Figure 2.

GM1 measurement in CLN7 knockout cells and patient fibroblasts

(A) Flow cytometric quantification of GM1 expression in HEK293T wt and ko cells (line 99-4) using fluorescence-coupled cholera toxin, subunit B. CLN7-deficient cells exhibited a 87% reduction in GM1 expression.

(B) GM1 measurement in fibroblasts from a healthy individual (Control) and from three patients with CLN7. Here, quantification was performed using a fluorescence microscope. Consistent with the experiment on HEK293T cells, cells from patients exhibited a 60% reduction in GM1 expression. ∗ Significant compared to wild-type cells in a) and compared to fibroblasts from a healthy individual in b).

(C) GM1 staining (green) on fixed CLN7 wt (top row) and ko cells (line 94-5 middle row, line 99-4 bottom row). F-actin was also stained (red) to visualize the cells. The last image of a row shows the merged image. Microscopic evaluation of the cells also showed that CLN7 ko cells expressed significantly less GM1 in their cell membrane. Scale bar: 20 μm.

(D) GM1 staining (green) of human fibroblasts from a healthy individual (top row) and from three patients with CLN7 (bottom three rows). As in the HEK293T cell model, GM1 expression was reduced by approximately 33% in patients. Nuclei are shown in blue. Scale bars of left column: 200 μm, right column: 50 μm.