Figure 4. Defective ATG7-Dependent Autophagy in Patients with ATG7 Variants.

Panel A shows a representative Western blot analysis from duplicate or triplicate assessment of autophagy-related proteins in primary fibroblasts cultured in basal conditions and immunoblotted against ATG7, p62, ATG5-ATG12, ATG3, and ULK1. β-Actin was used as a loading control. Panel B shows autophagic flux analyzed by Western blotting after treatment of immortalized or primary fibroblasts with (+) or without (-) chloroquine (CQ) (60 μmol per liter) and in the presence or absence of AZD8055 (1 μmol per liter) for 2 hours before immunoblot detection of LC3 and β-actin (loading control). Western blots are representative of triplicate experiments, except for those in the analysis of fibroblasts from Patient 5, which were completed in duplicate. Panel C shows estimated autophagic cargo sequestration, which was assessed as sequestered lactate dehydrogenase (LDH) in the presence or absence of autophagy induction (starvation with Earl’s balanced salt solution [EBSS]) and late-stage blockade (bafilomycin A1 [BafA1, 100 nmol per liter]) for 3 hours in primary dermal fibroblasts derived from Control 1 and Patients 1 and 4. This assay was performed in triplicate. Adjusted P values are based on ordinary one-way analysis of variance and Sidak’s multiple-comparison test. Horizontal bars indicate mean values, and I bars indicate standard deviations.