Quiescent G0 - phase Rabin8 + / + and Rabin8 − / − hTERT - RPE1 cells were immunostained with anti - ARL13B ( blue ) , anti - phospho - S473 - Akt ( red ) , anti - ninein ( green ) , and anti - PMP70 ( white ) antibodies . Arrow indicates the peroxisomes contacting the ciliary pocket . Scale bar , 5 μm . Quantification of ( C ) indicating that disruption of the Rabin8 gene significantly inhibited the spatial interaction between peroxisomes and primary cilia ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 cells per experiment ) .

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Quiescent G0-phase Rabin8+/+ and Rabin8−/− hTERT-RPE1 cells were immunostained with anti-ARL13B (blue), anti-phospho-S473-Akt (red), anti-ninein (green), and anti-PMP70 (white) antibodies. Arrow indicates the peroxisomes contacting the ciliary pocket. Scale bar, 5 μm. Quantification of (C) indicating that disruption of the Rabin8 gene significantly inhibited the spatial interaction between peroxisomes and primary cilia (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 45-50 cells per experiment).


Quiescent G0 - phase hTERT - RPE1 cells transiently expressing PEX3 - GFP - FRB ( green ) and tdTomato - BicD2 - FKBP ( red ) treated with rapamycin were imaged live for 10 min by confocal microscopy ( Movie EV10 ) . Scale bar , 5 μm .

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Quiescent G0-phase hTERT-RPE1 cells transiently expressing PEX3-GFP-FRB (green) and tdTomato-BicD2-FKBP (red) treated with rapamycin were imaged live for 10 min by confocal microscopy (Movie EV10). Scale bar, 5 μm.


Quiescent G0 - phase Rabin8 + / + and Rabin8 − / − hTERT - RPE1 cells transfected with PEX3 - GFP - FRB ( red ) and tdTomato - BicD2 - FKBP ( white ) were treated with or without 100 μM rapamycin for 30 min , and then immunostained with anti - acetylated - tubulin ( blue ) antibody . Cholesterol was stained with Filipin III ( Green ) . Arrows indicate the enrichment of ciliary cholesterol . The scale bars represent 5 μm . Quantification of the Filipin III intensity at primary cilia from ( G ) . Rapamycin - induced peroxisome targeting to the ciliary pocket restored the reduction of ciliary cholesterol in the Rabin8 − / − hTERT - RPE1 cells ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 cells per experiment ) . In the boxplot , medians , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

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

Quiescent G0-phase Rabin8+/+ and Rabin8−/− hTERT-RPE1 cells transfected with PEX3-GFP-FRB (red) and tdTomato-BicD2-FKBP (white) were treated with or without 100 μM rapamycin for 30 min, and then immunostained with anti-acetylated-tubulin (blue) antibody. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 μm. Quantification of the Filipin III intensity at primary cilia from (G). Rapamycin-induced peroxisome targeting to the ciliary pocket restored the reduction of ciliary cholesterol in the Rabin8−/− hTERT-RPE1 cells (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Rabin8 − / − hTERT - RPE1 cells were transfected with AcGFP1 - tagged Rabin8 , GFP - tagged Rab8A , GFP - tagged Rab8A - Q67L ( constitutively active form ) , GFP - Rab8B , GFP - Rab8B - Q67L ( constitutively active form ) , AcGFP1 - tagged Rab10 , or AcGFP1 - tagged Rab10 - Q68L ( constitutively active form ) , and then immunostained with anti - GFP ( red ) , anti - acetylated - tubulin ( blue ) , and anti - ninein ( white ) antibodies . Cholesterol was stained with Filipin III ( Green ) . Arrows indicate the enrichment of ciliary cholesterol . The scale bars represent 5 μm . Quantification of ( I ) showing that AcGFP1 - tagged Rabin8 and AcGFP1 - tagged Rab10 - Q68L effectively restored the defect in ciliary enrichment of cholesterol in Rabin8 − / − hTERT - RPE1 cells ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 35 - 40 cells per experiment ) . In the boxplot , medians , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

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Rabin8−/− hTERT-RPE1 cells were transfected with AcGFP1-tagged Rabin8, GFP-tagged Rab8A, GFP-tagged Rab8A-Q67L (constitutively active form), GFP-Rab8B, GFP-Rab8B-Q67L (constitutively active form), AcGFP1-tagged Rab10, or AcGFP1-tagged Rab10-Q68L (constitutively active form), and then immunostained with anti-GFP (red), anti-acetylated-tubulin (blue), and anti-ninein (white) antibodies. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 μm. Quantification of (I) showing that AcGFP1-tagged Rabin8 and AcGFP1-tagged Rab10-Q68L effectively restored the defect in ciliary enrichment of cholesterol in Rabin8−/− hTERT-RPE1 cells (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 35-40 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Whole - cell lysates from HEK293T cells expressing AcGFP1 or AcGFP1 - tagged PEX14 and 3 × FLAG - tagged KIFC3 were immunoprecipitated with anti - FLAG antibody and immunoblotted with anti - GFP or anti - FLAG antibody .

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Whole-cell lysates from HEK293T cells expressing AcGFP1 or AcGFP1-tagged PEX14 and 3×FLAG-tagged KIFC3 were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibody.


Whole - cell lysates from HEK293T cells expressing AcGFP1 - tagged KIFC3 and 3 × FLAG - tagged Rab10 or Rab10 - Q68L were immunoprecipitated with anti - FLAG antibody and immunoblotted with anti - GFP or anti - FLAG antibody .

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Whole-cell lysates from HEK293T cells expressing AcGFP1-tagged KIFC3 and 3×FLAG-tagged Rab10 or Rab10-Q68L were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibody.


Whole - cell lysates from HEK293T cells expressing AcGFP1 - tagged Pex14 and 3 × FLAG - tagged Rab10 or Rab10 - Q68L were immunoprecipitated with anti - FLAG antibody and immunoblotted with anti - GFP or anti - FLAG antibody .

Whole-cell lysates from HEK293T cells expressing AcGFP1-tagged Pex14 and 3×FLAG-tagged Rab10 or Rab10-Q68L were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibody.


Western blot analysis of the ciliary cholesterol trafficking - associated components in Pex1 + / + and Pex1 − / − hTERT - RPE1 cells . Total cell lysates were separated to crude peroxisomal ( Crude Pex ) , lysosomal and mitochondrial ( Lyso / Mito ) , and peroxisomal ( PEX ) fractions . CYPOR , a lysosomal and mitochondrial protein , served as a positive control for the Lyso / Mito fractionation . Total cell lysates were gradually injected at 20 µg and 5 µg into a gel for SDS - PAGE , while equal amounts ( 5 µg ) of protein from each fraction were loaded . Rabin8 , Rab10 , and KIFC3 were concentrated in the PEX fraction in Pex1 + / + hTERT - RPE1 cells . CYPOR ( cytochrome P450 reductase ) is a mitochondrial protein .

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Western blot analysis of the ciliary cholesterol trafficking-associated components in Pex1+/+ and Pex1−/− hTERT-RPE1 cells. Total cell lysates were separated to crude peroxisomal (Crude Pex), lysosomal and mitochondrial (Lyso/Mito), and peroxisomal (PEX) fractions. CYPOR, a lysosomal and mitochondrial protein, served as a positive control for the Lyso/Mito fractionation. Total cell lysates were gradually injected at 20 µg and 5 µg into a gel for SDS-PAGE, while equal amounts (5 µg) of protein from each fraction were loaded. Rabin8, Rab10, and KIFC3 were concentrated in the PEX fraction in Pex1+/+ hTERT-RPE1 cells. CYPOR (cytochrome P450 reductase) is a mitochondrial protein.


Quiescent G0 - phase hTERT - RPE1 cells transfected with 3 × FLAG - tagged KIFC3 were immunostained with anti - FLAG ( green ) , anti - PEX14 ( red ) , and anti - α - tubulin ( white ) antibodies . Magnified 3D - constituted images of the boxed regions showing peroxisomes are located on the microtubule arrays via KIFC3 ( arrows ) . The scale bars indicate 5 μm .

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Quiescent G0-phase hTERT-RPE1 cells transfected with 3×FLAG-tagged KIFC3 were immunostained with anti-FLAG (green), anti-PEX14 (red), and anti-α-tubulin (white) antibodies. Magnified 3D-constituted images of the boxed regions showing peroxisomes are located on the microtubule arrays via KIFC3 (arrows). The scale bars indicate 5 μm.


Western blot analysis showing depletion of KIFC3 in the KIFC3 − / − hTERT - RPE1 cell clones . GAPDH served as a loading control .

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Western blot analysis showing depletion of KIFC3 in the KIFC3−/− hTERT-RPE1 cell clones. GAPDH served as a loading control.


KIFC3 + / + and KIFC3 − / − hTERT - RPE1 cells incubated for 24 h without serum were immunostained with anti - pericentrin ( red ) and anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( green ) . Scale bar , 5 μm . Quantification of ( G ) indicating that KIFC3 − / − hTERT - RPE1 cells significantly reduced the ciliary accumulation of cholesterol ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 cells per experiment ) . In the boxplot , medians , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

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KIFC3+/+ and KIFC3−/− hTERT-RPE1 cells incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Scale bar, 5 μm. Quantification of (G) indicating that KIFC3−/− hTERT-RPE1 cells significantly reduced the ciliary accumulation of cholesterol (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Quiescent G0 - phase KIFC3 + / + and KIFC3 − / − hTERT - RPE1 cells were immunostained with anti - ARL13B ( blue ) , anti - phospho - S473 - Akt ( red ) , anti - ninein ( green ) , and anti - PMP70 ( white ) antibodies . Arrows represent the peroxisomes contacting the ciliary pocket . Scale bar , 5 μm . Quantification of ( I ) showing that disruption of the KIFC3 gene significantly interfered with the contact between peroxisomes and primary cilia ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 cells per experiment ) .

Quiescent G0-phase KIFC3+/+ and KIFC3−/− hTERT-RPE1 cells were immunostained with anti-ARL13B (blue), anti-phospho-S473-Akt (red), anti-ninein (green), and anti-PMP70 (white) antibodies. Arrows represent the peroxisomes contacting the ciliary pocket. Scale bar, 5 μm. Quantification of (I) showing that disruption of the KIFC3 gene significantly interfered with the contact between peroxisomes and primary cilia (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 45-50 cells per experiment).


KIFC3 − / − hTERT - RPE1 cells were transfected with AcGFP1 , AcGFP1 - tagged KIFC3 , rod domain - deleted KIFC3 mutant ( ΔRod ) , or motor domain - deleted KIFC3 mutant ( ΔMotor ) and cultured without serum for 24 h before Filipin - III ( green ) - mediated cholesterol staining and immunostaining with anti - GFP ( red ) , anti - pericentrin ( white ) , and anti - acetylated - tubulin ( blue ) antibodies . Arrows represent ciliary localization of cholesterol . The scale bars indicate 5 μm . Quantification of the Filipin III intensity at primary cilia from ( K ) . AcGFP1 - tagged KIFC3 deletion mutants did not restore the ciliary cholesterol insufficiency in the KIFC3 − / − hTERT - RPE1 cells ( mean + / - s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 40 - 50 cells per experiment ) . In the boxplot , medians , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

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KIFC3−/− hTERT-RPE1 cells were transfected with AcGFP1, AcGFP1-tagged KIFC3, rod domain-deleted KIFC3 mutant (ΔRod), or motor domain-deleted KIFC3 mutant (ΔMotor) and cultured without serum for 24 h before Filipin-III (green)-mediated cholesterol staining and immunostaining with anti-GFP (red), anti-pericentrin (white), and anti-acetylated-tubulin (blue) antibodies. Arrows represent ciliary localization of cholesterol. The scale bars indicate 5 μm. Quantification of the Filipin III intensity at primary cilia from (K). AcGFP1-tagged KIFC3 deletion mutants did not restore the ciliary cholesterol insufficiency in the KIFC3−/− hTERT-RPE1 cells (mean +/- s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Quiescent G0 - phase wild - type , Rab10 − / − , KIFC3 − / − , ORP3 − / − , and EHD3 − / − hTERT - RPE1 cells transfected with PEX3 - GFP - FRB ( red ) and tdTomato - BicD2 - FKBP ( white ) were treated with or without 100 μM rapamycin for 30 min , and then immunostained with anti - acetylated - tubulin ( blue ) antibody . Cholesterol was stained with Filipin III ( Green ) . Arrows indicate the enrichment of ciliary cholesterol . The scale bars represent 5 μm .

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Quiescent G0-phase wild-type, Rab10−/−, KIFC3−/−, ORP3−/−, and EHD3−/− hTERT-RPE1 cells transfected with PEX3-GFP-FRB (red) and tdTomato-BicD2-FKBP (white) were treated with or without 100 μM rapamycin for 30 min, and then immunostained with anti-acetylated-tubulin (blue) antibody. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 μm.


Quantification of the Filipin III intensity at primary cilia from ( A ) . Rapamycin - induced peroxisome targeting to the ciliary pocket restored the insufficiency of ciliary cholesterol in the Rab10 − / − and KIFC3 − / − hTERT - RPE1 cells , but not in ORP3 − / − and EHD3 − / − hTERT - RPE1 cells ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 35 - 40 cells per experiment ) . In the boxplot , medians , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

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Quantification of the Filipin III intensity at primary cilia from (A). Rapamycin-induced peroxisome targeting to the ciliary pocket restored the insufficiency of ciliary cholesterol in the Rab10−/−and KIFC3−/−hTERT-RPE1 cells, but not in ORP3−/−and EHD3−/− hTERT-RPE1 cells (*p<0.05, **p<0.01, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 35-40 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


( B ) Bar graph representing residual thrombin activity ( ΔOD / min ) in presence of the sdAbs without heparin .

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(B) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs without heparin.


( C ) Bar graph representing residual thrombin activity ( ΔOD / min ) in presence of the sdAbs and heparin .

(C) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs and heparin.


( D ) Bar graph representing residual activated factor X ( FXa ) activity ( ΔOD / min ) in presence of the sdAbs and heparin .

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(D) Bar graph representing residual activated factor X (FXa) activity (ΔOD/min) in presence of the sdAbs and heparin.


( B - C ) Graphs reporting the recovery of thrombin - and FXa - mediated substrate conversion in the presence of increasing doses of KB - AT - 23 , in presence of antithrombin and heparin .

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(B-C) Graphs reporting the recovery of thrombin- and FXa-mediated substrate conversion in the presence of increasing doses of KB-AT-23, in presence of antithrombin and heparin.


( D - E ) Thrombin generation profiles of FVIII - deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB - AT - 23 .

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(D-E) Thrombin generation profiles of FVIII-deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB-AT-23.


( B ) Purified KB - AT - 23 - fus and KB - hFX - 11 - fus were given intravenously to wild - type C57B6 mice ( 10 mg / kg ) . At indicated time - points , blood was collected and residual protein was measured . Plotted is residual protein versus time after injection . Green circles : KB - AT - 23 - fus ; Red circles : KB - hFX - 11 - fus .

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(B) Purified KB-AT-23-fus and KB-hFX-11-fus were given intravenously to wild-type C57B6 mice (10 mg/kg). At indicated time-points, blood was collected and residual protein was measured. Plotted is residual protein versus time after injection. Green circles: KB-AT-23-fus; Red circles: KB-hFX-11-fus.


( D ) Graph reporting the volume of blood loss ( µL ) in treated mice ( n = 4 - 9 per group ) observed during 30 minutes post TVT . Dosing was 10 mg / kg for KB - AT - 23 and 1 mg / kg for FVIIa . The grey area represents the range of blood loss in FVIII - treated mice ( 33 - 245 μL ) , which is similar to that of wild - type C57BL / 6 mice ( 49 - 308 μL ; Johansen et al . 2016 ) .

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(D) Graph reporting the volume of blood loss (µL) in treated mice (n=4-9 per group) observed during 30 minutes post TVT. Dosing was 10 mg/kg for KB-AT-23 and 1 mg/kg for FVIIa. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016).


( B ) Western blot analysis of KB - AT - 23 protein secretion in conditioned media 72 hours post - transfection of HuH7 cells . In the lower panel the western blot quantification via densitometry analysis is depicted . Data are presented as mean ± SD , and were analysed via 1 - way ANOVA with Dunnett ' s correction for multiple comparisons . * * * * p < 0 . 001 .

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(B) Western blot analysis of KB-AT-23 protein secretion in conditioned media 72 hours post-transfection of HuH7 cells. In the lower panel the western blot quantification via densitometry analysis is depicted. Data are presented as mean ±SD, and were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons. ****p<0.001.


( C ) HuH7 - celles were collected 72 hours post - transfection . Presented is the Western blot analysis of lysed cells for the presence of KB - AT - 23 . Cntr - refers to non - transfected cells , while Cntr + refers to purified recombinant KB - AT - 23 .

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(C) HuH7-celles were collected 72 hours post-transfection. Presented is the Western blot analysis of lysed cells for the presence of KB-AT-23. Cntr- refers to non-transfected cells, while Cntr+ refers to purified recombinant KB-AT-23.


( B ) Representative western blot analyses of KB - AT - 23 and antithrombin expression on plasma samples ( n = 4 ) collected from animals 27 weeks post - AAV injection . kDa , molecular weight marker .

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(B) Representative western blot analyses of KB-AT-23 and antithrombin expression on plasma samples (n=4) collected from animals 27 weeks post-AAV injection. kDa, molecular weight marker.


( C ) Measurement of D - dimers ( D2D ) plasma concentration 27 weeks post vector administration . Data are presented as mean ± SD ( n = 6 - 9 / group ) .

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(C) Measurement of D-dimers (D2D) plasma concentration 27 weeks post vector administration. Data are presented as mean ±SD (n=6-9/group).


( D ) Measurement of anti - KB - AT - 23 mouse IgG in plasma samples collected from mice ( n = 6 - 9 / group ) 8 weeks post - AAV administration . Data represent mean ± SD .

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(D) Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=6-9/group) 8 weeks post-AAV administration. Data represent mean±SD.


( E ) TVT test on males and females ( n = 4 - 5 per group ) performed 29 weeks post vector administration . The volume of blood loss ( µL ) during 30 minutes observation time is reported . The grey area represents the range of blood loss in FVIII - treated mice ( 33 - 245 μL ) , which is similar to that of wild - type C57BL / 6 mice ( 49 - 308 μL ; Johansen et al . 2016 ) . Boxes represent 25 - 75 % quartiles ; the central line indicates the median . Whiskers span min to max . Data were analysed via 1 - way ANOVA with Dunnett ' s correction for multiple comparisons .

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(E) TVT test on males and females (n=4-5 per group) performed 29 weeks post vector administration. The volume of blood loss (µL) during 30 minutes observation time is reported. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016). Boxes represent 25-75% quartiles; the central line indicates the median. Whiskers span min to max. Data were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons.


( F ) Vector genome copy number ( VGCN ) per cell in liver samples collected at sacrifice ( n = 8 / group ) . Data represent mean ± SD .

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(F) Vector genome copy number (VGCN) per cell in liver samples collected at sacrifice (n=8/group). Data represent mean±SD.


( C ) Measurement of anti - hFIX mouse IgG in plasma samples collected from mice injected with FIX or PBS ( n = 5 / group ) at different time points . Data represent mean ± SEM .

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(C) Measurement of anti-hFIX mouse IgG in plasma samples collected from mice injected with FIX or PBS (n=5/group) at different time points. Data represent mean±SEM.


( D ) Representative western blot analyses on plasma samples ( n = 4 ) collected from animals 8 weeks post - AAV injection .

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(D) Representative western blot analyses on plasma samples (n=4) collected from animals 8 weeks post-AAV injection.


( E ) Antithrombin activity in plasma samples collected from mice at different time points . Data are presented as mean ± SD ( n = 3 - 4 per datapoint ) . Data were analyzed in a 2 - way ANOVA with Tukey ' s correction for multiple comparisons . * * * p < 0 . 01 * * * * p < 0 . 001 ( 2 - way ANOVA with Tukey ' s multiple comparison test )

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(E) Antithrombin activity in plasma samples collected from mice at different time points. Data are presented as mean±SD (n=3-4 per datapoint). Data were analyzed in a 2-way ANOVA with Tukey's correction for multiple comparisons. ***p<0.01 ****p<0.001 (2-way ANOVA with Tukey's multiple comparison test)


( F ) Graph reporting the volume of blood loss ( µL ) over 30 minutes following the tail clip . WT : wild - type littermates of F9 - / - mice . The grey area represents the range of blood loss of F9 - / - mice that received AAV - vectors expressing human FIX . Data are presented as mean ± SD ( n = 3 - 4 / group ) , and were analyzed in a 1 - way ANOVA with Tukey ' s correction for multiple comparisons . * * p < 0 . 01 Boxes represent 25 % - 75 % quartiles , with the line indicating the median . Whiskers span min to max .

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

(F) Graph reporting the volume of blood loss (µL) over 30 minutes following the tail clip. WT: wild-type littermates of F9-/- mice. The grey area represents the range of blood loss of F9-/- mice that received AAV-vectors expressing human FIX. Data are presented as mean±SD (n=3-4/group), and were analyzed in a 1-way ANOVA with Tukey's correction for multiple comparisons. **p<0.01 Boxes represent 25%-75% quartiles, with the line indicating the median. Whiskers span min to max.


G Measurement of anti - KB - AT - 23 mouse IgG in plasma samples collected from mice ( n = 5 / group ) 8 weeks post - AAV administration . Data represent mean ± SD .

G Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=5/group) 8 weeks post-AAV administration. Data represent mean±SD.


( C ) Representative western blot analyses on plasma samples ( n = 4 ) collected from animals 4 weeks post - AAV injection . Cntrl + : positive controls represented by the loading of the purified KB - AT - 23 or KB - AT - 113 proteins .

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

(C) Representative western blot analyses on plasma samples (n=4) collected from animals 4 weeks post-AAV injection. Cntrl +: positive controls represented by the loading of the purified KB-AT-23 or KB-AT-113 proteins.


( D - H ) Measurement of anti - sdAbs mouse IgG in plasma samples collected at days 28 and 57 post - AAV injection . The ELISA plate was coated with the purified KB - AT - 01 ( D )

(D-H) Measurement of anti-sdAbs mouse IgG in plasma samples collected at days 28 and 57 post-AAV injection. The ELISA plate was coated with the purified KB-AT-01 (D)


Measurement of anti - sdAbs mouse IgG in plasma samples collected at days 28 and 57 post - AAV injection . The ELISA plate was coated with the purified KB - AT - 01 , - 02 ( E ) , - 03 ( F ) , - 23 ( G ) or - 113 ( H ) sdAbs .

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Measurement of anti-sdAbs mouse IgG in plasma samples collected at days 28 and 57 post-AAV injection. The ELISA plate was coated with the purified KB-AT-01 , -02 (E), -03 (F), -23 (G) or -113 (H) sdAbs.


( A ) RT - qPCR analysis of Myh7b / miR - 499 and Myh7 / miR - 208b levels during differentiation of myoblasts into mature myotubes ( n = 3 independent experiments ) . * p = 0 . 0005 ( Myh7b ) , * p < 0 . 0001 ( miR - 499 ) , * p = 0 . 0002 ( Myh7 ) , * p < 0 . 0001 ( miR - 208b ) .

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

(A) RT-qPCR analysis of Myh7b/miR-499 and Myh7/miR-208b levels during differentiation of myoblasts into mature myotubes (n = 3 independent experiments). *p = 0.0005 (Myh7b), *p < 0.0001 (miR-499), *p = 0.0002 (Myh7), *p < 0.0001 (miR-208b).


( B ) Oxygen consumption rates ( OCR ) in primary myoblasts and differentiated myotubes . Basal OCR was first measured , followed by administration of 10 mM sodium pyruvate , and 2 μM oligomycin ( to inhibit ATP synthase ) , uncoupler FCCP ( 2 μM ) , or rotenone / antimycin ( Rot / A ) ( 1 μM ) as indicted . n = 3 separate experiments done with 10 biological replicates . * p < 0 . 0001 ( FCCP ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Oxygen consumption rates (OCR) in primary myoblasts and differentiated myotubes. Basal OCR was first measured, followed by administration of 10 mM sodium pyruvate, and 2 μM oligomycin (to inhibit ATP synthase), uncoupler FCCP (2 μM), or rotenone/antimycin (Rot/A) (1 μM) as indicted. n = 3 separate experiments done with 10 biological replicates. *p < 0.0001 (FCCP).


( C ) Mean expression levels ( RT - qPCR ) in white vastus ( WV ) and soleus ( Sol ) muscle from 8 - week - old male wild type mice ( n = 5 mice per group ) . * p < 0 . 0001 ( Myh7b ) , * p < 0 . 0001 ( miR - 499 ) , * p < 0 . 0001 ( Myh7 ) , * p < 0 . 0001 ( miR - 208b ) .

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(C) Mean expression levels (RT-qPCR) in white vastus (WV) and soleus (Sol) muscle from 8-week-old male wild type mice (n = 5 mice per group). *p < 0.0001 (Myh7b), *p < 0.0001 (miR-499), *p < 0.0001 (Myh7), *p < 0.0001 (miR-208b).


( D ) Mitochondrial respiration rates were determined from mitochondria isolated from indicated muscle using pyruvate as substrate . ADP - dependent respiration , oligomycin - induced ( oligo ) , uncoupler FCCP , and antimycin A ( AA ) are shown . n = 3 separate experiments done with 7 - 8 biological replicates . * p < 0 . 05 ( ADP ) .

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

(D) Mitochondrial respiration rates were determined from mitochondria isolated from indicated muscle using pyruvate as substrate. ADP-dependent respiration, oligomycin-induced (oligo), uncoupler FCCP, and antimycin A (AA) are shown. n = 3 separate experiments done with 7-8 biological replicates. *p < 0.05 (ADP).


( E ) ( Left ) Schematic depicts the increments of speed over time . ( Right ) Respiratory exchange ratio ( RER ) during a graded exercise regimen as described in Materials and Methods ( n = 5 mice per group ) . Notably , MCK - miR - 499 mice were able to exercise at a higher speed before exhaustion . * p < 0 . 05 .

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

(E) (Left) Schematic depicts the increments of speed over time. (Right) Respiratory exchange ratio (RER) during a graded exercise regimen as described in Materials and Methods (n = 5 mice per group). Notably, MCK-miR-499 mice were able to exercise at a higher speed before exhaustion. *p < 0.05.


( F ) Bars represent mean blood lactate levels for 3 - month and 15 - month old male MCK - miR - 499 and NTG mice following a 25 min run on a motorized treadmill . For 3 - month blood lactate , NTG , n = 9 ; MCK - miR - 499 , n = 10 . For 15 - month blood lactate , NTG , n = 7 ; MCK - miR - 499 , n = 5 . * p = 0 . 039 ( 3 - month ) , * p = 0 . 018 ( 15 - month ) .

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

(F) Bars represent mean blood lactate levels for 3-month and 15-month old male MCK-miR-499 and NTG mice following a 25 min run on a motorized treadmill. For 3-month blood lactate, NTG, n = 9; MCK-miR-499, n = 10. For 15-month blood lactate, NTG, n = 7; MCK-miR-499, n = 5. *p = 0.039 (3-month), *p = 0.018 (15-month).


( G ) Mitochondrial respiration rates were determined from the plantaris muscle of the indicated genotypes using pyruvate / malate as substrate . Pyruvate / malate ( Py / M ) - stimulated , ADP - dependent respiration , oligomycin - induced ( oligo ) and the respiratory control ratio ( RC ) are shown . NTG , n = 6 ; MCK - miR - 499 , n = 4 . * p = 0 . 015 ( Py / M ) , * p = 0 . 017 ( ADP ) , * p = 0 . 001 ( Oligo ) .

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

(G) Mitochondrial respiration rates were determined from the plantaris muscle of the indicated genotypes using pyruvate/malate as substrate. Pyruvate/malate (Py/M)-stimulated, ADP-dependent respiration, oligomycin-induced (oligo) and the respiratory control ratio (RC) are shown. NTG, n = 6; MCK-miR-499, n = 4. *p = 0.015 (Py/M), *p = 0.017 (ADP), *p = 0.001 (Oligo).


( B ) Expression of the nuclear - encoded and mitochondrial - encoded genes ( RT - qPCR ) in the gastrocnemius muscle from the indicated genotypes ( n = 5 mice per group ) . * p < 0 . 05 .

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(B) Expression of the nuclear-encoded and mitochondrial-encoded genes (RT-qPCR) in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.05.


( C ) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using myoglobin , cytochrome c , and β - actin ( control ) antibodies ( n = 8 mice per group ) .

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(C) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using myoglobin, cytochrome c, and β-actin (control) antibodies (n = 8 mice per group).


( D ) Expression of genes ( RT - qPCR ) involved in muscle glucose and fatty acids metabolism in the gastrocnemius muscle from the indicated genotypes ( n = 5 mice per group ) . * p < 0 . 05 .

(D) Expression of genes (RT-qPCR) involved in muscle glucose and fatty acids metabolism in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.05.


( E ) LDH isoenzymes were separated by polyacrylamide gel electrophoresis using whole cell extracts from NTG heart ( Ht , control ) and gastrocnemius muscle from the indicated mice . A representative gel showing 3 - 4 independent mice per group is shown . ( F ) Quantification of LDH isoenzyme activity gel electrophoresis in E . Values represent the mean % ( + / - SEM ) total LDH activity . * p < 0 . 05 .

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(E) LDH isoenzymes were separated by polyacrylamide gel electrophoresis using whole cell extracts from NTG heart (Ht, control) and gastrocnemius muscle from the indicated mice. A representative gel showing 3-4 independent mice per group is shown.(F) Quantification of LDH isoenzyme activity gel electrophoresis in E. Values represent the mean % (+/- SEM) total LDH activity. *p < 0.05.


( G ) Cross - section of the gastrocnemius muscle from a 3 - month - old male NTG and MCK - miR - 499 stained for myosin I ATPase activity ( Top ) , SDH ( Middle ) , and α - GPDH ( Bottom ) . Representative images were shown . Scale bar : 500 μm .

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(G) Cross-section of the gastrocnemius muscle from a 3-month-old male NTG and MCK-miR-499 stained for myosin I ATPase activity (Top), SDH (Middle), and α-GPDH (Bottom). Representative images were shown. Scale bar: 500 μm.


( A ) ( Left ) Representative western blot analysis of PGC - 1α protein expression in the gastrocnemius muscle from the indicated genotypes , heart ( Ht ) lysate from WT mice and PGC - 1α mKO ( MCK - Cre ) mice as positive and negative controls , respectively . ( Right ) Quantification of the PGC - 1α / Tubulin signal ratios normalized ( = 1 . 0 ) to the NTG control ( n = 5 mice per group ) . * p = 0 . 00017 .

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

(A) (Left) Representative western blot analysis of PGC-1α protein expression in the gastrocnemius muscle from the indicated genotypes, heart (Ht) lysate from WT mice and PGC-1α mKO (MCK-Cre) mice as positive and negative controls, respectively. (Right) Quantification of the PGC-1α/Tubulin signal ratios normalized (=1.0) to the NTG control (n = 5 mice per group). *p = 0.00017.


( B ) Expression of the Ldhb , Ldha , Mb ( myoglobin ) , and Cox5a genes ( RT - qPCR ) in the gastrocnemius muscle from the indicated genotypes ( n = 5 mice per group ) . * p < 0 . 001 ( vs . NTG ) , ‡ p < 0 . 01 ( vs . 499Tg ) .

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(B) Expression of the Ldhb, Ldha, Mb (myoglobin), and Cox5a genes (RT-qPCR) in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.001 (vs. NTG), ‡p < 0.01 (vs. 499Tg).


( C ) ( Left ) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using cytochrome c , myoglobin , and α - tubulin ( control ) antibodies . ( Right ) Quantification of the Cyt c / Tubulin signal ratios normalized ( = 1 . 0 ) to the NTG control . NTG , n = 5 ; 499Tg , n = 6 ; PGC - 1α mKO , n = 5 ; 499Tg / PGC - 1α mKO , n = 5 . * p < 0 . 01 ( vs . NTG ) , ‡ p = 0 . 00018 ( vs . 499Tg ) .

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

(C) (Left) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using cytochrome c, myoglobin, and α-tubulin (control) antibodies. (Right) Quantification of the Cyt c/Tubulin signal ratios normalized (=1.0) to the NTG control. NTG, n = 5; 499Tg, n = 6; PGC-1α mKO, n = 5; 499Tg/PGC-1α mKO, n = 5. *p < 0.01 (vs. NTG), ‡p = 0.00018 (vs. 499Tg).


( D ) Cross - section of gastrocnemius muscle from the indicated mice stained for SDH activity ( Top ) as well as MHC1 immunofluorescence ( IF ) ( Bottom ) , Representative images were shown . Scale bar : 500 μm .

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(D) Cross-section of gastrocnemius muscle from the indicated mice stained for SDH activity (Top) as well as MHC1 immunofluorescence (IF) (Bottom), Representative images were shown. Scale bar: 500 μm.


( E ) Respiratory exchange ratio ( RER ) during a graded exercise regimen as described in Materials and Methods . Notably , no significant increase in exhaust speed was observed in 499Tg / PGC - 1α mKO mice compared to PGC - 1α mKO mice . PGC - 1α mKO , n = 5 ; 499Tg / PGC - 1α mKO , n = 8 .

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(E) Respiratory exchange ratio (RER) during a graded exercise regimen as described in Materials and Methods. Notably, no significant increase in exhaust speed was observed in 499Tg/PGC-1α mKO mice compared to PGC-1α mKO mice. PGC-1α mKO, n = 5; 499Tg/PGC-1α mKO, n = 8.


( F ) The bars represent the mean blood lactate levels from the indicated mice following a 25 min run on a motorized treadmill . NTG , n = 9 ; 499Tg , n = 7 ; PGC - 1α mKO , n = 6 ; 499Tg / PGC - 1α mKO , n = 9 . * p = 0 . 0009 ( NTG vs . 499Tg ) , p = 0 . 316 ( NS , not significant ) .

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(F) The bars represent the mean blood lactate levels from the indicated mice following a 25 min run on a motorized treadmill. NTG, n = 9; 499Tg, n = 7; PGC-1α mKO, n = 6; 499Tg/PGC-1α mKO, n = 9. *p = 0.0009 (NTG vs. 499Tg), p = 0.316 (NS, not significant).


( B ) Luciferase reporters containing the wild - type Fnip1 3 ' UTR or Fnip1 3 ' UTR mutated in the predicted binding site of miR - 499 were used in cotransfection studies in HEK293T cells in the presence or absence of plasmid expressing miR - 499 ( n = 3 independent experiments ) . * p < 0 . 0001 ( Fnip1 3 ' UTR ) ; * p = 0 . 023 ( Mutated ) .

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(B) Luciferase reporters containing the wild-type Fnip1 3'UTR or Fnip1 3'UTR mutated in the predicted binding site of miR-499 were used in cotransfection studies in HEK293T cells in the presence or absence of plasmid expressing miR-499 (n = 3 independent experiments). *p < 0.0001 (Fnip1 3'UTR); *p = 0.023 (Mutated).


( C ) RT - qPCR analysis of Fnip1 and Fnip2mRNA levels in the gastrocnemius muscle of the indicated genotypes ( n = 5 mice per group ) .

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(C) RT-qPCR analysis of Fnip1 and Fnip2mRNA levels in the gastrocnemius muscle of the indicated genotypes (n = 5 mice per group).


( D ) ( Top ) Fnip1 protein expression in the gastrocnemius muscle from the indicated mice . ( Bottom ) Quantification of the Fnip1 / Tubulin signal ratio ( n = 4 mice per group ) . * p = 0 . 014 .

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(D) (Top) Fnip1 protein expression in the gastrocnemius muscle from the indicated mice. (Bottom) Quantification of the Fnip1/Tubulin signal ratio (n = 4 mice per group). *p = 0.014.


( A ) ( Left ) Representative western blot analysis performed on extracts of myotubes subjected to Fnip1 siRNA or control ( Con ) siRNA using phospho - AMPKα ( Thr172 ) and AMPKα antibodies . ( Right ) Quantification of the p - AMPKα / AMPKα signal ratios ( n = 3 independent experiments ) . * p = 0 . 037 .

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(A) (Left) Representative western blot analysis performed on extracts of myotubes subjected to Fnip1 siRNA or control (Con) siRNA using phospho-AMPKα (Thr172) and AMPKα antibodies. (Right) Quantification of the p-AMPKα/AMPKα signal ratios (n = 3 independent experiments). *p = 0.037.


( B , C ) Results of RT - qPCR analysis for WT primary mouse myotubes after transfection with Fnip1 siRNAs or scrambled Con siRNAs as indicated . For C , 48 h post - siRNA transfection , myotubes were treated for 24 h with DMSO or 10 μm compound C before harvest ( n = 3 independent experiments ) . * p < 0 . 0001 ( Fnip1 in B ) , * p = 0 . 0001 ( PGC - 1α in B ) ; * p < 0 . 0001 ( vs . Con siRNA in C ) , ‡ p < 0 . 0001 ( vs . Fnip1 siRNA in C ) .

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(B, C) Results of RT-qPCR analysis for WT primary mouse myotubes after transfection with Fnip1 siRNAs or scrambled Con siRNAs as indicated. For C, 48 h post-siRNA transfection, myotubes were treated for 24 h with DMSO or 10 μm compound C before harvest (n = 3 independent experiments). *p < 0.0001 (Fnip1 in B), *p = 0.0001 (PGC-1α in B); *p < 0.0001 (vs. Con siRNA in C), ‡p < 0.0001 (vs. Fnip1 siRNA in C ).


( D ) ( Top ) Representative western blot analysis performed on extracts of the gastrocnemius muscle isolated from NTG or MCK - miR - 499 mice using phospho - AMPKα ( Thr172 ) , AMPKα , phospho - ACC ( Ser79 ) , or total ACC antibodies . ( Bottom ) Quantification of the p - AMPKα / AMPKα and p - ACC / ACC signal ratios normalized ( = 1 . 0 ) to the NTG control ( n = 8 mice per group ) . * p < 0 . 01 .

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(D) (Top) Representative western blot analysis performed on extracts of the gastrocnemius muscle isolated from NTG or MCK-miR-499 mice using phospho-AMPKα (Thr172), AMPKα, phospho-ACC (Ser79), or total ACC antibodies. (Bottom) Quantification of the p-AMPKα/AMPKα and p-ACC/ACC signal ratios normalized (=1.0) to the NTG control (n = 8 mice per group). *p < 0.01.


( E ) Oxygen consumption rates ( OCR ) in primary mouse myotubes transfected with Fnip1 siRNA or Con siRNA . n = 7 separate experiments done with 5 biological replicates . * p < 0 . 05 ( Pyruvate ) , * p < 0 . 01 ( FCCP ) .

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(E) Oxygen consumption rates (OCR) in primary mouse myotubes transfected with Fnip1 siRNA or Con siRNA. n = 7 separate experiments done with 5 biological replicates. *p < 0.05 (Pyruvate), *p < 0.01 (FCCP).


( F ) Results of RT - qPCR analysis for WT primary mouse myotubes subjected to inhibition of both miR - 499 and miR - 208b ( Anti - miRs ) ( n = 4 independent experiments ) . * p = 0 . 037 ( Ppargc1a ) , * p = 0 . 0019 ( Fnip1 ) .

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(F) Results of RT-qPCR analysis for WT primary mouse myotubes subjected to inhibition of both miR-499 and miR-208b (Anti-miRs) (n = 4 independent experiments). *p = 0.037 (Ppargc1a), *p = 0.0019 (Fnip1).


( G ) Oxygen consumption rates ( OCR ) in primary mouse myotubes transfected with miR - 499 / miR - 208b inhibitors alone and together with the presence of Fnip1 siRNA . n = 4 separate experiments done with 5 biological replicates . * p < 0 . 01 ( Anti - miRs vs . Control ) .

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(G) Oxygen consumption rates (OCR) in primary mouse myotubes transfected with miR-499/miR-208b inhibitors alone and together with the presence of Fnip1 siRNA. n = 4 separate experiments done with 5 biological replicates. *p < 0.01 (Anti-miRs vs. Control).


( A ) Mean expression levels ( RT - qPCR ) in gastrocnemius muscle of 8 - week - old male WT and mdx mice ( n = 5 mice per group ) . * p = 0 . 009 ( Myh7b ) , * p = 0 . 0007 ( miR - 499 ) , * p = 0 . 0143 ( Myh7 ) , * p < 0 . 0011 ( miR - 208b ) .

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(A) Mean expression levels (RT-qPCR) in gastrocnemius muscle of 8-week-old male WT and mdx mice (n = 5 mice per group). *p = 0.009 (Myh7b), *p = 0.0007 (miR-499), *p = 0.0143 (Myh7), *p < 0.0011 (miR-208b).


( B ) Mitochondrial respiration rates were determined from mitochondria isolated from the hindlimb muscle of the indicated mice using pyruvate as substrate . n = 3 separate experiments done with 7 - 8 biological replicates . * p < 0 . 01 ( ADP ) .

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(B) Mitochondrial respiration rates were determined from mitochondria isolated from the hindlimb muscle of the indicated mice using pyruvate as substrate. n = 3 separate experiments done with 7-8 biological replicates. *p < 0.01 (ADP).


( C ) ( Left ) Representative western blot analysis of PGC - 1α ( Top ) and Fnip1 ( Bottom ) protein expression in the gastrocnemius muscle from the indicated genotypes with α - tubulin as the loading control . ( Right ) Quantification of the PGC - 1α / Tubulin and Fnip1 / Tubulin signal ratios normalized ( = 1 . 0 ) to the WT control . WT , n = 8 ; mdx , n = 5 ; mdx / 499Tg , n = 7 . * p < 0 . 0001 ( vs . WT ) , ‡ p < 0 . 0001 ( vs . mdx ) .

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(C) (Left) Representative western blot analysis of PGC-1α (Top) and Fnip1 (Bottom) protein expression in the gastrocnemius muscle from the indicated genotypes with α-tubulin as the loading control. (Right) Quantification of the PGC-1α/Tubulin and Fnip1/Tubulin signal ratios normalized (=1.0) to the WT control. WT, n = 8; mdx, n = 5; mdx/499Tg, n = 7. *p < 0.0001 (vs. WT), ‡p < 0.0001 (vs. mdx).


( D ) Representative western blot analysis of myoglobin protein expression in the gastrocnemius muscle from the indicated genotypes with α - tubulin as the loading control ( n = 5 mice per group ) .

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(D) Representative western blot analysis of myoglobin protein expression in the gastrocnemius muscle from the indicated genotypes with α-tubulin as the loading control (n = 5 mice per group).


( E ) ( Top ) Representative MHC1 immunofluorescence ( IF ) in the soleus of the indicated genotypes . Scale bar : 500 μm . ( Bottom ) Representative H & amp ; amp ; E staining of the gastrocnemius muscle of the indicated genotypes . Scale bar : 100 μm .

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(E) (Top) Representative MHC1 immunofluorescence (IF) in the soleus of the indicated genotypes. Scale bar: 500 μm. (Bottom) Representative H&amp;E staining of the gastrocnemius muscle of the indicated genotypes. Scale bar: 100 μm.


( F ) 5 - week - old male WT , mdx , and mdx / MCK - miR - 499 mice were euthanized and serum creatine kinase activity was determined . WT , n = 10 ; mdx , n = 10 ; mdx / 499Tg , n = 14 . * p < 0 . 0001 ( vs . WT ) , ‡ p = 0 . 0003 ( vs . mdx ) .

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(F) 5-week-old male WT, mdx, and mdx/MCK-miR-499 mice were euthanized and serum creatine kinase activity was determined. WT, n = 10; mdx, n = 10; mdx/499Tg, n = 14. *p < 0.0001 (vs. WT), ‡p = 0.0003 (vs. mdx).


( G ) The bars represent the mean running time and distance ( ± SEM ) for 8 - week - old male mice on a motorized treadmill . WT , n = 10 ; mdx , n = 13 ; mdx / 499Tg , n = 8 . * p = 0 . 01406 ( Running time , vs . WT ) , ‡ p = 0 . 005756 ( Running time , vs . mdx ) . * p = 0 . 004558 ( Running distance , vs . WT ) , ‡ p = 0 . 001668 ( Running distance , vs . mdx ) .

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(G) The bars represent the mean running time and distance (± SEM) for 8-week-old male mice on a motorized treadmill. WT, n = 10; mdx, n = 13; mdx/499Tg, n = 8. *p = 0.01406 (Running time, vs. WT), ‡p = 0.005756 (Running time, vs. mdx). *p = 0.004558 (Running distance, vs. WT), ‡p = 0.001668 (Running distance, vs. mdx).


( H ) Respiratory exchange ratio ( RER ) during a graded exercise regimen on a motorized treadmill . mdx , n = 8 ; mdx / 499Tg , n = 6 . ‡ p < 0 . 05 .

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(H) Respiratory exchange ratio (RER) during a graded exercise regimen on a motorized treadmill. mdx, n = 8; mdx/499Tg, n = 6. ‡p < 0.05.


J Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals from the indicated time point ( in hours ) after the L4 stage . Animals were exposed to the indicated knockdown RNAi bacterial strains or a strain that only contained the empty RNAi vector since hatching . * P < 0 . 001 using ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point , with N = 20 animals per genotype and time point . Error bars indicate SEM .

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J Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) after the L4 stage. Animals were exposed to the indicated knockdown RNAi bacterial strains or a strain that only contained the empty RNAi vector since hatching. *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Error bars indicate SEM.


L Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals from the indicated time point ( in hours ) and the indicated genotypes after the L4 stage , as per ( J ) . * P < 0 . 001 using ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point , with N = 20 animals per genotype and time point . Error bars indicate SEM .

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L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) and the indicated genotypes after the L4 stage, as per (J). *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Error bars indicate SEM.


K , L Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4 + 48 hours . * P < 0 . 001 using ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point , with N = 20 animals per genotype and time point . Data has been normalized to wild type . Error bars indicate SEM .

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K,L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4+48 hours. *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Data has been normalized to wild type. Error bars indicate SEM.


B A separate SDS - PAGE and Western blot of the same lysates from ( A ) , probed with antibodies recognizing ubiquitin or actin as a loading control . The position of molecular weight markers is shown on the left of each blot . The position of UbG76V - GFP protein , as well as UbG76V - GFP with the indicated number of additional ubiquitin moieties , based on molecular weight , is indicated to the right of each blot . Endogenous poly - ubiquitinated proteins are also detected in the 80 - 200 kDa range , as indicated by the bracket . Twenty animals were loaded per lane for each indicated genotype .

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B A separate SDS-PAGE and Western blot of the same lysates from (A), probed with antibodies recognizing ubiquitin or actin as a loading control. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated to the right of each blot. Endogenous poly-ubiquitinated proteins are also detected in the 80-200 kDa range, as indicated by the bracket. Twenty animals were loaded per lane for each indicated genotype.


B Heat map of log fold change ( logFC , color scale at graph bottom ) in the expression of C . elegans CYP genes between the indicated mutant and wild type . Genes whose expression is lower in a mutant relative to wild type are colored blue , whereas genes whose expression is higher in a mutant relative to wild type are colored yellow . Rows are labeled for the different CYP families and subfamilies . Specific genes ( rows ) are clustered according to sequence alignment ( CLUSTAL W ) to maintain the relationship of their molecular evolution

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B Heat map of log fold change (logFC, color scale at graph bottom) in the expression of C. elegans CYP genes between the indicated mutant and wild type. Genes whose expression is lower in a mutant relative to wild type are colored blue, whereas genes whose expression is higher in a mutant relative to wild type are colored yellow. Rows are labeled for the different CYP families and subfamilies. Specific genes (rows) are clustered according to sequence alignment (CLUSTAL W) to maintain the relationship of their molecular evolution


D Abundance of the indicated free ω6 PUFA - derived eicosanoid , normalized to total protein , for each indicated genotype . Red , gray , and blue stacked bars indicate hydroxides , epoxide , and epoxide diol forms of eicosanoids , respectively . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA with Dunnett ' s posthoc comparison to wild type . N = 3 trials . Error bars indicate SEM . E Abundance of the indicated free ω3 PUFA - derived eicosanoid , as in ( D ) , normalized to total protein , for each indicated genotype . Red , gray , and blue stacked bars indicate hydroxides , epoxide , and epoxide diol forms of eicosanoids , respectively . * * * * P < 0 . 0001 , ANOVA with Dunnett ' s posthoc comparison to wild type . N = 3 trials . Error bars indicate SEM

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D Abundance of the indicated free ω6 PUFA-derived eicosanoid, normalized to total protein, for each indicated genotype. Red, gray, and blue stacked bars indicate hydroxides, epoxide, and epoxide diol forms of eicosanoids, respectively. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM. E Abundance of the indicated free ω3 PUFA-derived eicosanoid, as in (D), normalized to total protein, for each indicated genotype. Red, gray, and blue stacked bars indicate hydroxides, epoxide, and epoxide diol forms of eicosanoids, respectively. ****P<0.0001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM


B Relative abundance of mRNA for the cyp - 34A4 gene . Values are based on qRT - PCR analysis , with the relative levels for each gene normalized to the value observed in the wild - type control . * * * P < 0 . 001 , ANOVA with Dunnett ' s posthoc comparison to wild type . N = 3 trials . Error bars indicate SEM .

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B Relative abundance of mRNA for the cyp-34A4 gene. Values are based on qRT-PCR analysis, with the relative levels for each gene normalized to the value observed in the wild-type control. ***P<0.001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM.


C Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4 + 48 hours . * * * * P < 0 . 001 , ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point . N = 20 animals per genotype and time point . Error bars indicate SEM .

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C Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4+48 hours. ****P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.


F Quantified fluorescence as in ( B ) for animals exposed to the indicated RNAi vector or empty vector ( EV ) control , and in either a wild type ( WT ) or fat - 1 ( wa9 ) genetic background . * P < 0 . 001 , ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point . N = 20 animals per genotype and time point . Error bars indicate SEM .

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F Quantified fluorescence as in (B) for animals exposed to the indicated RNAi vector or empty vector (EV) control, and in either a wild type (WT) or fat-1(wa9) genetic background. *P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.


G Quantified fluorescence as in ( B ) for animals of the indicated genotype at the indicated time point . * P < 0 . 001 , ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point . N = 20 animals per genotype and time point . Error bars indicate SEM .

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G Quantified fluorescence as in (B) for animals of the indicated genotype at the indicated time point. *P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.


a ) EGF enhances colocalization of phosphorylated ( P ) - bRAF , P - MEK and P - ERK with LC3 . Immunofluorescence ( IF ) showing colocalization of P - bRAF ( green ) , P - MEK ( green ) and P - ERK ( green ) with LC3 ( red ) in NIH / 3T3 cells in presence or absence of EGF ( 10 min as shown in scheme ) . The bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test , 60 cells analysed from two experiments . Scale bars , 10 μm .

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a) EGF enhances colocalization of phosphorylated (P)-bRAF, P-MEK and P-ERK with LC3. Immunofluorescence (IF) showing colocalization of P-bRAF (green), P-MEK (green) and P-ERK (green) with LC3 (red) in NIH/3T3 cells in presence or absence of EGF (10 min as shown in scheme). The bars represent mean±s.e.m. **P0.01, ***P0.001; Student's t-test, 60 cells analysed from two experiments. Scale bars, 10 μm.


( b ) P - ERK colocalizes with pre - autophagosomal and autophagosomal structures . IF depicting colocalization of P - ERK ( green or red ) with LC3 ( red ) , ATG5 - ATG12 ( red ) , ATG16 ( green ) , vps34 ( red ) , WIPI1 ( red ) , WIPI2 ( red ) and P - ULK1 ( red ) in EGF - treated NIH / 3T3 cells . Scale bars , 10 μm . The bars represent mean ± s . e . m . 50 cells analysed from two experiments . ( c ) Growth factors in serum maintain P - ERK / LC3 colocalization . IF depicting colocalization of P - ERK ( green ) with LC3 ( red ) in 2 h serum - fed NIH / 3T3 cells .

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(b) P-ERK colocalizes with pre-autophagosomal and autophagosomal structures. IF depicting colocalization of P-ERK (green or red) with LC3 (red), ATG5-ATG12 (red), ATG16 (green), vps34 (red), WIPI1 (red), WIPI2 (red) and P-ULK1 (red) in EGF-treated NIH/3T3 cells. Scale bars, 10 μm. The bars represent mean±s.e.m. 50 cells analysed from two experiments. (c) Growth factors in serum maintain P-ERK/LC3 colocalization. IF depicting colocalization of P-ERK (green) with LC3 (red) in 2 h serum-fed NIH/3T3 cells.


( a - c ) Native merged images or images with colocalization highlighted in white pixels are shown . Nuclei are blue ( DAPI ) . Scale bar , 10 μm .

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(a-c) Native merged images or images with colocalization highlighted in white pixels are shown. Nuclei are blue (DAPI). Scale bar, 10 μm.


( a ) P - and total MEK and ERK are enriched in APh in vivo . Immunoblots for indicated proteins in homogenate ( Hom ) , APh , APL and Lys fractions from livers of fed mice , n = 4 .

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(a) P- and total MEK and ERK are enriched in APh in vivo. Immunoblots for indicated proteins in homogenate (Hom), APh, APL and Lys fractions from livers of fed mice, n=4.


( b ) EGF enhances enrichment of P - MEK and ERK in APh fractions in vitro . Immunoblots for indicated proteins in Hom , APh and APL fractions from NIH / 3T3 cells in presence or absence of EGF ( 10 min ) . The bars represent mean ± s . e . m . * P0 . 05 , * * P0 . 01 ; Student ' s t - test , n = 3 .

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

(b) EGF enhances enrichment of P-MEK and ERK in APh fractions in vitro. Immunoblots for indicated proteins in Hom, APh and APL fractions from NIH/3T3 cells in presence or absence of EGF (10 min). The bars represent mean±s.e.m. *P0.05, **P0.01; Student's t-test, n=3.


( c ) P - and total MEK and ERK localize on to the cytoplasmic / extra - luminal face of APh in vivo . Left ; model depicting localization of MEK and ERK on the extra - luminal face of autophagic vesicles and the ability of trypsin to degrade extra - luminal MEK and ERK , and right ; immunoblots for indicated proteins in APh fractions from mice livers untreated ( − ) or treated with increasing amounts of trypsin for 15 min . The bars represent mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 compared with corresponding trypsin - untreated value ; ANOVA - Bonferroni post hoc test , n = 3 . The p44 , p42 forms of ERK , LC3 - I and membrane - associated LC3 - II , and 37 kDa and 25 kDa forms of cathepsin ( Cath ) B are indicated .

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

(c) P- and total MEK and ERK localize on to the cytoplasmic/extra-luminal face of APh in vivo. Left; model depicting localization of MEK and ERK on the extra-luminal face of autophagic vesicles and the ability of trypsin to degrade extra-luminal MEK and ERK, and right; immunoblots for indicated proteins in APh fractions from mice livers untreated (−) or treated with increasing amounts of trypsin for 15 min. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with corresponding trypsin-untreated value; ANOVA-Bonferroni post hoc test, n=3. The p44, p42 forms of ERK, LC3-I and membrane-associated LC3-II, and 37 kDa and 25 kDa forms of cathepsin (Cath) B are indicated.


( a ) EGF enhances nuclear LC3 - II content in hepatocytes . Immunofluorescence ( IF ) depicting nuclear LC3 - II in RALA hepatocytes in presence or absence of EGF ( 10 min ) . Native / inverted images are shown . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * * P0 . 001 compared with control ( Con ) ; Student ' s t - test , 50 cells from two experiments .

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

(a) EGF enhances nuclear LC3-II content in hepatocytes. Immunofluorescence (IF) depicting nuclear LC3-II in RALA hepatocytes in presence or absence of EGF (10 min). Native/inverted images are shown. Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with control (Con); Student's t-test, 50 cells from two experiments.


( b ) EGF enhances nuclear P - ERK / LC3 - II colocalization . IF depicting P - ERK ( green ) / LC3 - II ( red ) colocalization in untreated ( Con ) or EGF - treated NIH / 3T3 cells . Native images ( top ) / images with colocalization in white pixels ( bottom ) are shown . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , 50 cells from two experiments .

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

(b) EGF enhances nuclear P-ERK/LC3-II colocalization. IF depicting P-ERK (green)/LC3-II (red) colocalization in untreated (Con) or EGF-treated NIH/3T3 cells. Native images (top)/images with colocalization in white pixels (bottom) are shown. Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, 50 cells from two experiments.


( c ) Adipogenic differentiation increases nuclear LC3 - II . Images depict nuclear LC3 - II in 3T3 - L1 preadipocytes in maintenance or differentiation medium ( 2 h ) . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * P0 . 01 compared with Con ; Student ' s t - test , 50 cells from two experiments .

O O O O 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

(c) Adipogenic differentiation increases nuclear LC3-II. Images depict nuclear LC3-II in 3T3-L1 preadipocytes in maintenance or differentiation medium (2 h). Scale bar, 5 μm. Bars represent mean±s.e.m. **P0.01 compared with Con; Student's t-test, 50 cells from two experiments.


( d ) Nuclear P - ERK / LC3 - II colocalization in serum - fed cells . IF showing P - ERK ( green ) / LC3 ( red ) colocalization in 2 h serum - fed NIH / 3T3 cells . Native images ( top ) / images with colocalization in white pixels ( bottom ) are shown . Scale bar , 5 μm .

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

(d) Nuclear P-ERK/LC3-II colocalization in serum-fed cells. IF showing P-ERK (green)/LC3 (red) colocalization in 2 h serum-fed NIH/3T3 cells. Native images (top)/images with colocalization in white pixels (bottom) are shown. Scale bar, 5 μm.


( e ) LC3 interacts with ERK in vivo . Immunoblots showing co - immunoprecipitation of LC3 with ERK , MEK and bRAF in homogenate ( Hom ) ( e , top ) , and of LC3 with P - and total ERK in nuclear fractions from mouse livers ( e , bottom ) .

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

(e) LC3 interacts with ERK in vivo. Immunoblots showing co-immunoprecipitation of LC3 with ERK, MEK and bRAF in homogenate (Hom) (e, top), and of LC3 with P- and total ERK in nuclear fractions from mouse livers (e, bottom).


( f ) Blocking nuclear transport decreases EGF - induced increase in nuclear LC3 - II . LC3 IF ( red ) in EGF - treated NIH / 3T3 cells pre - exposed ( 30 min ) or not to WGA . Bars represent mean ± s . e . m . * * * P0 . 001 compared to with ; Student ' s t - test , 60 cells from n = 3 . Scale bar , 10 μm .

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

(f) Blocking nuclear transport decreases EGF-induced increase in nuclear LC3-II. LC3 IF (red) in EGF-treated NIH/3T3 cells pre-exposed (30 min) or not to WGA. Bars represent mean±s.e.m. ***P0.001 compared to with; Student's t-test, 60 cells from n=3. Scale bar, 10 μm.


( g ) Blocking nuclear transport decreases nuclear ERK content . ERK IF ( green ) in EGF - treated NIH / 3T3 cells pre - exposed ( 30 min ) or not to WGA . Bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test .

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

(g) Blocking nuclear transport decreases nuclear ERK content. ERK IF (green) in EGF-treated NIH/3T3 cells pre-exposed (30 min) or not to WGA. Bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test.


( h ) Blocking nuclear transport does not modify P - ERK / LC3 - II colocalization . IF depicting nuclear P - ERK ( green ) / LC3 ( red ) colocalization in EGF - treated NIH / 3T3 cells pre - exposed or not to WGA . For ( g ) and ( h ) : Scale bar , 10 μm , bars are mean ± s . e . m . 50 cells from n = 2 . Nuclei are blue ( DAPI ) . Arrows indicate LC3 puncta , ERK or colocalization .

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

(h) Blocking nuclear transport does not modify P-ERK/LC3-II colocalization. IF depicting nuclear P-ERK (green)/LC3 (red) colocalization in EGF-treated NIH/3T3 cells pre-exposed or not to WGA. For (g) and (h): Scale bar, 10 μm, bars are mean±s.e.m. 50 cells from n=2. Nuclei are blue (DAPI). Arrows indicate LC3 puncta, ERK or colocalization.


( a ) Atg7 − / − livers display decreased ERK phosphorylation . Immunoblots for the indicated proteins in liver homogenate ( Hom ) and cytosolic ( Cyt ) fractions of control ( Con ) and liver - specific Atg7 − / − mice are shown . The bars represent mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 compared with Con ; Student ' s t - test , n = 4 .

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

(a) Atg7−/− livers display decreased ERK phosphorylation. Immunoblots for the indicated proteins in liver homogenate (Hom) and cytosolic (Cyt) fractions of control (Con) and liver-specific Atg7−/− mice are shown. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with Con; Student's t-test, n=4.


( b ) Atg7 − / − livers display decreased ERK2 dimers . Immunoblots show the indicated proteins in Hom from Con and Atg7 − / − livers . The bars represent mean ± s . e . m . * P0 . 05 , * * * P0 . 001 compared to Con ; Student ' s t - test , n = 4 - 5 . Arrows indicate p42 monomers and dimers .

O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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) Atg7−/− livers display decreased ERK2 dimers. Immunoblots show the indicated proteins in Hom from Con and Atg7−/− livers. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared to Con; Student's t-test, n=4-5. Arrows indicate p42 monomers and dimers.


( c ) Atg7 − / − brown adipose tissues ( BAT ) display decreased ERK phosphorylation . Immunoblots for indicated proteins in Hom from Con and Atg7 − / − BAT . The bars represent mean ± s . e . m . * * P0 . 01 compared with Con ; Student ' s t - test , n = 4 - 6 .

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

(c) Atg7−/− brown adipose tissues (BAT) display decreased ERK phosphorylation. Immunoblots for indicated proteins in Hom from Con and Atg7−/−BAT. The bars represent mean±s.e.m. **P0.01 compared with Con; Student's t-test, n=4-6.


( d ) Atg7 − / − livers exhibit decreased nuclear ERK phosphorylation . Immunoblots show the indicated proteins in homogenate ( Hom , lane 1 ) and nuclear fractions ( lanes 2 - 5 ) from Con and Atg7 − / − livers . The bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , n = 4 - 5 .

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

(d) Atg7−/− livers exhibit decreased nuclear ERK phosphorylation. Immunoblots show the indicated proteins in homogenate (Hom, lane 1) and nuclear fractions (lanes 2-5) from Con and Atg7−/− livers. The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=4-5.


( e ) LC3 C terminus glycine - deleted ( ΔG ) mutants exhibit decreased ERK phosphorylation . Immunoblots for indicated proteins in total lysates from CFP - LC3 - and CFP - LC3ΔG - transfected NIH / 3T3 cells exposed or not to EGF ( 10 min ) . The bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , n = 8 .

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

(e) LC3 C terminus glycine-deleted (ΔG) mutants exhibit decreased ERK phosphorylation. Immunoblots for indicated proteins in total lysates from CFP-LC3- and CFP-LC3ΔG-transfected NIH/3T3 cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=8.


( a ) Atg5 − / − MEFs display reduced ERK phosphorylation during nutrient deprivation . Quantification of P - ERK levels normalized to total ERK ( detected by immunoblotting ) in WT and Atg5 − / − MEFs cultured in the absence of serum for indicated times . The bars represent mean ± s . e . m . * P0 . 05 compared with corresponding WT value ; * * * P0 . 001 , * * * * P0 . 0001 compared with 10 min serum - starved Atg5 − / − MEFs ; ANOVA - Bonferroni post hoc test , n = 3 .

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

(a) Atg5−/− MEFs display reduced ERK phosphorylation during nutrient deprivation. Quantification of P-ERK levels normalized to total ERK (detected by immunoblotting) in WT and Atg5−/− MEFs cultured in the absence of serum for indicated times. The bars represent mean±s.e.m. *P0.05 compared with corresponding WT value; ***P0.001, ****P0.0001 compared with 10 min serum-starved Atg5−/− MEFs; ANOVA-Bonferroni post hoc test, n=3.


( b ) Serum - deprived Atg5 − / − MEFs display decreased ERK and p90RSK phosphorylation . Immunoblots for the indicated proteins in total lysates from 2 h serum - deprived WT and Atg5 − / − MEFs . The bars represent mean ± s . e . m . * P0 . 05 , * * * P0 . 001 compared with Con ; Student ' s t - test , n = 3 .

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

(b) Serum-deprived Atg5−/− MEFs display decreased ERK and p90RSK phosphorylation. Immunoblots for the indicated proteins in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared with Con; Student's t-test, n=3.


( c ) Atg5 − / − MEFs display decreased nuclear ERK activity . Immunoblots for phosphorylated HA ( haemagglutinin ) - tagged Elk1 in total lysates from 2 h serum - deprived WT and Atg5 − / − MEFs cells exposed or not to EGF ( 10 min ) .

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

(c) Atg5−/− MEFs display decreased nuclear ERK activity. Immunoblots for phosphorylated HA (haemagglutinin)-tagged Elk1 in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min).


( d ) Atg5 − / − MEFs display decreased Elk1 - driven gene expression . Elk1 - driven ZFP36 mRNA levels from 2 hserum - deprived WT and Atg5 − / − MEFs cells exposed or not to EGF ( 10 min ) . The bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , n = 3 . (

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 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

(d) Atg5−/− MEFs display decreased Elk1-driven gene expression. Elk1-driven ZFP36 mRNA levels from 2 hserum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=3. (


( e ) Decreased ERK phosphorylation in Atg5 − / − MEFs occurs independently of changes in ERK phosphatases . Immunoblots for the indicated proteins in 2 h serum - deprived WT MEFs transfected with scrambled siRNA ( scr ) , and Atg5 − / − MEFs transfected with scr or siRNAs against MKP3 or PP2A in the presence or absence of EGF . The bars represent mean ± s . e . m . , n = 3 .

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

(e) Decreased ERK phosphorylation in Atg5−/− MEFs occurs independently of changes in ERK phosphatases. Immunoblots for the indicated proteins in 2 h serum-deprived WT MEFs transfected with scrambled siRNA (scr), and Atg5−/− MEFs transfected with scr or siRNAs against MKP3 or PP2A in the presence or absence of EGF. The bars represent mean±s.e.m., n=3.


( a ) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5 - ATG12 , LC3 and WIPI1 . Immunofluorescence ( IF ) showing colocalization ( depicted as white pixels by ' colocalization finder application ' ) of WT - ERK2 - HA , FRS ERK2 mutants ( L198A - , L232A - , L198A / L232A - , Y261A - ERK2 - HA ) or common docking ( CD ) mutant ( D319N - ERK2 - HA ) with ATG5 - ATG12 ( panels 1 - 6 ) , LC3 ( panels 7 - 12 ) or WIPI1 ( panels 13 - 18 ) in EGF - treated NIH / 3T3 cells . ERK2 is stained in red , and autophagy proteins are stained in green . Scale bar , 10 μm . The bars represent mean ± s . e . m . * * P0 . 01 , * * * * P0 . 0001 compared with WT - ERK2 - transfected cells ; Student ' s t - test , 50 cells analysed from n = 2 . (

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

(a) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5-ATG12, LC3 and WIPI1. Immunofluorescence (IF) showing colocalization (depicted as white pixels by 'colocalization finder application') of WT-ERK2-HA, FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5-ATG12 (panels 1-6), LC3 (panels 7-12) or WIPI1 (panels 13-18) in EGF-treated NIH/3T3 cells. ERK2 is stained in red, and autophagy proteins are stained in green. Scale bar, 10 μm. The bars represent mean±s.e.m. **P0.01, ****P0.0001 compared with WT-ERK2-transfected cells; Student's t-test, 50 cells analysed from n=2. (


( b ) Mutations in FRS on ERK2 decrease colocalization of ERK2 with nuclearATG5 - ATG12 or LC3 . IF showing colocalization ( white pixels ) of WT - ERK2 - HA ( red ) , FRS ERK2 mutants ( L198A - , L232A - , L198A / L232A - , Y261A - ERK2 - HA ) or common docking ( CD ) mutant ( D319N - ERK2 - HA ) with ATG5 - ATG12 ( panels 1 - 6 ) or LC3 - II ( panels 7 - 12 ) in EGF - treated NIH / 3T3 cells . Scale bar , 5 μm . The bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 , * * * * P0 . 0001 compared with WT - ERK2 - transfected cells ; Student ' s t - test , 50 cells analysed from n = 2 . Arrows depict colocalization .

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

(b) Mutations in FRS on ERK2 decrease colocalization of ERK2 with nuclearATG5-ATG12 or LC3. IF showing colocalization (white pixels) of WT-ERK2-HA (red), FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5-ATG12 (panels 1-6) or LC3-II (panels 7-12) in EGF-treated NIH/3T3 cells. Scale bar, 5 μm. The bars represent mean±s.e.m. **P0.01, ***P0.001, ****P0.0001 compared with WT-ERK2-transfected cells; Student's t-test, 50 cells analysed from n=2. Arrows depict colocalization.


( c ) ATG5 - ATG12 conjugation is required for ERK phosphorylation . Immunoblots for P - ERK , total ERK and β - actin in NIH / 3T3 cells transfected with WT ATG5 or the conjugation - defective ATG5 K130R mutant and treated with EGF ( 10 min ) . The bars represent mean ± s . e . m . * P0 . 05 compared with WT ATG5 - transfected cells ; Student ' s t - test , n = 3 .

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

(c) ATG5-ATG12 conjugation is required for ERK phosphorylation. Immunoblots for P-ERK, total ERK and β-actin in NIH/3T3 cells transfected with WT ATG5 or the conjugation-defective ATG5 K130R mutant and treated with EGF (10 min). The bars represent mean±s.e.m. *P0.05 compared with WT ATG5-transfected cells; Student's t-test, n=3.


a ) ATG4B depletion increases steady - state LC3 - II levels . Immunoblots for indicated proteins in NIH / 3T3 cells transfected with scrambled siRNAs ( scr ) or siRNAs against ATG4B ( siATG4B ) in presence / absence of EGF . Bars represent mean ± s . e . m . * P0 . 05 , * * * P0 . 001 compared with scr ; Student ' s t - test , n = 4 .

O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

a) ATG4B depletion increases steady-state LC3-II levels. Immunoblots for indicated proteins in NIH/3T3 cells transfected with scrambled siRNAs (scr) or siRNAs against ATG4B (siATG4B) in presence/absence of EGF. Bars represent mean±s.e.m. *P0.05, ***P0.001 compared with scr; Student's t-test, n=4.


( b ) siATG4B cells exhibit increased autophagic vesicles . Representative electron micrographs depicting autophagic vesicles ( indicated by black arrows ) in scr and siATG4B NIH / 3T3 cells .

O O O O O O O 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) siATG4B cells exhibit increased autophagic vesicles. Representative electron micrographs depicting autophagic vesicles (indicated by black arrows) in scr and siATG4B NIH/3T3 cells.


( c ) ATG4B depletion does not increase autophagic flux in serum - fed cells . Immunoblots for indicated proteins in lysates from scr or siATG4B NIH / 3T3 cells in presence / absence of lysosomal inhibitors , ammonium chloride and leupeptin ( Inh ) for 2 h . Scale bars , 1 μm . Bars represent mean ± s . e . m . , n = 3 .

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

(c) ATG4B depletion does not increase autophagic flux in serum-fed cells. Immunoblots for indicated proteins in lysates from scr or siATG4B NIH/3T3 cells in presence/absence of lysosomal inhibitors, ammonium chloride and leupeptin (Inh) for 2 h. Scale bars, 1 μm. Bars represent mean±s.e.m., n=3.


( d ) siATG4B cells display increased nuclear LC3 - II . Images for LC3 - II ( red ) in scr or siATG4B NIH / 3T3 cells in presence / absence of EGF ( 10 min ) . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * * P0 . 001 compared with scr ; Student ' s t - test , 60 cells from n = 2 .

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

(d) siATG4B cells display increased nuclear LC3-II. Images for LC3-II (red) in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.


( e ) ATG4B deficiency increases P - ERK / LC3 - II colocalization . Immunofluorescence ( IF ) for P - ERK ( green ) / LC3 ( red ) colocalization in scr or siATG4B NIH / 3T3 cells in presence / absence of EGF ( 10 min ) . Scale bars , 10 μm . Bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 compared with scr ; Student ' s t - test , 60 cells from n = 2 .

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

(e) ATG4B deficiency increases P-ERK/LC3-II colocalization. Immunofluorescence (IF) for P-ERK (green)/LC3 (red) colocalization in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bars, 10 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.