CI-MPR co-localizes partially in LC3 positive vesicle in Pompe disease fibroblasts. (A) Steady-state analysis of CI-MPR and LC3 positive vesicles shows co-localization in severe and intermediate Pompe disease (PD) fibroblasts. The cells were fixed, permeabilized and stained with a mouse monoclonal antibody to CI-MPR and a rabbit polyclonal antibody to LC3 and Alexa 594-conjugated donkey anti-mouse IgG (red channel) and Alexa 488-conjugated donkey anti-rabbit IgG (green channel) antibodies. Stained cells were visualized by confocal fluorescence microscopy. The right columns show the merged images of double staining of CI-MPR and LC3. Magnification 63×. Insets show high magnification views. (B) Quantitative analysis of CI-MPR and LC3 co-localization after internalization of CI-MPR. The cells grown on coverslips were incubated at 18°C for 1 hour with 15 ng/ml Alexa 488-anti-CI-MPR monoclonal antibody. The cells were washed and fixed (0 min) or warmed to 37°C for 5, 15, 30 minutes (as indicated) before being fixed, permeabilized and stained with anti-LC3. The rate of CI-MPR/LC3 co-localization was determined using performed by LSM 3.2 software (Zeiss). The analysis was performed on at least 20 cells per sample. In severe PD fibroblasts, co-localization is higher as compared with control and CI-MPR, indicating sequestration of CI-MPR in autophagosomes.