Figure 4

Pompe disease fibroblasts show abnormal intracellular localization of CI-MPR and reduced availability at the plasma membrane. (A) Immunofluorescence analysis of CI-MPR steady-state distribution in control and Pompe disease (PD) fibroblasts. The cells were grown on coverslips, fixed, permealized and stained with a mouse monoclonal antibody to CI-MPR. In intermediate and severe PD fibroblasts 55% and 75% of cells respectively showed reduced or mislocalized signal (right panel). Quantitative analysis was performed on 40 cells. (B) Co-staining of CI-MPR and TGN46. Cells were treated as above, stained with Alexa 594-conjugated donkey anti-mouse IgG (red channel) and Alexa 488-conjugated donkey anti-rabbit IgG (green channel) and visualized by confocal fluorescence microscopy. The right columns show the merged images of double staining of CI-MPR and TGN46. Intermediate and severe PD fibroblasts show abnormal distribution of both markers. (C) FACS analysis of CI-MPR expression at the plasma membrane of control and PD fibroblasts. Cells were incubated in ice with an anti-CI-MPR and a secondary antibody labelled with phycoerythrin (PE). The rate of cells expressing CI-MPR at the plasma membrane in PD fibroblasts is reduced in PD fibroblasts compared with controls and correlates with disease severity. The percentage is calculated by assuming that in control fibroblasts the rate is 100%. The data are representative of two independent experiments.