Figure 1

Pompe disease fibroblasts show expansion of the autophagic compartment. A) Electron microscopy of control and juvenile human fibroblasts (left panel) and intermediate and severe human fibroblasts (right panel). The latter two cell lines show increased number of multivesicular bodies (black asterisks) and of autophagosomes or autolysosomes (white asterisks) characterized by double membranes (black arrows) and cytoplasmic content. Also evident is the glycogen accumulation in Pompe disease (PD) cells (white arrows). Lysosomes are indicated. Bars 200 nm. (B) LC3 staining in control and PD fibroblasts grown in complete medium (left panel) or starved in amino acid-free medium for 2 hours (right panel). Magnification 63×. Cells were fixed, permeabilized and immunostained with a polyclonal antibody to endogenous LC3 and visualized by fluorescence microscopy. LC3 staining shows multiple vesicles in intermediate and severe PD patients consistent with autophagosomes. Insets show high magnification views of autophagosomes. (C) Western blot analysis of LC3 in control and PD fibroblasts. Lanes 1 to 4: cells grown in complete medium; lanes 5 to 8: cells starved in amino acid-free medium for 2 hours. Quantitative analysis by Image J of LC3-II peptides, normalized as ratio of LC3-II/β-actin (top) and LC3-II/LC3-I (bottom), showed that intermediate and severe PD fibroblasts have increased amounts of LC3-II as compared with control and juvenile PD fibroblasts.