Fig. 5

Flow cytometric analysis of apoptotic proteases. The activation of caspases 8, caspase 9, caspase 3 and cathepsin B were evaluated at 24, 48 and 72 h of Pyr (8 μg/ml) and MBP (0.8, 8 μg/ml) treatment on Mel501 cells. Both caspases (8, 9 and 3) and cathepsin B are involved in MBP induced apoptosis. Flow cytometric data showed: i) a significant increase of caspase-8 activity starting from 24 h, reaching a peak at 48 and the plateau at 72 h (a); ii) activation of caspase-9 and caspase-3 after 48 and 72 h treatment (b and c,); iii) a significant increase of expression cathepsin B starting from 48 h, which persisted at later time points (72 h) (d), after MBP treatment (0.8 μg/ml). The higher dose of MBP (8 μg/ml) did not induce more relevant effects on the above proteases. Columns, mean values of three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01; significance compared with untreated cells