RIP1-Independent Effects of Necrostatin-1 putative necrosis regulator in a RNA interference screen [6]. We therefore tested whether Nec-1 might affect PKA functions. Treatment of Jurkat 4E3 cells with Nec-1 led to reduced expression of PKA-C1 in a dose-dependent manner (Fig. 4E, lower panel). Although Nec-1 had minimal effects on calyculin A induced phosphorylation of PKA substrates (Fig. 4E, top panel), this could be due to the compensatory effects of PKA-Ca and other calyculin A sensitive kinases. Thus, PKA-C1 might be one of the RIP independent targets of Nec-1. In addition to inhibiting TNF-induced necrosis, Nec-1 has also been shown to rescue necrosis in caspase-82/2 [9] and FADD2/2 T cells [23] in response to T cell receptor (TCR) stimulation. Indeed, low doses of Nec-1 (,1 mM) were sufficient to inhibit necrosis and rescue defective proliferation of FADD2/2 T cells (Fig. 5A) [31]. Strikingly, we found that higher doses of Nec-1 (.20 mM) led to reduced proliferation of wild type and FADD2/2 T cells in a dose-dependent manner (Fig. 5A). The inhibition of T cell proliferation by high doses of Nec-1 might be due to inhibition of cell proliferation or increased cytotoxicity. Incubation of na¨ıve T cells in vitro without stimulation led to cell death by neglect. Consistently, the viability of untreated T cells 24 hours later was reduced to ,60% (Fig. 5B, left panel). Incubation of these na¨ıve T cells with 50 mM of Nec-1 did not further reduce the cell viability (Fig. 5B, right panel). Thus, the inhibitory effect of Nec-1 on T cell proliferation was not due to toxicity of the inhibitor. We next measured T cell proliferation by labeling cells with the fluorescent dye CellTracer Violet. CellTracer Violet partitions equally between daughter cells as the parent cell undergoes cell division. Thus, the reduction in fluorescence signal indicates the number of cell divisions the cells have undergone. Treatment of Figure 5. High doses of Nec-1 inhibit T cell proliferation. (A) Low dose Nec-1 inhibits TCR-induced necrosis, but high dose Nec-1 inhibits T cell proliferation. Purified T cells from wild type or FADD2/2 mice were stimulated with plate-bound anti-CD3, anti-CD28 and increasing doses of Nec-1. Three days later, cell proliferation was measured by incorporation of [3H]-thymidine. Results shown are mean 6 SEM. (B) Nec-1 did not compromised T cell viability. Na¨ıve CD3+ T cells were purified from the spleen of wild type C57BL/6 mice and incubated at 37uC for 24 hours in the presence or absence of 50 mM Nec-1. Viability of the cells was determined by flow cytometry using propidium iodide (PI) uptake as an indication of cell death. Note that Nec-1 increased the baseline fluorescence of the T cells. (C) Nec-1 inhibits T cell division. Purified CD3+ primary T cells were labeled with CellTracer Violet fluorescent dye and stimulated with 1 mg/ml plate-bound anti-CD3 and 200 ng/ml anti-CD28 antibodies. Three days later, cell division was analyzed using a BD LSR2 flow cytometer. The numbers above each peak represent the number of cell division the cells had undergone. The numbers on the left represent the percentages of cells in each peak. (D) Nec-1 inhibits T cell blast formation. Purified CD3+ T cells were similarly activated as in (C). Three days later, formation of T cell blast as measured by forward scatter was determined by flow cytometry. (E) FADD2/2RIP12/2 DKO T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the absence or presence of Nec-1 were measured for cell proliferation as in (C). doi:10.1371/journal.pone.0023209.g005 PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23209