( C ) Representative confocal microscopy pictures of the phenotype produced by the indicated mutations . Procedures were as in B .

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

(C) Representative confocal microscopy pictures of the phenotype produced by the indicated mutations. Procedures were as in B.


( D ) Simultaneous mutation of the four essential amino acids to alanine blocks HA - LC3 conversion induced by CD16 : 7 - 263 - 281 . 293 cells were transfected with the indicated chimeras ( 4M , quadruple mutant ) and HA - LC3A , aggregated and lysed for western blotting .

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

(D) Simultaneous mutation of the four essential amino acids to alanine blocks HA-LC3 conversion induced by CD16:7-263-281. 293 cells were transfected with the indicated chimeras (4M, quadruple mutant) and HA-LC3A, aggregated and lysed for western blotting.


( E ) Simultaneous mutation of the four essential amino acids blocks GFP - LC3 activation and colocalization with the endocytosed chimera . JAR cells were transfected with the indicated CD16 : 7 - 263 - 281 constructs and GFP - LC3A , aggregated and stained for the endocytosed chimera ( red ) . Representative confocal microscopy images are shown .

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

(E) Simultaneous mutation of the four essential amino acids blocks GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 constructs and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Representative confocal microscopy images are shown.


( F ) Quantification of the phenotype in E . Data gathering and expression were as in B . Source data for this figure is available on the online supplementary information page .

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

(F) Quantification of the phenotype in E. Data gathering and expression were as in B.Source data for this figure is available on the online supplementary information page.


( A ) Apposition events between GFP - ATG16L1 and endocytosed CD16 : 7 - 263 - 281 . JAR cells were transfected with CD16 : 7 - 263 - 281 and GFP - ATG16L1 or GFP - BECLIN ( as indicated ) , aggregated for 4 h and stained for the endocytosed chimera ( red ) . Representative confocal microscopy pictures are shown . Two different examples are provided for GFP - ATG16L1 .

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

(A) Apposition events between GFP-ATG16L1 and endocytosed CD16:7-263-281. JAR cells were transfected with CD16:7-263-281 and GFP-ATG16L1 or GFP-BECLIN (as indicated), aggregated for 4 h and stained for the endocytosed chimera (red). Representative confocal microscopy pictures are shown. Two different examples are provided for GFP-ATG16L1.


( B - D ) 293T cells were transfected with the indicated constructs , lysed and subjected to GST immunoprecipitation using agarose beads coupled to glutathione ( IP , immunoprecipitation ; TL , total lysate ) . Shown are WBs against the indicated molecules . ( B ) AU - ATG16L1 co ‐ precipitates with wild ‐ type ( WT ) TMEM59 - GST but not with a mutated version where the four essential amino acids were mutated to alanine ( TMEM59 - 4M - GST ) . ( C ) Full ‐ length TMEM59 and TMEM59 - Δ282 co ‐ precipitate with GST - ATG16L1 , whereas the respective 4M versions do not . ( D ) AU - ATG16L1 co ‐ precipitates with a fusion protein between GST and the minimal active peptide of TMEM59 ( GST - 263 - 281 ) , but not with a 4M version ( GST - 263 - 281 - 4M ) or a GST fusion protein with the inactive portion of TMEM59 - ID ( GST - 282 - 323 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation using agarose beads coupled to glutathione (IP, immunoprecipitation; TL, total lysate). Shown are WBs against the indicated molecules. (B) AU-ATG16L1 co‐precipitates with wild‐type (WT) TMEM59-GST but not with a mutated version where the four essential amino acids were mutated to alanine (TMEM59-4M-GST). (C) Full‐length TMEM59 and TMEM59-Δ282 co‐precipitate with GST-ATG16L1, whereas the respective 4M versions do not. (D) AU-ATG16L1 co‐precipitates with a fusion protein between GST and the minimal active peptide of TMEM59 (GST-263-281), but not with a 4M version (GST-263-281-4M) or a GST fusion protein with the inactive portion of TMEM59-ID (GST-282-323).


( E ) HA - ATG16L1 expressed in bacteria co − precipitates with a GST - 263 - 281 recombinant protein purified from bacterial cultures , but not with a 4M version of the same construct or GST - 282 - 323 . The indicated GST partners were expressed in bacteria and purified using agarose beads coupled to glutathione . The loaded beads were then used for HA - ATG16L1 pull − down from crude bacterial lysates . Shown are WBs against the indicated molecules ( PD , pull − down ) . A Coomasie staining of a protein gel with the purified GST fusion proteins is shown . The right panel compares the amount of HA - ATG16L1 pulled down by GST - 263 - 281 with the signal provided by direct anti − HA immunoprecipitation . This result shows that about 20 - 25 % of the available HA - ATG16L1 protein is precipitated by GST - 263 - 281 . Asterisks indicate irrelevant bands in B and D . Source data for this figure is available on the online supplementary information page .

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

(E) HA-ATG16L1 expressed in bacteria co−precipitates with a GST-263-281 recombinant protein purified from bacterial cultures, but not with a 4M version of the same construct or GST-282-323. The indicated GST partners were expressed in bacteria and purified using agarose beads coupled to glutathione. The loaded beads were then used for HA-ATG16L1 pull−down from crude bacterial lysates. Shown are WBs against the indicated molecules (PD, pull−down). A Coomasie staining of a protein gel with the purified GST fusion proteins is shown. The right panel compares the amount of HA-ATG16L1 pulled down by GST-263-281 with the signal provided by direct anti−HA immunoprecipitation. This result shows that about 20-25% of the available HA-ATG16L1 protein is precipitated by GST-263-281. Asterisks indicate irrelevant bands in B and D.Source data for this figure is available on the online supplementary information page.


( A - F ) 293T cells were transfected with the indicated constructs , lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione . Shown are WBs against the indicated molecules . ( A ) A deleted version of ATG16L1 lacking the WD domain ( HA - ATG16L1 - ΔWD ) does not co ‐ precipitate with TMEM59 - GST . ( B ) TMEM59 does not co ‐ precipitate with ATG16L1 - ΔWD fused to GST . ( C ) The WD domain of ATG16L1 ( HA - ATG16L1 - WD ) suffices to co ‐ precipitate with TMEM59 - GST . ( D ) TMEM59 co ‐ precipitates with ATG16L1 - WD fused to GST . ( E ) HA - ATG16L1 - WD does not bind a 4M version of TMEM59 - GST . ( F ) TMEM59 - 4M does not co ‐ precipitate with the ATG16L1 - WD fused to GST . Source data for this figure is available on the online supplementary information page .

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

(A-F) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (A) A deleted version of ATG16L1 lacking the WD domain (HA-ATG16L1-ΔWD) does not co‐precipitate with TMEM59-GST. (B) TMEM59 does not co‐precipitate with ATG16L1-ΔWD fused to GST. (C) The WD domain of ATG16L1 (HA-ATG16L1-WD) suffices to co‐precipitate with TMEM59-GST. (D) TMEM59 co‐precipitates with ATG16L1-WD fused to GST. (E) HA-ATG16L1-WD does not bind a 4M version of TMEM59-GST. (F) TMEM59-4M does not co‐precipitate with the ATG16L1-WD fused to GST.Source data for this figure is available on the online supplementary information page.


( C , D , E , G , H ) 293T cells were transfected with the indicated constructs , lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione . Shown are WBs against the indicated molecules . ( C ) The N ‐ terminal CARD of NOD2 ( NOD2 - CARD1 - HA ) , but not the CARD of NOD1 ( NOD1 - CARD - HA ) , co ‐ precipitates with GST - ATG16L1 . ( D ) The ID of TLR2 ( HA - TLR2 - ID ) co ‐ precipitates with GST - ATG16L1 . ( E ) Mutated versions ( MUT ) of NOD2 - CARD1 - HA and HA - TLR2 - ID ( as shown ) do not co ‐ precipitate with GST - ATG16L1 . ( F ) Amino ‐ acid sequences of T3JAM and DEDD2 including the motif . Residues identified by the Prosite algorithm are highlighted . ( G ) Wild ‐ type ( WT ) versions of HA - T3JAM and HA - DEDD2 ( as indicated ) co ‐ precipitate with GST - ATG16L1 . ( H ) Mutated versions ( MUT ) of HA - T3JAM and HA - DEDD2 do not co ‐ precipitate with GST - ATG16L1 . Asterisks indicate irrelevant bands in C , D and G . Source data for this figure is available on the online supplementary information page .

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

(C, D, E, G, H) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (C) The N‐terminal CARD of NOD2 (NOD2-CARD1-HA), but not the CARD of NOD1 (NOD1-CARD-HA), co‐precipitates with GST-ATG16L1. (D) The ID of TLR2 (HA-TLR2-ID) co‐precipitates with GST-ATG16L1. (E) Mutated versions (MUT) of NOD2-CARD1-HA and HA-TLR2-ID (as shown) do not co‐precipitate with GST-ATG16L1. (F) Amino‐acid sequences of T3JAM and DEDD2 including the motif. Residues identified by the Prosite algorithm are highlighted. (G) Wild‐type (WT) versions of HA-T3JAM and HA-DEDD2 (as indicated) co‐precipitate with GST-ATG16L1. (H) Mutated versions (MUT) of HA-T3JAM and HA-DEDD2 do not co‐precipitate with GST-ATG16L1. Asterisks indicate irrelevant bands in C, D and G.Source data for this figure is available on the online supplementary information page.


( A ) The right panel shows successful TMEM59 depletion ( rabbit anti ‐ TMEM59immunoprecipitation plus chicken anti ‐ TMEM59WB ) .

(A) The right panel shows successful TMEM59 depletion (rabbit anti‐TMEM59immunoprecipitation plus chicken anti‐TMEM59WB).


( A ) Depletion of TMEM59 inhibits LC3II generation by SA at early infection times . HeLa cells were transfected with the indicated siRNAs and , 48 h later , infected with the bacteria ( SA ) for the shown times before lysing them for western blotting against the indicated molecules . The right panel shows successful TMEM59 depletion ( rabbit anti ‐ TMEM59 immunoprecipitation plus chicken anti ‐ TMEM59 WB ) .

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

(A) Depletion of TMEM59 inhibits LC3II generation by SA at early infection times. HeLa cells were transfected with the indicated siRNAs and, 48 h later, infected with the bacteria (SA) for the shown times before lysing them for western blotting against the indicated molecules. The right panel shows successful TMEM59 depletion (rabbit anti‐TMEM59 immunoprecipitation plus chicken anti‐TMEM59 WB).


( B ) Colocalization events between bacteria ( GFP labelled ) , TMEM59 - HA and Cherry - LC3A in infected cells . HeLa cells stably expressing TMEM59 - HA and Cherry - LC3A were infected for the indicated times ( moi = 10 ) , and processed for anti ‐ HA immunofluorescence . Representative confocal images are shown . The GFP , anti ‐ HA ( Cy5 ) and DAPI signals were pseudocolored in blue , green and white , respectively . Asterisks indicate overlay images : ( * ) , Cherry - LC3A / TMEM59 ; ( * * ) , Cherry - LC3A / TMEM59 / bacteria ; ( * * * ) , Cherry - LC3A / TMEM59 / bacteria / DAPI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization events between bacteria (GFP labelled), TMEM59-HA and Cherry-LC3A in infected cells. HeLa cells stably expressing TMEM59-HA and Cherry-LC3A were infected for the indicated times (moi=10), and processed for anti‐HA immunofluorescence. Representative confocal images are shown. The GFP, anti‐HA (Cy5) and DAPI signals were pseudocolored in blue, green and white, respectively. Asterisks indicate overlay images: (*), Cherry-LC3A/TMEM59; (**), Cherry-LC3A/TMEM59/bacteria; (***), Cherry-LC3A/TMEM59/bacteria/DAPI.


( C ) Colocalization between bacteria , TMEM59 - HA and Cherry - ATG16L1 . HeLa cells stably expressing TMEM59 - HA and Cherry - ATG16L1 were infected with SA ( 2 h , moi = 10 ) , and processed for anti ‐ HA immunofluorescence . Shown are confocal pictures displaying two representative examples . Asterisks indicate overlays as in B .

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

(C) Colocalization between bacteria, TMEM59-HA and Cherry-ATG16L1. HeLa cells stably expressing TMEM59-HA and Cherry-ATG16L1 were infected with SA (2 h, moi=10), and processed for anti‐HA immunofluorescence. Shown are confocal pictures displaying two representative examples. Asterisks indicate overlays as in B.


( D ) SA promotes co ‐ precipitation of endogenous ATG16L1 with endogenous TMEM59 . HeLa cells were infected with SA ( 2 h , moi = 10 ) , lysed and subjected to immunoprecipitation with anti ‐ TMEM59 antibodies or irrelevant protein G beads ( as indicated ) . Immunoprecipitates were processed for western blotting . The right panel shows control WBs of the lysates used for immunoprecipitation .

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

(D) SA promotes co‐precipitation of endogenous ATG16L1 with endogenous TMEM59. HeLa cells were infected with SA (2 h, moi=10), lysed and subjected to immunoprecipitation with anti‐TMEM59 antibodies or irrelevant protein G beads (as indicated). Immunoprecipitates were processed for western blotting. The right panel shows control WBs of the lysates used for immunoprecipitation.


( E ) Depletion of TMEM59 reduces recovery of SA from infected cells . HeLa cells were transfected with the indicated siRNAs , infected with SA 96 h later ( 5 h , moi = 0 . 1 ) and lysed for CFU evaluation . The experiment was carried out three independent times . The right panel shows successful TMEM59 depletion ( anti ‐ TMEM59 immunoprecipitation plus anti ‐ TMEM59 WB ) . Source data for this figure is available on the online supplementary information page .

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

(E) Depletion of TMEM59 reduces recovery of SA from infected cells. HeLa cells were transfected with the indicated siRNAs, infected with SA 96 h later (5 h, moi=0.1) and lysed for CFU evaluation. The experiment was carried out three independent times. The right panel shows successful TMEM59 depletion (anti‐TMEM59 immunoprecipitation plus anti‐TMEM59 WB).Source data for this figure is available on the online supplementary information page.


( E ) Depletion of TMEM59 reduces recovery of SA from infected cells . HeLa cells were transfected with the indicated siRNAs , infected with SA 96 h later ( 5 h , moi = 0 . 1 ) and lysed for CFU evaluation . The experiment was carried out three independent times . The graph displays average CFU counts obtained for the 10 − 1 dilution of the relevant extracts , ± s . d . of the three data sets . Asterisks indicate significant differences ( P < 0 . 01 , paired Student ' s t ‐ test ) .

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

(E) Depletion of TMEM59 reduces recovery of SA from infected cells. HeLa cells were transfected with the indicated siRNAs, infected with SA 96 h later (5 h, moi=0.1) and lysed for CFU evaluation. The experiment was carried out three independent times. The graph displays average CFU counts obtained for the 10−1 dilution of the relevant extracts, ±s.d. of the three data sets. Asterisks indicate significant differences (P<0.01, paired Student's t‐test).


A , B . Height ( A ) and body mass index ( B ) of the participants by age in the diet groups compared to 2 SD intervals ( shaded area ) and median in the Finnish reference population .

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

A, B. Height (A) and body mass index (B) of the participants by age in the diet groups compared to 2 SD intervals (shaded area) and median in the Finnish reference population.


C . Dietary intake of selected macro - and micronutrients of participants . The shaded areas represent Finnish dietary recommendations for 2 to 5 years olds that covers majority of participants ( THL , 2019 ) . Energy intake recommendations vary by weight and therefore unambiguous limits could not be represented .

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

C. Dietary intake of selected macro- and micronutrients of participants. The shaded areas represent Finnish dietary recommendations for 2 to 5 years olds that covers majority of participants (THL, 2019). Energy intake recommendations vary by weight and therefore unambiguous limits could not be represented.


A . Serum , plasma or urine levels of vitamin D , vitamin A , folate , zinc , iron and iodine status biomarkers in diet groups . Solid lines indicate cut - offs for deficiency , and dashed lines indicate cut - offs for insufficiency . The RBP limits for vitamin A insufficiency and deficiency are validated for the method at 1 . 17 µmol / l and 0 . 83 µmol / l , respectively . Vitamin A status was calculated based on RBP , transthyretin , and CRP . ( Talsma et al , 2015 ) .

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

A. Serum, plasma or urine levels of vitamin D, vitamin A, folate, zinc, iron and iodine status biomarkers in diet groups. Solid lines indicate cut-offs for deficiency, and dashed lines indicate cut-offs for insufficiency. The RBP limits for vitamin A insufficiency and deficiency are validated for the method at 1.17 µmol/l and 0.83 µmol/l, respectively. Vitamin A status was calculated based on RBP, transthyretin, and CRP.(Talsma et al, 2015).


B . Cholesterol metabolism biomarkers in the diet groups . The subfigure summarizes cholesterol metabolism in humans and highlights cholesterol - related metabolites measured in this study with black font . ( Nelson & Cox , 2012 ; Risley , 2002 ) .

O O O O O O O O O O O O O O O O O O O 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. Cholesterol metabolism biomarkers in the diet groups. The subfigure summarizes cholesterol metabolism in humans and highlights cholesterol-related metabolites measured in this study with black font. (Nelson & Cox, 2012; Risley, 2002).


B . Targeted analysis of bile acid concentrations in serum of omnivores , vegetarians and vegans .

B. Targeted analysis of bile acid concentrations in serum of omnivores, vegetarians and vegans.


C . Amino acid concentrations in untargeted metabolomics . Numeric means and standard deviations in this figure can be found in Appendix Table S4 . # Valine and betaine are two major components under the same m / z - peak , causing uncertainty in the interpretation of valine levels .

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

C. Amino acid concentrations in untargeted metabolomics. Numeric means and standard deviations in this figure can be found in Appendix Table S4. #Valine and betaine are two major components under the same m/z-peak, causing uncertainty in the interpretation of valine levels.


A . Fatty acid analysis from untargeted MS metabolomics including free alpha - linolenic acid ( ALA ) and docosahexaenoic acid ( DHA ) .

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

A. Fatty acid analysis from untargeted MS metabolomics including free alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA).


B . Carnitine - bound fatty acids commonly found in serum that were detected in mass spectrometry .

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

B. Carnitine-bound fatty acids commonly found in serum that were detected in mass spectrometry.


C . Lysophosphatidylcholine and lysophosphatidylethanolamine with C16 : 0 ( palmitic acid ) and C18 : 1 ( oleic acid ) .

C. Lysophosphatidylcholine and lysophosphatidylethanolamine with C16:0 (palmitic acid) and C18:1 (oleic acid).


D . Combined analysis of all found triglycerides correlating to medium - chain fatty acid lengths and long or very long chain fatty acid lengths .

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

D. Combined analysis of all found triglycerides correlating to medium-chain fatty acid lengths and long or very long chain fatty acid lengths.


p53 ChIPseq in MEFs of the indicated genotype treated with or without 10 µM nutlin - 3a ( Nutlin ) for 16 hours . Shown are 2 kb regions surrounding the summit of the 468 p53 binding peaks called in Nutlin - treated p53 + / + , but not p53 - / - or p53EE / EE MEFs . For p53EE / EE MEFs three independent replicates are shown .

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

p53 ChIPseq in MEFs of the indicated genotype treated with or without 10 µM nutlin-3a (Nutlin) for 16 hours. Shown are 2 kb regions surrounding the summit of the 468 p53 binding peaks called in Nutlin-treated p53+/+, but not p53-/- or p53EE/EE MEFs. For p53EE/EE MEFs three independent replicates are shown.


RNAseq was performed with MEFs of the indicated genotype untreated or treated with 10 µM Nutlin for 16 hours . Shown are all Nutlin - regulated genes from the MSigDB gene set Hallmarks _ P53 _ Pathway with a mean log2FC ≥ 1 in p53 + / + cells . Shown are the z - transformed RNA expression values ( FPKM ) .

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

RNAseq was performed with MEFs of the indicated genotype untreated or treated with 10 µM Nutlin for 16 hours. Shown are all Nutlin-regulated genes from the MSigDB gene set Hallmarks_P53_Pathway with a mean log2FC≥1 in p53+/+ cells. Shown are the z-transformed RNA expression values (FPKM).


RNAseq data were subjected to gene set enrichment analysis ( GSEA ) . Shown are enrichment plots for the indicated set of p53 downstream genes in pairwise comparisons of Nutlin - treated MEFs with the indicated p53 genotypes .

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

RNAseq data were subjected to gene set enrichment analysis (GSEA). Shown are enrichment plots for the indicated set of p53 downstream genes in pairwise comparisons of Nutlin-treated MEFs with the indicated p53 genotypes.


Reverse transcription quantitative PCR ( RTqPCR ) analysis of p53 target genes in MEFs treated for 24 hours with 1 µg / ml doxorubicin ( Doxo ) . Shown are expression values normalized to β - actin as mean ± SD ( n = 6 ) .

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

Reverse transcription quantitative PCR (RTqPCR) analysis of p53 target genes in MEFs treated for 24 hours with 1 µg/ml doxorubicin (Doxo). Shown are expression values normalized to β-actin as mean ± SD (n=6).


Western blots of protein lysates prepared from MEFs treated for 24 hours with ( G ) 10 µM

Western blots of protein lysates prepared from MEFs treated for 24 hours with (G) 10 µM Nutlin


Western blots of protein lysates prepared from MEFs treated for 24 hours with 0 . 4 µg / ml Doxo .

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

Western blots of protein lysates prepared from MEFs treated for 24 hours with 0.4 µg/ml Doxo.


Proliferation of primary MEFs . Cells were treated o / n with 0 . 2 µg / ml doxorubicin ( Doxo ) and pulse - labeled with 32 µM 5 - Bromo - 2 - deoxyuridine ( BrdU ) , fixed and processed for flow cytometry analysis . n = 4 .

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

Proliferation of primary MEFs. Cells were treated o/n with 0.2 µg/ml doxorubicin (Doxo) and pulse-labeled with 32 µM 5-Bromo-2-deoxyuridine (BrdU), fixed and processed for flow cytometry analysis. n=4.


Long - term proliferation assay with primary MEFs of indicated genotypes . + / + and - / - : n = 3 ; EE / EE : n = 6 .

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Long-term proliferation assay with primary MEFs of indicated genotypes. +/+ and -/-: n=3; EE/EE: n=6.


MEFs were immortalized with the adenoviral oncogene E1A . 12S ( E1A MEF ) and treated with 0 . 4 µg / ml Doxo for 17 hours . Apoptosis ( Annexin V ) was quantified by flow cytometry . + / + and - / - : n = 3 ; EE / EE : n = 6 . Western blots show expression of E1A and β - actin as loading control .

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MEFs were immortalized with the adenoviral oncogene E1A.12S (E1A MEF) and treated with 0.4 µg/ml Doxo for 17 hours. Apoptosis (Annexin V) was quantified by flow cytometry. +/+ and -/-: n=3; EE/EE: n=6. Western blots show expression of E1A and β-actin as loading control.


Primary thymocytes were irradiated ex vivo with 6 Gy or treated with 1 µM dexamethasone as a control for p53 - independent apoptosis . Cell survival relative to untreated samples was analyzed using CellTiter Glow assay ( Promega ) . n = 11 for each time point and genotype .

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Primary thymocytes were irradiated ex vivo with 6 Gy or treated with 1 µM dexamethasone as a control for p53-independent apoptosis. Cell survival relative to untreated samples was analyzed using CellTiter Glow assay (Promega). n=11 for each time point and genotype.


mRNA expression analysis ( RTqPCR ) of p53 target genes in thymocytes 6 hours after 6 Gy irradiation . Shown are expression values normalized to β - actin .

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mRNA expression analysis (RTqPCR) of p53 target genes in thymocytes 6 hours after 6 Gy irradiation. Shown are expression values normalized to β-actin.


Mice of indicated genotype were subjected to 6 Gy whole - body irradiation and pulse - labeled with 120 mg / kg BrdU two hours before sacrifice at different time points . Small intestines were stained for ( F ) apoptosis ( TUNEL )

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Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for (F) apoptosis (TUNEL)


Mice of indicated genotype were subjected to 6 Gy whole - body irradiation and pulse - labeled with 120 mg / kg BrdU two hours before sacrifice at different time points . Small intestines were stained for proliferation ( BrdU ) ; scale bars 50 µm .

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Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for proliferation (BrdU); scale bars 50 µm.


Mice of indicated genotype were subjected to 6 Gy whole - body irradiation and pulse - labeled with 120 mg / kg BrdU two hours before sacrifice at different time points . Small intestines were stained for ( F ) apoptosis ( TUNEL ) Quantification for n = 3 mice / genotype ( 150 crypts / mouse ) .

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Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for (F) apoptosis (TUNEL) Quantification for n=3 mice/genotype (150 crypts/mouse).


Mice of indicated genotype were subjected to 6 Gy whole - body irradiation and pulse - labeled with 120 mg / kg BrdU two hours before sacrifice at different time points . Small intestines were stained for proliferation ( BrdU ) ; scale bars 50 µm . Quantification for n = 3 mice / genotype ( 150 crypts / mouse ) .

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Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for proliferation (BrdU); scale bars 50 µm. Quantification for n=3 mice/genotype (150 crypts/mouse).


Observed and expected genotype distribution of newborn offspring from mating Mdm2 + / - Trp53EE / + mice ( total number of pups n = 185 ; contingency test P = 0 . 0029 ) . Observed and expected genotype distribution of newborn offspring from mating Mdm2 + / - Trp53 + / - mice ( total number of pups n = 435 ; contingency test P = 0 . 912 ) .

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Observed and expected genotype distribution of newborn offspring from mating Mdm2+/-Trp53EE/+ mice (total number of pups n=185; contingency test P=0.0029). Observed and expected genotype distribution of newborn offspring from mating Mdm2+/-Trp53+/- mice (total number of pups n=435; contingency test P=0.912).


Representative picture of an E9 . 5 Mdm2 - / - Trp53EE / EE embryo ( right ) displays characteristic phenotypic abnormalities compared to an Mdm2 + / - ; Trp53EE / EE embryo ( left ) .

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Representative picture of an E9.5 Mdm2-/-Trp53EE/EE embryo (right) displays characteristic phenotypic abnormalities compared to an Mdm2+/-;Trp53EE/EE embryo (left).


IHC for p53 and cleaved caspase - 3 shows strong accumulation of p53EE protein and massive apoptosis in Mdm2 - / - Trp53EE / EE compared to Mdm2 + / - ; Trp53EE / EE control embryos .

IHC for p53 and cleaved caspase-3 shows strong accumulation of p53EE protein and massive apoptosis in Mdm2-/-Trp53EE/EE compared to Mdm2+/-;Trp53EE/EE control embryos.


Mdm2 - / - Trp53 - / - ( double knock - out , DKO ) MEFs were transduced with pInducer20 - p53EE lentivirus to enable tetracyclin ( Tet ) - inducible expression of mouse p53EE . Induction of p53EE sensitized cells to apoptosis induced by 24 hour treatment with 1 µg / ml doxorubicin ( Doxo ) as detected by flow cytometry for Annexin V .

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Mdm2-/-Trp53-/- (double knock-out, DKO) MEFs were transduced with pInducer20-p53EE lentivirus to enable tetracyclin (Tet)-inducible expression of mouse p53EE. Induction of p53EE sensitized cells to apoptosis induced by 24 hour treatment with 1 µg/ml doxorubicin (Doxo) as detected by flow cytometry for Annexin V.


mRNA expression of p53 target genes was measured in cells from ( A ) relative to β - actin by RTqPCR following treatment with Tet ± 1 µg / ml Doxo . Expression of the pro - apoptotic p53 target genes Bbc3 ( Puma ) and Bax is not significantly induced . n = 6 .

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mRNA expression of p53 target genes was measured in cells from (A) relative to β-actin by RTqPCR following treatment with Tet ± 1 µg/ml Doxo. Expression of the pro-apoptotic p53 target genes Bbc3 (Puma) and Bax is not significantly induced. n=6.


Proliferation of MEFs with indicated genotypes was analyzed by live - cell imaging in the presence of 10 µM nutlin - 3a ( Nutlin ) and / or 0 . 05 µg / ml doxorubicin ( Doxo ) . Shown is the median confluence ± SEM ( n = 12 ) .

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Proliferation of MEFs with indicated genotypes was analyzed by live-cell imaging in the presence of 10 µM nutlin-3a (Nutlin) and/or 0.05 µg/ml doxorubicin (Doxo). Shown is the median confluence ±SEM (n=12).


Cell viability assays for MEFs with indicated genotypes treated with Mdm2 inhibitors ± 0 . 4 µg / ml Doxo . n = 3 .

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Cell viability assays for MEFs with indicated genotypes treated with Mdm2 inhibitors ± 0.4 µg/ml Doxo. n=3.


Western blot of E1A - MEFs with indicated genotypes treated with 10 µM Nutlin ± 0 . 4 µg / ml Doxo for 18 hours reveals induction of apoptosis ( cCasp - 3 , cleaved caspase - 3 ; cParp , cleaved Parp ) in double - treated p53EE / EE MEFs in the absence of p53 target gene ( p21 , Mdm2 ) activation . * , 10 hours 0 . 4 µg / ml Doxo ; * * , 10 hours 0 . 2 µg / ml Doxo .

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Western blot of E1A-MEFs with indicated genotypes treated with 10 µM Nutlin ± 0.4 µg/ml Doxo for 18 hours reveals induction of apoptosis (cCasp-3, cleaved caspase-3; cParp, cleaved Parp) in double-treated p53EE/EE MEFs in the absence of p53 target gene (p21, Mdm2) activation. *, 10 hours 0.4 µg/ml Doxo; **, 10 hours 0.2 µg/ml Doxo.


mRNA expression of p53 target genes was measured in E1A MEFs of indicated genotype relative to β - actin by RTqPCR following combined treatment with 10 µM Nutlin and 1 µg / ml Doxo . n = 12 .

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mRNA expression of p53 target genes was measured in E1A MEFs of indicated genotype relative to β-actin by RTqPCR following combined treatment with 10 µM Nutlin and 1 µg/ml Doxo. n=12.


Western blot of p53EE / EE E1A - MEFs with indicated genotypes treated for 18 hours with 10 µM Nutlin , 0 . 4 µg / ml Doxo and 30 - 100 - 500 nM Ganetespib as indicated . Comb , combined treatment with 3 drugs for 18 hours . Pre , pre - treatment with ganetespib for 24 hours followed by combined treatment with Nutlin and Doxorubicin for 18 hours .

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Western blot of p53EE/EE E1A-MEFs with indicated genotypes treated for 18 hours with 10 µM Nutlin, 0.4 µg/ml Doxo and 30-100-500 nM Ganetespib as indicated. Comb, combined treatment with 3 drugs for 18 hours. Pre, pre-treatment with ganetespib for 24 hours followed by combined treatment with Nutlin and Doxorubicin for 18 hours.


Western blot of cellular fractions from unstressed and Doxo - treated MEFs of indicated genotype identifies p53EE protein in the mitochondrial fraction . PCNA , Tom20 / Bak are shown as nuclear and mitochondrial marker proteins .

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Western blot of cellular fractions from unstressed and Doxo-treated MEFs of indicated genotype identifies p53EE protein in the mitochondrial fraction. PCNA, Tom20/Bak are shown as nuclear and mitochondrial marker proteins.


Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet - inducible expression of human p53EE . Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0 . 5 µg / ml Doxo .

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Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet-inducible expression of human p53EE. Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0.5 µg/ml Doxo.


Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet - inducible expression of human p53EE . Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0 . 5 µg / ml Doxo . This effect is enhanced by addition of Nutlin and blocked by the pan - caspase inhibitor QVDOph , but not the ferroptosis inhibitor Ferrostatin - 1 ( Ferr ) as control . ( A ) n = 3 ; ( B ) n = 2 .

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Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet-inducible expression of human p53EE. Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0.5 µg/ml Doxo. This effect is enhanced by addition of Nutlin and blocked by the pan-caspase inhibitor QVDOph, but not the ferroptosis inhibitor Ferrostatin-1 (Ferr) as control. (A) n=3; (B) n=2.


Apoptosis analysis using Annexin V staining in cells from ( A ) . n = 3 .

Apoptosis analysis using Annexin V staining in cells from (A). n=3.


mRNA expression analysis ( RTqPCR ) of p53 target genes following treatment with 0 . 5 µg / ml Doxo ± 10 µM Nutlin . KO , tet - induced H1299 - pInd cells ; EE , tet - induced H1299 - pInd - p53EE cells ; WT , H460 cells . mRNA expression was normalized to β - actin ; n = 3 .

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mRNA expression analysis (RTqPCR) of p53 target genes following treatment with 0.5 µg/ml Doxo ± 10 µM Nutlin. KO, tet-induced H1299-pInd cells; EE, tet-induced H1299-pInd-p53EE cells; WT, H460 cells. mRNA expression was normalized to β-actin; n=3.


Proximity ligation assay for p53 with Tom20 in indicated untreated ( Mock ) or Doxo - treated ( 0 . 1 µg / ml , 24 hours ) H1299 cells following tet - induction . Shown are representative cells with PLA signals ( red ) counterstained with DAPI ( blue ) . Quantification of proximity ligation assays for p53EE with Tom20 , Bcl - 2 , Bcl - xL and Bak in H1299 cells . Doxo treatment ( 0 . 1 µg / ml , 24 hours ) as indicated . Plotted is the average number of PLA signals per cell for 10 fields of view .

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Proximity ligation assay for p53 with Tom20 in indicated untreated (Mock) or Doxo-treated (0.1 µg/ml, 24 hours) H1299 cells following tet-induction. Shown are representative cells with PLA signals (red) counterstained with DAPI (blue). Quantification of proximity ligation assays for p53EE with Tom20, Bcl-2, Bcl-xL and Bak in H1299 cells. Doxo treatment (0.1 µg/ml, 24 hours) as indicated. Plotted is the average number of PLA signals per cell for 10 fields of view.


Mitochondrial membrane potential of control and p53EE expressing H1299 cells in the absence and presence of increasing concentrations of BID BH3 peptide . Treatment with the mitochondrial depolarizer CCCP is shown as a positive control ; n = 3 .

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Mitochondrial membrane potential of control and p53EE expressing H1299 cells in the absence and presence of increasing concentrations of BID BH3 peptide. Treatment with the mitochondrial depolarizer CCCP is shown as a positive control; n=3.


Apoptosis measured by flow cytometry using Annexin V in H1299 cells with tet - inducible expression of the indicated human p53 mutants ; n = 3 .

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Apoptosis measured by flow cytometry using Annexin V in H1299 cells with tet-inducible expression of the indicated human p53 mutants; n=3.


mRNA expression analysis ( RTqPCR ) of p53 target genes normalized to β - actin ; n = 3 .

mRNA expression analysis (RTqPCR) of p53 target genes normalized to β-actin; n=3.


Kaplan - Meier survival plots for mice with indicated genotypes , number of mice , and median survival . Right panel , analysis of survival differences using the Log - rank ( Mantel - Cox ) test .

Kaplan-Meier survival plots for mice with indicated genotypes, number of mice, and median survival. Right panel, analysis of survival differences using the Log-rank (Mantel-Cox) test.


p53 immunohistochemistry ( IHC ) in representative spontaneous tumors ; scale bars 25 µm .

p53 immunohistochemistry (IHC) in representative spontaneous tumors; scale bars 25 µm.


Kaplan - Meier survival plots for Eµ - Myc transgenic mice with different p53 genotype . Kaplan - Meier survival plots for wild - type recipient mice transplanted with AML1 / ETO9a + NrasG12D - transduced fetal liver cells ( 5 donors / genotype ) .

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Kaplan-Meier survival plots for Eµ-Myc transgenic mice with different p53 genotype. Kaplan-Meier survival plots for wild-type recipient mice transplanted with AML1/ETO9a + NrasG12D-transduced fetal liver cells (5 donors/genotype).


p53 IHC for representative samples from ( D ) and ( E ) illustrates constitutive stabilization of p53EE in cancer cells ; scale bars 25 µm .

p53 IHC for representative samples from (D) and (E) illustrates constitutive stabilization of p53EE in cancer cells; scale bars 25 µm.


Apoptosis in Eµ - Myc lymphoma cells of indicated genotype was measured by flow cytometry ( FITC - VAD - FMK staining ) at indicated time points after treatment with 3 µg / ml MAF . Significance was calculated versus untreated . n ≥ 5 .

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Apoptosis in Eµ-Myc lymphoma cells of indicated genotype was measured by flow cytometry (FITC-VAD-FMK staining) at indicated time points after treatment with 3 µg/ml MAF. Significance was calculated versus untreated. n≥5.


Enrichment of p53 shRNA - expressing Eµ - Myc ; p53EE lymphoma cells under MAF treatment . Non - silencing control shRNA served as control . n = 3 .

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Enrichment of p53 shRNA-expressing Eµ-Myc;p53EE lymphoma cells under MAF treatment. Non-silencing control shRNA served as control. n=3.


mRNA expression of p53 target genes was measured relative to β - actin by RTqPCR following treatment of Eµ - Myc lymphoma cells of indicated genotype with 3 µg / ml MAF . n ≥ 3 .

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mRNA expression of p53 target genes was measured relative to β-actin by RTqPCR following treatment of Eµ-Myc lymphoma cells of indicated genotype with 3 µg/ml MAF. n≥3.


Kaplan - Meier survival plots for cyclophosphamide - treated versus untreated control mice transplanted with Eµ - Myc lymphoma cells of indicated genotype at day 0 . Treatment started ~ 10 days later when peripheral lymph nodes became palpable . The time course of the experiment is illustrated in a scheme .

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Kaplan-Meier survival plots for cyclophosphamide-treated versus untreated control mice transplanted with Eµ-Myc lymphoma cells of indicated genotype at day 0. Treatment started ~10 days later when peripheral lymph nodes became palpable. The time course of the experiment is illustrated in a scheme.


Residual disease in the spleen of mice from ( D ) was quantified 24 hours and 7 days after therapy using a quantitative PCR assays with primers specific to the Eµ - Myc transgene ( present only in lymphoma cells ) . Shown is the copy number of the Eµ - Myc transgene relative to a control locus present in all cells . Untreated Eµ - Myc mice ( Myc tg ) were used as positive control . Non - transgenic mice ( no Myc tg ) served as negative controls to define the detection limit . Significance was tested by t - test ( two - sided , unpaired ) .

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Residual disease in the spleen of mice from (D) was quantified 24 hours and 7 days after therapy using a quantitative PCR assays with primers specific to the Eµ-Myc transgene (present only in lymphoma cells). Shown is the copy number of the Eµ-Myc transgene relative to a control locus present in all cells. Untreated Eµ-Myc mice (Myc tg) were used as positive control. Non-transgenic mice (no Myc tg) served as negative controls to define the detection limit. Significance was tested by t-test (two-sided, unpaired).


Representative bioluminescence imaging ( BLI ) pictures of mice transplanted with AE9 + Nras AML cells . Day 0 indicates the start of combination therapy with cytarabine ( AraC ) + doxorubicin ( Doxo ) .

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Representative bioluminescence imaging (BLI) pictures of mice transplanted with AE9+Nras AML cells. Day 0 indicates the start of combination therapy with cytarabine (AraC) + doxorubicin (Doxo).


Kaplan - Meier survival plots for animals from ( G ) . Statistical significance of differences calculated by Log - rank ( Mantel - Cox ) test between treated Eµ - Myc ; p53EE / EE AML and all other groups is indicated in the table . C , untreated control ; T , therapy ( AraC + Doxo ) .

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Kaplan-Meier survival plots for animals from (G). Statistical significance of differences calculated by Log-rank (Mantel-Cox) test between treated Eµ-Myc;p53EE/EE AML and all other groups is indicated in the table. C, untreated control; T, therapy (AraC+Doxo).


( A , B ) Representative Western blot ( A ) and quantification ( B ) of p62 associated with mitochondria from spinal cords . Protein levels are normalized by subunit ATPβ of mitochondria ( Complex V ) . Mitochondrial p62 levels are increased at 120 days ( symptomatic stage ) in SOD1 - G93A spinal cord relative to Non Tg and wild type SOD1 ( wtSOD1 ) . Results are expressed as mean ± SEM relative to Non Tg at 30 days ; n = 8 mice ( 4 males and 4 females ) . At 120 days , * * p = 0 . 0018 ( Non Tg vs . SOD1 - G93A ) and * * p = 0 . 0011 ( SOD1 - G93A vs . wtSOD1 ) by paired one - way ANOVA with Tukey ' s correction . No other statistically significant differences were found ( paired Friedman ' s test with Dunn ' s correction at 30 days and paired one - way ANOVA with Tukey ' s correction at 60 and 90 days ) .

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(A, B) Representative Western blot (A) and quantification (B) of p62 associated with mitochondria from spinal cords. Protein levels are normalized by subunit ATPβ of mitochondria (Complex V). Mitochondrial p62 levels are increased at 120 days (symptomatic stage) in SOD1-G93A spinal cord relative to Non Tg and wild type SOD1 (wtSOD1). Results are expressed as mean ± SEM relative to Non Tg at 30 days; n=8 mice (4 males and 4 females). At 120 days, **p= 0.0018 (Non Tg vs. SOD1-G93A) and **p= 0.0011 (SOD1-G93A vs. wtSOD1) by paired one-way ANOVA with Tukey's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 days and paired one-way ANOVA with Tukey's correction at 60 and 90 days).


( C , D ) Representative Western blot ( C ) and quantification ( D ) of Tim23 in homogenates from spinal cords using β - actin as normalizer . Results are expressed as mean ± SEM and relative to Non Tg at 30 days ; n = 6 mice ( 3 males and 3 females ) . * * * p = 0 . 0002 ( Non Tg vs . SOD1 - G93A at 120 days ) by paired one - way ANOVA with Tukey ' s correction . No other statistically significant differences were found ( paired Friedman ' s test with Dunn ' s correction at 30 days and paired one - way ANOVA with Tukey ' s correction at 60 and 90 days ) . Tim23 is decreased in SOD1 - G93A spinal cord relative to Non Tg at 120 days .

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(C, D) Representative Western blot (C) and quantification (D) of Tim23 in homogenates from spinal cords using β-actin as normalizer. Results are expressed as mean ± SEM and relative to Non Tg at 30 days; n=6 mice (3 males and 3 females). ***p= 0.0002 (Non Tg vs. SOD1-G93A at 120 days) by paired one-way ANOVA with Tukey's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 days and paired one-way ANOVA with Tukey's correction at 60 and 90 days). Tim23 is decreased in SOD1-G93A spinal cord relative to Non Tg at 120 days.


( E , F ) Western blot ( E ) and quantification ( F ) of COXI in homogenates from spinal cords . Protein levels are normalized by β - actin . Results are expressed as mean ± SEM and relative to Non Tg at 30 days ; n = 6 mice ( 3 males and 3 females ) . * p = 0 . 017 ( Non Tg vs . SOD1 - G93A at 120 days ) by paired Friedman ' s test with Dunn ' s correction . No other statistically significant differences were found ( paired Friedman ' s test with Dunn ' s correction at 30 and 60 days and paired one - way ANOVA with Tukey ' s correction at 90 days ) . COX1 is decreased in SOD1 - G93A spinal cord relative to Non Tg at 120 days .

(E, F) Western blot (E) and quantification (F) of COXI in homogenates from spinal cords. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and relative to Non Tg at 30 days; n=6 mice (3 males and 3 females). *p= 0.017 (Non Tg vs. SOD1-G93A at 120 days) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 and 60 days and paired one-way ANOVA with Tukey's correction at 90 days). COX1 is decreased in SOD1-G93A spinal cord relative to Non Tg at 120 days.


G ) qPCR ( fold change ) of PGC1α mRNA normalized by β - actin mRNA . Results are expressed as mean ± SEM and fold change of Non Tg at 30 days ; n = 6 ( 3 males and 3 females ) for 30 and 120 days , n = 4 ( 2 males and 2 females ) for 60 days and n = 5 ( 3 males and 2 females ) for 90 days . * p = 0 . 035 at 90 days and * * * p = 0 . 0007 at 120 days . Paired Student ' s t test ( for 60 , 90 and 120 days ) and paired Wilcoxon ' s test ( for 30 days ) were used for comparisons .

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

G) qPCR (fold change) of PGC1α mRNA normalized by β-actin mRNA. Results are expressed as mean ± SEM and fold change of Non Tg at 30 days; n= 6 (3 males and 3 females) for 30 and 120 days, n= 4 (2 males and 2 females) for 60 days and n= 5 (3 males and 2 females) for 90 days. *p= 0.035 at 90 days and ***p= 0.0007 at 120 days. Paired Student's t test (for 60, 90 and 120 days) and paired Wilcoxon's test (for 30 days) were used for comparisons.


A ) Representative images of mt - Keima expressing spinal cord sections of SOD1 - G93A and Non Tg mice at 90 days . Mt - Keima fluorescence with excitation at 458nm is pseudocolored in green and at 543nm is pseudocolored in red . Emissions were recorded sequentially at 600 - 650nm . Scale bar , 150 µm .

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

A) Representative images of mt-Keima expressing spinal cord sections of SOD1-G93A and Non Tg mice at 90 days. Mt-Keima fluorescence with excitation at 458nm is pseudocolored in green and at 543nm is pseudocolored in red. Emissions were recorded sequentially at 600-650nm. Scale bar, 150 µm.


B ) Representative images of mt - Keima expressing motor neurons in the ventral horn of SOD1 - G93A and Non Tg mice at 90 days , imaged and pseudocolored Scale bar , 20 µm .

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

B) Representative images of mt-Keima expressing motor neurons in the ventral horn of SOD1-G93A and Non Tg mice at 90 days, imaged and pseudocolored Scale bar, 20 µm.


C ) Quantification of the rate of mitophagy in motor neurons expressed as the percentage of area with high ratio ( 543 / 458nm ) signal normalized by the total mitochondria area . Data were collected from mice at 60 days ( n = 3 , 1 male and 2 females ) , 90 days ( n = 4 , 2 males and 2 females ) , and 120 days ( n = 4 , 2 males and 2 females ) . Number of neurons imaged : 60 days , n = 77 Non Tg and 87 SOD1 - G93A ; 90 days , n = 118 Non Tg and 173 SOD1 - G93A ; 120 days , n = 110 Non Tg and 130 SOD1 - G93A . Comparisons within pairs showed that mitophagy is increased in SOD1 - G93A spinal cords at 60 and 90 days compared to Non Tg . Results are expressed as mean ± SEM . For 60 days , * * p = 0 . 0022 ; for 90 days , * * * p = 0 . 0001 ; for 120 days , p = 0 . 094 , all by unpaired Mann - Whitney test . Comparisons along time showed that mitophagy increases over time in both genotypes . For Non Tg , by unpaired Kruskal - Wallis ' s test with Dunn ' s correction , * * p = 0 . 0046 and * * * p = 0 . 0001 . For SOD1 - G93A , by unpaired Kruskal - Wallis ' s test with Dunn ' s correction , * * p = 0 . 0077 and * * * p = 0 . 0001 .

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

C) Quantification of the rate of mitophagy in motor neurons expressed as the percentage of area with high ratio (543/458nm) signal normalized by the total mitochondria area. Data were collected from mice at 60 days (n= 3, 1 male and 2 females), 90 days (n= 4, 2 males and 2 females), and 120 days (n= 4, 2 males and 2 females). Number of neurons imaged: 60 days, n= 77 Non Tg and 87 SOD1-G93A; 90 days, n= 118 Non Tg and 173 SOD1-G93A; 120 days, n= 110 Non Tg and 130 SOD1-G93A. Comparisons within pairs showed that mitophagy is increased in SOD1-G93A spinal cords at 60 and 90 days compared to Non Tg. Results are expressed as mean ± SEM. For 60 days, **p= 0.0022; for 90 days, ***p= 0.0001; for 120 days, p= 0.094, all by unpaired Mann-Whitney test. Comparisons along time showed that mitophagy increases over time in both genotypes. For Non Tg, by unpaired Kruskal-Wallis's test with Dunn's correction, **p= 0.0046 and ***p= 0.0001. For SOD1-G93A, by unpaired Kruskal-Wallis's test with Dunn's correction, **p=0.0077 and *** p= 0.0001.


Representative Western blot ( A ) of Parkin levels in spinal cord homogenates . Protein levels are normalized by β - actin . Results are expressed as mean ± SEM and relative to Non Tg control at 30 days ; n = 8 ( 4 males and 4 females ) . Parkin levels are decreased in SOD1 - G93A at 90 and 120 days compared to Non Tg .

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

Representative Western blot (A) of Parkin levels in spinal cord homogenates. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and relative to Non Tg control at 30 days; n= 8 (4 males and 4 females). Parkin levels are decreased in SOD1-G93A at 90 and 120 days compared to Non Tg.


quantification ( B ) of Parkin levels in spinal cord homogenates . Protein levels are normalized by β - actin . Results are expressed as mean ± SEM and relative to Non Tg control at 30 days ; n = 8 ( 4 males and 4 females ) . Paired Wilcoxon ' s test at 90 days , * * p = 0 . 0078 ; paired Student ' s t test at 120 days , * * p = 0 . 0054 . Paired Wilcoxon ' s test for 30 and 60 days showed no statistically significant differences . Parkin levels are decreased in SOD1 - G93A at 90 and 120 days compared to Non Tg .

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

quantification (B) of Parkin levels in spinal cord homogenates. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and relative to Non Tg control at 30 days; n= 8 (4 males and 4 females). Paired Wilcoxon's test at 90 days, **p= 0.0078; paired Student's t test at 120 days, **p= 0.0054. Paired Wilcoxon's test for 30 and 60 days showed no statistically significant differences. Parkin levels are decreased in SOD1-G93A at 90 and 120 days compared to Non Tg.


C ) qPCR ( fold change ) of Parkin mRNA normalized by β - actin mRNA . Results are expressed as mean ± SEM and fold change relative to Non Tg at 30 days of age ; n = 6 ( 3 males and 3 females ) for 30 and 120 days , n = 4 ( 2 males and 2 females ) for 60 days and n = 5 ( 3 males and 2 females ) for 90 days . No statistically significant differences were found by paired Student ' s t test ( 90 days ) and Wilcoxon ' s test ( 30 , 60 and 120 days ) .

C) qPCR (fold change) of Parkin mRNA normalized by β-actin mRNA. Results are expressed as mean ± SEM and fold change relative to Non Tg at 30 days of age; n= 6 (3 males and 3 females) for 30 and 120 days, n= 4 (2 males and 2 females) for 60 days and n= 5 (3 males and 2 females) for 90 days. No statistically significant differences were found by paired Student's t test (90 days) and Wilcoxon's test (30, 60 and 120 days).


D , E ) Representative Western blot ( D ) and quantification ( E ) of Parkin levels in spinal cord mitochondria at 120 days . Protein levels are normalized by Complex V . Parkin is reduced in spinal cord mitochondria of SOD1 - G93A mice at 120 days . Results are expressed as mean ± SEM and shown as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * * p = 0 . 0078 , by Wilcoxon ' s t test .

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

D, E) Representative Western blot (D) and quantification (E) of Parkin levels in spinal cord mitochondria at 120 days. Protein levels are normalized by Complex V. Parkin is reduced in spinal cord mitochondria of SOD1-G93A mice at 120 days. Results are expressed as mean ± SEM and shown as percent of Non Tg; n=8 (4 males and 4 females); **p= 0.0078, by Wilcoxon's t test.


A ) Representative Western blot of spinal cord homogenates at 130 days , showing that Parkin is absent in PKO and PKO / G93A mice .

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

A) Representative Western blot of spinal cord homogenates at 130 days, showing that Parkin is absent in PKO and PKO/G93A mice.


B ) Parkin quantification indicates that Parkin is decreased in G93A mice relative to Non Tg at 130 days . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * * p = 0 . 0078 , by Wilcoxon ' s t test .

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

B) Parkin quantification indicates that Parkin is decreased in G93A mice relative to Non Tg at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); **p=0.0078, by Wilcoxon's t test.


C ) Quantification of SOD1 at 130 days indicates that Parkin knockout does not affect the levels of transgenic mutant human SOD1 expressed in SOD1 - G93A spinal cord . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; no statistically significant differences relative to SOD1 expression were found between G93A and PKO / G93A , p = 0 . 214 , by paired Student ' s t test . Differences between Non Tg and G93A ( by paired Friedman ' s test with Dunn ' s correction ) , * p = 0 . 020 and PKO and PKO / G93A ( by paired one - way ANOVA with Tukey ' s correction ) , * * * p = 0 . 0001 .

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

C) Quantification of SOD1 at 130 days indicates that Parkin knockout does not affect the levels of transgenic mutant human SOD1 expressed in SOD1-G93A spinal cord. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); no statistically significant differences relative to SOD1 expression were found between G93A and PKO/G93A, p= 0.214, by paired Student's t test. Differences between Non Tg and G93A (by paired Friedman's test with Dunn's correction), *p= 0.020 and PKO and PKO/G93A (by paired one-way ANOVA with Tukey's correction), ***p= 0.0001.


D , E ) SOD1 filter trap assay ( D ) of spinal cord homogenates at 130 days and quantification of dot intensities ( E ) , showing that PKO / G93A mice have less SOD1 aggregates than G93A . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * * p = 0 . 0078 by paired Wilcoxon ' s test .

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

D, E) SOD1 filter trap assay (D) of spinal cord homogenates at 130 days and quantification of dot intensities (E), showing that PKO/G93A mice have less SOD1 aggregates than G93A. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); **p= 0.0078 by paired Wilcoxon's test.


F ) Representative images of 130 days old lumbar spinal cords sections immunostained for p62 . Scale bar , 200 µm .

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

F) Representative images of 130 days old lumbar spinal cords sections immunostained for p62. Scale bar, 200 µm.


G ) Quantification of the average number of large p62 - positive inclusions per section at 130 days indicates that PKO PKO / G93A mice have less SOD1 aggregates than G93A . Results are expressed as mean ± SEM ; n = 7 ( 3 males and 4 females ) mice for Non Tg and PKO , n = 10 ( 5 males and 5 females ) mice for G93A and n = 9 ( 4 males and 5 females ) mice for PKO / G93A ; * p = 0 . 010 by unpaired Mann - Whitney test .

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

G) Quantification of the average number of large p62-positive inclusions per section at 130 days indicates that PKO PKO/G93A mice have less SOD1 aggregates than G93A. Results are expressed as mean ± SEM; n= 7 (3 males and 4 females) mice for Non Tg and PKO, n= 10 (5 males and 5 females) mice for G93A and n= 9 (4 males and 5 females) mice for PKO/G93A; *p= 0.010 by unpaired Mann-Whitney test.


A ) Immunostaining of anterior horn of lumbar spinal cord for ChAT ( in green ) and p62 ( in red ) at 130 days . Scale bar , 10 µm .

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

A) Immunostaining of anterior horn of lumbar spinal cord for ChAT (in green) and p62 (in red) at 130 days. Scale bar, 10 µm.


B ) Quantification of the percentage of ChAT - positive motor neurons ( MN ) containing p62 - positive inclusions at 130 days . PKO PKO / G93A MN have less SOD1 aggregates than G93A , while Non Tg and PKO MN have none . Data were collected from n = 4 ( 2 males and 2 females ) mice for Non Tg and PKO and n = 6 ( 3 males and 3 females ) for G93A and PKO / G93A ( males and females ) . Number of images recorded for each genotype : n = 9 for Non Tg and PKO , n = 16 for G93A and n = 12 for PKO / G93A . Results are expressed as mean ± SEM ; * p = 0 . 032 , by unpaired one - tailed Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the percentage of ChAT-positive motor neurons (MN) containing p62-positive inclusions at 130 days. PKO PKO/G93A MN have less SOD1 aggregates than G93A, while Non Tg and PKO MN have none. Data were collected from n= 4 (2 males and 2 females) mice for Non Tg and PKO and n= 6 (3 males and 3 females) for G93A and PKO/G93A (males and females). Number of images recorded for each genotype: n= 9 for Non Tg and PKO, n= 16 for G93A and n= 12 for PKO/G93A. Results are expressed as mean ± SEM; *p= 0.032, by unpaired one-tailed Student's t test.


C ) Representative Western blots of OPTN , p62 and VCP in spinal cord mitochondrial fractions at 130 days . Protein levels are normalized by Complex V . The quantifications show that mitochondria of PKO / G93A mice have less mitophagy adaptor proteins than G93A relative to Complex V .

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

C) Representative Western blots of OPTN, p62 and VCP in spinal cord mitochondrial fractions at 130 days. Protein levels are normalized by Complex V. The quantifications show that mitochondria of PKO/G93A mice have less mitophagy adaptor proteins than G93A relative to Complex V.


D ) Quantification of OPTN relative to Complex V at 130 days . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * p = 0 . 039 by paired Student ' s t test .

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

D) Quantification of OPTN relative to Complex V at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); *p= 0.039 by paired Student's t test.


E ) Quantification of p62 , relative to Complex V at 130 days . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * * p = 0 . 0029 by paired Student ' s t test .

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

E) Quantification of p62, relative to Complex V at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); **p= 0.0029 by paired Student's t test.


F ) VCP was quantified using Complex V as loading reference at 130 days . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) ; * p = 0 . 044 by paired Student ' s t test .

F) VCP was quantified using Complex V as loading reference at 130 days. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females); *p= 0.044 by paired Student's t test.


A ) Kaplan - Meier survival analysis ( Log - Rank test with Holm - Sidak ' s correction for multiple comparisons ) . Mean age of death ( ± SEM ) for G93A was 169 . 3 ± 2 . 5 days and for PKO / G93A was 185 . 6 ± 2 . 2 days , indicating that Parkin knockout prolongs lifespan of SOD1 - G93A mice ; n = 19 for Non Tg ( 9 males and 10 females ) , n = 20 for PKO ( 10 males and 10 females ) , n = 18 for G93A ( 8 males and 10 females ) and n = 19 for PKO / G93A ( 9 males and 10 females ) ; p = 0 . 0000285 .

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

A) Kaplan-Meier survival analysis (Log-Rank test with Holm-Sidak's correction for multiple comparisons). Mean age of death (± SEM) for G93A was 169.3 ± 2.5 days and for PKO/G93A was 185.6 ± 2.2 days, indicating that Parkin knockout prolongs lifespan of SOD1-G93A mice; n=19 for Non Tg (9 males and 10 females), n= 20 for PKO (10 males and 10 females), n= 18 for G93A (8 males and 10 females) and n= 19 for PKO/G93A (9 males and 10 females); p= 0.0000285.


B ) Body weight changes over time , relative to body weight at 45 days that is set at 100 % for each mouse . Parkin knockout delays body weight loss in SOD1 - G93A mice . Results are expressed as mean ± SEM ; n = 19 for Non Tg ( 9 males and 10 females ) , n = 20 for PKO ( 10 males and 10 females ) , n = 18 for G93A ( 8 males and 10 females ) and n = 19 for PKO / G93A ( 9 males and 10 females ) ; * p = 0 . 017 ( at 150 days ) , * p = 0 . 004 ( at 164 days ) , * p = 0 . 002 ( at 171 days ) and * * p = 0 . 001 ( at 178 days ) , by two - way ANOVA with repeated measures and with Holm - Sidak ' s correction for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Body weight changes over time, relative to body weight at 45 days that is set at 100% for each mouse. Parkin knockout delays body weight loss in SOD1-G93A mice. Results are expressed as mean ± SEM; n=19 for Non Tg (9 males and 10 females), n=20 for PKO (10 males and 10 females), n=18 for G93A (8 males and 10 females) and n=19 for PKO/G93A (9 males and 10 females); *p= 0.017 (at 150 days), *p= 0.004 (at 164 days), *p= 0.002 (at 171 days) and **p= 0.001 (at 178 days), by two-way ANOVA with repeated measures and with Holm-Sidak's correction for multiple comparisons.


C ) Clasping score changes over time . Parkin knockout does not affect clasping onset in SOD1 - G93A mice . An intermediate score of 2 . 5 is indicated by the dotted line . Results are expressed as mean ± SEM ; n = 19 for Non Tg ( 9 males and 10 females ) , n = 20 for PKO ( 10 males and 10 females ) , n = 18 for G93A ( 8 males and 10 females ) and n = 19 for PKO / G93A ( 9 males and 10 females ) .

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

C) Clasping score changes over time. Parkin knockout does not affect clasping onset in SOD1-G93A mice. An intermediate score of 2.5 is indicated by the dotted line. Results are expressed as mean ± SEM; n=19 for Non Tg (9 males and 10 females), n=20 for PKO (10 males and 10 females), n=18 for G93A (8 males and 10 females) and n=19 for PKO/G93A (9 males and 10 females).


D ) Representative images of NMJs in the soleus muscle at 130 days . Muscles were immunostained for neurofilament H ( NF - H , in red ) and stained with α - bungarotoxin ( α - BTX , in green ) . Scale bar , 20 µm .

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

D) Representative images of NMJs in the soleus muscle at 130 days. Muscles were immunostained for neurofilament H (NF-H, in red) and stained with α-bungarotoxin (α-BTX, in green). Scale bar, 20 µm.


E ) Quantification of the percentage of innervated NMJs at 130 days indicates that Parkin knockout decreases denervation in SOD1 - G93A mice . Results are expressed as mean ± SEM and were collected from n = 6 mice per group ( 3 males and 3 females ) ; n of images evaluated = 49 for Non Tg ( with 483 NMJs counted ) , 46 for PKO ( 396 NMJs counted ) , 47 for G93A ( 413 NMJs assessed ) and 38 for PKO / G93A ( 325 NMJs counted ) ; * * * p = 0 . 0001 , by unpaired Mann - Whitney test .

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

E) Quantification of the percentage of innervated NMJs at 130 days indicates that Parkin knockout decreases denervation in SOD1-G93A mice. Results are expressed as mean ± SEM and were collected from n= 6 mice per group (3 males and 3 females); n of images evaluated = 49 for Non Tg (with 483 NMJs counted), 46 for PKO (396 NMJs counted), 47 for G93A (413 NMJs assessed) and 38 for PKO/G93A (325 NMJs counted); ***p= 0.0001, by unpaired Mann-Whitney test.


F ) Representative images of lumbar spinal cord sections at 130 days immunostained with ChAT . Scale bar , 200 µm . G ) Quantification of ChAT - positive MN in the spinal cord ventral horn ( dotted areas ) at 130 days shows that Parkin knockout decreases MN loss in the spinal cord of SOD1 - G93A mice . Results are expressed as mean ± SEM ; n = 10 mice per group in Non Tg , PKO and G93A ( 5 males and 5 females ) and n = 9 for PKO / G93A ( 4 males and 5 females ) ; * p = 0 . 031 , by unpaired Student ' s t test . H

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

F) Representative images of lumbar spinal cord sections at 130 days immunostained with ChAT. Scale bar, 200 µm. G) Quantification of ChAT-positive MN in the spinal cord ventral horn (dotted areas) at 130 days shows that Parkin knockout decreases MN loss in the spinal cord of SOD1-G93A mice. Results are expressed as mean ± SEM; n = 10 mice per group in Non Tg, PKO and G93A (5 males and 5 females) and n= 9 for PKO/G93A (4 males and 5 females); *p=0.031, by unpaired Student's t test. H


H ) Representative images of lumbar spinal cords sections immunostained for GFAP at 130 days . Scale bar , 200 µm . I ) Quantification of the average GFAP staining intensity at 130 days indicates that Parkin knockout decreases spinal cord astrogliosis in SOD1 - G93Amice . Results are expressed as mean ± SEM ; n = 10 mice per group ( 5 males and 5 females ) ; * p = 0 . 033 , by paired Student ' s t test .

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

H) Representative images of lumbar spinal cords sections immunostained for GFAP at 130 days. Scale bar, 200 µm. I) Quantification of the average GFAP staining intensity at 130 days indicates that Parkin knockout decreases spinal cord astrogliosis in SOD1-G93Amice. Results are expressed as mean ± SEM; n = 10 mice per group (5 males and 5 females); *p= 0.033, by paired Student's t test.


A ) Citrate synthase activity in spinal cord homogenates at disease end stage . Results are expressed as mean ± SEM ; n = 8 ( 4 males and 4 females ) mice per group ; * p = 0 . 024 , by paired Student ' s t test . Activities are expressed as nmoles of substrate oxidized per minute per mg of protein .

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

A) Citrate synthase activity in spinal cord homogenates at disease end stage. Results are expressed as mean ± SEM; n= 8 (4 males and 4 females) mice per group; *p= 0.024, by paired Student's t test. Activities are expressed as nmoles of substrate oxidized per minute per mg of protein.


B ) Cytochrome oxidase ( COX ) activity at end stage . Results are expressed as mean ± SEM ; n = 8 ( 4 males and 4 females ) mice per group ; * p = 0 . 032 , by paired Student ' s t test . Activities are expressed as nmoles of substrate oxidized per minute per mg of protein .

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B) Cytochrome oxidase (COX) activity at end stage. Results are expressed as mean ± SEM; n = 8 (4 males and 4 females) mice per group; *p= 0.032, by paired Student's t test. Activities are expressed as nmoles of substrate oxidized per minute per mg of protein.


C ) qPCR ( fold change ) of PGC1α mRNA normalized by β - actin mRNA at 130 days of age . Results are expressed as mean ± SEM and as fold change of the Non Tg ; n = 8 ( 4 males and 4 females ) . No statistically significant differences were found between G93A and PKO / G93A ( p = 0 . 250 by paired Wilcoxon ' s test ) ; * p = 0 . 035 paired Friedman ' s test with Dunn ' s correction ( Non Tg vs . G93A ) . No other statistically significant differences were found . D ) qPCR ( fold change ) of PGC1α mRNA normalized by β - actin mRNA at end stage . Results are expressed as mean ± SEM and as fold change of the Non Tg ; n = 8 ( 4 males and 4 females ) . No statistically significant differences were found between G93A and PKO / G93A ( p = 0 . 613 by paired Student ' s t test ) . * * p = 0 . 0058 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A comparison ) and * * * p < 0 . 0001 by paired one - way ANOVA with Tukey ' s correction ( for PKO vs . PKO / G93A comparison ) . Parkin knockout improves PGC1α expression at 130 days ( C ) but the effect is lost at end stage ( D ) . E

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

C) qPCR (fold change) of PGC1α mRNA normalized by β-actin mRNA at 130 days of age. Results are expressed as mean ± SEM and as fold change of the Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A (p= 0.250 by paired Wilcoxon's test); *p= 0.035 paired Friedman's test with Dunn's correction (Non Tg vs. G93A). No other statistically significant differences were found. D) qPCR (fold change) of PGC1α mRNA normalized by β-actin mRNA at end stage. Results are expressed as mean ± SEM and as fold change of the Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A (p= 0.613 by paired Student's t test). **p= 0.0058 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A comparison) and ***p<0.0001 by paired one-way ANOVA with Tukey's correction (for PKO vs. PKO/G93A comparison). Parkin knockout improves PGC1α expression at 130 days (C) but the effect is lost at end stage (D). E


E , F ) Representative Western blots ( E ) and quantification ( F ) of PARIS at disease end stage . Protein levels are normalized by β - actin and results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) . No statistically significant differences were found between G93A and PKO / G93A by paired Wilcoxon ' s test ( p = 0 . 99 ) . PARIS levels are unchanged in SOD1 - G93A mice relative to Non Tg at disease end stage .

E, F) Representative Western blots (E) and quantification (F) of PARIS at disease end stage. Protein levels are normalized by β-actin and results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.99). PARIS levels are unchanged in SOD1-G93A mice relative to Non Tg at disease end stage.


A , B ) Western blots ( A ) and quantification ( B ) of Miro1 in spinal cord homogenates at disease end stage . β - actin is used as loading reference . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group ; * p = 0 . 011 by paired Student ' s t test ( for comparison G93A vs . PKO / G93A ) and * * p = 0 . 003 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A ) . Parkin knockout increases the levels of Miro1 in SOD1 - G93A mice at disease end stage .

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

A, B) Western blots (A) and quantification (B) of Miro1 in spinal cord homogenates at disease end stage. β-actin is used as loading reference. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (4 males and 4 females) mice per group; *p= 0.011 by paired Student's t test (for comparison G93A vs. PKO/G93A) and **p= 0.003 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A). Parkin knockout increases the levels of Miro1 in SOD1-G93A mice at disease end stage.


C , D ) Western blots ( C ) and quantification ( D ) of Mfn2 in spinal cord homogenates at disease end stage . Protein levels are normalized by β - actin . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group ; No statistically significant differences were found between G93A and PKO / G93A ( p = 0 . 078 by paired Wilcoxon ' s test ) ; * * * p = 0 . 0007 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A ) and * p = 0 . 037 by paired Friedman ' s test with Dunn ' s correction ( for PKO vs . PKO / G93A ) . Parkin knockout increases the levels of Mfn2 in SOD1 - G93A mice at disease end stage .

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

C, D) Western blots (C) and quantification (D) of Mfn2 in spinal cord homogenates at disease end stage. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females) mice per group; No statistically significant differences were found between G93A and PKO/G93A (p=0.078 by paired Wilcoxon's test); ***p= 0.0007 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and *p= 0.037 by paired Friedman's test with Dunn's correction (for PKO vs. PKO/G93A). Parkin knockout increases the levels of Mfn2 in SOD1-G93A mice at disease end stage.


E ) Western blots of spinal cord mitochondria at disease end stage , probed for lysine 48 ( left panel ) and lysine 63 ( right panel ) ubiquitin chains . Citrate synthase is used as loading control . Asterisks indicate ubiquitinated proteins with different abundance in SOD1 - G93A samples .

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E) Western blots of spinal cord mitochondria at disease end stage, probed for lysine 48 (left panel) and lysine 63 (right panel) ubiquitin chains. Citrate synthase is used as loading control. Asterisks indicate ubiquitinated proteins with different abundance in SOD1-G93A samples.


F , G ) Representative Western blot ( F ) and quantification ( G ) of LC3 II in spinal cord mitochondria at end stage . Protein levels are normalized by Complex V . LC3 II is increased in both G93A and PKO / G93A mitochondria and Parkin knockout does not affect LC3 II levels . Results are expressed as mean ± SEM and as percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group ; No statistically significant differences were found between G93A and PKO / G93A ( p = 0 . 504 by paired Student ' s t test ) . * p = 0 . 035 by paired Friedman ' s test with Dunn ' s correction ( for Non Tg vs . G93A ) and * * p = 0 . 0014 by paired one - way ANOVA with Tukey ' s correction ( for PKO vs . PKO / G93A )

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

F, G) Representative Western blot (F) and quantification (G) of LC3 II in spinal cord mitochondria at end stage. Protein levels are normalized by Complex V. LC3 II is increased in both G93A and PKO/G93A mitochondria and Parkin knockout does not affect LC3 II levels. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (4 males and 4 females) mice per group; No statistically significant differences were found between G93A and PKO/G93A (p=0.504 by paired Student's t test). *p= 0.035 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and **p= 0.0014 by paired one-way ANOVA with Tukey's correction (for PKO vs. PKO/G93A)


H ) Representative Western blot of March5 and Mul1 in spinal cord mitochondria at end stage .

H) Representative Western blot of March5 and Mul1 in spinal cord mitochondria at end stage.


I ) Quantification of March5 at disease end stage . Results are expressed as mean ± SEM and percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group . No statistically significant differences were found between G93A and PKO / G93A by paired Wilcoxon ' s test ( p = 0 . 742 ) . Protein levels are normalized by Complex V . March5 levels are unaffected by Parkin knockout .

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

I) Quantification of March5 at disease end stage. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.742). Protein levels are normalized by Complex V. March5 levels are unaffected by Parkin knockout.


J ) Quantification of Mul1 at disease end stage . Results are expressed as mean ± SEM and percent of Non Tg ; n = 8 ( 4 males and 4 females ) mice per group . No statistically significant differences were found between G93A and PKO / G93A by paired Wilcoxon ' s test ( p = 0 . 94 ) . Protein levels are normalized by Complex V . Mul1 levels are unaffected by Parkin knockout .

J) Quantification of Mul1 at disease end stage. Results are expressed as mean ± SEM and percent of Non Tg; n= 8 (4 males and 4 females) mice per group. No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.94). Protein levels are normalized by Complex V. Mul1 levels are unaffected by Parkin knockout.


B . Beta - hydroxybutyrate ( ketone body ) concentrations , plasma .

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B. Beta-hydroxybutyrate (ketone body) concentrations, plasma.


C . Creatine kinase ( CK ) concentrations , plasma .

C. Creatine kinase (CK) concentrations, plasma.


D . Myoglobin concentrations , plasma . Dashed line : upper limit of control range . The individual patients are shown separately , and controls as mean with standard deviation ( n = 10 ) . Arrow : mAD endpoint in PEO patients ; lines under graphs : duration of mAD .

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D. Myoglobin concentrations, plasma. Dashed line: upper limit of control range. The individual patients are shown separately, and controls as mean with standard deviation (n=10). Arrow: mAD endpoint in PEO patients; lines under graphs: duration of mAD.


E - G . PEO patient ( P1 ) muscle before diet : E . Ultrathin section of abnormal muscle fiber ( arrowhead ) with subsarcolemmal accumulations of mitochondria . Scale bar 50 µmF . Electron micrograph of subsarcolemmal accumulation of abnormal mitochondria ( same cell as in E ) . G . Enlargement of the area marked in F ; mitochondria with paracrystalline " parking lot " inclusions ( arrow ) and concentric cristae ( asterisk ) . Scale bar 1µmH - J . PEO patient ( P1 ) muscle after mAD ( H - J ) : H . Ultrathin section , lytic muscle fiber with centrally located organellar debris ( arrow ) . Scale bar 50 µmI . Electron micrograph of a degrading muscle fiber with an invaded macrophage ( arrow ) ( same cell as in H ) . J . Enlargement of the fiber in I : paracrystalline inclusions within mitochondria ( asterisk ) . Scale bar 1µmK . Quantification of necrotic or apoptotic cells in PEO muscle from ultrathin sections . n . a . , not available .

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

E-G. PEO patient (P1) muscle before diet:E. Ultrathin section of abnormal muscle fiber (arrowhead) with subsarcolemmal accumulations of mitochondria. Scale bar 50 µmF. Electron micrograph of subsarcolemmal accumulation of abnormal mitochondria (same cell as in E).G. Enlargement of the area marked in F; mitochondria with paracrystalline "parking lot" inclusions (arrow) and concentric cristae (asterisk). Scale bar 1µmH-J. PEO patient (P1) muscle after mAD (H-J):H. Ultrathin section, lytic muscle fiber with centrally located organellar debris (arrow). Scale bar 50 µmI. Electron micrograph of a degrading muscle fiber with an invaded macrophage (arrow) (same cell as in H).J. Enlargement of the fiber in I: paracrystalline inclusions within mitochondria (asterisk). Scale bar 1µmK. Quantification of necrotic or apoptotic cells in PEO muscle from ultrathin sections. n.a., not available.


A - B . Cleaved caspase - 3 positive muscle fibers ( * ) . A . PEO patient ( P3 ) on normal diet ( inset : caspase staining in healthy control individual ) B . PEO patient ( P3 ) after modified Atkins diet . Arrowhead : caspase negative necrotic fiber .

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A-B. Cleaved caspase-3 positive muscle fibers (*).A. PEO patient (P3) on normal diet (inset: caspase staining in healthy control individual)B. PEO patient (P3) after modified Atkins diet. Arrowhead: caspase negative necrotic fiber.


C . Western blot analysis of autophagy receptor p62 and pax7 marking muscle satellite cell activation ; left , blot ; right , signal quantification in each patient .

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C. Western blot analysis of autophagy receptor p62 and pax7 marking muscle satellite cell activation; left, blot; right, signal quantification in each patient.


D - E . Immunohistochemistry analysis of p62 and mitochondrial mass marker , consecutive sections . Asterisks indicate p62 - positive fibers and corresponding affected ragged - red fibers . F - G . p62 and mitochondrial mass after mAD ; consecutive sections . Arrowheads indicate fibers that show p62 positivity but have normal mitochondrial mass .

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D - E. Immunohistochemistry analysis of p62 and mitochondrial mass marker, consecutive sections. Asterisks indicate p62-positive fibers and corresponding affected ragged-red fibers.F - G. p62 and mitochondrial mass after mAD; consecutive sections. Arrowheads indicate fibers that show p62 positivity but have normal mitochondrial mass.


H . p62 and LC3 positive ragged - red fibers ( * ) in PEO patient ( P2 ) , normal diet . I . Muscle fibers showing granular LC3 - positivity , PEO patient ( P1 ) , after mAD . Immunohistochemistry on frozen sections ; bars : 50 µm , except for H , 100 µm .

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H. p62 and LC3 positive ragged-red fibers (*) in PEO patient (P2), normal diet.I. Muscle fibers showing granular LC3-positivity, PEO patient (P1), after mAD. Immunohistochemistry on frozen sections; bars: 50 µm, except for H, 100 µm.


A - C . Muscle strength and performance of PEO patients ( P1 - P4 ) on normal diet ( ND ) and after modified Atkins diet ( mAD ) ( one and six months and 2 . 5 years after initiation of diet ) .

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A-C. Muscle strength and performance of PEO patients (P1-P4) on normal diet (ND) and after modified Atkins diet (mAD) (one and six months and 2.5 years after initiation of diet).


D - G . Spiroergometry results from PEO patients and control subjects on normal diet ( ND ) , as well as one month and six months after mAD initiation . PEO patients had higher serumlactate levels , and attenuated respiratory response to exercise . These effects were further enhanced after mAD and remained after 6 months of the study . mAD increased patients ' serumammonium ( NH4 + ) levels . Data presented as mean and SD .

D-G. Spiroergometry results from PEO patients and control subjects on normal diet (ND), as well as one month and six months after mAD initiation. PEO patients had higher serumlactate levels, and attenuated respiratory response to exercise. These effects were further enhanced after mAD and remained after 6 months of the study. mAD increased patients' serumammonium (NH4+) levels. Data presented as mean and SD.


A . PEO patients , relative amino acid levels on normal diet ( PEO ) , immediately after finishing mAD ( PEO _ mAD ) , and one month after mAD . B . Control subjects , relative amino acid levels on normal diet ( Control ) and immediately after finishing mAD ( Control _ mAD ) .

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A. PEO patients, relative amino acid levels on normal diet (PEO), immediately after finishing mAD (PEO_mAD), and one month after mAD.B. Control subjects, relative amino acid levels on normal diet (Control) and immediately after finishing mAD (Control_mAD).


C . Relative values of plasma metabolites in creatine pathway , compared to controls on ND . Right : illustration of major metabolites and pathway of creatine synthesis .

C. Relative values of plasma metabolites in creatine pathway, compared to controls on ND. Right: illustration of major metabolites and pathway of creatine synthesis.


D . Relative values of plasma purine and pyrimidine degradation pathway intermediates , compared to controls on ND . Right : illustration of major metabolites and synthesis of pyrimidine and purines .

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D. Relative values of plasma purine and pyrimidine degradation pathway intermediates, compared to controls on ND. Right: illustration of major metabolites and synthesis of pyrimidine and purines.