Figure 5

Bi-allelic variants in SNF8 in individual A2 lead to loss of ESCRT II complex resulting in an autophagic phenotype in fibroblasts

(A) Fibroblasts derived from individual A2 (top) or control individuals (bottom) were analyzed by transmission electron microscopy. Arrows indicate large vesicular structures that contain cytoplasmic content, consistent with being autolysosomes. Scale bars: 1 μm.

(B) Fibroblasts were incubated with BSA-gold (10 nm) for 24 h and washed and then the gold was chased overnight to accumulate in lysosomal compartments. Cells were then studied by transmission electron microscopy. Images of untreated fibroblasts are found on the left (Ctrl) whereas images on the right side (24 h) represent fibroblasts stained with BSA-gold. In BJ control fibroblasts, most of the gold tracer is found in uniformly dense lysosomes. In patient-derived cells, the gold is found in the dense part of bigger lysosomal compartments, which mostly consist of an electron-lucent lumen. This phenotype is similar for the various patient-derived fibroblasts. Scale bars: 1 μm.

(C) Fibroblasts derived from individual A2 or control individuals were left untreated of incubated with Bafilomycin A1 and then fixed and processed for immunofluorescence confocal microscopy with antibodies against LC3 and LAMP1. Co-localization between the two markers after Bafilomycin A1 treatment is indicative of autolysosomes. Scale bar: 10 μm.