Fig. 4: Atg4d disruption leads to defects in delipidation of mammalian ATG8 proteins.

A Immunoblotting analyses showing lipidation status of all murine ATG8 proteins in MEFs deficient for each of the ATG4 members, either in full media (left) or upon 4 h of nutrient deprivation (right). B Immunoblotting analyses against Myc epitope in MEFs deficient for each ATG4 member, stably-expressing a version of Myc-tagged LC3B exposing the reactive glycine at its carboxyl end amino-acid (LC3B∆C22). C Schematic representation of the different possible autophagosome-related structures containing mKeima-LC3B. D Representative images for WT and Atg4d^−/− MEFs stably expressing Keima-LC3B, cultured in the indicated conditions. E Representative images showing the fluorescence intensity ratio from both mKeima-LC3B signals (Ex586-Em620)/(Ex440-Em620). F Left, quantification of mKeima-LC3B ratio from (Ex586-Em620)/(Ex440-Em620) signals. Each point value represents the average ratio of mKeima-LC3B positive structures of a single cell. Right, relative abundance of mKeima-LC3B positive structures in both WT and Atg4d^−/− MEFs in the indicated culture conditions. G Schematic strategy of the SNAPtag®-LC3B assay developed to specifically monitor LC3B present at the cytosolic leaflet of the autolysosomal membrane. H Schematic representation of the expected results for either normal or defective LC3 delipidation. I Representative pictures and quantification of WT and Atg4d^−/− MEFs stably expressing SNAPtag®-LC3B and double-stained with MIL (green) and Alexa594®-conjugated anti-LAMP1 antibody (red) in the indicated conditions. Graphs show intensity profiles for fluorescent signals along the direction indicated in the insets (α−ω). Scale bars 10 μm. The percentage of LAMP1-positive dots which are also positive for MIL labelling is shown. LOAD: β-actin. Scale bars: 10 μm, 3 μm (E) and 2 μm (I) in insets. Measurements were done with 30 cells per genotype and treatment. *P < 0.05, 2-tailed unpaired Student’s t test.