Tumor necrosis factor-α (TNF) can induce either apoptosis or programmed necrosis (necroptosis) when different cell lines are used 1, 2. While receptor-interacting protein 1 (RIP1) can participate in signaling for both apoptosis and necrosis 3, 4, recent studies by us and by others have shown that RIP3 is essential for TNF-induced necroptosis 5, 6, 7 and has no role in TNF-induced apoptosis 6, 7. Classification of TNF-induced cell death in a number of cell lines, such as L929 and murine embryonic fibroblasts (MEFs), has been made based on morphological and/or biochemical parameters 2, 7, 8, 9, 10. It is noteworthy that some apoptotic features, such as testing positive for Annexin V, can also be found in cells that show features of necrosis 11, and, therefore, only major parameters such as caspase dependence can be used to distinguish apoptosis from necrosis. The pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD) has been used to determine the type of cell death induced by TNF (e.g., apoptosis can be inhibited by inhibition of caspases but necroptosis cannot) 4, 12, 13. In response to TNF treatment, the fibroblast cell lines – the N line of NIH3T3, the A line of NIH3T3, L929 and MEF cells – showed differences in the caspase dependence of their cell death 2, 7, 8, 9, 10. To obtain an overall picture of the pathways that mediate TNF-induced cell death signaling, we need to know the caspase dependence of the cell death occurring in these cell lines under different conditions, and the dependence of RIP1 and/or RIP3 in these cell death pathways.

RIP1 is known to play a crucial role in TNF-induced apoptosis 3. TNF treatment in the A line of NIH3T3 cells induces caspase-dependent apoptosis, and expression of RIP1 causes caspase-dependent cell death in the A line of NIH3T3 cells (Supplementary information, Figures S1A, S1B and S2A), supporting the role of RIP1 in apoptosis. However, there must also be a RIP1-independent apoptotic pathway, since TNF+CHX can induce RIP1-independent apoptosis in an immortalized RIP1−/− cell line (named as MEF-ZG line in our figures; Lin et al. 4 and Supplementary information, Figure S2B and S2C). For an unknown reason, both RIP1 and RIP3 were undetectable in this cell line. This cell line is abnormal in comparison with other immortalized MEF lines, in that it is ∼100-fold more sensitive to TNF+CHX-induced cell death, although it cannot be killed by TNF alone. Nevertheless, the data obtained using this cell line demonstrate that there is a RIP1-independent apoptotic pathway.

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