Figure 6

Presence of mutant GCase increases apoptotic stimuli induced cleavage of caspase 3 and caspase 9 leading to cell death. (A) SHSY5Y cells, stably expressing WT or N370S mutant GCase, were treated with 1.2 μM of staurosporine for 3 hours, after which cell lysates were prepared and subjected to western blot analysis. The corresponding blot was interacted with anti-cleaved caspase 3 antibody or anti-cleaved caspase 9 antibody and anti-ERK antibodies, as a loading control. (B) To quantify the results, the blots were scanned and intensity of cleaved caspase 3 or cleaved caspase 9 at each lane was divided by the intensity of ERK 1/2 in the same lane. The value obtained for cells expressing WT GCase was considered as 1. The results represent the mean ± SEM of five independent experiments. *P < 0.05, *P < 0.02 in Student t-test. (C) SHSY5Y cells, stably expressing either WT or N370S or L444P mutant GCase variants or naïve SHSY5Y cells (control), were treated with 1.2 μM of staurosporine for 3 hours or 1% hydrogen peroxide (H₂O₂) for 1 hour after which cells were subjected to XTT colorimetric assay (Biological industries, Beit Haemek, Israel). The results represent the mean ± SEM of three to five independent experiments. ***P < 0.001 in Student t-test.