Figure 5. Analysis of autophagic flux and formation of inclusion bodies containing ubiquitinated proteins.

(A) Primary fibroblasts were incubated with bovine serum albumin derivative (dye-conjugated [DQ] BSA) conjugated to a self-quenched fluorophore (10 µg/mL) for 1 hour at 37°C in complete culture medium and then left in a starvation medium for 5 and 16 hours to induce autophagy. Cells were mounted on coverslips and immediately analyzed by confocal microscopy. Dequenched DQ-BSA was not observed over the time period of 16 hours in patient cells. (B) Primary fibroblasts obtained from one of the 2 affected sibs (right) and an unaffected individual (left) were treated with puromycin (5 μg/mL) for 2 hours to induce formation of polyubiquitin-positive bodies. Cells were then fixed and stained with SQSTM1 (green) and FK1 (red) antibodies. Images are representative of 300 analyzed cells. In all panels, bars correspond to 40 μm.