The effect of 3-APS in neuronal cells. A) Expression of tau protein was determined by western blot in non differentiated (0 days) and after starting differentiation for four days (4) or after eight days (8) with 2 mM dibutyril cyclic cAMP (cAMP) in SH-SY5Y neuroblastoma cells. Actin was taken as a control of loading protein. B) Effect of 3-APS on undifferentiated SH-SY5Y cells. In the absence (control) of 3-APS, or in the presence of 100 μM 3-APS. Cells were stained with phalloidin to label actin cytoskeleton. C) Effect of 3-APS on differentiated SH-SY5Y cells. SH-SY5Y cells differentiated for 8 days were untreated (control) or treated with 100 μM 3-APS. The cells were stained with antibodies raised against tau protein and with phalloidin to label polymerized actin. D) In the presence of 3-APS there is an increase, in differentiated SH-SY5Y cells, in Th-S staining. Untreated, or cells treated with 100 μM 3-APS were stained with Th-S and visualized by an immunofluorescence microscopy (see methods). An increase in Th-S staining upon 3-APS treatment was found. E) Detergent insoluble tau aggregates were isolated from neuroblastoma cells untreated or treated with 100 μM and 500 μM 3-APS. The amount of tau in detergent insoluble material was measured by testing its reaction with tau antibody 7.51. Actin was used as a loading control (see methods). F) Th-S staining in hippocampal neurons treated with 3-APS. Primary cultures of hippocampal neurons were stained with Th-S in the absence (control) or the presence of 100μM 3-APS or 500 μM 3-APS. The quantitation of the fluorescent intensity is shown. (G) Effect of 3-APS in hippocampal neurons viability. Primary hippocampal cultures were incubated in the absence (control) or the presence of 100 μM, 500 μM and 1000 μM, for 24, 48 or 72 hours and the percentage of cell survival was determined.