Fig 2: Autophagy analyses in atg4c atg4d DKO cells and mice. (A) Immunoblotting analyses against mATG8 proteins in heart, liver and muscle tissues from WT, atg4d-/- and atg4c atg4d DKO mice under basal conditions and upon 24 h of fasting. ACTB/ß-actin was used as sample processing controls. (B) Autophagic flux analyses in WT, atg4d-/-, atg4c-/- and atg4c atg4d DKO MEFS cultured in rich medium (control) of starved during 4 h either in the presence or absence of bafilomycin A1.

Fig 3: Sequence and phylogenetic analyses of ATG4 proteins. (A) Amino acid sequence of yeast, D. melanogaster, C. elegans, human and mouse ATG4 proteins. The multiple alignment was performed with the ClustalX program. Gaps are indicated by hyphens. The analysis of the alignment was performed with jalview (version 2.2). Coloring was applied according to both amino-acid identity percentage and conservation (threshold value = 7.5). Amino-acids highlighted in red represent conserved residues with 100% of identity among all depicted species. Amino-acids highlighted in orange represent residues with a high degree of similarity involving only conservative substitutions. Amino-acids highlighted in yellow show residues in which a high degree of similarity is found, even when non-conservative substitutions occur in some species. Dark blue and dark green represent residues with 100% of identity in the ATG4AB and ATG4C ATG4D subfamilies, respectively. Light blue and light green represent residues which high degree of conservation (involving only conservative substitutions) in the ATG4AB and ATG4C ATG4D subfamilies, respectively. (B) Phylogenetic tree of the ATG4 family. Amino acid sequences of the different A. thaliana, C. elegans, D. melanogaster, human and mouse ATG4 proteins were aligned using the Phylip program package (version 3.6). Numbers represent reliability values after bootstrapping the data. Percentage of identity between the different ATG4s members: ATG4A vs ATG4B: 57%; ATG4A vs ATG4C: 30%; ATG4A vs ATG4D: 32.76%; ATG4B vs ATG4C: 28.38%; ATG4B vs ATG4D: 30.73 and ATG4C vs ATG4D: 46.39%.