J . Representative WB of Skm lipid peroxidation and redox system proteins . Two samples per condition are shown . Each sample contains extracts from 3 mice . Histograms represent quantification ( wt , n = 6 ; LowOXPHOS , n = 6 ) . 4 - Hydroxynonenal ( 4HN ) , peroxiredoxin 2 , 3 and 6 ( PRX ) , superoxide dismutase 1 and 2 ( SOD ) , catalase ( CATA ) and glutathione reductase ( GSR ) immunoblots are shown . βF1 is presented as a loading control . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

J. Representative WB of Skm lipid peroxidation and redox system proteins. Two samples per condition are shown. Each sample contains extracts from 3 mice. Histograms represent quantification (wt, n= 6; LowOXPHOS, n= 6). 4-Hydroxynonenal (4HN), peroxiredoxin 2, 3 and 6 (PRX), superoxide dismutase 1 and 2 (SOD), catalase (CATA) and glutathione reductase (GSR) immunoblots are shown. βF1 is presented as a loading control. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


K . Representative WB of Skm OXPHOS and related system proteins . Two samples per condition are shown . Each sample contains extracts from 3 mice . Histograms represent quantification ( wt , n = 6 ; LowOXPHOS , n = 6 ) . NDUFA9 ( CI ) , SDHA and B ( CII ) , ETF subunits A and B , ETFDH , CoreII ( CIII ) , subunit 1 ( CIV ) , βF1 ( CV ) and hATPIF1 immunoblots are shown . Tubulin is presented as a loading control . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

K. Representative WB of Skm OXPHOS and related system proteins. Two samples per condition are shown. Each sample contains extracts from 3 mice. Histograms represent quantification (wt, n= 6; LowOXPHOS, n= 6). NDUFA9 (CI), SDHA and B (CII), ETF subunits A and B, ETFDH, CoreII (CIII), subunit 1 (CIV), βF1(CV) and hATPIF1 immunoblots are shown. Tubulin is presented as a loading control. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


L . Skm iTRAQ ratio of FADH2 - binding proteins ( wt , n = 12 ; LowOXPHOS , n = 12 ) . A higher intensity of red color represents a higher LowOXPHOS / wt expression ratio .

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L. Skm iTRAQ ratio of FADH2-binding proteins (wt, n= 12; LowOXPHOS, n= 12). A higher intensity of red color represents a higher LowOXPHOS/wt expression ratio.


M . Oxidized FAD levels and the reduced / oxidized FAD ratio in Skm extracts ( wt , n = 8 ; LowOXPHOS n = 8 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

M. Oxidized FAD levels and the reduced/oxidized FAD ratio in Skm extracts (wt, n= 8; LowOXPHOS n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


A . Enzymatic activity of respiratory complex CII in Skm isolated mitochondria from wt and LowOXPHOS mice fed with chow or HFD ( wt , n = 4 ; LowOXPHOS , n = 4 ; wt + HFD , n = 4 ; LowOXPHOS + HFD , n = 4 ) .

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A. Enzymatic activity of respiratory complex CII in Skm isolated mitochondria from wt and LowOXPHOS mice fed with chow or HFD (wt, n= 4; LowOXPHOS, n= 4; wt + HFD, n= 4; LowOXPHOS + HFD, n= 4).


B . Polarographic profiles of isolated mitochondria from wt ( lower trace ) and LowOXPHOS ( upper trace ) animals using succinate ( left graph ) or palmitoyl - carnitine ( right graph ) as a substrate . Quantification of maximal respiration in the right histogram . Bars are the mean ± s . e . m . of n = 3 mice / genotype , 3 traces / mouse ; OL , oligomycin ; Ant A , antimycin A . Data information : * , # p < 0 . 05 when compared to wt or LowOXPHOS , respectively , by ANOVA and Student ' s t - test .

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

B. Polarographic profiles of isolated mitochondria from wt (lower trace) and LowOXPHOS (upper trace) animals using succinate (left graph) or palmitoyl-carnitine (right graph) as a substrate. Quantification of maximal respiration in the right histogram. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.


( C - E ) 2D - PAGE of Skm extracts ( C , D ) and acetylated proteins ( E ) from wt ( n = 4 ) and LowOXPHOS ( n = 3 ) mice . The isoelectric point ( pI ) of the CII subunits SDHB ( C ) and SDHA ( D ) was calculated by protein migration in pH 3 - 10 NL strips .

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(C-E) 2D-PAGE of Skm extracts (C, D) and acetylated proteins (E) from wt (n= 4) and LowOXPHOS (n= 3) mice. The isoelectric point (pI) of the CII subunits SDHB (C) and SDHA (D) was calculated by protein migration in pH 3-10 NL strips.


F . Immunocapture ( IP ) of SDHA blotted with anti - acetyl - K antibody ( upper panel ) and of acetylated proteins blotted with anti - SDHA antibody ( middle panel ) in Skm isolated mitochondria from wt ( n = 3 ) and LowOXPHOS ( n = 3 ) mice .

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F. Immunocapture (IP) of SDHA blotted with anti-acetyl-K antibody (upper panel) and of acetylated proteins blotted with anti-SDHA antibody (middle panel) in Skm isolated mitochondria from wt (n= 3) and LowOXPHOS (n= 3) mice.


I . MitoSox staining in myocytes expressing or not ATPIF1H49K . The left scheme illustrates where each ETC inhibitor works . The right histogram shows the quantification of mitochondrial ROS . Bars are the mean ± s . e . m . of n = 3 experiments , 12 replicas / condition . Data information : * , # p < 0 . 05 when compared to wt or LowOXPHOS , respectively , by ANOVA and Student ' s t - test .

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

I. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each ETC inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.


J . MitoSox staining in myocytes expressing or not ATPIF1H49K . The left scheme illustrates where each CII inhibitor works . The right histogram shows the quantification of mitochondrial ROS . Bars are the mean ± s . e . m . of n = 3 experiments , 12 replicas / condition . Data information : * , # p < 0 . 05 when compared to wt or LowOXPHOS , respectively , by ANOVA and Student ' s t - test .

J. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each CII inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.


K . Representative blue native immunoblots ( BN ) and clear native in - gel activity ( CN ) of Skm mitochondrial membrane proteins from wt ( n = 3 ) and LowOXPHOS ( n = 3 ) 2 - month - old mice . The migration of the respiratory complexes / supercomplexes CI - V is indicated . Subassembly of CI ( with no activity , suggesting degradation ) was observed in LowOXPHOS mice . VDAC is shown as a loading control .

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K. Representative blue native immunoblots (BN) and clear native in-gel activity (CN) of Skm mitochondrial membrane proteins from wt (n= 3) and LowOXPHOS (n= 3) 2-month-old mice. The migration of the respiratory complexes/supercomplexes CI-V is indicated. Subassembly of CI (with no activity, suggesting degradation) was observed in LowOXPHOS mice. VDAC is shown as a loading control.


( A , B ) The schematic illustrates the screening process of 702 drugs related to the mitochondrial respiratory capacity assessed by Seahorse XFe96 in C2C12 myocytes . Palmitate is used as a substrate . A total of 41 hits were identified . Maximum respiration ( Max R , % of untreated cells ) of compounds categorized by their therapeutic use ( B ) .

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(A, B) The schematic illustrates the screening process of 702 drugs related to the mitochondrial respiratory capacity assessed by Seahorse XFe96 in C2C12 myocytes. Palmitate is used as a substrate. A total of 41 hits were identified. Maximum respiration (Max R, % of untreated cells) of compounds categorized by their therapeutic use (B).


( C , D ) The schematic illustrates the screening process of the 41 hits on FFA β - oxidation using 9 , 103H ( N ) palmitic acid as a substrate in C2C12 myocytes . Ten enhancers were identified . D . Correlation between the maximum respiration ( x ) and palmitate β - oxidation ( y ) . Colors identify classes of pharmaceuticals as in B . Edaravone was selected as a hit .

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(C, D) The schematic illustrates the screening process of the 41 hits on FFA β-oxidation using 9,103H(N) palmitic acid as a substrate in C2C12 myocytes. Ten enhancers were identified. D. Correlation between the maximum respiration (x) and palmitate β-oxidation (y). Colors identify classes of pharmaceuticals as in B. Edaravone was selected as a hit.


E . Representative respiratory profile of C2C12 myocytes treated ( green trace ) or not treated ( black trace ) with 2 µM edaravone . The CPT1 inhibitor etomoxir was used as a negative control . OCR , oxygen consumption rate ; OL , oligomycin ; DNP , 2 , 4 - dinitrophenol ; Rot , rotenone ; Ant A , antimycin A . Quantification in right histogram . Bars are the mean ± s . e . m . of 12 replicas / condition . Data information ; 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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E. Representative respiratory profile of C2C12 myocytes treated (green trace) or not treated (black trace) with 2 µM edaravone. The CPT1 inhibitor etomoxir was used as a negative control. OCR, oxygen consumption rate; OL, oligomycin; DNP, 2,4-dinitrophenol; Rot, rotenone; Ant A, antimycin A. Quantification in right histogram. Bars are the mean ± s.e.m. of 12 replicas/condition. Data information ;0.05 when compared to wt by ANOVA and Student's t-test.


( F , G ) FFA β - oxidation in C2C12 myocytes transfected with CRL or ATPIF1H49K plasmids ( F ) and in primary myotubes from wt and LowOXPHOS mice ( G ) , treated ( green bars ) or not with 2 µM edaravone . Bars are the mean ± s . e . m . of n = 3 experiments , 9 replicas / condition . Data information : * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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

(F, G) FFA β-oxidation in C2C12 myocytes transfected with CRL or ATPIF1H49K plasmids (F) and in primary myotubes from wt and LowOXPHOS mice (G), treated (green bars) or not with 2 µM edaravone. Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition. Data information: *p <0.05 when compared to wt by ANOVA and Student's t-test.


A . Mitochondrial ROS after 3 hours treatment with 2 μM edaravone ( green bars ) , 10 nM MitoQ ( purple bars ) or 1 mM NAC ( blue bars ) . Bars are the mean ± s . e . m . of n = 3 experiments , 9 replicas / condition .

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A. Mitochondrial ROS after 3 hours treatment with 2 μM edaravone (green bars), 10 nM MitoQ (purple bars) or 1 mM NAC (blue bars). Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition.


B . Representative WB expression of Skm ROS system proteins in mice administered ( 30 days ) edaravone . Two samples per condition are shown . Each sample contains extracts from 3 mice ( wt + edaravone , n = 6 ; LowOXPHOS + edaravone n = 6 ) . Peroxiredoxin 2 and 3 ( PRX ) , catalase ( CATA ) and glutathione reductase ( GSR ) immunoblots are shown . GAPDH is presented as a loading control .

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

B. Representative WB expression of Skm ROS system proteins in mice administered (30 days) edaravone. Two samples per condition are shown. Each sample contains extracts from 3 mice (wt + edaravone, n= 6; LowOXPHOS + edaravone n= 6). Peroxiredoxin 2 and 3 (PRX), catalase (CATA) and glutathione reductase (GSR) immunoblots are shown. GAPDH is presented as a loading control.


C . qPCR relative expression of TNFα and PGC1α in myocytes treated for 24 h with 2μM edaravone ( green bars ) , 10nM MitoQ ( purple bars ) or 1 mM NAC ( blue bars ) . 6 replicas / condition .

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C. qPCR relative expression of TNFα and PGC1α in myocytes treated for 24 h with 2μM edaravone (green bars), 10nM MitoQ (purple bars) or 1 mM NAC (blue bars). 6 replicas/condition.


Representative WB expression of Skm de novo lipid synthesis ( D ) One month of edaravone treatment downregulated FASN and ACLY in both wt and LowOXPHOS mice ( D ) Two samples per condition are shown . Each sample contains extracts from 3 mice . βF1 is presented as a loading control . Quantifications in lateral histograms ( wt , n = 6 ; LowOXPHOS , n = 6 ; wt + edaravone , n = 6 ; LowOXPHOS + edaravone , n = 6 ) .

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

Representative WB expression of Skm de novo lipid synthesis (D) One month of edaravone treatment downregulated FASN and ACLY in both wt and LowOXPHOS mice (D) Two samples per condition are shown. Each sample contains extracts from 3 mice. βF1 is presented as a loading control. Quantifications in lateral histograms (wt, n= 6; LowOXPHOS, n= 6; wt + edaravone, n= 6; LowOXPHOS + edaravone, n= 6).


Representative OXPHOS ( E ) proteins . One month of edaravone treatment in both wt and LowOXPHOS mice and rewired the upregulation of ETF - A , ETF - B and SDHA proteins to the levels of wt ( E ) . Two samples per condition are shown . Each sample contains extracts from 3 mice . Tubulin is presented as a loading control . Quantifications in lateral histograms ( wt , n = 6 ; LowOXPHOS , n = 6 ; wt + edaravone , n = 6 ; LowOXPHOS + edaravone , n = 6 ) .

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

Representative OXPHOS (E) proteins. One month of edaravone treatment in both wt and LowOXPHOS mice and rewired the upregulation of ETF-A, ETF-B and SDHA proteins to the levels of wt (E). Two samples per condition are shown. Each sample contains extracts from 3 mice. Tubulin is presented as a loading control. Quantifications in lateral histograms (wt, n= 6; LowOXPHOS, n= 6; wt + edaravone, n= 6; LowOXPHOS + edaravone, n= 6).


F . 2D - PAGE of Skm extracts from wt ( n = 3 ) and LoxOXPHOS ( n = 3 ) mice treated with edaravone . The pI of SDHA , calculated by protein migration in pH 3 - 10 NL strips , was the same for both genotypes .

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F. 2D-PAGE of Skm extracts from wt (n= 3) and LoxOXPHOS (n= 3) mice treated with edaravone. The pI of SDHA, calculated by protein migration in pH 3-10 NL strips, was the same for both genotypes.


G . Representative blue native immunoblots ( BN ) of Skm mitochondrial membrane proteins from 2 - month - old mice treated with edaravone for 30 days ( wt + edaravone , n = 3 ; LowOXPHOS + edaravone , n = 3 ) . The migration of the respiratory complexes / supercomplexes CI - CIV and hATPIF1 is indicated . VDAC is shown as a loading control .

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G. Representative blue native immunoblots (BN) of Skm mitochondrial membrane proteins from 2-month-old mice treated with edaravone for 30 days (wt + edaravone, n= 3; LowOXPHOS + edaravone, n= 3). The migration of the respiratory complexes/supercomplexes CI-CIV and hATPIF1 is indicated. VDAC is shown as a loading control.


H . Mouse body weight following the administration of HFD and edaravone . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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H. Mouse body weight following the administration of HFD and edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.


I . v - WAT amounts at day 60th of HFD in mice treated with edaravone . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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I. v-WAT amounts at day 60th of HFD in mice treated with edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.


J . Insulin ( ITT ) and glucose ( GTT ) tolerance tests after 60 days of HFD in mice treated with edaravone . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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J. Insulin (ITT) and glucose (GTT) tolerance tests after 60 days of HFD in mice treated with edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.


K . GTT after 80 days of HFD ( left ) and ITT after 90 days of HFD ( right ) in wt and LowOXPHOS mice treated with edaravone , showing that the compound has an improving effect itself . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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K. GTT after 80 days of HFD (left) and ITT after 90 days of HFD (right) in wt and LowOXPHOS mice treated with edaravone, showing that the compound has an improving effect itself. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.


L . Blood glucose following the administration of HFD . T2D onset occurred on day 90 in wt and LowOXPHOS mice treated with edaravone . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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L. Blood glucose following the administration of HFD. T2D onset occurred on day 90 in wt and LowOXPHOS mice treated with edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.


B Interaction of Atg18 with retromer subunits . Genomically tagged yomCherry - fusions of each retromer subunit were expressed in SEY6210 atg18 ∆ , atg21 ∆ , hsv2 ∆ cells , together with a plasmid expressing Atg18HA3yEGFP . Cell lysates were subjected to immunoprecipitation using RFP - Trap magnetic beads and analyzed by SDS - PAGE and Western blotting against the indicated proteins .

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B Interaction of Atg18 with retromer subunits. Genomically tagged yomCherry-fusions of each retromer subunit were expressed in SEY6210 atg18∆, atg21∆, hsv2∆ cells, together with a plasmid expressing Atg18HA3yEGFP. Cell lysates were subjected to immunoprecipitation using RFP-Trap magnetic beads and analyzed by SDS-PAGE and Western blotting against the indicated proteins.


D Atg18 - Vps35 interaction . Genomically tagged Vps35yomCherry was pulled down from lysates of SEY6210 WT or SEY6210 vps26 ∆ strains carrying genomically tagged Atg18HA3yEGFP . Adsorbed proteins were analyzed by SDS - PAGE and Western blotting using the antibodies indicated in brackets . E Signals from the blots in d were quantified on an infrared fluorescence scanner and normalized to the amount of pulled - down Vps35yomCherry . n = 3 independent experiments were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM . * * * P < 0 . 001 .

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D Atg18-Vps35 interaction. Genomically tagged Vps35yomCherry was pulled down from lysates of SEY6210 WT or SEY6210 vps26∆ strains carrying genomically tagged Atg18HA3yEGFP. Adsorbed proteins were analyzed by SDS-PAGE and Western blotting using the antibodies indicated in brackets. E Signals from the blots in d were quantified on an infrared fluorescence scanner and normalized to the amount of pulled-down Vps35yomCherry. n=3 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. *** P < 0.001.


F Atg18 - Vps26 interaction . Genomically tagged Vps26yomCherry was pulled down from lysates of SEY6210 WT , SEY6210 vps35 ∆ or SEY6210 vps29 ∆ strains carrying genomically tagged Atg18HA3yEGFP . Adsorbed proteins were analyzed as in D . G Proteins from F were quantified as in E . n = 3 independent experiments were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM . * * * p < 0 . 001 ) .

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F Atg18-Vps26 interaction. Genomically tagged Vps26yomCherry was pulled down from lysates of SEY6210 WT, SEY6210 vps35∆ or SEY6210 vps29∆ strains carrying genomically tagged Atg18HA3yEGFP. Adsorbed proteins were analyzed as in D. G Proteins from F were quantified as in E. n=3 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. *** p < 0.001).


B Salt - induced vacuole fission . The indicated cells were logarithmically grown in YPD and stained with FM4 - 64 . Vacuole morphology was imaged as in A , before and after a mild salt shock with 0 . 5 M of NaCl for 15 min . The look - up table has been inverted to allow better representation of the clusters of extremely small vacuolar fragments in the SNX mutants . Scale bar : 5 µm . C The number of vacuoles per cell was quantified for the samples from B . n = 3 experiments with at least 100 cells per condition were evaluated ; Error bars represent the SEM .

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B Salt-induced vacuole fission. The indicated cells were logarithmically grown in YPD and stained with FM4-64. Vacuole morphology was imaged as in A, before and after a mild salt shock with 0.5 M of NaCl for 15 min. The look-up table has been inverted to allow better representation of the clusters of extremely small vacuolar fragments in the SNX mutants. Scale bar: 5 µm. C The number of vacuoles per cell was quantified for the samples from B. n=3 experiments with at least 100 cells per condition were evaluated; Error bars represent the SEM.


A Vps17 labilizes the Atg18 - Vps26 interaction . Wildtype or vps17 ∆ cells expressing ATG18HA3 - yEGFP from a centromeric plasmid were logarithmically grown in YPD . Genomically tagged Vps26yomCherry was pulled down from whole - cell extracts and analyzed for associated Atg18HA3 - yEGFP by SDS - PAGE and Western blotting . Glucose - 6 - phosphate dehydrogenase ( Zwf1 ) serves as a loading control . The intensity of the interacting Atg18HA3 - yEGFP was quantified on a LICOR fluorescence imager and normalized to the amount of Vps26yomCherry . Values of the wildtype interaction were used as the reference and set to 1 . n = 4 independent experiments were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM , * * p < 0 . 01 )

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A Vps17 labilizes the Atg18-Vps26 interaction. Wildtype or vps17∆ cells expressing ATG18HA3-yEGFP from a centromeric plasmid were logarithmically grown in YPD. Genomically tagged Vps26yomCherry was pulled down from whole-cell extracts and analyzed for associated Atg18HA3-yEGFP by SDS-PAGE and Western blotting. Glucose-6-phosphate dehydrogenase (Zwf1) serves as a loading control. The intensity of the interacting Atg18HA3-yEGFP was quantified on a LICOR fluorescence imager and normalized to the amount of Vps26yomCherry. Values of the wildtype interaction were used as the reference and set to 1. n = 4 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM, ** p < 0.01)


B Epistasis of ATG18 and retromer genes concerning vacuolar morphology . The indicated cells were logarithmically grown in YPD medium , stained with FM4 - 64 and calcofluor white C Quantification of vacuole morphology . The number of vacuoles per cell was measured in the cells from B . The graph shows the fractions of cells displaying the indicated numbers of vacuolar vesicles ( n = 3 biological experiments with at least 100 cells per condition and experiment were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM . * * p < 0 .

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B Epistasis of ATG18 and retromer genes concerning vacuolar morphology. The indicated cells were logarithmically grown in YPD medium, stained with FM4-64 and calcofluor white C Quantification of vacuole morphology. The number of vacuoles per cell was measured in the cells from B. The graph shows the fractions of cells displaying the indicated numbers of vacuolar vesicles (n = 3 biological experiments with at least 100 cells per condition and experiment were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. ** p < 0.01


B Purified Atg18 and CSC - GFP were mixed in a 1 : 1 ratio , incubated together in PBS and analyzed by native PAGE and Western blotting using the antibodies indicated in brackets .

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B Purified Atg18 and CSC-GFP were mixed in a 1:1 ratio, incubated together in PBS and analyzed by native PAGE and Western blotting using the antibodies indicated in brackets.


C Pull - down . Cells ( SEY6210 atg18 ∆ , atg21 ∆ ) expressing genomically tagged Vps26yomCherry and the indicated Atg18HA3 - yEGFP variants were logarithmically grown in SC - URA media . Vps26yomCherry was pulled down from whole - cell extracts with RFP - trap magnetic beads and analyzed by SDS - PAGE and Western blotting against the indicated proteins . Bands were quantified on a LICOR fluorescence imager . Signals of Atg18HA3yEGFP were normalized relative to those of Vps26yomCherry . Vps1 served as a loading control . n = 3 independent replicates . Data were subjected to an unpaired t - test . Bars represent the mean and errors bars the SEM , * * * P < 0 . 001 ) .

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C Pull-down. Cells (SEY6210 atg18∆, atg21∆) expressing genomically tagged Vps26yomCherry and the indicated Atg18HA3-yEGFP variants were logarithmically grown in SC-URA media. Vps26yomCherry was pulled down from whole-cell extracts with RFP-trap magnetic beads and analyzed by SDS-PAGE and Western blotting against the indicated proteins. Bands were quantified on a LICOR fluorescence imager. Signals of Atg18HA3yEGFP were normalized relative to those of Vps26yomCherry. Vps1 served as a loading control. n = 3 independent replicates. Data were subjected to an unpaired t-test. Bars represent the mean and errors bars the SEM, *** P < 0.001).


A , Effect of Atg18T56E on salt - induced vacuole fission . Cells expressing Atg18WTHA3 - yEGFP or Atg18T56E - HA3 - yEGFP from centromeric plasmids in a SEY6210 atg18 ∆ , atg21 ∆ strain were logarithmically grown in SD - URA medium . They were stained and imaged before and after the induction of vacuole fission by a short salt shock Calcofluor white - stained cell walls are only represented in the merge ( blue ) . Scale bar : 5 µm . B , Quantification of the number of vacuoles per cell from A , n = 3 biological experiments with at least 100 cells per condition were scored ; bars represent the mean and error bars the SEM . C , Epistasis of atg18T56E over a vps17 ∆ mutation . The indicated variants of Atg18 - HA3 - yEGFP were expressed from plasmids in a SEY6210 atg18 ∆ , atg21 ∆ , vps17 ∆ strain . Cells were logarithmically grown in SD - URA and imaged Scale bar : 5 µm . D , Quantification of the experiments from C , n = 3 independent experiments with at least 100 cells per condition scored ; bars represent the mean and error bars the SEM .

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A, Effect of Atg18T56E on salt-induced vacuole fission. Cells expressing Atg18WTHA3-yEGFP or Atg18T56E-HA3-yEGFP from centromeric plasmids in a SEY6210 atg18∆, atg21∆ strain were logarithmically grown in SD-URA medium. They were stained and imaged before and after the induction of vacuole fission by a short salt shock Calcofluor white-stained cell walls are only represented in the merge (blue). Scale bar: 5 µm. B, Quantification of the number of vacuoles per cell from A, n = 3 biological experiments with at least 100 cells per condition were scored; bars represent the mean and error bars the SEM. C, Epistasis of atg18T56E over a vps17∆ mutation. The indicated variants of Atg18-HA3-yEGFP were expressed from plasmids in a SEY6210 atg18∆, atg21∆, vps17∆ strain. Cells were logarithmically grown in SD-URA and imaged Scale bar: 5 µm. D, Quantification of the experiments from C, n = 3 independent experiments with at least 100 cells per condition scored; bars represent the mean and error bars the SEM .


A . Atg18 - dependent recruitment of CSC - GFP to small liposomes . SUVs were incubated ( 10 min , 25 ° C ) with purified CSC ( 1 . 5 µM ) alone or in combination with the indicated recombinant Atg18 variants ( 1 . 5 µM ) . The vesicles were sedimented by centrifugation and supernatants ( Sup . ) and pellets were analyzed by SDS - PAGE and Coomassie staining . B . Quantification of the Coomassie signals for Vps35 by densitometry on a LICOR Odyssey scanner . n = 3 independent experiments . Bars represent the mean and error bars the SEM , * * p < 0 . 01 ; * p < 0 . 05 . C . Quantification as in B , but for the Atg18 variants .

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A. Atg18-dependent recruitment of CSC-GFP to small liposomes. SUVs were incubated (10 min, 25°C) with purified CSC (1.5 µM) alone or in combination with the indicated recombinant Atg18 variants (1.5 µM). The vesicles were sedimented by centrifugation and supernatants (Sup.) and pellets were analyzed by SDS-PAGE and Coomassie staining. B. Quantification of the Coomassie signals for Vps35 by densitometry on a LICOR Odyssey scanner. n = 3 independent experiments. Bars represent the mean and error bars the SEM, ** p < 0.01; * p < 0.05. C. Quantification as in B, but for the Atg18 variants.


A Colocalization of WIPI1 with Vps26A . The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells , from which endogenous WIPI1 had been deleted ( WIPI1 - KO ) . The cells were analyzed by confocal microscopy . Scale bars : 10 μm . Insets show enlargements of the outlined areas . B Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders ' colocalization coefficient M2 . The red line indicates the mean ; n = 120 cells per condition , pooled from three independent experiments . P - values were calculated by t - test ( analysis performed with 99 % confidence * * * p < 0 . 0001 ) . C Expression levels of WIPI1mCherry variants . Lysates ( 50 μg of protein per sample ) from the cells in A were analyzed by SDS - PAGE and Western blot against WIPI1 and tubulin .

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A Colocalization of WIPI1 with Vps26A. The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells, from which endogenous WIPI1 had been deleted (WIPI1-KO). The cells were analyzed by confocal microscopy. Scale bars: 10 μm. Insets show enlargements of the outlined areas. B Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders' colocalization coefficient M2. The red line indicates the mean; n = 120 cells per condition, pooled from three independent experiments. P-values were calculated by t-test (analysis performed with 99% confidence ***p < 0.0001). C Expression levels of WIPI1mCherry variants. Lysates (50 μg of protein per sample) from the cells in A were analyzed by SDS-PAGE and Western blot against WIPI1 and tubulin.


D Depletion of VPS35 . HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 ( VPS35 KD ) or a control siRNA pool . Lysates ( 50 μg per sample ) from the cells were analyzed by SDS - PAGE and Western blot against Vps35 and tubulin . E Blots from D were quantified on a LICOR Odyssey fluorescence imager . n = 3 . Red lines indicate the means , n = 3 independent experiments , using a Welch ' s t - test statistical analysis . Bars represent the mean and error bars the SD .

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D Depletion of VPS35. HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 (VPS35 KD) or a control siRNA pool. Lysates (50 μg per sample) from the cells were analyzed by SDS-PAGE and Western blot against Vps35 and tubulin. E Blots from D were quantified on a LICOR Odyssey fluorescence imager. n=3. Red lines indicate the means, n = 3 independent experiments, using a Welch's t-test statistical analysis. Bars represent the mean and error bars the SD.


F Cells from D were analyzed by confocal microscopy 18 h after transfection . Scale bar : 10 μm . G Quantification of tubule length in the cells from F . Data are means ± SD . of n = 210 tubules per condition , from three independent experiments . p values were calculated by unpaired Student ' s t - test . 99 % confidence : * * * = p < 0 . 0001 .

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F Cells from D were analyzed by confocal microscopy 18 h after transfection. Scale bar: 10 μm. G Quantification of tubule length in the cells from F. Data are means ± SD. of n=210 tubules per condition, from three independent experiments. p values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.


A , Tf recycling . WIPI1 - KO cells were transfected with WIPI1WT - eGFP , WIPI1S69E - eGFP or WIPI1S69A - eGFP for 18h . Then , they were serum - starved for 1 h , loaded with Alexa Fluor 568 - conjugated Tf , chased at 37 ° C for 1 h without labeled Tf , and analyzed by confocal microscopy . Scale bar : 10 μm . White dashed lines delineate the circumference of the cells . B , Quantification of Tf - fluorescence in the cells from a that expressed WIPI1 variants . Total cell fluorescence was integrated and corrected for background fluorescence . Mean values ± SD are shown . n = 3 independent experiments with a total of 150 cells analyzed per condition . P values were calculated by unpaired Student ' s t - test . 99 % confidence : * * * = p < 0 . 0001 .

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A, Tf recycling. WIPI1-KO cells were transfected with WIPI1WT-eGFP, WIPI1S69E-eGFP or WIPI1S69A-eGFP for 18h. Then, they were serum-starved for 1 h, loaded with Alexa Fluor 568-conjugated Tf, chased at 37°C for 1 h without labeled Tf, and analyzed by confocal microscopy. Scale bar: 10 μm. White dashed lines delineate the circumference of the cells. B, Quantification of Tf-fluorescence in the cells from a that expressed WIPI1 variants. Total cell fluorescence was integrated and corrected for background fluorescence. Mean values ± SD are shown. n = 3 independent experiments with a total of 150 cells analyzed per condition. P values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.


A . Confocal microscopy analysis showing the localization of endogenous Rab22a ( green ) and different endocytic markers ( red ) : early endosomal marker EEA1 , late endosome / lysosomal marker Lamp1 and the recycling compartment marker TfR in BMDCs at steady state . The means ± SEM of the Pearson correlation coefficients ( Pcc ) from Rab22a and EEA1 ( 0 . 4317 ± 0 . 05101 ) , Lamp1 ( 0 . 8412 ± 0 . 03562 ) and TfR ( 0 . 6409 ± 0 . 02999 ) were estimated from 15 images analyzed for each marker . Scale bars : 5 µm .

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A. Confocal microscopy analysis showing the localization of endogenous Rab22a (green) and different endocytic markers (red): early endosomal marker EEA1, late endosome/lysosomal marker Lamp1 and the recycling compartment marker TfR in BMDCs at steady state. The means ± SEM of the Pearson correlation coefficients (Pcc) from Rab22a and EEA1 (0.4317 ± 0.05101), Lamp1 (0.8412 ± 0.03562) and TfR (0.6409 ± 0.02999) were estimated from 15 images analyzed for each marker. Scale bars: 5 µm.


B . Colocalization of endogenous Rab22a ( red ) and EEA1 ( magenta ) around endosomes containing fluorescent soluble OVA ( OVA - FITC , green ) after 30 min of internalization by BMDCs . More than 60 % of the endosomes were positive for Rab22a and EEA1 . The indicated boxes are shown at higher magnification in the insets . Scale bars : 5 µm .

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B. Colocalization of endogenous Rab22a (red) and EEA1 (magenta) around endosomes containing fluorescent soluble OVA (OVA-FITC, green) after 30 min of internalization by BMDCs. More than 60% of the endosomes were positive for Rab22a and EEA1. The indicated boxes are shown at higher magnification in the insets. Scale bars: 5 µm.


C . IF detection of endogenous Rab22a ( green ) and Lamp1 ( red ) after 1 h of phagocytosis of 3 µm latex beads ( LB ) in BMDCs . Around 50 % of the phagosomes were double positive for Rab22a and Lamp1 . Asterisks indicate the LB . Scale bars : 5 µm .

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C. IF detection of endogenous Rab22a (green) and Lamp1 (red) after 1 h of phagocytosis of 3 µm latex beads (LB) in BMDCs. Around 50% of the phagosomes were double positive for Rab22a and Lamp1. Asterisks indicate the LB. Scale bars: 5 µm.


D . BMDCs were incubated with 3 μm magnetic beads for 15 min at 37 ° C and chased for 0 or 45 min . The panel shows immunoblotting of purified phagosomes and the total cell lysates ( TCL ) analyzed for Lamp1 and Rab22a . A total protein amount of 10 µg and 50 µg was loaded for purified phagosomes and TCL , respectively . The blot is representative of three independent experiments .

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D. BMDCs were incubated with 3 μm magnetic beads for 15 min at 37°C and chased for 0 or 45 min. The panel shows immunoblotting of purified phagosomes and the total cell lysates (TCL) analyzed for Lamp1 and Rab22a. A total protein amount of 10 µg and 50 µg was loaded for purified phagosomes and TCL, respectively. The blot is representative of three independent experiments.


A . Immunoblotting and densitometry quantification of Rab22a in JAWS - IIDCs infected with lentiviruses encoding a random sequence ( Scramble ) and a shRNA specific for silencing Rab22a ( Rab22a KD ) . Data are representative of at least three independent experiments .

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A. Immunoblotting and densitometry quantification of Rab22a in JAWS-IIDCs infected with lentiviruses encoding a random sequence (Scramble) and a shRNA specific for silencing Rab22a (Rab22a KD). Data are representative of at least three independent experiments.


B . RNA extraction and qPCR quantification of Rab22a in the same cells analyzed in A . Data show mean ± SEM of triplicate values and are representative of two independent experiments . * * P = 0 . 0047 .

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B. RNA extraction and qPCR quantification of Rab22a in the same cells analyzed in A. Data show mean ± SEM of triplicate values and are representative of two independent experiments. ** P = 0.0047.


C . IF labeling and confocal microscopy analysis showing the distribution of MHC - I molecules ( H - 2Kb , green ) and Rab22a ( red ) in Scramble and Rab22a KD JAWS - II DCs . Nuclei stained with DAPI and DIC images are shown on the left . Overlay is shown in the right panels . Scale bars : 5 µm . Data are representative of at least 30 images analyzed for each experimental condition from three independent experiments .

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C. IF labeling and confocal microscopy analysis showing the distribution of MHC-I molecules (H-2Kb, green) and Rab22a (red) in Scramble and Rab22a KD JAWS-II DCs. Nuclei stained with DAPI and DIC images are shown on the left. Overlay is shown in the right panels. Scale bars: 5 µm. Data are representative of at least 30 images analyzed for each experimental condition from three independent experiments.


D . FACS analysis of MHC - I labeled in intact ( cell surface ) and permeabilized ( total ) Scramble and Rab22a KD JAWS - II cells . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . P = 0 . 1082 ( ns ) and * * * P = 0 . 0003 .

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D. FACS analysis of MHC-I labeled in intact (cell surface) and permeabilized (total) Scramble and Rab22a KD JAWS-II cells. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. P = 0.1082 (ns) and ***P = 0.0003.


E . MHC - I staining on isolated phagosomes was measured by FACS at the indicated time periods after 3 µm LB internalization by Scramble and Rab22a KD JAWS - II DCs . Data represent mean ± SEM of three independent experiments . * * * P < 0 . 001 at 3 h and * P < 0 . 05 at 5 h between both DC types .

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E. MHC-I staining on isolated phagosomes was measured by FACS at the indicated time periods after 3 µm LB internalization by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. ***P < 0.001 at 3 h and *P < 0.05 at 5 h between both DC types.


F . MHC - I molecules recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS - II DCs . Data represent mean ± SEM of three independent experiments . * P < 0 . 05 at 10 min and * * P < 0 . 01 at 20 and 40 min .

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F. MHC-I molecules recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. *P < 0.05 at 10 min and **P < 0.01 at 20 and 40 min.


G . The transferrin ( Tfn ) recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS - II DCs . Data represent mean ± SEM of three independent experiments . P > 0 . 05 ( ns ) at 10 , 20 and 40 min .

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G. The transferrin (Tfn) recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. P > 0.05 (ns) at 10, 20 and 40 min.


A - C . The cross - presentation ability after incubation with A ) soluble OVA , B ) OVA / BSA - coated beads and C ) the SIINFEKL control peptide at the indicated concentrations by Scramble and Rab22a KD JAWS - II DCs was evaluated with the B3Z hybridoma . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . A ) * * * P = 0 . 0001 and B ) P = 0 . 1432 ( ns ) ; * * P = 0 . 0044 . The two - tailed Student ' s unpaired t test was performed .

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A-C. The cross-presentation ability after incubation with A) soluble OVA, B) OVA/BSA-coated beads and C) the SIINFEKL control peptide at the indicated concentrations by Scramble and Rab22a KD JAWS-II DCs was evaluated with the B3Z hybridoma. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. A) ***P = 0.0001 and B) P = 0.1432 (ns); **P = 0.0044. The two-tailed Student's unpaired t test was performed.


D - E . Evaluation of endocytosis and phagocytosis in Scramble and Rab22a KD JAWS - II DCs . D ) The endocytosis of fluorescent OVA after 1 h of internalization and E ) the phagocytosis of 3 µm fluorescent LB at different times of internalization were assessed by FACS analysis . The antigen internalization was conducted at 37 ° C for effective uptake and at 4 ° C as negative control . Data represent mean ± SEM of triplicate values and are representative of three independent experiments .

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D-E. Evaluation of endocytosis and phagocytosis in Scramble and Rab22a KD JAWS-II DCs. D) The endocytosis of fluorescent OVA after 1 h of internalization and E) the phagocytosis of 3 µm fluorescent LB at different times of internalization were assessed by FACS analysis. The antigen internalization was conducted at 37°C for effective uptake and at 4°C as negative control. Data represent mean ± SEM of triplicate values and are representative of three independent experiments.


F . The kinetics of OVA degradation , as percentage of proteases inhibitors , in isolated phagosomes at the indicated time periods post - internalization from Scramble and Rab22a KD JAWS - II DCs was assessed by FACS analysis . Data represent mean ± SEM of three independent experiments .

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F. The kinetics of OVA degradation, as percentage of proteases inhibitors, in isolated phagosomes at the indicated time periods post-internalization from Scramble and Rab22a KD JAWS-II DCs was assessed by FACS analysis. Data represent mean ± SEM of three independent experiments.


G . Immunoblotting of Rab22a and Actin in BMDCs infected with lentiviruses encoding a random sequence ( Scramble ) and two shRNA specific for silencing Rab22a ( Rab22a KD # 1 and # 2 ) .

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G. Immunoblotting of Rab22a and Actin in BMDCs infected with lentiviruses encoding a random sequence (Scramble) and two shRNA specific for silencing Rab22a (Rab22a KD #1 and #2).


H - I . The cross - presentation capacity after the incubation with H ) soluble OVA at the indicated concentrations by Scramble , Rab22a KD # 1 and Rab22a KD # 2 BMDCs was evaluated as described before for JAWS - II DCs . Data represent mean ± SEM of triplicate values and are representative of two independent experiments . * * * P = 0 . 0001 . The two - tailed Student ' s unpaired t test was performed .

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H-I. The cross-presentation capacity after the incubation with H) soluble OVA at the indicated concentrations by Scramble, Rab22a KD #1 and Rab22a KD #2 BMDCs was evaluated as described before for JAWS-II DCs. Data represent mean ± SEM of triplicate values and are representative of two independent experiments. ***P = 0.0001. The two-tailed Student's unpaired t test was performed.


H - I . The cross - presentation capacity after the incubation with I ) the SIINFEKL control peptide at the indicated concentrations by Scramble , Rab22a KD # 1 and Rab22a KD # 2 BMDCs was evaluated as described before for JAWS - II DCs . Data represent mean ± SEM of triplicate values and are representative of two independent experiments . * * * P = 0 . 0001 . The two - tailed Student ' s unpaired t test was performed .

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H-I. The cross-presentation capacity after the incubation with I) the SIINFEKL control peptide at the indicated concentrations by Scramble, Rab22a KD #1 and Rab22a KD #2 BMDCs was evaluated as described before for JAWS-II DCs. Data represent mean ± SEM of triplicate values and are representative of two independent experiments. ***P = 0.0001. The two-tailed Student's unpaired t test was performed.


J . Soluble OVA was electroporated into the cytosol of Scramble and Rab22a KD JAWS - II DCs and T cell activation was determined 2 h later with the B3Z hibridoma . To control endogenous MHC - I antigen presentation specificity , DCs were also treated with brefreldin A ( BFA ) . The use of this drug markedly reduced CD8 + T cell response to similar levels obtained by DCs without any antigen ( ∅ ) . Data represent mean ± SEM of triplicate values and are representative of three independent experiments .

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J. Soluble OVA was electroporated into the cytosol of Scramble and Rab22a KD JAWS-II DCs and T cell activation was determined 2 h later with the B3Z hibridoma. To control endogenous MHC-I antigen presentation specificity, DCs were also treated with brefreldin A (BFA). The use of this drug markedly reduced CD8+ T cell response to similar levels obtained by DCs without any antigen (∅). Data represent mean ± SEM of triplicate values and are representative of three independent experiments.


K . Immunoblotting showing the amount of OVA incorporated by Scramble ( a ) and Rab22a KD ( b ) JAWS - II DCs after electroporation and BFA treatment .

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K. Immunoblotting showing the amount of OVA incorporated by Scramble (a) and Rab22a KD (b) JAWS-II DCs after electroporation and BFA treatment.


A . Scramble and Rab22a KD JAWS - II DCs were infected with OVA - YFP - expressing T . gondii ( TgRHYFPSAG1 - OVA ) for 8 h and confocal images detecting the parasite ( green ) , endogenous Rab22a ( red ) and GRA6 ( magenta ) were taken . Top panels : Scramble cells ; bottom panels : Rab22a KD cells . White boxes are shown at higher magnification in the insets . The nuclear marker DAPI ( blue ) and DIC images are shown in the left panels . Overlays are shown in the right panels . Scale bars : 5 µm . Images are representative of at least 30 analyzed from three independent experiments .

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A. Scramble and Rab22a KD JAWS-II DCs were infected with OVA-YFP-expressing T. gondii (TgRHYFPSAG1-OVA) for 8 h and confocal images detecting the parasite (green), endogenous Rab22a (red) and GRA6 (magenta) were taken. Top panels: Scramble cells; bottom panels: Rab22a KD cells. White boxes are shown at higher magnification in the insets. The nuclear marker DAPI (blue) and DIC images are shown in the left panels. Overlays are shown in the right panels. Scale bars: 5 µm. Images are representative of at least 30 analyzed from three independent experiments.


B . The cross - presentation of OVA secreted by T . gondii ( TgRHYFPSAG1 - OVA ) after 8 h of infection at the indicated MOI was evaluated by B3Z T cell activation . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . * * * P < 0 . 001 . A two - way ANOVA and the Bonferroni post - test were performed .

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B. The cross-presentation of OVA secreted by T. gondii (TgRHYFPSAG1-OVA) after 8 h of infection at the indicated MOI was evaluated by B3Z T cell activation. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. ***P < 0.001. A two-way ANOVA and the Bonferroni post-test were performed.


C . The efficiency of infection ( 8 h ) of TgRHYFPSAG1 - OVA in Scramble and Rab22a KD JAWS - II DCs was measured by FACS analysis at the indicated MOI . Data represent mean ± SEM of duplicate values and are representative of three independent experiments .

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C. The efficiency of infection (8 h) of TgRHYFPSAG1-OVA in Scramble and Rab22a KD JAWS-II DCs was measured by FACS analysis at the indicated MOI. Data represent mean ± SEM of duplicate values and are representative of three independent experiments.


D . Immunoblotting showing the total amount of OVA in Scramble and Rab22a KD JAWS - II DCs after 90 min and 7 h of infection with TgRHYFPSAG1 - OVA parasite at MOI 2 and 6 . Fifty µg of total cell lysates were loaded onto each lane . Data are representative of two independent experiments .

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D. Immunoblotting showing the total amount of OVA in Scramble and Rab22a KD JAWS-II DCs after 90 min and 7 h of infection with TgRHYFPSAG1-OVA parasite at MOI 2 and 6. Fifty µg of total cell lysates were loaded onto each lane. Data are representative of two independent experiments.


A ) Purified His - α - synuclein protein was agitated for 0 - 2 weeks ( lane 1 , 0weeks ; lanes 2 and 3 , 1week ; lanes 4 and 5 , 2weeks ) . Aliquots from each sample were separated into supernatant ( lanes 2 and 4 ) and pellet ( lanes3 and 5 ) fractions . These fractions were subjected to immunoblotting with anti - α - synuclein antibody . Numbers at left are molecular weight markers ( MW ) in kilodaltons .

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A) Purified His-α-synuclein protein was agitated for 0-2 weeks (lane 1, 0weeks; lanes 2 and 3, 1week; lanes 4 and 5, 2weeks). Aliquots from each sample were separated into supernatant (lanes 2 and 4) and pellet (lanes3 and 5) fractions. These fractions were subjected to immunoblotting with anti-α-synuclein antibody. Numbers at left are molecular weight markers (MW) in kilodaltons.


( B ) Fibril formation of α - synuclein was measured with ProteoStat , an aggregation detection dye . a . u . , arbitrary units .

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(B) Fibril formation of α-synuclein was measured with ProteoStat, an aggregation detection dye. a.u., arbitrary units.


( C ) AFM images of α - synuclein agitated for 0 or 2 weeks were acquired in air using the dynamic mode . A high - magnification view of the boxed area is shown in the lower panel . The scale on the right represents the height of pixels in the image . Scale bars , 100 nm .

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(C) AFM images of α-synuclein agitated for 0 or 2 weeks were acquired in air using the dynamic mode. A high-magnification view of the boxed area is shown in the lower panel. The scale on the right represents the height of pixels in the image. Scale bars, 100 nm.


( D ) α - Synuclein inclusions in fibril - introduced HEK293 cells were detected immunocytochemically with anti - phosphorylated α - synuclein ( P - αSyn ) , anti - ubiquitin ( Ub ) , and anti - p62 ( p62 ) antibodies . Confocal images reconstructed in the z - axis along the white lines are shown in the right panel ( Z - axis ) . Blue , DAPI . Scale bar , 10 µm .

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(D) α-Synuclein inclusions in fibril-introduced HEK293 cells were detected immunocytochemically with anti-phosphorylated α-synuclein (P-αSyn), anti-ubiquitin (Ub), and anti-p62 (p62) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm.


( A ) Autophagic clearance of α - synuclein inclusions was verified by immunocytochemical analysis with anti - phosphorylated α - synuclein ( P - αSyn ) and anti - LC3 ( LC3 ) antibodies . Confocal images reconstructed in the z - axis along the white lines are shown in the right panel ( Z - axis ) . Blue , DAPI . Scale bar , 10 µm . Phosphorylated α - synuclein - positive inclusions were sequestrated into LC3 - positive autophagosomes .

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(A) Autophagic clearance of α-synuclein inclusions was verified by immunocytochemical analysis with anti-phosphorylated α-synuclein (P-αSyn) and anti-LC3 (LC3) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm. Phosphorylated α-synuclein-positive inclusions were sequestrated into LC3-positive autophagosomes.


( B ) Similarly , this sequestration was confirmed in HEK293 cells stably expressing DsRed - LC3 . The upper and lower panels show α - synuclein fibrils - and mock - introduced cells , respectively . Blue , DAPI . Scale bar , 10 µm .

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(B) Similarly, this sequestration was confirmed in HEK293 cells stably expressing DsRed-LC3. The upper and lower panels show α-synuclein fibrils-and mock-introduced cells, respectively. Blue, DAPI. Scale bar, 10 µm.


( A ) HEK293 cells stably expressing GFP - LC3 were transfected with control ( upper ) or Atg - 5 siRNA ( lower ) . After 36 h , α - synuclein fibrils were introduced into them for 4 h , followed by immunostaining with anti - phospho - α - synuclein antibody ( P - αSyn ) . Blue , DAPI . Scale bar , 10 µm .

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(A) HEK293 cells stably expressing GFP-LC3 were transfected with control (upper) or Atg-5 siRNA (lower). After 36 h, α-synuclein fibrils were introduced into them for 4 h, followed by immunostaining with anti-phospho-α-synuclein antibody (P-αSyn). Blue, DAPI. Scale bar, 10 µm.


( B ) The number of GFP - LC3 - positive α - synuclein inclusions ( % ) were assessed in randomly chosen fields ( n = 5 ) with 48 - 141 inclusions . Statistical analysis was performed with t - test ( * p < 0 . 01 ) .

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(B) The number of GFP-LC3-positive α-synuclein inclusions (%) were assessed in randomly chosen fields (n = 5) with 48-141 inclusions. Statistical analysis was performed with t-test (*p<0.01).


( C ) Clearance of α - synuclein inclusions in control ( solid bar ) or Atg - 5 ( open bar ) knockdown cells was measured at 4 h and 24 h after α - synuclein fibrils . The number of phosphorylated α - synuclein - positive cells was assessed in randomly chosen fields ( n = 5 ) . Data from each experiment are were normalized to Cont - siRNA - 4 h ( 100 % ) and represented as relative value of α - synuclein inclusions ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 .

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(C) Clearance of α-synuclein inclusions in control (solid bar) or Atg-5 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment are were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01


( A ) Autophagy flux was analyzed by LC3 protein monitoring . α - Synuclein fibrils - introduced HEK293 cells were treated with ( + ) / without ( − ) bafilomycin A1 ( BafA1 ) for 2 h . Cell lysates were subjected to immunoblotting analysis using anti - LC3 ( upper panel ) or anti - actin ( lower panel ) antibodies . Quantification of the relative levels of LC3 - II / Actin or autophagic flux ( Baf + / Baf - ) is shown with the ratio . Relative level of LC3 - II / Actin was represented by mean ± s . d . as a graph . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 05 . This experiment was repeated three times .

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(A) Autophagy flux was analyzed by LC3 protein monitoring. α-Synuclein fibrils-introduced HEK293 cells were treated with (+)/without (−) bafilomycin A1 (BafA1) for 2 h. Cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin or autophagic flux (Baf+/Baf-) is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.05. This experiment was repeated three times.


( B ) α - Synuclein fibrils were introduced into HEK293 cells , followed by addition of DMSO , 100 nM bafilomycin ( BafA1 ) , or 200 nM rapamycin ( Rapa ) . Cells were stained by anti - phosphorylated α - synuclein antibody ( green ) and DAPI ( blue ) 1 h ( left panels ) or 24 h ( right panels ) after introduction .

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(B) α-Synuclein fibrils were introduced into HEK293 cells, followed by addition of DMSO, 100 nM bafilomycin (BafA1), or 200 nM rapamycin (Rapa). Cells were stained by anti-phosphorylated α-synuclein antibody (green) and DAPI (blue) 1 h (left panels) or 24 h (right panels) after introduction.


( C ) The number of phosphorylated α - synuclein - positive cells per unit area ( 0 . 6 mm2 ) was assessed in randomly chosen fields ( n = 6 ) . Data represent mean ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 05 , * * p < 0 . 001 .

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(C) The number of phosphorylated α-synuclein-positive cells per unit area (0.6 mm2) was assessed in randomly chosen fields (n = 6). Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.05, **p<0.001.


( D ) Colocalization of α - synuclein inclusions and lysosomes . α - Synuclein inclusions ( P - αSyn ) and lysosomes ( LAMP1 ) were immunostained with anti - phospho - α - synuclein polyclonal antibody ( green ) and anti - LAMP1 antibody ( red ) 1 h ( left panel ) or 4 h ( middle panel ) after fibril introduction . Fibril - unintroduced cells were used as mock control ( right panel ) . Arrowheads and arrows indicate localized or unlocalized α - synuclein inclusions to lysosomes , respectively . Blue , DAPI . Scale bars , 10 µm .

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(D) Colocalization of α-synuclein inclusions and lysosomes. α-Synuclein inclusions (P-αSyn) and lysosomes (LAMP1) were immunostained with anti-phospho-α-synuclein polyclonal antibody (green) and anti-LAMP1 antibody (red) 1 h (left panel) or 4 h (middle panel) after fibril introduction. Fibril-unintroduced cells were used as mock control (right panel). Arrowheads and arrows indicate localized or unlocalized α-synuclein inclusions to lysosomes, respectively. Blue, DAPI. Scale bars, 10 µm.


( E ) Colocalization of α - synuclein inclusions ( n = 41 - 73 ) to lysosomes were measured in randomly chosen fields . The experiment was repeated three times . Data represent mean ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 01 .

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(E) Colocalization of α-synuclein inclusions (n = 41-73) to lysosomes were measured in randomly chosen fields. The experiment was repeated three times. Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01.


( A ) HEK293 cells stably expressing GFP - LC3 were transfected with p62 - siRNA ( right panels ) or control - siRNA ( left panels ) . Phosphorylated α - synuclein - positive inclusions ( P - αSyn ) are indicated by arrowheads . In p62 - knockdown cells , the expression and accumulation of p62 were not detected , and inclusions were never sequestrated by GFP - LC3 autophagosomes . Scale bars , 20 µm .

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(A) HEK293 cells stably expressing GFP-LC3 were transfected with p62-siRNA (right panels) or control-siRNA (left panels). Phosphorylated α-synuclein-positive inclusions (P-αSyn) are indicated by arrowheads. In p62-knockdown cells, the expression and accumulation of p62 were not detected, and inclusions were never sequestrated by GFP-LC3 autophagosomes. Scale bars, 20 µm.


( B ) The number of GFP - LC3 - positive α - synuclein inclusions ( % ) in Fig . 5A were assessed in randomly chosen fields with 32 - 44 inclusions ( n = 3 ) . Data represent mean ± s . e . m . Statistical analysis was performed with t - test ( * p < 0 . 05 ) .

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(B) The number of GFP-LC3-positive α-synuclein inclusions (%) in Fig. 5A were assessed in randomly chosen fields with 32-44 inclusions (n = 3). Data represent mean ± s.e.m. Statistical analysis was performed with t-test (*p<0.05).


( C ) α - Synuclein fibrils were introduced into control ( Cont ) or p62 - knockdown ( p62 ) cells . Four hours after introduction , cell lysates were subjected to immunoblotting analysis using anti - LC3 ( upper panel ) or anti - actin ( lower panel ) antibodies . Quantification of the relative levels of LC3 - II / Actin is shown with the ratio . Relative level of LC3 - II / Actin was represented by mean ± s . d . as a graph . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 01 . This experiment was repeated three times .

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(C) α-Synuclein fibrils were introduced into control (Cont) or p62-knockdown (p62) cells. Four hours after introduction, cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01. This experiment was repeated three times.


( D ) Clearance of α - synuclein inclusions in control ( solid bar ) or p62 ( open bar ) knockdown cells was measured at 4 h and 24 h after α - synuclein fibrils . The number of phosphorylated α - synuclein - positive cells was assessed in randomly chosen fields ( n = 5 ) . Data from each experiment were normalized to Cont - siRNA - 4 h ( 100 % ) and represented as relative value of α - synuclein inclusions ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 01 .

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(D) Clearance of α-synuclein inclusions in control (solid bar) or p62 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01.


( A ) α - Synuclein fibrils were introduced into HEK293 cells stably expressing both DsRed - LC3 and EGFP - parkin . After 24 h , the cells were immunostained with anti - phosphorylated α - synuclein ( P - αSyn ) . There was no alteration in the localization of EGFP - parkin . The arrows indicate α - synuclein inclusion . Scale bar , 10 µm .

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(A) α-Synuclein fibrils were introduced into HEK293 cells stably expressing both DsRed-LC3 and EGFP-parkin. After 24 h, the cells were immunostained with anti-phosphorylated α-synuclein (P-αSyn). There was no alteration in the localization of EGFP-parkin. The arrows indicate α-synuclein inclusion. Scale bar, 10 µm.


( B ) Mitochondrial clearance was confirmed in mock - ( upper panels ) or α - synuclein fibrils - introduced cells ( lower panels ) . After introduction , EGFP - parkin cells were treated with DMSO ( left ) or 10 µM CCCP for 4 h ( middle ) or 16 h ( right ) . Cells were examined immunocytochemically with anti - Tom20 ( red ) and anti - phosphorylated α - synuclein antibody ( P - αSyn ; aqua ) . Figures were presented as merged images . Individual images were provided in supplemental figure ( Fig . S8 ) . Scale bar , 10 µm .

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(B) Mitochondrial clearance was confirmed in mock- (upper panels) or α-synuclein fibrils-introduced cells (lower panels). After introduction, EGFP-parkin cells were treated with DMSO (left) or 10 µM CCCP for 4 h (middle) or 16 h (right). Cells were examined immunocytochemically with anti-Tom20 (red) and anti-phosphorylated α-synuclein antibody (P-αSyn; aqua). Figures were presented as merged images. Individual images were provided in supplemental figure (Fig. S8). Scale bar, 10 µm.


( Left panel ) A10 cells were transfected with Flag - FMRP for 3 hr then cultured for 24 hr prior to fixation and staining for β - catenin ( green ) , Flag ( FMRP , red ) , and nucleus ( blue ) . ( Bottom right panel ) A line was drawn through a representative cell to indicate relative intensity of RGB signals . ( Top right panel ) An orthogonal projection was generated by a Z - stack ( 240 nm interval ) image set using the ZEN program ( Zeiss ) depicting the region surrounding the nucleus in the XY , XZ , and YZ

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(Left panel) A10 cells were transfected with Flag-FMRP for 3 hr then cultured for 24 hr prior to fixation and staining for β-catenin (green), Flag (FMRP, red), and nucleus (blue). (Bottom right panel) A line was drawn through a representative cell to indicate relative intensity of RGB signals. (Top right panel) An orthogonal projection was generated by a Z-stack (240 nm interval) image set using the ZEN program (Zeiss) depicting the region surrounding the nucleus in the XY, XZ, and YZ planes


B . ( i ) HEK 293T cells were transfected with Flag - FMRP as in A , and endogenous β - catenin protein complexes were immunoprecipitated with β - catenin antibody . Co - immunoprecipitated Flag - FMRP protein was detected by Western blot analysis with the Flag antibody . Non - programmed rabbit IgG was used as a control . ( ii ) Primary VSMCs were harvested and subjected to an endogenous coIP analysis with either β - catenin or FMRP antibody . The corresponding protein was detected in the precipitated immunocomplex by Western blot . Non - programmed rabbit IgG was used as a

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B. (i) HEK 293T cells were transfected with Flag-FMRP as in A, and endogenous β-catenin protein complexes were immunoprecipitated with β-catenin antibody. Co-immunoprecipitated Flag-FMRP protein was detected by Western blot analysis with the Flag antibody. Non-programmed rabbit IgG was used as a control. (ii) Primary VSMCs were harvested and subjected to an endogenous coIP analysis with either β-catenin or FMRP antibody. The corresponding protein was detected in the precipitated immunocomplex by Western blot. Non-programmed rabbit IgG was used as a control


( i ) Purified 6x - His - FMRP and various regions of β - catenin proteins fused to GST were incubated with glutathione - agarose beads . Purified GST or GST - fusion proteins were visualized by Western blotting using the GST antibody , and sizes of corresponding GST - fusion proteins were confirmed . Pulled - down FMRP proteins by GST fused β - catenin were detected by immunoblotting with the FMRP antibody . ( ii ) Interaction between FMRP and β - catenin was assessed in the presence or absence of RNA . HEK 293T cells were subjected to an endogenous coIP analysis using either β - catenin or FMRP antibody , in the presence or absence of RNase A ( 10 µg / mL ) . The corresponding protein was detected in the precipitated immunocomplex by Western blot , and non - programmed rabbit IgG was used as a control . RNA was extracted from parallel cultured cells and total RNA content was analyzed by agarose gel electrophoresis in the presence or absence of RNase

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(i) Purified 6x-His-FMRP and various regions of β-catenin proteins fused to GST were incubated with glutathione-agarose beads. Purified GST or GST-fusion proteins were visualized by Western blotting using the GST antibody, and sizes of corresponding GST-fusion proteins were confirmed. Pulled-down FMRP proteins by GST fused β-catenin were detected by immunoblotting with the FMRP antibody. (ii) Interaction between FMRP and β-catenin was assessed in the presence or absence of RNA. HEK 293T cells were subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody, in the presence or absence of RNase A (10 µg/mL). The corresponding protein was detected in the precipitated immunocomplex by Western blot, and non-programmed rabbit IgG was used as a control. RNA was extracted from parallel cultured cells and total RNA content was analyzed by agarose gel electrophoresis in the presence or absence of RNase A


( Top panel ) A10 cells were transfected with GBP - Lifeact and either EYFP - β - catenin , Flag - FMRP , or mCherry - β - catenin . The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag ( red ) , as indicated . ( Bottom panel ) A10 cells were transfected with GBP - Lifeact similar to above , but with both EYFP - β - catenin and Flag - FMRP , and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag ( red )

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(Top panel) A10 cells were transfected with GBP-Lifeact and either EYFP-β-catenin, Flag-FMRP, or mCherry-β-catenin. The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag (red), as indicated. (Bottom panel) A10 cells were transfected with GBP-Lifeact similar to above, but with both EYFP-β-catenin and Flag-FMRP, and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag (red)


( Top panel ) A10 cells were transfected wit EYFP - β - catenin , Flag - FMRP , or mCherry - β - catenin . The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag ( red ) , as indicated . ( Bottom panel ) A10 cells were transfected with GBP - Lifeact similar to above , but with both EYFP - β - catenin and Flag - FMRP , and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag ( red ) E . A10 cells were transfected as in ( D ) with GBP - Lamin

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(Top panel) A10 cells were transfected wit EYFP-β-catenin, Flag-FMRP, or mCherry-β-catenin. The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag (red), as indicated. (Bottom panel) A10 cells were transfected with GBP-Lifeact similar to above, but with both EYFP-β-catenin and Flag-FMRP, and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag (red) E. A10 cells were transfected as in (D) with GBP-Lamin B1


A . Co - sedimentation of protein / RNA complexes on a 10 - 50 % sucrose gradient . Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients ; peaks corresponding to the 40S and 60S subunits , 80S monosome , and polysomes were detected by UV absorbance at 254 nm , and indicated proteins in these fractions were detected by Western blot . Corresponding total cell lysate was used as

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A. Co-sedimentation of protein/RNA complexes on a 10-50% sucrose gradient. Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients; peaks corresponding to the 40S and 60S subunits, 80S monosome, and polysomes were detected by UV absorbance at 254 nm, and indicated proteins in these fractions were detected by Western blot. Corresponding total cell lysate was used as input


( i ) Precipitated proteins in A10 cell lysates with m7GTP - agarose beads were identified by Western blot analysis . eIF4E and tubulin were shown as positive and negative controls , respectively . Total lysates were used as input . ( ii ) A10 cell lysates were incubated with GTP - agarose beads , and none of the proteins tested were precipitated with the beads . Total lysates were used as input

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(i) Precipitated proteins in A10 cell lysates with m7GTP-agarose beads were identified by Western blot analysis. eIF4E and tubulin were shown as positive and negative controls, respectively. Total lysates were used as input. (ii) A10 cell lysates were incubated with GTP-agarose beads, and none of the proteins tested were precipitated with the beads. Total lysates were used as input control


( i ) HEK 293T cells were transfected with empty vector or Flag - FMRP , and lysates were subjected to m7GTP - agarose pull - down as in ( C )

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(i) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to m7GTP-agarose pull-down as in (C)


( ii ) HEK 293T cells were transfected with empty vector or Flag - FMRP , and lysates were subjected to GTP - agarose pull - down as in ( i ) . E . HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP - agarose pull - downs as in ( C )

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(ii) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to GTP-agarose pull-down as in (i). E. HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP-agarose pull-downs as in (C)


F . HEK 293T cells lysates were subjected to m7GTP - agarose pull - downs as in ( C ) in the presence or absence of RNase A ( 10 µg / mL ) . RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis .

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F. HEK 293T cells lysates were subjected to m7GTP-agarose pull-downs as in (C) in the presence or absence of RNase A (10 µg/mL). RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis.


Primary VSMCs ( left panel ) , A10 ( middle panel ) , and HEK 293T cells ( right panel ) were transfected with siRNAs targeting ctnnb1 ( β - catenin ) or scrambled RNA . After 36 hr , the cells were pulsed with 0 . 5 µM puromycin for 15 min and then harvested . The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody . β - catenin blots indicated efficacy of siRNA and actin was used as a loading control . The average puromycin / actin ratio ( n = 3 ) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation . A one - way ANOVA and Tukey post - hoc test was performed , with p - values indicated above the error

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Primary VSMCs (left panel), A10 (middle panel), and HEK 293T cells (right panel) were transfected with siRNAs targeting ctnnb1 (β-catenin) or scrambled RNA. After 36 hr, the cells were pulsed with 0.5 µM puromycin for 15 min and then harvested. The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody. β-catenin blots indicated efficacy of siRNA and actin was used as a loading control. The average puromycin/actin ratio (n=3) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation. A one-way ANOVA and Tukey post-hoc test was performed, with p-values indicated above the error bars