was not due to inefficient silencing of RIP1 expression (Fig. 2C). By contrast, similar siRNA-mediated silencing of RIP1 in NIH 3T3 fibroblasts, which was less efficient compared with that achieved in L929 cells (Fig. 2D), effectively inhibited TNF-induced necrosis (Fig. 2E). Furthermore, siRNA-mediated silencing of another essential programmed necrosis mediator, RIP3, effectively inhibited zVAD-fmk and TNF-induced necrosis in L929 cells (Fig. 2A–B), and TNF-induced necrosis in NIH 3T3 cells (Fig. 2E). Our results are consistent with those of Hitomi et al, which show that siRNA knock-down of RIP1 in L929 cells exhibited disparate effects on necrosis induced by zVAD-fmk or TNF [26]. Inhibition of the pro-survival transcription factor NF-kappa B sensitizes cells to TNF-induced apoptosis. Because RIP1 is required for activation of NF-kappa B, knock-down of RIP1 could have sensitized L929 cells to TNF-induced apoptosis. In addition, RIP1 competes with TRADD for binding to TNFR1 [27]. Since TRADD promotes FADD recruitment and apoptosis, loss of RIP1 could activate caspase-dependent apoptosis. To evaluate this possibility, we treated RIP1-silenced L929 cells with the pan-caspase inhibitor zVAD-fmk and/or the RIP1 inhibitor Nec-1. Consistent with previous results, zVAD-fmk increased TNF-induced necrosis in RIP1 siRNA (Fig. 3A) and control siRNA transfected cells (Fig. 3B). In contrast, Nec-1 effectively inhibited TNF-induced necrosis in RIP1-Independent Effects of Necrostatin-1 RIP1 siRNA (Fig. 3A) and control siRNA transfected cells (Fig. 3B). Nec-1 also inhibited cell death induced by TNF and zVAD-fmk in RIP1 siRNA transfected cells (Fig. 3A). Interestingly, residual death could be detected in RIP1 knock-down L929 cells treated with TNF, zVAD-fmk, and Nec-1, suggesting the possibility of caspase- and RIP1-independent cell death under these conditions. PARP-1 cleavage by caspases was not detected in RIP1 siRNA transfected L929 cells, but was readily detected in TNF-treated Jurkat cells (Fig. 3C). Furthermore, caspase activity as measured by cleavage of the fluorogenic peptide substrate DEVD-AMC was not detected in RIP1 or control siRNA transfected L929 cells, but was readily detected in TNF-treated Jurkat cells (Fig. 3D). Thus, TNF-induced necrosis in L929 cells is RIP1-independent and yet inhibitable by Nec-1. A recent report indicates that Nec-1 inhibits Erk activation in TNF and Smac mimetic treated L929 cells [28]. Although the authors found that Erk inhibition did not protect L929 cells against TNF/Smac mimetic induced necrosis [28], we found that the Erk inhibitor U0126 inhibited TNF-induced necrosis in L929 cells, albeit at a lower level than that by Nec-1 (Fig. 4A). Surprisingly, phospho-Erk was readily detected in untreated L929 cells, and TNF treatment led to reduction in phosphorylated Erk (Fig. 4B, top two panels). By contrast, TNF-induced a transient phosphor- Figure 3. Knock-down of RIP1 expression in L929 cells did not result in caspase-dependent apoptosis. L929 cells were transfected with siRNA against (A) RIP1 or (B) TR4. Transfected cells were treated with zVAD-fmk, Nec-1 and TNF as indicated. Cell death was determined by MTS assay. (C) L929 cells transfected with the indicated siRNA or Jurkat 4E3 cells were treated with TNF. PARP-1 cleavage was determined by Western blot. (D) Cell lysates from (C) were tested for active caspase 3 as measured by cleavage of the substrate DEVD-AMC. Release of AMC was determined by increases in AMC fluorescence as described in materials and methods. doi:10.1371/journal.pone.0023209.g003 PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23209