Figure 3.

Disulfiram selectively increases ADAM10 expression and leads to decrease in A-beta production. SH-SY5Y cells were incubated with disulfiram at a concentration of 2.2 µM, or DMSO as a solvent-control (values set to 100%) for 48 h. (a) The cell lysate was used for measurement of ADAM10 activity by using a pro-fluorescent specific substrate. Additionally, aliquots of cell lysate were subjected to SDS-PAGE and Western blotting for analysis of levels of TACE and BACE-1. GAPDH served as a loading control. Soluble proteins from cell supernatants after 5 h of secretion were precipitated by trichloroacetic acid and subjected to SDS-PAGE and Western blotting with a sAPP-beta specific antibody (b). All measured densitometric values were normalized to values obtained for GAPDH of the respective sample (c) (n ≥ 5 for all quantifications). For TACE, the sum of mature and proform was quantified. Cropped blot pictures are separated by a white space, for full-length blot pictures please see Suppl. Figure 2. A-beta peptide secretion (A-beta 1-x) was measured by ELISA in samples from a 16 h secretion period (d). Statistical analysis: unpaired Student’s t-test (***p < 0.001; *p < 0.05; ns, p > 0.05). (e) Cells treated for 48 h with solvent (control), human A-beta₄₂ peptides or peptides in combination with disulfiram (A-beta/Dis) were analyzed for viability (n = 15, two independent experiments). Statistical analysis: One Way ANOVA **p < 0.01; ***p < 0.001).