purified murine CD3+ T cells with plate-bound anti-CD3 antibody, which mimics TCR signaling, and the costimulatory molecule CD28 potently stimulated cell proliferation as shown by the dilution of the CellTracer Violet dye (Fig. 5C, panel a). At doses that are commonly used in other studies (30 mM), Nec-1 severely impeded cell division (Fig. 5C, compare panels a and e). In fact, cell division was completely abrogated by 50 mM of Nec-1 (Fig. 5C, panel f). Importantly, lower doses of Nec-1 (1 mM) that were sufficient to inhibit FADD2/2 T cell necrosis had little effect on T cell division (Fig. 5C, panel b). In addition to cell proliferation, T cell activation also leads to increase in cell size, which can be measured as an increase in forward light scatter by flow cytometry. Consistent with the cell proliferation results, Nec-1 inhibited the formation of ‘‘T cell blasts’’ in response to TCR/ CD28 stimulation in a dose-dependent manner (Fig. 5D). RIP12/2 T cells exhibit a modest defect in T cell proliferation [32]. Thus, the effect of Nec-1 on T cell proliferation could be due to inhibition of RIP1. To evaluate this possibility, we examined the effect of Nec-1 on FADD2/2RIP12/2 double knock-out (DKO) T cells. Unlike the FADD2/2 or RIP12/2 T cells, the DKO T cells exhibited normal T cell proliferation [10]. We found that proliferation of DKO T cells was similarly inhibited by high doses of Nec-1 (Fig. 5E). Taken together, these results indicate that Nec- 1 inhibits T cell proliferation independent of RIP1. The effect of Nec-1 on T cell proliferation suggests that it might target proximal signaling of the TCR. A signature of proximal TCR signaling is the rapid phosphorylation at tyrosine residues for many adaptor proteins and kinases. Western blot with phospho- tyrosine antibody showed that the phosphorylation of several protein species was differentially affected by Nec-1. In particular, the TCR-induced phosphorylation of a 30–40 kDa species that resembled the size of the essential adaptor LAT (linker for activation of T cells) was visibly reduced by Nec-1 (Fig. 6A, right panel). To confirm the effect of Nec-1 on LAT phosphorylation, we immunoprecipitated the T cell lysates with the phospho- tyrosine antibody, followed by Western blot analysis of LAT. We found that while Nec-1 did not dramatically affect the initial phosphorylation of LAT, it abolished sustained LAT phosphor- ylation (Fig. 6B, compare lanes 3 and 6). In contrast, phosphor- ylation of Lck, an upstream tyrosine kinase that is phosphorylated RIP1-Independent Effects of Necrostatin-1 at the inhibitory tyrosine Y505 before TCR activation and Y394 after TCR stimulation [33], was not affected by Nec-1 (Fig. 6B). Furthermore, phosphorylation of Erk was not diminished by Nec- 1 (Fig. 6C). Thus, we conclude that the abrogation of LAT phosphorylation is one of the possible mechanisms by which Nec-1 inhibits T cell proliferation. Discussion The emerging role of RIP1 and RIP3-dependent necrosis in many disease pathologies have led to the wide use of Nec-1 and its derivatives by many investigators. Nec-1 was thought to specifically inhibit the pro-necrotic kinase function of RIP1 [15,18] without affecting the kinase activity of RIP3 [6]. In this report, we show that Nec-1 exhibits an unexpected anti-necrotic effect that is independent of RIP1. In L929 cells, necrosis could be induced by treatment with zVAD-fmk or exogenous TNF. However, our data indicated that zVAD-fmk induced necrosis, but not TNF-induced necrosis, was dependent on RIP1. This is surprising because a recent report shows that zVAD-fmk triggers autocrine TNF production that in turn activates necrosis downstream of TNFR1 [25]. The requirement for RIP1 in zVAD-fmk induced necrosis might be explained by its effect on MAP kinase activation [29]. Furthermore, a recent report shows that zVAD-fmk activated a PKC-MAPK signaling axis that is responsible for autocrine production of TNF in L929 cells [25]. Thus, we speculate that the requirement for RIP1 in zVAD-fmk induced necrosis might be attributed to its role in MAP kinase induced TNF production. In addition to TNF-induced necrosis in L929 cells, it is noteworthy that RIP1-independent but RIP3-dependent necrosis has also been observed in murine cytomegalovirus (MCMV) infection [34]. Therefore, although RIP1 is the upstream activator of RIP3 in most cases, necrosis can proceed in a RIP1- independent manner. Whether RIP3 acts alone or in concert with another RIP1-like factor in these situations will be the subject of further investigation. Importantly, the death of L929 cells with knocked-down expression of RIP1 was inhibited by Nec-1, but not zVAD-fmk. This result rules out the possibility of conversion of necrosis to apoptosis upon RIP1 silencing. While these results Figure 6. Nec-1 blocks sustained phosphorylation of LAT during T cell activation. (A) Whole cell extracts (WCE) were examined from T cells stimulated with anti-CD3 antibody in the presence or absence of 50 mM Nec-1. Western blot was performed with phospho-tyrosine specific antibody. Short and long exposures were shown to highlight the different phosphorylated species. (B) Nec-1 impairs TCR-induced LAT phosphorylation. Total lysates from activated T cells were subjected to immunoprecipitation with phospho-tyrosine antibody. The resulting immune complex was evaluated for presence of LAT and Lck by Western blot. (C) Nec-1 does not affect Erk phosphorylation. Purified T cells were stimulated as in (A). Western blot was performed with phospo-Erk or total Erk specific antibodies. doi:10.1371/journal.pone.0023209.g006 PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23209