Figure 3

Molecular effects of γ-secretase inhibition on signalling proteins involved in regulating cell growth or death. CCK-81 cells were either left untreated (0), treated with DMSO as a control (DM) or incubated with the γ-secretase inhibitors as indicated. Cells were lysed in RIPA buffer and 50 μg of total protein was subjected to immunoblotting analysis as specified. Both inhibitors result in the down-regulation of Val1744-NICD, which is paralleled by a loss of Hes1 expression, an effect that is evident already after 3 to 6 h. Cleaved PARP, an indicator of cell death, was also analysed and more prominent with L-685,458, which induces cell killing in CCK-81 by an unknown mechanism (Table 1). With both GSI, an increase in pErk (pT202pY204) and pAkt (pS473) is evident. In addition, a downregulation of anti-apoptotic Bcl-2 protein was detectable, to a moderate degree by DBZ and more pronounced with L-685458. Actin was analysed as a loading control.