Fig. 3.

Increased lysosomal G_M2 and reduced G_M3 ganglioside in the patient's cultured fibroblasts.

(A) Fibroblasts from the patient and two normal healthy controls cultured on glass coverslips were fixed and stained with monoclonal humanized anti-G_M2 and mouse anti-LAMP-2 antibodies followed by anti-human IgG Alexa 488-labeled (green) and anti-mouse IgG Alexa 555-labeled (red) secondary antibodies. Nuclei were stained with Draq5 (blue). The patient's fibroblasts show a higher intensity of G_M2 staining (green) than control fibroblasts and increased co-localization of G_M2 and LAMP-2 staining.

(B) Fibroblasts from the patient and two normal healthy controls were stained with monoclonal mouse anti-G_M3 antibody followed by anti-mouse IgG Alexa-555-labeled secondary antibody (red). Nuclei were stained with Draq5 (blue).

The images were acquired in a Leica SP8 confocal microscope with a 63 × objective. Bar represents 10 μM. The panels show representative images of at least 40 studied for each cell type.

(C) Mean G_M2 and G_M3 staining intensity and a fraction of G_M2-positive cells in control and patient's fibroblasts. Mean staining intensities per μm² were measured with ImageJ software. Data show mean (± SEM) of individual values measured for 35 randomly selected cells. **P < 0.01, *P < 0.05 in unpaired two-tailed t-test. G_M2-positive cells were manually counted in three randomly selected microscope fields (~ 150 cells each). Data show mean values (± SEM) of 3 independent experiments. *P < 0.05 in unpaired two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)