Fig 1: Autophagy in the back skin of GFP-LC3 reporter mice. (A) LSM of GFP-LC3 transgenic Atg7f/f and atg7−/–deficient skin samples. Atg7f/f skin displays a discrete number of GFP puncta reminiscent of autophagosomes (→) in all layers of the epidermis (epi). Based on co-immunofluorescence with the epidermal cornification marker IVL (magenta), most of the GFP is found in the transitional layer at the stratum corneum in Atg7f/f and atg7−/- skin. Note that the corneal layer above the transitional layer is negative for GFP. In atg7−/–deficient skin, GFP-LC3 is diffusely distributed, indicative of free GFP-LC3 not incorporated into autophagosomes. In addition, some aggregates are found (►) and both the intensity and thickness of the GFP-positive transitional layer is increased (→←). Below: Quantification of transitional layer thickness, with 0.8 µm Atg7f/f vs. 2.2 µm atg7−/-, p < 0.01, N = 3. Quantification of un-degraded DNA in the corneum, p < 0.05, N = 3. (B) Immunoblot for autophagy and differentiation markers. SQSTM1, at the detection limit with a high variability (11.2 ± 5-fold, p = 0,1); GFP-LC3 levels increased by 2.2-fold in atg7−/- samples due to the abrogation of LC3-I/II conversion (13-fold inhibition). Basal keratin KRT14 (K14) and suprabasal KRT10 (K10) show no alterations. The lysosome marker LAMP1 (2.7-fold, p*<0.05), the intermediate (48 kDa; 1.4-fold, p*<0.05) and the mature (32 kDa) forms of CTSD accumulate in atg7−/- epidermis. The CTSD pro-form is barely detectable. No statistically significant changes are detected in the levels of the terminal keratinization markers FLG (filaggrin) and IVL (involucrin), and RPL26 (ribosomal protein L26). (C) SR-LSM images of the back skin (i) and ear (iii) of Atg7f/f and atg7−/- mice. (i) High abundance of peri-nuclear autophagosomes (open arrowhead) in a basally located cell (α) and the cell above (β). In a higher magnification (ii) the β cell displaying a reticular pattern of GFP-LC3 with one pole of the net being connected to the nucleus (nuclear border is marked by a dotted line). Note: the β cell nucleus is shrunken. (iii) An example of an extremely GFP bright, flattened cell (γ) in the final stage of CDA at the corneum. (iv) Higher magnification images revealing large bubbles of GFP-positive autophagosomes surrounding the hydrolyzed nuclear DNA. (v) Absence of GFP-LC3-positive autophagosomes in atg7−/- cells, showing only occasional spots of aggregated GFP (►). (D) Quantification of keratinocytes undergoing CDA at the SG-SC border in the BS (N = 3). Autophagosomes (→), ACD (open arrowhead), GFP-LC3 aggregates (►). Abbreviations and labels: apical corneal border (dashed line), basal cell (α), basal membrane (fine dashed line), cell moving upward (β), epidermis (epi), fold change relative to KRT10 (fc/K10), IVL (involucrin), GFP-LC3 (green), stratum corneum (SC), stratum granulosum (SG). Nuclear stain: DAPI. Scale bars: 5 µm.