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 .

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 O O O O O 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 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 .

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

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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 .

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

(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 .

O O O O O O O O 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-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 .

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

(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 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 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 B-CONTROLLED_VAR 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 . (

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(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 .

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(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 .

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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 .

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(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 .

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(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 .

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(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.


( f ) ATG4B - deficient cells display increased nuclear P - ERK / LC3 colocalization . IF depicting P - ERK ( green ) / LC3 ( red ) colocalization in nuclei of scr or siATG4B NIH / 3T3 cells in presence / absence of EGF . Scale bars , 5 μm . Bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 compared with scr ; Student ' s t - test , 60 cells from n = 2 .

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(f) ATG4B-deficient cells display increased nuclear P-ERK/LC3 colocalization. IF depicting P-ERK (green)/LC3 (red) colocalization in nuclei of scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.


( g ) ATG4B deficiency augments EGF - induced MEK and ERK phosphorylation . Immunoblots for P - and total MEK and ERK , LC3 and GAPDH in total lysates from scr or siATG4B NIH / 3T3 cells in presence / absence of EGF . Bars represent mean ± s . e . m . , n = 3 .

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(g) ATG4B deficiency augments EGF-induced MEK and ERK phosphorylation. Immunoblots for P- and total MEK and ERK, LC3 and GAPDH in total lysates from scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Bars represent mean±s.e.m., n=3.


h ) ATG4B deficiency increases nuclear P - ERK levels . Native ( top ) / inverted images ( bottom ) showing nuclear P - ERK content . P - ERK fluorescence in untreated ( Con ) / EGF - treated scr or siATG4B NIH / 3T3 cells is shown . Scale bars , 5 μm . Bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 compared with scr ; Student ' s t - test , 60 cells from n = 2 . Nuclei are blue ( DAPI ) .

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h) ATG4B deficiency increases nuclear P-ERK levels. Native (top)/inverted images (bottom) showing nuclear P-ERK content. P-ERK fluorescence in untreated (Con)/EGF-treated scr or siATG4B NIH/3T3 cells is shown. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2. Nuclei are blue (DAPI).


A HEK 293 cells were transfected with mCherry - tagged Parkin and FLAG - tagged UBQLN2 for 24 h , and then were treated with 5 µg / mL Antimycin A and Oligomycin A ( A / O ) for 4 h and 24 h . The cells were stained with anti - FLAG ( green ) antibody and anti - TOM20 ( gray ) , and then were visualized using confocal microscopy . Scale bars , 10 µm . Insets were higher magnifications of the dashed box area . Scale bars , 2 . 5 µm . B , C The dashed square is magnified as shown in the boxed region in A with corresponding line scan indicating Parkin and UBQLN2 co - localization on the depolarized mitochondria ( the line scan indicates pixel intensity of Parkin and UBQLN2 ) .

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A HEK 293 cells were transfected with mCherry-tagged Parkin and FLAG-tagged UBQLN2 for 24 h, and then were treated with 5 µg/mL Antimycin A and Oligomycin A (A/O) for 4 h and 24 h. The cells were stained with anti-FLAG (green) antibody and anti-TOM20 (gray), and then were visualized using confocal microscopy. Scale bars, 10 µm. Insets were higher magnifications of the dashed box area. Scale bars, 2.5 µm. B, C The dashed square is magnified as shown in the boxed region in A with corresponding line scan indicating Parkin and UBQLN2 co-localization on the depolarized mitochondria (the line scan indicates pixel intensity of Parkin and UBQLN2).


D HEK 293 cells were transfected with mCherry - tagged Parkin and EGFP - tagged UBQLN2 . 24 h after transfection , cells were treated with 1 µg / mL A / O for 4 h , and then were harvested for the isolation of mitochondria . Whole cell ( Total ) , cytosol ( Cyto ) and mitochondria ( Mito ) were lysed and analyzed by western blotting with the indicated antibodies .

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D HEK 293 cells were transfected with mCherry-tagged Parkin and EGFP-tagged UBQLN2. 24 h after transfection, cells were treated with 1 µg/mL A/O for 4 h, and then were harvested for the isolation of mitochondria. Whole cell (Total), cytosol (Cyto) and mitochondria (Mito) were lysed and analyzed by western blotting with the indicated antibodies.


A Domain structure of Parkin in upper region . HEK 293 cells were transfected with HA - tagged UBQLN2 and FLAG - tagged Parkin WT or mutants ( K161N , T240R , C418R , C431F and P437L ) for 24 h , and then were treated with 5 µg / mL A / O for 5 h . The cells were stained with anti - HA ( green ) and FLAG ( red ) antibodies . Then the cells were visualized using confocal microscopy . Scale bars , 10 µm . Lower panel were higher magnifications of the dashed box area . Scale bars , 2 . 5 µm . B , C The quantification data of the Parkin or UBQLN2 with mitochondrial localization in A . At least 60 anti - HA - UBQLN2 or anti - FLAG - Parkin positive cells were counted for each sample . Data from three independent experiments were represented as means ± SD . , ns , not significantly different ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , one - way ANOVA .

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A Domain structure of Parkin in upper region. HEK 293 cells were transfected with HA-tagged UBQLN2 and FLAG-tagged Parkin WT or mutants (K161N, T240R, C418R, C431F and P437L) for 24 h, and then were treated with 5 µg/mL A/O for 5 h. The cells were stained with anti-HA (green) and FLAG (red) antibodies. Then the cells were visualized using confocal microscopy. Scale bars, 10 µm. Lower panel were higher magnifications of the dashed box area. Scale bars, 2.5 µm. B, C The quantification data of the Parkin or UBQLN2 with mitochondrial localization in A. At least 60 anti-HA-UBQLN2 or anti-FLAG-Parkin positive cells were counted for each sample. Data from three independent experiments were represented as means ± SD., ns, not significantly different; **, p<0.01; ***, p<0.001, one-way ANOVA.


B HEK 293 cells were transfected with indicated siRNA . After 48 h , the cells were re - transfected with FLAG - Parkin and mt - mKeima for 17 h . Then the cells were treated with 5 µg / mL A / O for 5 h , and visualized using confocal microscopy . Intracellular mt - mKeima signal excited at 458 nm ( measuring mitochondria with a neutral pH ) was shown in green color , while red color indicated the mt - mKeima fluorescence excited at 561 nm ( measuring mitochondria with an acidic pH ) in the same cell . Scale bars , 10 µm .

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B HEK 293 cells were transfected with indicated siRNA. After 48 h, the cells were re-transfected with FLAG-Parkin and mt-mKeima for 17 h. Then the cells were treated with 5 µg/mL A/O for 5 h, and visualized using confocal microscopy. Intracellular mt-mKeima signal excited at 458 nm (measuring mitochondria with a neutral pH) was shown in green color, while red color indicated the mt-mKeima fluorescence excited at 561 nm (measuring mitochondria with an acidic pH) in the same cell. Scale bars, 10 µm.


D The cells were transfected and treated as B . Cells were analyzed by FACS . The percentage of cells with lysosomal positive mt - mKeima are shown on the representative FACS data . Data from three independent experiments were represented as means ± SD . , * * , p < 0 . 01 ; * * * , p < 0 . 001 , one - way ANOVA .

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D The cells were transfected and treated as B. Cells were analyzed by FACS. The percentage of cells with lysosomal positive mt-mKeima are shown on the representative FACS data. Data from three independent experiments were represented as means ± SD., **, p<0.01; ***, p<0.001, one-way ANOVA.


G HEK 293 cells were transfected with indicated siRNAs . After 24 h , the cells were re - transfected with FLAG tag or FLAG - tagged UBQLN2WT or ∆ UBA , along with mCherry - Parkin for 24 h . The cells then were treated with or without 1 µg / mL A / O for 24 h . The cell lysates were subjected to immunoblot with indicated antibodies .

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G HEK 293 cells were transfected with indicated siRNAs. After 24 h, the cells were re-transfected with FLAG tag or FLAG-tagged UBQLN2WT or ∆UBA, along with mCherry-Parkin for 24 h. The cells then were treated with or without 1 µg/mL A/O for 24 h. The cell lysates were subjected to immunoblot with indicated antibodies.


A Domain structure of UBQLN2 in upper region . HEK 293 cells were transfected with FLAG - tagged UBQLN2 - WT or ALS - linked mutants ( ∆ UBA , ∆ UBL , P497H or P509S ) , along with mCherry - Parkin . After 24 h , the cells were treated with 5 µg / mL A / O for 5 h . The cells were stained with anti - FLAG ( green ) and anti - TOM20 ( gray ) antibodies , and then were visualized using confocal microscopy . Scale bars , 10 µm . Insets were higher magnifications of the dashed box area . Scale bars , 2 . 5 µm .

O O O O B-MEASURED_VAR 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 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

A Domain structure of UBQLN2 in upper region. HEK 293 cells were transfected with FLAG-tagged UBQLN2-WT or ALS-linked mutants (∆UBA, ∆UBL, P497H or P509S), along with mCherry-Parkin. After 24 h, the cells were treated with 5 µg/mL A/O for 5 h. The cells were stained with anti-FLAG (green) and anti-TOM20 (gray) antibodies, and then were visualized using confocal microscopy. Scale bars, 10 µm. Insets were higher magnifications of the dashed box area. Scale bars, 2.5 µm.


C The cells were transfected with mCherry - Parkin and HA - tagged UBQLN2 - WT or ALS - mutants as A and subjected to immunofluorescent assay as in A , but were treated with 1 µg / mL A / O for 24 h . Scale bar , 10 µm . Yellow arrow indicates uncleared mitochondria .

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C The cells were transfected with mCherry-Parkin and HA-tagged UBQLN2-WT or ALS-mutants as A and subjected to immunofluorescent assay as in A, but were treated with 1 µg/mL A/O for 24 h. Scale bar, 10 µm. Yellow arrow indicates uncleared mitochondria.


E HEK 293T cells were transfected with indicated siRNAs . After 24 h , the cells were re - transfected with FLAG - tagged UBQLN2 - WT or mutants , along with mCherry - Parkin for 24h . Then the cells were treated with or without 1 µg / mL A / O for 18 h . The cell lysates were subjected to immunoblot with indicated antibodies .

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E HEK 293T cells were transfected with indicated siRNAs. After 24 h, the cells were re-transfected with FLAG-tagged UBQLN2-WT or mutants, along with mCherry-Parkin for 24h. Then the cells were treated with or without 1 µg/mL A/O for 18 h. The cell lysates were subjected to immunoblot with indicated antibodies.


A HEK 293 cells were transfected with mCherry - tagged Parkin , HA - tagged UBQLN2 , FLAG - tagged HSP70 and BFP - mito for 24 h , and were then treated with 5 µg / mL A / O for 4 h . The cells were stained with anti - HA ( green ) and FLAG ( gray ) antibodies , and were visualized using confocal microscopy . Scale bar , 10 µm . Right panel were higher magnifications of the dashed box area . Scale bars , 2 . 5 µm .

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A HEK 293 cells were transfected with mCherry-tagged Parkin, HA-tagged UBQLN2, FLAG-tagged HSP70 and BFP-mito for 24 h, and were then treated with 5 µg/mL A/O for 4 h. The cells were stained with anti-HA (green) and FLAG (gray) antibodies, and were visualized using confocal microscopy. Scale bar, 10 µm. Right panel were higher magnifications of the dashed box area. Scale bars, 2.5 µm.


D HEK 293T cells expressing HA - tagged UBQLN2 and FLAG - tagged HSP70 ( WT or ΔC ) were incubated with 5 µg / mL A / O for 5 h . The cells were then subjected to immunoprecipitation assay using anti - FLAG antibody .

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D HEK 293T cells expressing HA-tagged UBQLN2 and FLAG-tagged HSP70 (WT or ΔC) were incubated with 5 µg/mL A/O for 5 h. The cells were then subjected to immunoprecipitation assay using anti-FLAG antibody.


E HEK 293T cells expressing HA tagged UBQLN2 - WT , ΔUBA or P497H ) and FLAG - tagged HSP70 were incubated with 5 µg / mL A / O for 5 h . Immunoprecipitation assay were performed using UBQLN2 antibody .

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E HEK 293T cells expressing HA tagged UBQLN2-WT, ΔUBA or P497H) and FLAG-tagged HSP70 were incubated with 5 µg/mL A/O for 5 h. Immunoprecipitation assay were performed using UBQLN2 antibody.


G The quantification data of UBQLN2 recruitment to mitochondria after 5 µg / mL A / O treatment . HEK 293T cells were transfected with indicated siRNAs . The cells were re - transfected with HA - tagged UBQLN2 - WT , mCherry - Parkin , along with BFP - mito , and treated with 5 µg / mL A / O for 5 h . The cells were subjected to immunofluorescent assay using anti - HA antibody . Data from three independent experiments were represented as means ± SD . , Sixty cells were counted for each sample . * * , p < 0 . 01 , one - way ANOVA .

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G The quantification data of UBQLN2 recruitment to mitochondria after 5 µg/mL A/O treatment. HEK 293T cells were transfected with indicated siRNAs. The cells were re-transfected with HA-tagged UBQLN2-WT, mCherry-Parkin, along with BFP-mito, and treated with 5 µg/mL A/O for 5 h. The cells were subjected to immunofluorescent assay using anti-HA antibody. Data from three independent experiments were represented as means ± SD., Sixty cells were counted for each sample. **, p<0.01, one-way ANOVA.


A HEK 293T cells expressing mCherry - Parkin were pre - treated with proteasome inhibitor ( 20 µM MG132 ) for 12 h , and treated with 1 µg / mL A / O for 2 h . Then the cells were fractionated and proteins were immunoblotted for relative antibodies . B The level of Mfn2 ( OMM protein ) , TOM20 , COXIV and HSP60 was quantified and normalized relative to GAPDH , and was shown in A . The data from three independent experiments were presented as mean ± SD . , ns , not significantly different ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , one - way ANOVA .

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A HEK 293T cells expressing mCherry-Parkin were pre-treated with proteasome inhibitor (20 µM MG132) for 12 h, and treated with 1 µg/mL A/O for 2 h. Then the cells were fractionated and proteins were immunoblotted for relative antibodies. B The level of Mfn2 (OMM protein), TOM20, COXIV and HSP60 was quantified and normalized relative to GAPDH, and was shown in A. The data from three independent experiments were presented as mean± SD., ns, not significantly different; **, p<0.01; ***, p<0.001, one-way ANOVA.


HEK 293T cells were transfected with indicated siRNAs for 48 h . C The cells were re - transfected with mCherry - Parkin for 12 h and were pre - treated with or without 20 µM MG132 for 12 h and treated with1 µg / mL A / O for 2 h . The cell lysates were subjected to immunoblot with indicated antibodies . The data from three independent experiments were presented as mean ± SD . , ns , not significantly different ; * , p < 0 . 05 ; * * , p < 0 . 01 , one - way ANOVA .

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HEK 293T cells were transfected with indicated siRNAs for 48 h. C The cells were re-transfected with mCherry-Parkin for 12 h and were pre-treated with or without 20 µM MG132 for 12 h and treated with1 µg/mL A/O for 2 h. The cell lysates were subjected to immunoblot with indicated antibodies. The data from three independent experiments were presented as mean± SD., ns, not significantly different; *, p<0.05; **, p<0.01, one-way ANOVA.


HEK 293T cells were transfected with indicated siRNAs for 48 h . The cells were transfected with HA - tagged UBQLN2 - WT or ∆ 379 - 462 , along with mCherry - Parkin for 12 h . Then the cells were treated with 1 µg / mL A / O for 2 h . The cell lysates were subjected to immunoblot using indicated antibodies . G Relative densities from F were shown in G . The data from three independent experiments were presented as means ± SD . , ns , not significantly different ; * , p < 0 . 05 ; * * , p < 0 . 01 , one - way ANOVA .

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HEK 293T cells were transfected with indicated siRNAs for 48 h. The cells were transfected with HA-tagged UBQLN2-WT or ∆379-462, along with mCherry-Parkin for 12 h. Then the cells were treated with 1 µg/mL A/O for 2 h. The cell lysates were subjected to immunoblot using indicated antibodies. G Relative densities from F were shown in G. The data from three independent experiments were presented as means ± SD., ns, not significantly different; *, p<0.05; **, p<0.01, one-way ANOVA.


HEK 293T cells were transfected with indicated siRNAs for 48 h . H The cells were re - transfected with HA - tagged UBQLN2 - WT , along with mCherry - Parkin for 12 h . Then the cells were pretreated with 20 µM MG132 for 12 h and treated with 5 µg / mL A / O for 4 h . Mitochondria isolated from cell lysates were treated with or without 10 µg / mL proteinase K ( Pro . K ) and then were subjected to immunoblot with indicated antibodies . Mito : the isolated mitochondria ; Mito ( Pro . K ) : mitochondria after Pro . K treatment . COXIV level in " Mito ( Pro . K ) " group was quantified and normalized relative to HSP60 in " Mito group " . The data from three independent experiments were presented as mean ± SD . , * * p < 0 . 01 , * * * , p < 0 . 001 , one - way ANOVA .

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HEK 293T cells were transfected with indicated siRNAs for 48 h. H The cells were re-transfected with HA-tagged UBQLN2-WT, along with mCherry-Parkin for 12 h. Then the cells were pretreated with 20 µM MG132 for 12 h and treated with 5 µg/mL A/O for 4 h. Mitochondria isolated from cell lysates were treated with or without 10 µg/mL proteinase K (Pro.K) and then were subjected to immunoblot with indicated antibodies. Mito: the isolated mitochondria; Mito (Pro.K): mitochondria after Pro.K treatment. COXIV level in "Mito (Pro.K)" group was quantified and normalized relative to HSP60 in "Mito group". The data from three independent experiments were presented as mean± SD., ** p<0.01, ***, p<0.001, one-way ANOVA.


HEK 293T cells were transfected with indicated siRNAs for 48 h . I The cells were re - transfected with HA , HA - UBQLN2 - WT or P497H , along with GFP - Parkin and FLAG - PHB2 for 20 h , and then were treated with or without 5 µg / mL A / O for 4 h . Images of situ PLA using rabbit anti - LC3B and mouse anti - FLAG antibodies . Blue : nuclei ( DAPI ) ; white dots : PLA positive puncta . Scale bar , 10 µm . J Over 500 cells were counted for each sample in I . The data from three independent experiments were presented as mean ± SD . , * , p < 0 . 05 ; * * p < 0 . 01 , * * * , p < 0 . 001 , one - way ANOVA .

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 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 O

HEK 293T cells were transfected with indicated siRNAs for 48 h. I The cells were re-transfected with HA, HA-UBQLN2-WT or P497H, along with GFP-Parkin and FLAG-PHB2 for 20 h, and then were treated with or without 5 µg/mL A/O for 4 h. Images of situ PLA using rabbit anti-LC3B and mouse anti-FLAG antibodies. Blue: nuclei (DAPI); white dots: PLA positive puncta. Scale bar, 10 µm. J Over 500 cells were counted for each sample in I. The data from three independent experiments were presented as mean± SD., *, p<0.05; ** p<0.01, ***, p<0.001, one-way ANOVA.


A Mouse cortical neurons ( DIV 7 ) were infected with lentivirus carrying mCherry - Parkin for 45 h , and then the neurons were treated with 5 µg / mL A / O for 5 h and subjected to immunofluorescent assay using anti - UBQLN2 ( green ) and anti - HSP60 ( gray ) antibodies . Scale bars , 10 µm . Lower panel were higher magnifications of the dashed box area . Scale bar , 2 . 5 µm .

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A Mouse cortical neurons (DIV 7) were infected with lentivirus carrying mCherry-Parkin for 45 h, and then the neurons were treated with 5 µg/mL A/O for 5 h and subjected to immunofluorescent assay using anti-UBQLN2 (green) and anti-HSP60 (gray) antibodies. Scale bars, 10 µm. Lower panel were higher magnifications of the dashed box area. Scale bar, 2.5 µm.


Mouse cortical neurons ( DIV 7 ) were transfected with indicated siRNAs for 24 h . B The neurons were infected with lentivirus carrying mCherry - Parkin for 24 h and then were treated with 1 µg / mL A / O for 48 h . The neurons were stained with DAPI , anti - UBQLN2 ( green ) and anti - HSP60 ( gray ) antibodies and visualized . Scale bars , 10 µm . Yellow arrows indicate uncleared mitochondria .

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Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. B The neurons were infected with lentivirus carrying mCherry-Parkin for 24 h and then were treated with 1 µg/mL A/O for 48 h. The neurons were stained with DAPI, anti-UBQLN2 (green) and anti-HSP60 (gray) antibodies and visualized. Scale bars, 10 µm. Yellow arrows indicate uncleared mitochondria.


Mouse cortical neurons ( DIV 7 ) were transfected with indicated siRNAs for 24 h . F The neurons were infected with lentivirus carrying mCherry - Parkin for 36 h and were treated with 100 ng / mL Antimycin A ( Antimycin A alone , AA ) and 1 µM DFP for 10 h . The neurons were subjected to immunofluorescent assay using anti - TOM20 ( magenta ) and anti - LAMP 2 ( gray ) antibodies . Scale bars , 10 µm . Right panel were higher magnifications of the dashed box area . Scale bars , 2 . 5 µm . Yellow arrows indicate the co - localization between mitochondria and lysosomes .

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Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. F The neurons were infected with lentivirus carrying mCherry-Parkin for 36 h and were treated with 100 ng/mL Antimycin A (Antimycin A alone, AA) and 1 µM DFP for 10 h. The neurons were subjected to immunofluorescent assay using anti-TOM20 (magenta) and anti-LAMP 2 (gray) antibodies. Scale bars, 10 µm. Right panel were higher magnifications of the dashed box area. Scale bars, 2.5 µm. Yellow arrows indicate the co-localization between mitochondria and lysosomes.


Mouse cortical neurons ( DIV 7 ) were transfected with indicated siRNAs for 24 h . H The neurons were infected with lentivirus carrying mCherry - Parkin for 24 h . Then , the neurons were treated with 100 ng / mL Antimycin A and 1 µM DFP for another 10 h and subjected to immunofluorescent assay using anti - MAP2 antibody . Scale bars , 10 µm . I , J The neurite length ( I ) and cell death ( J ) in more than 30 neurons were quantified from each group in H . The data from three independent experiments were presented as the means ± SD . , * , p < 0 . 05 ; * * * , p < 0 . 001 , one - way ANOVA .

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Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. H The neurons were infected with lentivirus carrying mCherry-Parkin for 24 h. Then, the neurons were treated with 100 ng/mL Antimycin A and 1 µM DFP for another 10 h and subjected to immunofluorescent assay using anti-MAP2 antibody. Scale bars, 10 µm. I, J The neurite length (I) and cell death (J) in more than 30 neurons were quantified from each group in H. The data from three independent experiments were presented as the means ± SD., *, p<0.05; ***, p<0.001, one-way ANOVA.


a , b , LDH release , IL - 1β secretion ( a ) and immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 and IL - 1α ( b )

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a, b, LDH release, IL-1β secretion (a) and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α(b)


and immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 and IL - 1α ( b ) from unprimed BMDMs infected for 16h with the indicated bacteria ( grown to stationary phase ) .

and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α (b) from unprimed BMDMs infected for 16h with the indicated bacteria (grown to stationary phase).


c , Time course measuring LDH release from unprimed or IFN - γ - primed BMDMs infected with S .

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c, Time course measuring LDH release from unprimed or IFN-γ-primed BMDMs infected with S. typhimurium


d , e , LDH release and IL - 1β secretion from primed BMDMs infected with SPI - 1 - expressing logarithmic phase S . typhimurium , treated with monosodium urate , alum and nigericin or transfected with poly ( dA : dT ) and LPS .

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d, e, LDH release and IL-1β secretion from primed BMDMs infected with SPI-1-expressing logarithmic phase S. typhimurium, treated with monosodium urate, alum and nigericin or transfected with poly(dA:dT) and LPS.


LDH release and IL - 1β secretion from unprimed wild - type and Gbp2 − / − BMDMs infected for 16h with the indicated bacteria ( grown to stationary phase ) . Graphs show mean and s . d . of quadruplicate wells and data are representative of two ( b ) and three ( a , c - f ) independent experiments . * Crossreactive band ; * * P < 0 . 01 ; NS , not significant ( two - tailed t - test ) .

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LDH release and IL-1β secretion from unprimed wild-type and Gbp2−/− BMDMs infected for 16h with the indicated bacteria (grown to stationary phase). Graphs show mean and s.d. of quadruplicate wells and data are representative of two (b) and three (a, c-f) independent experiments. *Crossreactive band; **P<0.01; NS, not significant (two-tailed t-test).


a , b , Quantification of live ( mCherry - positive ) and dead ( mCherry - negative ) S . typhimurium per cell by immunofluorescence ( a ) or as percent of total by flow -

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a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry


a , b , Quantification of live ( mCherry - positive ) and dead ( mCherry - negative ) S . typhimurium per cell by immunofluorescence ( a ) or as percent of total by flow - cytometry ( b ) in unprimed BMDMs at 16 h post - infection .

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a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry (b) in unprimed BMDMs at 16 h post-infection.


c , Immunostaining for GBP2 and quantification of live and dead Salmonella at 4 h post - infection . Arrowheads , bacteria shown in insets .

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c, Immunostaining for GBP2 and quantification of live and dead Salmonella at 4 h post-infection. Arrowheads, bacteria shown in insets.


d , Quantification of GBP - positive bacteria , LDH release and IL - 1β secretion at indicated time points from BMDMs infected with Salmonella , live or killed by different means .

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d, Quantification of GBP-positive bacteria, LDH release and IL-1β secretion at indicated time points from BMDMs infected with Salmonella, live or killed by different means.


e , Immunohistochemistry for GBP2 and Salmonella on spleen tissue from Salmonella ( mCherry - positive ) - infected mice ( representative of n = 3 per group ) . S . tm . , S . typhimurium .

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e, Immunohistochemistry for GBP2 and Salmonella on spleen tissue from Salmonella (mCherry-positive)-infected mice (representative of n = 3 per group). S. tm., S. typhimurium.


f , g , Quantification of GBP - positive Salmonella in anti - IFN - γ - treated or control animals ( f ) and live and dead bacteria among GBP2 - negative / - positive Salmonella ( g ) ( n = 3 per group ) . Scale bars , 10 µm ( c ) , 1 µm ( e ) . Graphs show mean and 5 - 95 percentile ( box plots ) or s . d . of technical triplicates , and data are representative of three independent experiments . * P 0 . 05 , * * P 0 . 01 ( two - tailed t - test ) .

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f, g, Quantification of GBP-positive Salmonella in anti-IFN-γ-treated or control animals (f) and live and dead bacteria among GBP2-negative/-positive Salmonella (g) (n = 3 per group). Scale bars, 10 µm (c), 1 µm (e). Graphs show mean and 5-95 percentile (box plots) or s.d. of technical triplicates, and data are representative of three independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).


a , b , Unprimed BMDMs infected with S . typhimurium for 4 h and immunostained for LC3 and GBP2 . Arrowheads , bacteria shown in insets . Scale bars , 10 & amp ; amp ; mgr ; m .

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a, b, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for LC3 and GBP2. Arrowheads, bacteria shown in insets. Scale bars, 10 &amp;mgr;m.


. c , Quantification of results from b .

. c, Quantification of results from b.


b . d - g , LDH release and immunoblots for caspase - 1 and caspase - 11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3 - methyladenine ( 3 - MA ) .

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b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).


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b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).


h , i , LDH release and IL - 1b secretion from BMDMs infected for 16 h or transfected with LPS . Graphs show mean and s . d . of quadruplicate wells and data are representative of two ( e , i ) and three ( a - d , f - h ) independent experiments . * P 0 . 05 , * * P 0 . 01 ; NS , not significant ( two - tailed t - test ) .

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h, i, LDH release and IL-1b secretion from BMDMs infected for 16 h or transfected with LPS. Graphs show mean and s.d. of quadruplicate wells and data are representative of two (e, i) and three (a-d, f-h) independent experiments. *P  0.05, **P  0.01; NS, not significant (two-tailed t-test).


a , Quantification of galectin - 8 - positive Salmonella in unprimed BMDMs at 4 h post - infection .

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a, Quantification of galectin-8-positive Salmonella in unprimed BMDMs at 4 h post-infection. b


c , Unprimed BMDMs infected with S . typhimurium for 4 h and immunostained for galectin - 8 , GBP2 and LC3 . Arrowheads , bacteria shown in insets . Scale bars , 10 µm . d , Quantification of galectin - 8 / LC3 - double - positive Salmonella at indicated time points post - infection .

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c, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for galectin-8, GBP2 and LC3. Arrowheads, bacteria shown in insets. Scale bars, 10 µm. d, Quantification of galectin-8/LC3-double-positive Salmonella at indicated time points post-infection.


e - h , Quantification of cytosolic and vacuolar bacteria by flow cytometry in BMDMs infected with mCherry - positive S . typhimurium ( e - g ) or S . flexneri ( h , wild - type or ΔT3SS ) for 4 h . Graphs show mean and s . d . or 5 - 95 percentile ( Box plots ) of technical triplicates . Data are representative of 2 ( g , h ) , 3 ( a - d ) and 4 ( e , f ) independent experiments . * P 0 . 05 , * * P 0 . 01 ( two - tailed 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 O O O O O O O O O O O O O O O O O O O O O O O O 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-h, Quantification of cytosolic and vacuolar bacteria by flow cytometry in BMDMs infected with mCherry-positive S. typhimurium (e-g) or S. flexneri (h, wild-type or ΔT3SS) for 4 h. Graphs show mean and s.d. or 5-95 percentile (Box plots) of technical triplicates. Data are representative of 2 (g, h), 3 (a-d) and 4 (e, f) independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).


Determination of serum testosterone ( B ) and 17β - estradiol levels , in post - pubescent male , castrated male ( CtM ) , and female Hanwoo ( n = 5 in each group ) .

O O O B-MEASURED_VAR 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

Determination of serum testosterone (B) and 17β-estradiol levels, in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group).


Determination of the body weight , in post - pubescent male , castrated male ( CtM ) , and female Hanwoo ( n = 5 in each group ) .

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

Determination of the body weight , in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group).


D . PCoA of the rectal bacterial 16S rRNA gene sequences based on the weighted UniFrac distance matrix .

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

D. PCoA of the rectal bacterial 16S rRNA gene sequences based on the weighted UniFrac distance matrix.


E . Discriminant taxa of the male and CtM rectal microbiota , as determined by the linear discriminant analysis effect size ( LEfSe ) . The different abundances are represented by LDA scores .

O O O O O O O O O O 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. Discriminant taxa of the male and CtM rectal microbiota, as determined by the linear discriminant analysis effect size (LEfSe). The different abundances are represented by LDA scores.


F . The serum metabolome profiles were analyzed using GC - TOF - MS and clustered by PCoA based on the Bray - Curtis dissimilarity matrix .

O O O 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. The serum metabolome profiles were analyzed using GC-TOF-MS and clustered by PCoA based on the Bray-Curtis dissimilarity matrix.


G . The relative abundances of serum metabolites displayed as bar graphs ( n = 5 in each group ) .

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

G. The relative abundances of serum metabolites displayed as bar graphs (n = 5 in each group).


H . The BCAA serum levels quantified enzymatically , as described in the materials and methods section .

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

H. The BCAA serum levels quantified enzymatically, as described in the materials and methods section.


A . Serum testosterone levels in the adult male and CtM Hanwoo ( n = 10 in each group ) . The scattered dots in the bar graph represent the individual replicates .

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

A. Serum testosterone levels in the adult male and CtM Hanwoo (n = 10 in each group). The scattered dots in the bar graph represent the individual replicates.


PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the rumen , ileum , and colon , shown for the different segments of the gastrointestinal tract ( B )

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

PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the rumen, ileum, and colon, shown for the different segments of the gastrointestinal tract (B)


C . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data or the rumen , ileum , and colon , combined data ( C ) . D . Microbial dissimilarity ( calculated based on the weighted UniFrac distance matrix , 100 values in each group ) between the male and CtM groups in the different intestinal compartments . The lines , boxes , and whiskers in the box plot diagrams represent the median , first and third quartiles , and min - to - max distribution of replicate values , respectively .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data or the rumen, ileum, and colon, combined data (C). D. Microbial dissimilarity (calculated based on the weighted UniFrac distance matrix, 100 values in each group) between the male and CtM groups in the different intestinal compartments. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively.


E . The relative abundances of abundant bacterial taxa ( > 0 . 5 % of the mean abundance ) .

E. The relative abundances of abundant bacterial taxa (> 0.5% of the mean abundance).


B . The intestinal branched - chain amino acid ( BCAA ) levels in the adult male and CtM Hanwoo ( n = 10 in each group ) . BCAA levels in the luminal contents of the ileum , cecum , and colon were quantified by an enzymatic method using a BCAA assay kit . The lines , boxes , and whiskers in the box plot diagrams represent the median , first and third quartiles , and min - to - max distribution of replicate values , respectively . The scattered dots in the box plot diagrams represent the individual replicates .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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. The intestinal branched-chain amino acid (BCAA) levels in the adult male and CtM Hanwoo (n = 10 in each group). BCAA levels in the luminal contents of the ileum, cecum, and colon were quantified by an enzymatic method using a BCAA assay kit. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively. The scattered dots in the box plot diagrams represent the individual replicates.


A . Representative images of dressed bodies of the adult male and CtM Hanwoo ( n = 10 in each group ) .

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

A. Representative images of dressed bodies of the adult male and CtM Hanwoo (n = 10 in each group).


B . Dressed body weight , thickness of the dorsal subcutaneous fat , total weight of the extramuscular fat ( posterior subcutaneous fat , mesenteric fat , and retroperitoneal fat ) , and serum levels of the branched - chain amino acids ( BCAAs ) .

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

B. Dressed body weight, thickness of the dorsal subcutaneous fat, total weight of the extramuscular fat (posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat), and serum levels of the branched-chain amino acids (BCAAs).


C . Representative images of fresh striploin muscle from the adult male and CtM carcasses ( n = 5 in each group ) .

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 images of fresh striploin muscle from the adult male and CtM carcasses (n = 5 in each group).


D . The intramuscular fat area in size - normalized muscle .

D. The intramuscular fat area in size-normalized muscle.


E . The intramuscular metabolome profiles analyzed using GC - TOF - MS and clustered by PCoA based on the Bray - Curtis dissimilarity

E. The intramuscular metabolome profiles analyzed using GC-TOF-MS and clustered by PCoA based on the Bray-Curtis dissimilarity matrix


F , G . The relative abundances of intramuscular metabolites ( F ) and β - hydroxybutyrate ( 3 - HB ) ( G ) in the male and CtM samples .

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

F, G. The relative abundances of intramuscular metabolites (F) and β-hydroxybutyrate (3-HB) (G) in the male and CtM samples.


H . The serum and mucle ketone body levels , quantified enzymatically , as described in the Materials and Methods section ( n = 10 in each group ) .

H. The serum and mucle ketone body levels, quantified enzymatically, as described in the Materials and Methods section (n = 10 in each group).


C . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon , shown according to the different diets and antibiotic treatment .

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 B-CONTROLLED_VAR O O

C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon, shown according to the different diets and antibiotic treatment.


D . The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot .

D. The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot.


E . The serum branched - chain amino acid ( BCAA ) levels quantified enzymatically , as described in the Materials and Methods section .

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

E. The serum branched-chain amino acid (BCAA) levels quantified enzymatically, as described in the Materials and Methods section.


F . The total weight of the extramuscular fat , including posterior subcutaneous fat , mesenteric fat , and retroperitoneal fat .

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

F. The total weight of the extramuscular fat, including posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat.


G , H . Representative images ( G ) and weight ( H ) of the hindlimb intermuscular fat . The images are from a longitudinal section of the hind leg . The fat weight data are presented as a percentage of the body weight .

O O O O O O O O O O O O O O O O O O O O O O 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, H. Representative images (G) and weight (H) of the hindlimb intermuscular fat. The images are from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.


I . Serum and hindlimb muscle ketone body levels quantified enzymatically .

I. Serum and hindlimb muscle ketone body levels quantified enzymatically.


B . The serum testosterone levels in the Sham - R and castrated male ( CtM ) - R mice .

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

B. The serum testosterone levels in the Sham-R and castrated male (CtM)-R mice.


C . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon .

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

C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon.


E . The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot .

E. The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot.


F . Weights of the posterior subcutaneous fat , epididymal fat , mesenteric fat , and retroperitoneal fat in the Sham - R and CtM - R mice .

O O O O O 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. Weights of the posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat in the Sham-R and CtM-R mice.


G , H . Representative images ( G ) and weight ( H ) of the hindlimb intermuscular fat in the Sham - R and CtM - R mice . Images were obtained from a longitudinal section of the hind leg . The fat weight data are presented as a percentage of the body weight .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H. Representative images (G) and weight (H) of the hindlimb intermuscular fat in the Sham-R and CtM-R mice. Images were obtained from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.


I . Serum and hindlimb muscle ketone body levels , quantified enzymatically .

I. Serum and hindlimb muscle ketone body levels, quantified enzymatically.


B . Body weight gain in response to the different levels of dietary BCAAs .

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

B. Body weight gain in response to the different levels of dietary BCAAs.


C . Representative images of gross anatomy .

C. Representative images of gross anatomy.


D . The total weight of the extramuscular fat ( posterior subcutaneous fat , epididymal fat , mesenteric fat , and retroperitoneal fat ) in the mice fed high - fat diet ( HFD ) with or without BCAAs .

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

D. The total weight of the extramuscular fat (posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat) in the mice fed high-fat diet (HFD) with or without BCAAs.


E . The serum BCAA levels in the mice fed HFD with or without BCAAs .

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

E. The serum BCAA levels in the mice fed HFD with or without BCAAs.


F . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown . The body weight gain data are presented as a percentage of the initial body weight . The fat weight data are presented as a percentage of the body weight .

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

F. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown. The body weight gain data are presented as a percentage of the initial body weight. The fat weight data are presented as a percentage of the body weight.


G . Serum and hindlimb muscle ketone body levels , quantified enzymatically .

G. Serum and hindlimb muscle ketone body levels, quantified enzymatically.


A - E Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread spermatocytes at ( A ) zygotene ( Zyg . ) , ( B ) pachytene ( Pac . ) , ( C ) diplotene ( Dip . ) , ( D ) late diplotene ( L . dip . ) , and ( E ) prometaphase I ( ProM I ) .

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

A-E Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) zygotene (Zyg.), (B) pachytene (Pac.), (C) diplotene (Dip.), (D) late diplotene (L. dip.), and (E) prometaphase I (ProM I).


F - H Double - immunolabeling of Sororin ( green ) and SYCP1 ( pseudocoloured in pink ) on spread spermatocytes at ( F ) zygotene , ( G ) pachytene , and ( H ) diplotene .

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

F-H Double-immunolabeling of Sororin (green) and SYCP1 (pseudocoloured in pink) on spread spermatocytes at (F) zygotene, (G) pachytene, and (H) diplotene.


I - K Double - immunolabeling of Sororin ( green ) and HORMAD1 ( pseudocoloured in blue ) on spread spermatocytes at ( I ) zygotene , ( J ) pachytene , and ( K ) diplotene . The sex bivalent ( XY ) and their pseudoautosomal regions ( arrows ) , as well as the round body ( arrowheads ) are indicated . Scale bar : 10 µm .

I-K Double-immunolabeling of Sororin (green) and HORMAD1 (pseudocoloured in blue) on spread spermatocytes at (I) zygotene, (J) pachytene, and (K) diplotene. The sex bivalent (XY) and their pseudoautosomal regions (arrows), as well as the round body (arrowheads) are indicated. Scale bar: 10 µm.


A , C Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread spermatocytes at ( A ) early zygotene ( E . zyg . ) and ( C ) late zygotene ( L . zyg . ) . B , D Double - immunolabeling of SMC3 ( pseudocoloured in cyan ) and SYCP3 ( red ) on spread spermatocytes at ( B ) early zygotene and ( D ) late zygotene .

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 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

A,C Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) early zygotene (E. zyg.) and (C) late zygotene (L. zyg.).B,D Double-immunolabeling of SMC3 (pseudocoloured in cyan) and SYCP3 (red) on spread spermatocytes at (B) early zygotene and (D) late zygotene.


E Double - immunolabeling of Sororin ( green ) and REC8 - myc ( pseudocoloured in purple ) on a spread mid zygotene spermatocyte . The sex univalents ( X , Y ) are indicated . Scale bar : 10 µm .

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

E Double-immunolabeling of Sororin (green) and REC8-myc (pseudocoloured in purple) on a spread mid zygotene spermatocyte. The sex univalents (X,Y) are indicated. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread pachytene - like ( Pac . - like ) and zygotene - like ( Zyg . - like ) spermatocytes of different genotypes . Unsynapsed axial elements of sex chromosomes ( X , Y ) are indicated . Arrowheads indicate nucleolar - associated round bodies . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread pachytene-like (Pac.-like) and zygotene-like (Zyg.-like) spermatocytes of different genotypes. Unsynapsed axial elements of sex chromosomes (X,Y) are indicated. Arrowheads indicate nucleolar-associated round bodies. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) , and counterstaining of the chromatin with DAPI ( blue ) on spread spermatocytes at ( A ) metaphase I ( M I ) , ( G ) anaphase I ( A I ) , ( H ) interkinesis ( Int . ) , ( I ) prophase II ( Pro . II ) , and ( J ) metaphase II ( M II ) . The sex bivalent ( XY ) is indicated . Arrows indicate the centriolar area at cell poles . Arrowheads indicate the Sororin labeling at the centromere of Y chromosome . B - F Selected metaphase I autosomal ( B and C ) and sex ( E and F ) bivalents . The sex chromosomes ( X , Y ) are indicated . K , L Enlarged metaphase II centromeres . Scale bar : 10 µm .

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 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

Double-immunolabeling of Sororin (green) and SYCP3 (red), and counterstaining of the chromatin with DAPI (blue) on spread spermatocytes at (A) metaphase I (M I), (G) anaphase I (A I), (H) interkinesis (Int.), (I) prophase II (Pro. II), and (J) metaphase II (M II). The sex bivalent (XY) is indicated. Arrows indicate the centriolar area at cell poles. Arrowheads indicate the Sororin labeling at the centromere of Y chromosome.B-F Selected metaphase I autosomal (B and C) and sex (E and F) bivalents. The sex chromosomes (X,Y) are indicated.K,L Enlarged metaphase II centromeres. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and kinetochores ( ACA serum , red ) , and counterstaining of the chromatin with DAPI ( blue ) on spread spermatocytes at ( A ) metaphase I ( M I ) , ( B ) metaphase II ( M II ) , and ( C ) anaphase II ( A II ) . Arrowheads indicate the cell poles . The centromere of the Y chromosome and chromatids ( Y ) is indicated . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and kinetochores (ACA serum, red), and counterstaining of the chromatin with DAPI (blue) on spread spermatocytes at (A) metaphase I (M I), (B) metaphase II (M II), and (C) anaphase II (A II). Arrowheads indicate the cell poles. The centromere of the Y chromosome and chromatids (Y) is indicated. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) , and counterstaining of the chromatin with DAPI ( blue ) on spread Sgo2 - / - spermatocytes at ( A ) pachytene ( Pac . ) , ( B ) diplotene ( Dip . ) , ( C ) metaphase I ( M I ) , and ( D ) metaphase II - like ( M II - like ) . The sex bivalent ( XY ) is indicated . Arrowheads indicate round bodies . Arrows in ( A ) indicate the pseudoautomal region in the sex bivalent , and the centriolar area at cell poles in ( C , D ) . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red), and counterstaining of the chromatin with DAPI (blue) on spread Sgo2-/- spermatocytes at (A) pachytene (Pac.), (B) diplotene (Dip.), (C) metaphase I (M I), and (D) metaphase II-like (M II-like). The sex bivalent (XY) is indicated. Arrowheads indicate round bodies. Arrows in (A) indicate the pseudoautomal region in the sex bivalent, and the centriolar area at cell poles in (C,D). Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread cultured wild - type control ( WT Control ) , and okadaic acid treated wild - type ( WT + OA ) and Smc1β - / - ( Smc1β - / - + OA ) spermatocytes . A - B WT control cultured spermatocytes in ( A ) late zygotene ( L . zyg . ) and ( B ) metaphase I ( M I ) . Arrows indicate the cell poles . C - D WT cultured spermatocytes treated with okadaic acid in ( C ) late zygotene ( L . zyg . ) and ( D ) metaphase I - like ( M I - like ) . E - F Smc1β - / - cultured spermatocytes treated with okadaic acid in ( E ) pachytene - like ( Pac . - like ) and ( F ) metaphase I - like ( M I - like ) . Arrowheads indicate nucleolar - associated round bodies . The unsynapsed axial elements of the sex chromosomes ( X , Y ) in prophase I , as well as the sex bivalent ( XY ) in metaphases I are indicated . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread cultured wild-type control (WT Control), and okadaic acid treated wild-type (WT + OA) and Smc1β-/- (Smc1β-/- + OA) spermatocytes.A-B WT control cultured spermatocytes in (A) late zygotene (L. zyg.) and (B) metaphase I (M I). Arrows indicate the cell poles.C-D WT cultured spermatocytes treated with okadaic acid in (C) late zygotene (L. zyg.) and (D) metaphase I-like (M I-like).E-F Smc1β-/- cultured spermatocytes treated with okadaic acid in (E) pachytene-like (Pac.-like) and (F) metaphase I-like (M I-like). Arrowheads indicate nucleolar-associated round bodies. The unsynapsed axial elements of the sex chromosomes (X,Y) in prophase I, as well as the sex bivalent (XY) in metaphases I are indicated. Scale bar: 10 µm.


( A ) Western blot analysis of whole head homogenates from transgenic flies expressing a Myc - tagged PINK1 genomic construct and a UAS - RNAi construct driven by elav - GAL4 . The RNAi constructs used were as follows : the non - specific control mCherry - RNAi ( Control - R ) , Lon - RNAi1 ( Lon - R1 ) , and Lon - RNAi2 ( Lon - R2 ) . An anti - Myc antibody was used to detect PINK1 and an anti - Actin antibody was used as a protein loading control . ( B ) Densitometry of the PINK1 - Myc bands from the indicated genotypes was performed using Fiji software . The PINK1 - Myc band intensities were then normalized to their respective Actin loading controls , and these ratios were in turn normalized to the Control - R PINK1 / Actin ratio .

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

(A) Western blot analysis of whole head homogenates from transgenic flies expressing a Myc-tagged PINK1 genomic construct and a UAS-RNAi construct driven by elav-GAL4. The RNAi constructs used were as follows: the non-specific control mCherry-RNAi (Control-R), Lon-RNAi1 (Lon-R1), and Lon-RNAi2 (Lon-R2). An anti-Myc antibody was used to detect PINK1 and an anti-Actin antibody was used as a protein loading control. (B) Densitometry of the PINK1-Myc bands from the indicated genotypes was performed using Fiji software. The PINK1-Myc band intensities were then normalized to their respective Actin loading controls, and these ratios were in turn normalized to the Control-R PINK1/Actin ratio.


( C ) Western blot analysis of Lon protease ( Lon ) , Mitochondrial transcription factor A ( TFAM ) , Heat shock protein 60 ( Hsp60 ) and Actin in whole head homogenate from control and Lon deficient animals . ( D ) Quantification of the Lon band intensities from the indicated genotypes performed as described in B . ( E ) Quantification of the TFAM bands from the indicated genotypes performed as described in B . ( F ) Quantification of the Hsp60 bands from the indicated genotypes performed as described in B .

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

(C) Western blot analysis of Lon protease (Lon), Mitochondrial transcription factor A (TFAM), Heat shock protein 60 (Hsp60) and Actin in whole head homogenate from control and Lon deficient animals. (D) Quantification of the Lon band intensities from the indicated genotypes performed as described in B. (E) Quantification of the TFAM bands from the indicated genotypes performed as described in B. (F) Quantification of the Hsp60 bands from the indicated genotypes performed as described in B.