specifically inhibits the pro-necrotic kinase function of RIP1 [18], but has no effects on another pro-necrotic kinase RIP3 [6]. Moreover, Nec-1 does not interfere with TNF-induced NF-kappa B activation [15], an effect that is also driven by RIP1. Because of its relatively specific effect against the pro-necrotic activity of RIP1, Nec-1 has become a popular tool to probe the role of necrosis in different experimental models of cell injury. In this report, we show that Nec-1 inhibits necrosis in a RIP1- independent manner in L929 cells. In addition, at doses that are commonly used in many studies, Nec-1 impairs the sustained phosphorylation of linker for activation of T cells (LAT), a crucial adaptor in proximal T cell receptor (TCR) signaling. As a result, Nec-1 treated cells failed to expand and proliferate in response to TCR stimulation. These results reveal that Nec-1 exhibits RIP1- dependent and independent effects in necrosis and other signal transduction processes. Thus, care should be taken in interpreting results derived from using this inhibitor. Materials and Methods Ethics Statement All studies involving animals were approved by the University of Massachusetts Institutional Animal Care and Use Committee (IACUC) (protocol #1396). Animals were maintained in accor- dance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996). Cell death assays L929 and NIH 3T3 cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle medium. Jurkat cells [19,20] and primary T cells isolated from wild type and FADD2/2 mice [21] were cultured in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin, 30 mg/ml L-glutamine, and 60 mM 2- mercaptoethanol. For cell death assays, cells were seeded at 5,000– 10,000 cells per well on 96-well microtiter plates and stimulated as indicated. For L929 and NIH 3T3 cells, 10 ng/ml of recombinant mouse TNF was used (Invitrogen, Carlsbad, CA, USA). For NIH 3T3 cells were pre-treated with 0.5 mg/ml cycloheximide and 10 mM zVAD-fmk (Axxora, San Diego, CA, USA) for one hour before TNF stimulation. For Jurkat cells, necrosis was induced by pretreatment with 20 mM zVAD-fmk for 1 hour and 100 ng/ml human TNF (Invitrogen, Carlsbad, CA, USA). MAP kinase inhibitors (Enzo Life Sciences, Farmingdale, NY, USA) were used at the following concentrations: Erk inhibitor U0126 (50 mM), Jnk inhibitor II (30 mM), p38 inhibitor SB203580 (50 mM). Cell death was determined 6–14 hours post-TNF treatment by CellTiter aqueous non-radioactive cell proliferation assay (MTS) (Promega, Madison, WI, USA). In some experiments, we confirmed cell death by flow cytometry using propidium iodide uptake as an indication of cell death (data not shown). siRNA transfection Short oligonucleotide RNAs (27-mer siRNA) were purchased from IDT technologies (Coralville, IA, USA). For transfection of siRNA to L929 cells or NIH 3T3 cells, 300,000 cells were seeded on 6-well plate and transfected with 20 nM siRNA and 12 ml HiPerfect reagent (Qiagen, Valencia, CA, USA). Thirty-six hours after transfection, cells were replated on 96-well plate (5,000 cells per well) overnight before stimulation with recombinant mouse TNF. siRNA sequences used are: RIP1 #1 (59-gaggauauucucagg- cuucagguccuu-39), RIP1 #2 (59-ccuucguuuccuuuccuccucucuguu- 39), RIP3 (59-aagauuaaccauagccuucaccuccca-39). RIP1-Independent Effects of Necrostatin-1 Western blot Cell lysates or immune complexes were analyzed by standard SDS-PAGE. After transfer to nitrocellulose membranes, Western blots were performed with antibodies to RIP1, 1-actin, PARP-1 (BD Pharmingen, San Diego, CA, USA), RIP3 (ProSci, Poway, CA, USA), Erk, Jnk, PKA substrate (Cell Signaling, Danvers, MA, USA), PKA, LAT (Santa Cruz, Santa Cruz, CA, USA), and phospho-tyrosine (Millipore, Bellerica, MA, USA). Fluorogenic caspase 3 activity assay Fifty micrograms of cell extracts were diluted in 200 ml of iTFB buffer (10% sucrose, 30 mM HEPES [pH 7.4], 10 mM CaCl2 and 5 mM DTT) containing 25 mM of N-acetyl-Asp-Glu-Val-Asp-7- amino-4-methylcoumarin (Ac-DEVD-AMC) (Axxora, San Diego, CA, USA). The release of fluorescent AMC was measured in a FLUOSTAR multi-well plate reader at 340 nm for excitation and 450 nm for emission over 3 hours. The rate of conversion was calculated by including 25 mM of free AMC as control. T cell activation CD3+ T cells were purified from the spleens of 10–12 week old C57BL/6 mice using the Dynal negative T cell isolation kit (Invitrogen). Flow cytometry shows that the resulting cells were .98% CD3+ (data not shown). Unless otherwise stated, purified T cells were stimulated with plate-bound 1 mg/ml anti-CD3 and 200 ng/ml anti-CD28 antibodies (eBioscience, San Diego, CA, USA). For thymidine incorporation, 1 mCi of [3H]-thymidine was added to the cells on day 3 for 18 hours before harvesting. For CellTracer Violet dilution assay, cells were loaded with 5 mM CellTracer Violet as per manufacturer’s protocol (Invitrogen). Three days after stimulation, dilution of the dye was determined on a BD LSR2 flow cytometer and analyzed with Flowjo software (Treestar, Ashland, OR, USA). For Western blot and immuno- precipitations of T cell lysates, purified T cells were stimulated with 3 mg/ml biotinylated anti-CD3 antibody and 0.5 mg/ml streptavidin for the indicated times before lysis in buffer containing 150 mM NaCl, 20 mM Tris.Cl [pH 7.4], 0.2% NP-40, 1 mM EDTA, 3 mM NaF, 1 mM b-glycerophosphate, 1 mM sodium orthovanadate, 10% glycerol, 16 protease inhibitor cocktail (Roche), and 16 phosphatase inhibitor cocktail (Sigma). Results The murine fibrosarcoma L929 cells are unique in that they readily undergo necrosis in response to TNF [22]. TNF-induced necrosis in L929 cells did not require caspase inhibition, although the caspase inhibitor zVAD-fmk did further enhance TNF- induced necrosis (Fig. 1A). Regardless of whether zVAD-fmk was used, Nec-1 inhibited TNF-induced necrosis in L929 cells in a dose-dependent manner, reaching maximal inhibition at 20 mM or higher (Fig. 1B and data not shown). This dose of Nec-1 (20– 50 mM) was commonly used in many publications [9,15,18,23]. Although Nec-1 has been reported to inhibit the expression of ectopically expressed RIP1 in HEK293T cells [6], it did not affect endogenous RIP1 expression in L929 cells (Fig. 1C). These results are consistent with previous results that Nec-1 is an allosteric inhibitor of RIP1 kinase function [18]. In addition to exogenous TNF, treatment with the caspase inhibitor zVAD-fmk alone also causes necrosis in L929 cells [24]. zVAD-fmk induced necrosis in L929 cells is mediated by autocrine production of TNF. It exhibits a slower kinetics than TNF-induced necrosis and is optimally induced at high cell density and small volume of the culture medium [25]. Under these conditions, siRNA-mediated silencing of RIP1 by two different siRNAs PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23209