could be explained by incomplete silencing of RIP1, it is unlikely because zVAD-fmk induced necrosis was efficiently inhibited in the same cells. Interestingly, residual death could be detected in RIP1-silenced L929 cells treated with TNF, zVAD-fmk and Nec- 1. This result suggests the possibility that caspase- and RIP1- independent cell death might occur when both apoptosis and necrosis pathways are inhibited. Nec-1 modestly reduced expression of PKA-C1, but had little effects on Jnk or Erk activation. The effect of Nec-1 on PKA-C1 is intriguing because PKA-C1 was identified as a putative necrosis mediator previously [6,35]. Based on our findings, the anti- necrosis effect of Nec-1 previously attributed to RIP1 might have to be re-evaluated. Interestingly, low doses of Nec-1 (1 mM) were sufficient to rescue necrosis in FADD2/2 T cells. In contrast, higher doses of Nec-1 (20 mM) were required to fully rescue TNF- induced necrosis in L929 cells. The molecular basis for this differential sensitivity to Nec-1 is unknown, but could be due to different RIP1 expression level or Nec-1 inhibition of other necrosis mediators in L929 cells. Besides its effect on necrosis, Nec-1 also interferes with proximal TCR signaling. Importantly, the inhibition of TCR signaling and proliferation was only apparent at higher concentrations of Nec-1 that were commonly used in studies assessing the role of RIP1- dependent necrosis in different cells [9,23]. Nec-1 mediated inhibition of T cell proliferation was not reported in these studies. However, we have observed similar inhibition of T cell References 1. Moquin D, Chan FK (2010) The molecular regulation of programmed necrotic cell injury. Trends Biochem Sci 35: 434–441. 2. Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G (2010) Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol 11: 700–714. 3. 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