Figure 1 ( A ) RT - qPCR analysis of Myh7b / miR - 499 and Myh7 / miR - 208b levels during differentiation of myoblasts into mature myotubes ( n = 3 independent experiments ) . * p = 0 . 0005 ( Myh7b ) , * p < 0 . 0001 ( miR - 499 ) , * p = 0 . 0002 ( Myh7 ) , * p < 0 . 0001 ( miR - 208b ) . ( B ) Oxygen consumption rates ( OCR ) in primary myoblasts and differentiated myotubes . Basal OCR was first measured , followed by administration of 10 mM sodium pyruvate , and 2 μM oligomycin ( to inhibit ATP synthase ) , uncoupler FCCP ( 2 μM ) , or rotenone / antimycin ( Rot / A ) ( 1 μM ) as indicted . n = 3 separate experiments done with 10 biological replicates . * p < 0 . 0001 ( FCCP ) . ( C ) Mean expression levels ( RT - qPCR ) in white vastus ( WV ) and soleus ( Sol ) muscle from 8 - week - old male wild type mice ( n = 5 mice per group ) . * p < 0 . 0001 ( Myh7b ) , * p < 0 . 0001 ( miR - 499 ) , * p < 0 . 0001 ( Myh7 ) , * p < 0 . 0001 ( miR - 208b ) . ( D ) Mitochondrial respiration rates were determined from mitochondria isolated from indicated muscle using pyruvate as substrate . ADP - dependent respiration , oligomycin - induced ( oligo ) , uncoupler FCCP , and antimycin A ( AA ) are shown . n = 3 separate experiments done with 7 - 8 biological replicates . * p < 0 . 05 ( ADP ) . ( E ) ( Left ) Schematic depicts the increments of speed over time . ( Right ) Respiratory exchange ratio ( RER ) during a graded exercise regimen as described in Materials and Methods ( n = 5 mice per group ) . Notably , MCK - miR - 499 mice were able to exercise at a higher speed before exhaustion . * p < 0 . 05 . ( F ) Bars represent mean blood lactate levels for 3 - month and 15 - month old male MCK - miR - 499 and NTG mice following a 25 min run on a motorized treadmill . For 3 - month blood lactate , NTG , n = 9 ; MCK - miR - 499 , n = 10 . For 15 - month blood lactate , NTG , n = 7 ; MCK - miR - 499 , n = 5 . * p = 0 . 039 ( 3 - month ) , * p = 0 . 018 ( 15 - month ) . ( G ) Mitochondrial respiration rates were determined from the plantaris muscle of the indicated genotypes using pyruvate / malate as substrate . Pyruvate / malate ( Py / M ) - stimulated , ADP - dependent respiration , oligomycin - induced ( oligo ) and the respiratory control ratio ( RC ) are shown . NTG , n = 6 ; MCK - miR - 499 , n = 4 . * p = 0 . 015 ( Py / M ) , * p = 0 . 017 ( ADP ) , * p = 0 . 001 ( Oligo ) .

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

Figure 1(A) RT-qPCR analysis of Myh7b/miR-499 and Myh7/miR-208b levels during differentiation of myoblasts into mature myotubes (n = 3 independent experiments). *p = 0.0005 (Myh7b), *p < 0.0001 (miR-499), *p = 0.0002 (Myh7), *p < 0.0001 (miR-208b).(B) Oxygen consumption rates (OCR) in primary myoblasts and differentiated myotubes. Basal OCR was first measured, followed by administration of 10 mM sodium pyruvate, and 2 μM oligomycin (to inhibit ATP synthase), uncoupler FCCP (2 μM), or rotenone/antimycin (Rot/A) (1 μM) as indicted. n = 3 separate experiments done with 10 biological replicates. *p < 0.0001 (FCCP).(C) Mean expression levels (RT-qPCR) in white vastus (WV) and soleus (Sol) muscle from 8-week-old male wild type mice (n = 5 mice per group). *p < 0.0001 (Myh7b), *p < 0.0001 (miR-499), *p < 0.0001 (Myh7), *p < 0.0001 (miR-208b).(D) Mitochondrial respiration rates were determined from mitochondria isolated from indicated muscle using pyruvate as substrate. ADP-dependent respiration, oligomycin-induced (oligo), uncoupler FCCP, and antimycin A (AA) are shown. n = 3 separate experiments done with 7-8 biological replicates. *p < 0.05 (ADP).(E) (Left) Schematic depicts the increments of speed over time. (Right) Respiratory exchange ratio (RER) during a graded exercise regimen as described in Materials and Methods (n = 5 mice per group). Notably, MCK-miR-499 mice were able to exercise at a higher speed before exhaustion. *p < 0.05.(F) Bars represent mean blood lactate levels for 3-month and 15-month old male MCK-miR-499 and NTG mice following a 25 min run on a motorized treadmill. For 3-month blood lactate, NTG, n = 9; MCK-miR-499, n = 10. For 15-month blood lactate, NTG, n = 7; MCK-miR-499, n = 5. *p = 0.039 (3-month), *p = 0.018 (15-month).(G) Mitochondrial respiration rates were determined from the plantaris muscle of the indicated genotypes using pyruvate/malate as substrate. Pyruvate/malate (Py/M)-stimulated, ADP-dependent respiration, oligomycin-induced (oligo) and the respiratory control ratio (RC) are shown. NTG, n = 6; MCK-miR-499, n = 4. *p = 0.015 (Py/M), *p = 0.017 (ADP), *p = 0.001 (Oligo).


Figure 2 ( B ) Expression of the nuclear - encoded and mitochondrial - encoded genes ( RT - qPCR ) in the gastrocnemius muscle from the indicated genotypes ( n = 5 mice per group ) . * p < 0 . 05 . ( C ) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using myoglobin , cytochrome c , and β - actin ( control ) antibodies ( n = 8 mice per group ) . ( D ) Expression of genes ( RT - qPCR ) involved in muscle glucose and fatty acids metabolism in the gastrocnemius muscle from the indicated genotypes ( n = 5 mice per group ) . * p < 0 . 05 . ( E ) LDH isoenzymes were separated by polyacrylamide gel electrophoresis using whole cell extracts from NTG heart ( Ht , control ) and gastrocnemius muscle from the indicated mice . A representative gel showing 3 - 4 independent mice per group is shown . ( F ) Quantification of LDH isoenzyme activity gel electrophoresis in E . Values represent the mean % ( + / - SEM ) total LDH activity . * p < 0 . 05 . ( G ) Cross - section of the gastrocnemius muscle from a 3 - month - old male NTG and MCK - miR - 499 stained for myosin I ATPase activity ( Top ) , SDH ( Middle ) , and α - GPDH ( Bottom ) . Representative images were shown . Scale bar : 500 μm .

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

Figure 2(B) Expression of the nuclear-encoded and mitochondrial-encoded genes (RT-qPCR) in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.05.(C) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using myoglobin, cytochrome c, and β-actin (control) antibodies (n = 8 mice per group).(D) Expression of genes (RT-qPCR) involved in muscle glucose and fatty acids metabolism in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.05.(E) LDH isoenzymes were separated by polyacrylamide gel electrophoresis using whole cell extracts from NTG heart (Ht, control) and gastrocnemius muscle from the indicated mice. A representative gel showing 3-4 independent mice per group is shown.(F) Quantification of LDH isoenzyme activity gel electrophoresis in E. Values represent the mean % (+/- SEM) total LDH activity. *p < 0.05.(G) Cross-section of the gastrocnemius muscle from a 3-month-old male NTG and MCK-miR-499 stained for myosin I ATPase activity (Top), SDH (Middle), and α-GPDH (Bottom). Representative images were shown. Scale bar: 500 μm.


Figure 3 ( A ) ( Left ) Representative western blot analysis of PGC - 1α protein expression in the gastrocnemius muscle from the indicated genotypes , heart ( Ht ) lysate from WT mice and PGC - 1α mKO ( MCK - Cre ) mice as positive and negative controls , respectively . ( Right ) Quantification of the PGC - 1α / Tubulin signal ratios normalized ( = 1 . 0 ) to the NTG control ( n = 5 mice per group ) . * p = 0 . 00017 . ( B ) Expression of the Ldhb , Ldha , Mb ( myoglobin ) , and Cox5a genes ( RT - qPCR ) in the gastrocnemius muscle from the indicated genotypes ( n = 5 mice per group ) . * p < 0 . 001 ( vs . NTG ) , ‡ p < 0 . 01 ( vs . 499Tg ) . ( C ) ( Left ) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using cytochrome c , myoglobin , and α - tubulin ( control ) antibodies . ( Right ) Quantification of the Cyt c / Tubulin signal ratios normalized ( = 1 . 0 ) to the NTG control . NTG , n = 5 ; 499Tg , n = 6 ; PGC - 1α mKO , n = 5 ; 499Tg / PGC - 1α mKO , n = 5 . * p < 0 . 01 ( vs . NTG ) , ‡ p = 0 . 00018 ( vs . 499Tg ) . ( D ) Cross - section of gastrocnemius muscle from the indicated mice stained for SDH activity ( Top ) as well as MHC1 immunofluorescence ( IF ) ( Bottom ) , Representative images were shown . Scale bar : 500 μm . ( E ) Respiratory exchange ratio ( RER ) during a graded exercise regimen as described in Materials and Methods . Notably , no significant increase in exhaust speed was observed in 499Tg / PGC - 1α mKO mice compared to PGC - 1α mKO mice . PGC - 1α mKO , n = 5 ; 499Tg / PGC - 1α mKO , n = 8 . ( F ) The bars represent the mean blood lactate levels from the indicated mice following a 25 min run on a motorized treadmill . NTG , n = 9 ; 499Tg , n = 7 ; PGC - 1α mKO , n = 6 ; 499Tg / PGC - 1α mKO , n = 9 . * p = 0 . 0009 ( NTG vs . 499Tg ) , p = 0 . 316 ( NS , not significant ) .

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

Figure 3(A) (Left) Representative western blot analysis of PGC-1α protein expression in the gastrocnemius muscle from the indicated genotypes, heart (Ht) lysate from WT mice and PGC-1α mKO (MCK-Cre) mice as positive and negative controls, respectively. (Right) Quantification of the PGC-1α/Tubulin signal ratios normalized (=1.0) to the NTG control (n = 5 mice per group). *p = 0.00017.(B) Expression of the Ldhb, Ldha, Mb (myoglobin), and Cox5a genes (RT-qPCR) in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.001 (vs. NTG), ‡p < 0.01 (vs. 499Tg).(C) (Left) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using cytochrome c, myoglobin, and α-tubulin (control) antibodies. (Right) Quantification of the Cyt c/Tubulin signal ratios normalized (=1.0) to the NTG control. NTG, n = 5; 499Tg, n = 6; PGC-1α mKO, n = 5; 499Tg/PGC-1α mKO, n = 5. *p < 0.01 (vs. NTG), ‡p = 0.00018 (vs. 499Tg).(D) Cross-section of gastrocnemius muscle from the indicated mice stained for SDH activity (Top) as well as MHC1 immunofluorescence (IF) (Bottom), Representative images were shown. Scale bar: 500 μm.(E) Respiratory exchange ratio (RER) during a graded exercise regimen as described in Materials and Methods. Notably, no significant increase in exhaust speed was observed in 499Tg/PGC-1α mKO mice compared to PGC-1α mKO mice. PGC-1α mKO, n = 5; 499Tg/PGC-1α mKO, n = 8.(F) The bars represent the mean blood lactate levels from the indicated mice following a 25 min run on a motorized treadmill. NTG, n = 9; 499Tg, n = 7; PGC-1α mKO, n = 6; 499Tg/PGC-1α mKO, n = 9. *p = 0.0009 (NTG vs. 499Tg), p = 0.316 (NS, not significant).


Figure 4 ( B ) Luciferase reporters containing the wild - type Fnip1 3 ' UTR or Fnip1 3 ' UTR mutated in the predicted binding site of miR - 499 were used in cotransfection studies in HEK293T cells in the presence or absence of plasmid expressing miR - 499 ( n = 3 independent experiments ) . * p < 0 . 0001 ( Fnip1 3 ' UTR ) ; * p = 0 . 023 ( Mutated ) . ( C ) RT - qPCR analysis of Fnip1 and Fnip2mRNA levels in the gastrocnemius muscle of the indicated genotypes ( n = 5 mice per group ) . ( D ) ( Top ) Fnip1 protein expression in the gastrocnemius muscle from the indicated mice . ( Bottom ) Quantification of the Fnip1 / Tubulin signal ratio ( n = 4 mice per group ) . * p = 0 . 014 .

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

Figure 4(B) Luciferase reporters containing the wild-type Fnip1 3'UTR or Fnip1 3'UTR mutated in the predicted binding site of miR-499 were used in cotransfection studies in HEK293T cells in the presence or absence of plasmid expressing miR-499 (n = 3 independent experiments). *p < 0.0001 (Fnip1 3'UTR); *p = 0.023 (Mutated).(C) RT-qPCR analysis of Fnip1 and Fnip2mRNA levels in the gastrocnemius muscle of the indicated genotypes (n = 5 mice per group).(D) (Top) Fnip1 protein expression in the gastrocnemius muscle from the indicated mice. (Bottom) Quantification of the Fnip1/Tubulin signal ratio (n = 4 mice per group). *p = 0.014.


Figure 5 ( A ) ( Left ) Representative western blot analysis performed on extracts of myotubes subjected to Fnip1 siRNA or control ( Con ) siRNA using phospho - AMPKα ( Thr172 ) and AMPKα antibodies . ( Right ) Quantification of the p - AMPKα / AMPKα signal ratios ( n = 3 independent experiments ) . * p = 0 . 037 . ( B , C ) Results of RT - qPCR analysis for WT primary mouse myotubes after transfection with Fnip1 siRNAs or scrambled Con siRNAs as indicated . For C , 48 h post - siRNA transfection , myotubes were treated for 24 h with DMSO or 10 μm compound C before harvest ( n = 3 independent experiments ) . * p < 0 . 0001 ( Fnip1 in B ) , * p = 0 . 0001 ( PGC - 1α in B ) ; * p < 0 . 0001 ( vs . Con siRNA in C ) , ‡ p < 0 . 0001 ( vs . Fnip1 siRNA in C ) . ( D ) ( Top ) Representative western blot analysis performed on extracts of the gastrocnemius muscle isolated from NTG or MCK - miR - 499 mice using phospho - AMPKα ( Thr172 ) , AMPKα , phospho - ACC ( Ser79 ) , or total ACC antibodies . ( Bottom ) Quantification of the p - AMPKα / AMPKα and p - ACC / ACC signal ratios normalized ( = 1 . 0 ) to the NTG control ( n = 8 mice per group ) . * p < 0 . 01 . ( E ) Oxygen consumption rates ( OCR ) in primary mouse myotubes transfected with Fnip1 siRNA or Con siRNA . n = 7 separate experiments done with 5 biological replicates . * p < 0 . 05 ( Pyruvate ) , * p < 0 . 01 ( FCCP ) . ( F ) Results of RT - qPCR analysis for WT primary mouse myotubes subjected to inhibition of both miR - 499 and miR - 208b ( Anti - miRs ) ( n = 4 independent experiments ) . * p = 0 . 037 ( Ppargc1a ) , * p = 0 . 0019 ( Fnip1 ) . ( G ) Oxygen consumption rates ( OCR ) in primary mouse myotubes transfected with miR - 499 / miR - 208b inhibitors alone and together with the presence of Fnip1 siRNA . n = 4 separate experiments done with 5 biological replicates . * p < 0 . 01 ( Anti - miRs vs . Control ) .

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

Figure 5(A) (Left) Representative western blot analysis performed on extracts of myotubes subjected to Fnip1 siRNA or control (Con) siRNA using phospho-AMPKα (Thr172) and AMPKα antibodies. (Right) Quantification of the p-AMPKα/AMPKα signal ratios (n = 3 independent experiments). *p = 0.037.(B, C) Results of RT-qPCR analysis for WT primary mouse myotubes after transfection with Fnip1 siRNAs or scrambled Con siRNAs as indicated. For C, 48 h post-siRNA transfection, myotubes were treated for 24 h with DMSO or 10 μm compound C before harvest (n = 3 independent experiments). *p < 0.0001 (Fnip1 in B), *p = 0.0001 (PGC-1α in B); *p < 0.0001 (vs. Con siRNA in C), ‡p < 0.0001 (vs. Fnip1 siRNA in C ).(D) (Top) Representative western blot analysis performed on extracts of the gastrocnemius muscle isolated from NTG or MCK-miR-499 mice using phospho-AMPKα (Thr172), AMPKα, phospho-ACC (Ser79), or total ACC antibodies. (Bottom) Quantification of the p-AMPKα/AMPKα and p-ACC/ACC signal ratios normalized (=1.0) to the NTG control (n = 8 mice per group). *p < 0.01.(E) Oxygen consumption rates (OCR) in primary mouse myotubes transfected with Fnip1 siRNA or Con siRNA. n = 7 separate experiments done with 5 biological replicates. *p < 0.05 (Pyruvate), *p < 0.01 (FCCP).(F) Results of RT-qPCR analysis for WT primary mouse myotubes subjected to inhibition of both miR-499 and miR-208b (Anti-miRs) (n = 4 independent experiments). *p = 0.037 (Ppargc1a), *p = 0.0019 (Fnip1).(G) Oxygen consumption rates (OCR) in primary mouse myotubes transfected with miR-499/miR-208b inhibitors alone and together with the presence of Fnip1 siRNA. n = 4 separate experiments done with 5 biological replicates. *p < 0.01 (Anti-miRs vs. Control).


Figure 6 ( A ) Mean expression levels ( RT - qPCR ) in gastrocnemius muscle of 8 - week - old male WT and mdx mice ( n = 5 mice per group ) . * p = 0 . 009 ( Myh7b ) , * p = 0 . 0007 ( miR - 499 ) , * p = 0 . 0143 ( Myh7 ) , * p < 0 . 0011 ( miR - 208b ) . ( B ) Mitochondrial respiration rates were determined from mitochondria isolated from the hindlimb muscle of the indicated mice using pyruvate as substrate . n = 3 separate experiments done with 7 - 8 biological replicates . * p < 0 . 01 ( ADP ) . ( C ) ( Left ) Representative western blot analysis of PGC - 1α ( Top ) and Fnip1 ( Bottom ) protein expression in the gastrocnemius muscle from the indicated genotypes with α - tubulin as the loading control . ( Right ) Quantification of the PGC - 1α / Tubulin and Fnip1 / Tubulin signal ratios normalized ( = 1 . 0 ) to the WT control . WT , n = 8 ; mdx , n = 5 ; mdx / 499Tg , n = 7 . * p < 0 . 0001 ( vs . WT ) , ‡ p < 0 . 0001 ( vs . mdx ) . ( D ) Representative western blot analysis of myoglobin protein expression in the gastrocnemius muscle from the indicated genotypes with α - tubulin as the loading control ( n = 5 mice per group ) . ( E ) ( Top ) Representative MHC1 immunofluorescence ( IF ) in the soleus of the indicated genotypes . Scale bar : 500 μm . ( Bottom ) Representative H & amp ; amp ; E staining of the gastrocnemius muscle of the indicated genotypes . Scale bar : 100 μm . ( F ) 5 - week - old male WT , mdx , and mdx / MCK - miR - 499 mice were euthanized and serum creatine kinase activity was determined . WT , n = 10 ; mdx , n = 10 ; mdx / 499Tg , n = 14 . * p < 0 . 0001 ( vs . WT ) , ‡ p = 0 . 0003 ( vs . mdx ) . ( G ) The bars represent the mean running time and distance ( ± SEM ) for 8 - week - old male mice on a motorized treadmill . WT , n = 10 ; mdx , n = 13 ; mdx / 499Tg , n = 8 . * p = 0 . 01406 ( Running time , vs . WT ) , ‡ p = 0 . 005756 ( Running time , vs . mdx ) . * p = 0 . 004558 ( Running distance , vs . WT ) , ‡ p = 0 . 001668 ( Running distance , vs . mdx ) . ( H ) Respiratory exchange ratio ( RER ) during a graded exercise regimen on a motorized treadmill . mdx , n = 8 ; mdx / 499Tg , n = 6 . ‡ p < 0 . 05 .

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

Figure 6(A) Mean expression levels (RT-qPCR) in gastrocnemius muscle of 8-week-old male WT and mdx mice (n = 5 mice per group). *p = 0.009 (Myh7b), *p = 0.0007 (miR-499), *p = 0.0143 (Myh7), *p < 0.0011 (miR-208b).(B) Mitochondrial respiration rates were determined from mitochondria isolated from the hindlimb muscle of the indicated mice using pyruvate as substrate. n = 3 separate experiments done with 7-8 biological replicates. *p < 0.01 (ADP).(C) (Left) Representative western blot analysis of PGC-1α (Top) and Fnip1 (Bottom) protein expression in the gastrocnemius muscle from the indicated genotypes with α-tubulin as the loading control. (Right) Quantification of the PGC-1α/Tubulin and Fnip1/Tubulin signal ratios normalized (=1.0) to the WT control. WT, n = 8; mdx, n = 5; mdx/499Tg, n = 7. *p < 0.0001 (vs. WT), ‡p < 0.0001 (vs. mdx).(D) Representative western blot analysis of myoglobin protein expression in the gastrocnemius muscle from the indicated genotypes with α-tubulin as the loading control (n = 5 mice per group).(E) (Top) Representative MHC1 immunofluorescence (IF) in the soleus of the indicated genotypes. Scale bar: 500 μm. (Bottom) Representative H&amp;E staining of the gastrocnemius muscle of the indicated genotypes. Scale bar: 100 μm.(F) 5-week-old male WT, mdx, and mdx/MCK-miR-499 mice were euthanized and serum creatine kinase activity was determined. WT, n = 10; mdx, n = 10; mdx/499Tg, n = 14. *p < 0.0001 (vs. WT), ‡p = 0.0003 (vs. mdx).(G) The bars represent the mean running time and distance (± SEM) for 8-week-old male mice on a motorized treadmill. WT, n = 10; mdx, n = 13; mdx/499Tg, n = 8. *p = 0.01406 (Running time, vs. WT), ‡p = 0.005756 (Running time, vs. mdx). *p = 0.004558 (Running distance, vs. WT), ‡p = 0.001668 (Running distance, vs. mdx).(H) Respiratory exchange ratio (RER) during a graded exercise regimen on a motorized treadmill. mdx, n = 8; mdx/499Tg, n = 6. ‡p < 0.05.


Figure 1J Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals from the indicated time point ( in hours ) after the L4 stage . Animals were exposed to the indicated knockdown RNAi bacterial strains or a strain that only contained the empty RNAi vector since hatching . * P < 0 . 001 using ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point , with N = 20 animals per genotype and time point . Error bars indicate SEM . L Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals from the indicated time point ( in hours ) and the indicated genotypes after the L4 stage , as per ( J ) . * P < 0 . 001 using ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point , with N = 20 animals per genotype and time point . Error bars indicate SEM .

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

Figure 1J Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) after the L4 stage. Animals were exposed to the indicated knockdown RNAi bacterial strains or a strain that only contained the empty RNAi vector since hatching. *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Error bars indicate SEM.L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) and the indicated genotypes after the L4 stage, as per (J). *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Error bars indicate SEM.


Figure 2K , L Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4 + 48 hours . * P < 0 . 001 using ANOVA with Dunnett ' s posthoc comparison to the wild - type control equivalent time point , with N = 20 animals per genotype and time point . Data has been normalized to wild type . Error bars indicate SEM .

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

Figure 2K,L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4+48 hours. *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Data has been normalized to wild type. Error bars indicate SEM.


Figure 3B A separate SDS - PAGE and Western blot of the same lysates from ( A ) , probed with antibodies recognizing ubiquitin or actin as a loading control . The position of molecular weight markers is shown on the left of each blot . The position of UbG76V - GFP protein , as well as UbG76V - GFP with the indicated number of additional ubiquitin moieties , based on molecular weight , is indicated to the right of each blot . Endogenous poly - ubiquitinated proteins are also detected in the 80 - 200 kDa range , as indicated by the bracket . Twenty animals were loaded per lane for each indicated genotype .

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

Figure 3B A separate SDS-PAGE and Western blot of the same lysates from (A), probed with antibodies recognizing ubiquitin or actin as a loading control. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated to the right of each blot. Endogenous poly-ubiquitinated proteins are also detected in the 80-200 kDa range, as indicated by the bracket. Twenty animals were loaded per lane for each indicated genotype.


Figure 4 B Heat map of log fold change ( logFC , color scale at graph bottom ) in the expression of C . elegans CYP genes between the indicated mutant and wild type . Genes whose expression is lower in a mutant relative to wild type are colored blue , whereas genes whose expression is higher in a mutant relative to wild type are colored yellow . Rows are labeled for the different CYP families and subfamilies . Specific genes ( rows ) are clustered according to sequence alignment ( CLUSTAL W ) to maintain the relationship of their molecular evolution D Abundance of the indicated free ω6 PUFA - derived eicosanoid , normalized to total protein , for each indicated genotype . Red , gray , and blue stacked bars indicate hydroxides , epoxide , and epoxide diol forms of eicosanoids , respectively . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA with Dunnett ' s posthoc comparison to wild type . N = 3 trials . Error bars indicate SEM . E Abundance of the indicated free ω3 PUFA - derived eicosanoid , as in ( D ) , normalized to total protein , for each indicated genotype . Red , gray , and blue stacked bars indicate hydroxides , epoxide , and epoxide diol forms of eicosanoids , respectively . * * * * P < 0 . 0001 , ANOVA with Dunnett ' s posthoc comparison to wild type . N = 3 trials . Error bars indicate SEM

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

Figure 4 B Heat map of log fold change (logFC, color scale at graph bottom) in the expression of C. elegans CYP genes between the indicated mutant and wild type. Genes whose expression is lower in a mutant relative to wild type are colored blue, whereas genes whose expression is higher in a mutant relative to wild type are colored yellow. Rows are labeled for the different CYP families and subfamilies. Specific genes (rows) are clustered according to sequence alignment (CLUSTAL W) to maintain the relationship of their molecular evolution  D Abundance of the indicated free ω6 PUFA-derived eicosanoid, normalized to total protein, for each indicated genotype. Red, gray, and blue stacked bars indicate hydroxides, epoxide, and epoxide diol forms of eicosanoids, respectively. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM. E Abundance of the indicated free ω3 PUFA-derived eicosanoid, as in (D), normalized to total protein, for each indicated genotype. Red, gray, and blue stacked bars indicate hydroxides, epoxide, and epoxide diol forms of eicosanoids, respectively. ****P<0.0001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM


Figure 5B 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B 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.


Figure 6F 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6F 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.


f1a ) 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 . ( a - c ) Native merged images or images with colocalization highlighted in white pixels are shown . Nuclei are blue ( DAPI ) . Scale bar , 10 μm .

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

f1a) 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.(a-c) Native merged images or images with colocalization highlighted in white pixels are shown. Nuclei are blue (DAPI). Scale bar, 10 μm.


f2 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f2(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.(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.


f3 ( 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 . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(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.(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.(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.(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.


f4 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4(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.(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.(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.


f5 ( 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 . ( 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 . ( ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5(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.(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. ((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.


f6 ( 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 . ( c ) ATG5 - ATG12 conjugation is required for ERK phosphorylation . Immunoblots for P - ERK , total ERK and β - actin in NIH / 3T3 cells transfected with WT ATG5 or the conjugation - defective ATG5 K130R mutant and treated with EGF ( 10 min ) . The bars represent mean ± s . e . m . * P0 . 05 compared with WT ATG5 - transfected cells ; Student ' s t - test , n = 3 .

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

f6(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.(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.


f7a ) 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 . ( 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 . ( 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 . ( 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 ) .

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

f7a) 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.(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.(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.(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).


Figure 1A 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 .

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

Figure 1A 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.


Figure 2A 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 .

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

Figure 2A 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.


Figure 3B 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 . 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 .

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

Figure 3B 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.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.


Figure 4A 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 . 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 .

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

Figure 4A 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.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.


Figure 5A 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 . 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 .

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

Figure 5A 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.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.


Figure 6A 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A 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.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.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.


Figure 7A 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 . 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 .

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

Figure 7A 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.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.


f1a , 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 ) . c , Time course measuring LDH release from unprimed or IFN - γ - primed BMDMs infected with S . typhimuriumd , 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 ) .

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

f1a, 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).c, Time course measuring LDH release from unprimed or IFN-γ-primed BMDMs infected with S. typhimuriumd, 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).


f2a , b , Quantification of live ( mCherry - positive ) and dead ( mCherry - negative ) S . typhimurium per cell by immunofluorescence ( a ) or as percent of total by flow - cytometrya , 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 . 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 . 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 ) .

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

f2a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometrya, 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.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.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).