The uptake of rhGAA is defective in fibroblasts from severe and intermediate Pompe disease patients. (A) Pompe disease (PD) fibroblasts were incubated for 24 hours with different concentrations of rhGAA (0.5, 5, 50 μg/ml) in the medium and GAA activity was assayed. Correction of GAA activity in severe and intermediate fibroblasts from patients 1 to 3 is less efficient as compared with juvenile fibroblasts from patients 4 and 5. (B) Uptake of Alexa Fluor 546-labeled rhGAA in control and PD fibroblasts. The cells were grown on coverslips and were incubated with fluorescent rhGAA for different times (2, 4 and 8 hours), washed, fixed and visualized by fluorescence microscopy. The images show the results obtained after 2 hours of incubation. In severe PD fibroblasts a diffuse staining of labelled rhGAA is observed, in contrast with the granular pattern typical of lysosomal staining in control fibroblasts. Magnification 63×. (C) Western-blot analysis of GAA polypeptides shows reduced processing of rhGAA precursor into the 76 kDa mature enzyme, confirming impaired transport of rhGAA to the lysosomal compartment. Lysates from untreated control fibroblasts and control and PD fibroblasts treated with rhGAA for 4 hours were analyzed. (D) ImageJ quantitative analysis of the rhGAA molecular forms. The intensity of bands is normalized as ratio of rhGAA/β-actin.