Fig. 2: Autophagy regulation and flux analyses in Atg4d^−/− mice.

A Representative immunoblotting of SQSTM1/p62 in protein extracts from WT and mutant mice showing the effect of 24 h starvation on SQSTM1/p62 degradation. α-tubulin (skeletal muscle and heart) or β-actin (liver) were used as sample processing controls. N = 6 mice per genotype and condition. B Immunoblotting analyses against mATG8 proteins showing that the higher content of their lipidated forms in Atg4d^−/− MEFs is not due to autophagic flux block. Torin1 was used as autophagy inducer and BafA1 was used to inhibit lysosomal degradation of mATG8-membrane-bound forms. C Representative immunofluorescence images of endogenous SQSTM1/p62 and Ubiquitin in WT and knockout MEFs. Cells cultured in full medium are shown in the images. D Quantification of the data shown in (C) showing the effect of nutrient deprivation on p62 degradation and Ubiquitin accumulation in cultured MEFs (Co: Full medium and NF: Nutrient-free medium). E Representative images of immunofluorescence analysis of endogenous mATG8 proteins in WT, Atg4d^−/− MEFs and Atg4d^−/− MEFs expressing Flag-ATG4D. Images show cells cultured in full medium. F, G Quantification of the data from (E) either in Control (F) or upon Nutrient deprivation (G). H Immunoblotting analyses of mATG8 proteins in WT, Atg4d^−/− MEFs and Atg4d^−/− MEFs stably expressing Flag-ATG4D in the indicated conditions. I Representative fluorescence microscope images of WT and mutant MEFs stably expressing GFP-LC3B in basal and autophagy-inducing conditions. J–K Quantification of average GFP-LC3B content (J) and average GFP-LC3B dot size (K) from the data shown in (I). L Representative flow cytometry profiles for GFP-LC3B degradation in response to nutrient deprivation in the presence/absence of BafA1. M Quantification of the data from (L). Average WT values were set to 100%. N Autophagic-dependent degradation (sensitive to the autophagy inhibitor 3-MA) of isotope-labelled long-lived proteins upon nutrient deprivation in a pulse-chase analysis. O Immunoblotting analyses of the major autophagy-regulatory pathways in WT and Atg4d^−/− MEFs upon nutrient deprivation. P Representative images of WT and Atg4d^−/− MEFs stably expressing GFP-ULK1, GFP-ATG5, GFP-STX17TM, GFP-VAMP8 or stained with Lysotracker^(R) were cultured in regular or starvation medium for 4 h (NF). Q Quantification of the data represented in the images. LOAD: GAPDH (skeletal muscle, heart and liver) or β-actin (cells). Bars represent mean ± SEM (N > 80 cells per condition). Scale bars, 10 µm. *P < 0.05, 2-tailed unpaired Student’s t test (in D, J, K, M and Q) and one-way ANOVA followed by Tukey´s post hoc test (in F, G and N).