R8, grey bars) or at 30 µmol.m-2.s-1 660-nm red light (R30, pink bars) showing partial insensitivity of pap8-1 to the PBG overexpression. Measurements are given in source data; in the order of the graph n equals (50, 133, 58, 43, 36, 112, 25, 60) δ-test (comparison of the mean) R8: δPBG/PBGp8-1= 20.38 corresponding to p-value < 10-31 δwt/p8-1= 0.5 < 1.96 not significant at α set to 0.05).Nuclear accumulation of PBG observed under GFP excitation in the given genotypes. (C,D) Epi-fluorescence microscopy. (E,F) Confocal microscopy showing the size of the nuclear bodies. Scale bars equal 5 µm.(G) Box plot (Min, 1st quartile, median as the central band, 3rd quartile, max) on the diameter of the nuclear bodies (NBs); n equals the number of records.H Immuno-blots using a GFP antibody or a PAP8 antibody showing respectively the levels of PHYB-GFP and PAP8 in the given genotypes grown in the dark for 3 days or in light; n.a., not applicable as the pap8-1 mutant can only be visually distinguished from wild type after light exposure; 2 lines (L#06 and L#07) for PBG/pap8-1 were tested. Coomassie blue staining presented as loading; signals were quantified using ImageJ.I RT-qPCR analysis on wild type, pap8-1, PBG and PBG pap8-1. Seedlings were grown in the dark (D) or under white light (L, 30 µmol.m-2.s-1); levels of transcripts are given relative to EF1α; error bars correspond to standard errors on technical triplicates and the dark sample is the wild-type PBG line. δ-test (comparison of the mean) ***, P < 10-72.J Immuno-blots showing the levels of PIF1, PIF3, HY5 in given genotypes grown in the Dark or Light condition as noted: p5/+, mix of an heterozygous pap5-2 siblings progeny undistinguishable from wild type; p5-2, pap5-2 and pifq, quadruple pif1-1 pif3-3 pif4-2 pif5-3 mutant; Histone H3 (H3), RbcL and PAP8 were used as controls; n.a., not applicable.


Figure 6A Bimolecular fluorescence complementation tests using in combination PAP8ΔcTP - NY ( P8Δc - NY ) with PAP5ΔcTP - YC ( P5Δc - YC ) or PAP8ΔcTP - YC ( P8Δc - YC ) with PAP5ΔcTP - NY ( P5Δc - NY ) ; PAP10 - RFP ( P10 - RFP ) was used as internal positive control for transfection ; the ratio in grey depicts the number of green - fluorescent cells over red - fluorescent cells . Arrowheads indicate nuclei . See Appendix Fig . S6 for control experiments ; transgenes expressed under CaMV35S promoter . B Overlay of 1H - 15N correlation 2D NMR spectra of free 15N - labelled PAP8 alone ( blue ) or in complex with PAP5 ( red ) . Grey areas depict changes of signals in the PAP8 spectrum . C 15N - Filtered Diffusion Ordered Spectroscopy - NMR measurements to PAP8 . Exponential decay curves of PAP8 in absence or in presence of MBP - PAP5 are shown in red and black respectively . The units on the y - axis are normalized values of the integrals of the signal measured in the amide proton region .

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

Figure 6A Bimolecular fluorescence complementation tests using in combination PAP8ΔcTP-NY (P8Δc-NY) with PAP5ΔcTP-YC (P5Δc-YC) or PAP8ΔcTP-YC (P8Δc-YC) with PAP5ΔcTP-NY (P5Δc-NY); PAP10-RFP (P10-RFP) was used as internal positive control for transfection; the ratio in grey depicts the number of green-fluorescent cells over red-fluorescent cells. Arrowheads indicate nuclei. See Appendix Fig. S6 for control experiments; transgenes expressed under CaMV35S promoter.B Overlay of 1H-15N correlation 2D NMR spectra of free 15N-labelled PAP8 alone (blue) or in complex with PAP5 (red). Grey areas depict changes of signals in the PAP8 spectrum.C 15N-Filtered Diffusion Ordered Spectroscopy-NMR measurements to PAP8. Exponential decay curves of PAP8 in absence or in presence of MBP-PAP5 are shown in red and black respectively. The units on the y-axis are normalized values of the integrals of the signal measured in the amide proton region.


Figure 7A Immuno - blots showing the levels of PAP8 , PAP5 , Histone H3 ( H3 ) , Rubisco ( RbcL ) , and HY5 ; D , Dark ; Ph , photomorphogenic growth conditions from germination on . * , HY5 is detected as a modified form ( + 6 kDa ) ; the arrows indicate the two different post - translational modified forms of HY5 for which accumulation occurs upon light exposure . B Relative protein contents normalized to histone H3 during the transition from skotomorphogenesis to photomorphogenesis , we propose a phase of photo - initiation corresponding to the 5 minutes of light allowing PAP8 and PAP5 to rise to nearly 100 % of their maximum . The first macroscopic signs of greening are indicated around 3 hours while the photosynthetic apparatus bursts at 8 hours to be fully accumulated at 48 hours .

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

Figure 7A Immuno-blots showing the levels of PAP8, PAP5, Histone H3 (H3), Rubisco (RbcL), and HY5 ; D, Dark; Ph, photomorphogenic growth conditions from germination on. *, HY5 is detected as a modified form (+ 6 kDa); the arrows indicate the two different post-translational modified forms of HY5 for which accumulation occurs upon light exposure.B Relative protein contents normalized to histone H3 during the transition from skotomorphogenesis to photomorphogenesis, we propose a phase of photo-initiation corresponding to the 5 minutes of light allowing PAP8 and PAP5 to rise to nearly 100% of their maximum. The first macroscopic signs of greening are indicated around 3 hours while the photosynthetic apparatus bursts at 8 hours to be fully accumulated at 48 hours.


Figure 1qRT - PCR of mGPDH , myogenin and myosin heavy chain ( MyHC ) levels during C2C12 myocyte differentiationimmunoblot of mGPDH , myogenin and myosin heavy chain ( MyHC ) levels during C2C12 myocyte differentiation . Quantification represents the levels of indicated protein normalized to β - actinImmunoblot of mGPDH , voltage - dependent anion channel ( VDAC ) and cytochrome c ( Cyt C ) levels in mitochondrial lysate during C2C12 myocyte differentiation . Quantification represents the levels of indicated protein normalized to COX IVActivity assay of mGPDH at days 0 and 7 after C2C12 myocyte differentiationRepresentative images of MyHC immunofluorescence ( E ) of C2C12 myocyte transfected with the siRNA or the overexpression plasmid for mGPDC2C12 myocyte transfected with the siRNA or the overexpression plasmid for mGPDH ; the fusion index ( F ) and the distribution of nuclei per myotube ( G ) were calculated at day 5 after differentiation . Immunoblot of mGPDH , myogenin and MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH . Quantificatio represents the levels of indicated protein normalized to β - actin at indicated day after differentiationmyogenin and MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH . Quantificatio represents the levels of indicated protein normalized to β - actin at indicated day after differentiationqRT - PCR analysis of mGPDH , myogenin and MyHC in C2C12 myocytes transfected with the siRN for mGPDH at day 4 after differentiationqRT - PCR analysis of mGPDH , myogenin and MyHC in C2C12 myocytes transfected wit overexpression plasmid for mGPDH at day 4 after

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

Figure 1qRT-PCR of mGPDH, myogenin and myosin heavy chain (MyHC) levels during C2C12 myocyte differentiationimmunoblot of mGPDH, myogenin and myosin heavy chain (MyHC) levels during C2C12 myocyte differentiation. Quantification represents the levels of indicated protein normalized to β-actinImmunoblot of mGPDH, voltage-dependent anion channel (VDAC) and cytochrome c (Cyt C) levels in mitochondrial lysate during C2C12 myocyte differentiation. Quantification represents the levels of indicated protein normalized to COX IVActivity assay of mGPDH at days 0 and 7 after C2C12 myocyte differentiationRepresentative images of MyHC immunofluorescence (E) of C2C12 myocyte transfected with the siRNA or the overexpression plasmid for mGPDC2C12 myocyte transfected with the siRNA or the overexpression plasmid for mGPDH; the fusion index (F) and the distribution of nuclei per myotube (G) were calculated at day 5 after differentiation.Immunoblot of mGPDH, myogenin and MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH. Quantificatio represents the levels of indicated protein normalized to β-actin at indicated day after differentiationmyogenin and MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH. Quantificatio represents the levels of indicated protein normalized to β-actin at indicated day after differentiationqRT-PCR analysis of mGPDH, myogenin and MyHC in C2C12 myocytes transfected with the siRN for mGPDH at day 4 after differentiationqRT-PCR analysis of mGPDH, myogenin and MyHC in C2C12 myocytes transfected wit overexpression plasmid for mGPDH at day 4 after differentiation


Figure 2qRT - PC of mGPDH , myogenin and developmental myosin heavy chain ( myh8 , myl4 and myh3 ) in gastrocnemius ( GA ) muscle from C57BL / 6J mice at indicated day after CTX intramuscular injectionimmunoblo ( B ) of mGPDH , myogeni in gastrocnemius ( GA ) muscle from C57BL / 6J mice at indicated day after CTX intramuscular injectionActivity assay of mGPDH in GA muscle from C57BL / 6J mice at day 0 and 7 after CTX injectionRepresentative images of the H & E staining ( arrowhead , necrotic myofibers ; asterisks , regenerating fibers ) ( D ) , distribution of the fibers cross - section area ( CSA ) ( E ) , percentage of myofibers with central nuclei ( F ) in GA muscle from WT and mGPDH - / - mice at day 7 post CTX injectionimmunofluorescence staining of desmin ( green ) ( G ) in GA muscle from WT and mGPDH - / - mice at day 7 post CTX injectionMuscle weight ( H in GA muscle from WT and mGPDH - / - mice at day 14 post CTX injectiontrichrome staining ( I ) in GA muscle from WT and mGPDH - / - mice at day 14 post CTX injection . Quantification represents the fibrotic areasqRT - PCR ( J for mGPDH , myogenin and myh3 in GA muscle from WT and mGPDH - / - mice at day 7 post CTX injectionimmunoblot ( K ) for mGPDH , myogeni in GA muscle from WT and mGPDH - / - mice at day 7 post CTX injectionqRT - PCR for mGPDH , myogenin and myh3 ( L ) in GA muscle from mdx mice 4 weeks after AAV - mGPDH intramuscular injectionH & E staining ( M ) , distribution of the fibers CSA ( N in GA muscle from mdx mice 4 weeks after AAV - mGPDH intramuscular injection ) , qRT - PCR ( O in GA muscle from mdx mice 4 weeks after AAV - mGPDH intramuscular injectionimmunofluorescence staining ( P ) for utrophi in GA muscle from mdx mice 4 weeks after AAV - mGPDH intramuscular injectiontrichrome staining ( Q ) in GA muscle from mdx mice 4 weeks after AAV - mGPDH intramuscular injectionExercise capacity of mdx mice 6 weeks after AAV - mGPDH tail vein injection .

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

Figure 2qRT-PC of mGPDH, myogenin and developmental myosin heavy chain (myh8, myl4 and myh3) in gastrocnemius (GA) muscle from C57BL/6J mice at indicated day after CTX intramuscular injectionimmunoblo (B) of mGPDH, myogeni in gastrocnemius (GA) muscle from C57BL/6J mice at indicated day after CTX intramuscular injectionActivity assay of mGPDH in GA muscle from C57BL/6J mice at day 0 and 7 after CTX injectionRepresentative images of the H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (D), distribution of the fibers cross-section area (CSA) (E), percentage of myofibers with central nuclei (F) in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injectionimmunofluorescence staining of desmin (green) (G) in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injectionMuscle weight (H in GA muscle from WT and mGPDH-/- mice at day 14 post CTX injectiontrichrome staining (I) in GA muscle from WT and mGPDH-/- mice at day 14 post CTX injection. Quantification represents the fibrotic areasqRT-PCR (J for mGPDH, myogenin and myh3 in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injectionimmunoblot (K) for mGPDH, myogeni in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injectionqRT-PCR for mGPDH, myogenin and myh3 (L) in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injectionH&E staining (M), distribution of the fibers CSA (N in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injection), qRT-PCR (O in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injectionimmunofluorescence staining (P) for utrophi in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injectiontrichrome staining (Q) in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injectionExercise capacity of mdx mice 6 weeks after AAV-mGPDH tail vein injection.


Figure 3Mitochondrial DNA ( A ) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationnuclear - encoded OXPHOS genes ( B in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationrespirometry analysis ( C in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationimmunoblots of mGPDH , phospho - Thr172 AMPK ( p - AMPK ) , total AMPK ( AMPK ) , phospho - Ser79 - ACC ( p - ACC ) , total ACC and PGC1α and corresponding quantifications represent mGPDH , p - AMPK , p - ACC and PGC1α protein level in C2C12 myocytes transfected with siRN for mGPDH 24 h after differentiationimmunoblots of mGPDH , phospho - Thr172 AMPK ( p - AMPK ) , total AMPK ( AMPK ) , phospho - Ser79 - ACC ( p - ACC ) , total ACC and PGC1α and corresponding quantifications represent mGPDH , p - AMPK , p - ACC and PGC1α protein levels ( D - F ) in C2C12 myocytes transfecte wit plasmid for mGPDH 24 h after differentiationImmunoblots of p - AMPK , p - ACC and PGC1α and corresponding quantifications represent p - AMPK , p - ACC and PGC1α protein levels ( G in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C ( CC ) 24 h after differentiationmitochondrial DNA ( H in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C ( CC ) 24 h after differentiationnuclear - encoded OXPHOS genes combined by NDUFS8 , SDHb , Uqcrc1 , COX5b and ATP5a1 ( I ) in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C ( CC ) 24 h after differentiationNAD + / NADH ratio ( J in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationimmunoprecipitation analysis for PGC - 1α acetyl - lysine ( Ac - Lys ) level ( K ) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationImmunoblot of c - myc and myogenin ( L in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h ( L , M after differentiationcorresponding quantifications represent c - myc and myogenin protein levels ( M ) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h ( L , Mrepresentative images of MyHC immunofluorescence ( N ) , fusion index ( O ) and the distribution of nuclei per myotube ( P ) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC a 72 h ( N - P ) after differentiationImmunoblots of p - AMPK , p - ACC , PGC1α and myogenin in C2C12 myocytes transfected with mGPDH plasmid with the CaMKKβ inhibitor STO - 609 at 24 h after differentiation . Quantifications represent p - AMPK , p - ACC , PGC1α and myogenin protein levelsImmunoblots of p - AMPK and p - ACC in C2C12 myocytes transfected with mGPDH plasmid with the Ca2 + chelator BAPTA - AM at 24 h after differentiation . Quantifications represent p - AMPK and p - ACC protein levels .

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

Figure 3Mitochondrial DNA (A) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationnuclear-encoded OXPHOS genes (B in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationrespirometry analysis (C in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationimmunoblots of mGPDH, phospho-Thr172 AMPK (p-AMPK), total AMPK (AMPK), phospho-Ser79-ACC (p-ACC), total ACC and PGC1α and corresponding quantifications represent mGPDH, p-AMPK, p-ACC and PGC1α protein level in C2C12 myocytes transfected with siRN for mGPDH 24 h after differentiationimmunoblots of mGPDH, phospho-Thr172 AMPK (p-AMPK), total AMPK (AMPK), phospho-Ser79-ACC (p-ACC), total ACC and PGC1α and corresponding quantifications represent mGPDH, p-AMPK, p-ACC and PGC1α protein levels (D-F) in C2C12 myocytes transfecte wit plasmid for mGPDH 24 h after differentiationImmunoblots of p-AMPK, p-ACC and PGC1α and corresponding quantifications represent p-AMPK, p-ACC and PGC1α protein levels (G in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiationmitochondrial DNA (H in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiationnuclear-encoded OXPHOS genes combined by NDUFS8, SDHb, Uqcrc1, COX5b and ATP5a1 (I) in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiationNAD+/NADH ratio (J in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationimmunoprecipitation analysis for PGC-1α acetyl-lysine (Ac-Lys) level (K) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationImmunoblot of c-myc and myogenin (L in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L,M after differentiationcorresponding quantifications represent c-myc and myogenin protein levels (M) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L,Mrepresentative images of MyHC immunofluorescence (N), fusion index (O) and the distribution of nuclei per myotube (P) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC a 72 h (N-P) after differentiationImmunoblots of p-AMPK, p-ACC, PGC1α and myogenin in C2C12 myocytes transfected with mGPDH plasmid with the CaMKKβ inhibitor STO-609 at 24 h after differentiation. Quantifications represent p-AMPK, p-ACC, PGC1α and myogenin protein levelsImmunoblots of p-AMPK and p-ACC in C2C12 myocytes transfected with mGPDH plasmid with the Ca2+ chelator BAPTA-AM at 24 h after differentiation. Quantifications represent p-AMPK and p-ACC protein levels.