Figure 9.

TNF-induced A-SMase processing and activation, ceramide production, cathepsin D activation and apoptosis in caspase-7 knockout MEFs. (A, B) Wild-type MEFs or caspase-7 knockout MEFs were incubated with TNF for the indicated times and analysed by western blot and A-SMase activity assays. While in wild-type MEFs cleavage of endogenous pro-A-SMase and activation of A-SMase is observed, both events are completely blocked in caspase-7 knockout MEFs. (C) Wild-type MEFs or caspase-7 knockout MEFs were treated with TNF for indicated times and the production of ceramide analysed by TLC. TNF-induced ceramide production in caspase-7 knockout MEFs is completely prevented in contrast to wild-type MEFs. (D) Wild-type MEFs or caspase-7 knockout MEFs were treated with TNF for 12 h and analysed for expression of active 32 kDa cathepsin D protein. Caspase-7 deficiency in caspase-7 knockout MEFs prevents TNF induction of cathepsin D processing, which can be detected in wild-type MEFs. (E) Wild-type MEFs or caspase-7 knockout MEFs treated with CHX and TNF for 24 h were analysed for apoptosis by annexin-V/propidium iodide staining.