RIP1-Independent Effects of Necrostatin-1 Figure 1. Nec-1 inhibits necrosis in L929 cells in a dose-dependent manner. (A) Nec-1 inhibits TNF-induced necrosis in L929 cells. L929 cells were treated with 10 mM zVAD-fmk, 20 mM Nec-1 and 10 ng/ml mouse TNF (mTNF) as indicated. Cell death was determined by MTS assay as described in materials and methods. (B) L929 cells were treated with the indicated doses of Nec-1, followed by stimulation with 10 ng/ml mTNF. Cell Death was analyzed as in (A). (C) Nec-1 did not alter RIP1 or RIP3 protein expression in L929 cells. doi:10.1371/journal.pone.0023209.g001 efficiently protected L929 cells from zVAD-fmk induced necrosis (Fig. 2A). This is consistent with previous reports that RIP1 is required for necrosis induced by zVAD-fmk [24]. Strikingly, siRNA-mediated silencing of RIP1 had no effects on TNF-induced necrosis in L929 cells (Fig. 2B). Western blot analysis shows that the lack of protection against TNF-induced necrosis in L929 cells Figure 2. Nec-1 inhibits TNF-induced necrosis independent of RIP1 in L929 cells. (A–C) RIP1 is required for zVAD-fmk, but not TNF-induced necrosis in L929 cells. (A) L929 cells were transfected with the indicated siRNA oligonucleotides with HiPerfect (Qiagen). TR4 is the control siRNA against TRAIL-R4 [36]. Cells were treated with (A) 50 mM zVAD-fmk or (B) 10 ng/ml mTNF and cell death was measured by MTS assay. (C) Western blot shows RIP1 and RIP3 expression in cells transfected with the indicated siRNAs. (D–E) RIP1 is required for TNF-induced necrosis in 3T3 fibroblasts. NIH 3T3 fibroblasts were transfected with the indicated siRNA. (D) Expression of RIP1, RIP3 and ß-actin in cells transfected with the indicated siRNA was determined by Western blot. (E) Necrosis was induced with 10 mM zVAD-fmk, 0.5 mg/ml cycloheximide and 10 ng/ml mTNF for 20 hours. Cell death was assessed as in (A). doi:10.1371/journal.pone.0023209.g002 PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23209