Fig. 1: Induction of apoptosis and necroptosis in L929sAhFas cells by anti-Fas antibodies and mTNF, respectively.

A An overview of extrinsic apoptosis is shown schematically, where the added anti-Fas antibody binds to the anti-Fas receptor, activating Fas associated death domain (FADD). This in turn activates the caspase cascade. Starting at caspase 8 and 10 activation and resulting in activation of caspase 3 and 7 and in turn apoptosis. B An overview of induction of necroptosis by mTNF which binds to the mTNF receptor 1 and activates the intracellular TNF receptor associated death domain (TRADD). Activation of TRADD results in activation of RIPK1 and RIPK3, with the latter being responsible for the formation of the necrosome. The necrosome in turn phosphorylates MLKL (mixed lineage kinase domain like pseudokinase) to create a pore forming complex in the membrane and thus leading to necroptotic cell death. C, D Induction of regulated cell death in L929sAhFas cells using anti-Fas and mTNF analyzed by fluorescence microscopy. A representative fluorescent image of L929sAhFas cells induced to die by apoptosis (C, 125 ng/mL anti-Fas) or necroptosis (D, 250 ng/mL mTNF). The following fluorescence stainings are used in these images: Hoechst 33342 (nuclei), propidium iodide (PI, staining of the nuclei after cell membrane rupture) and annexin-V (phosphatidylserine (PS) exposure). PS exposure happens in early apoptosis and can be externalized in necroptosis. Thus cannot be used to differentiate between cell death types 47. Zooms of both conditions clearly show regulated cell death specific features, as membrane blebbing in apoptosis and swelling of the cell in necroptosis. E, F A quantification of cell death by fluorescent microscopy over time. Images were obtained after induction of each experiment and relative PI exposure (and thus percentage of cell death) was measured over time. Clearly the induction of both apoptosis and necroptosis (using anti-Fas and mTNF, respectively) work efficiently. The inhibitors for both apoptosis (10 μM of zVAD-fmk, pan-caspase inhibitor) as necroptosis (10 μM of Nec-1s) have been used. Blocking caspase activity using zVAD-fmk, after induction of apoptosis using anti-Fas, will lead to a switch to necroptosis and thus an increase in cell death 38,39 which can be further blocked by Nec-1s.