Figure 1.

TNF induces the secretion of multiple pro-inflammatory mediators irrespective of cell death. (a) HeLa cells were treated with TNF (10 ng/ml). After 6 h, cells were harvested and subjected to full genome microarray analysis. (b) HeLa cells were stimulated with TNF at the indicated concentrations. After 24 h, apoptosis was scored by morphology, and the cytokine/chemokine concentrations in the culture supernatants were determined by ELISA. (c) L929 cells were stimulated with TNF at the indicated concentrations. After 8 h, cell death was scored by morphology, and cytokine concentrations in the culture supernatants were determined by ELISA. (d) L929 cells were pretreated for 1 h in the presence or absence of zVAD (10 μM) followed by stimulation with TNF (10 ng/ml). After 8 h, cells were visualized by phase-contrast microscopy or H&E staining. (e) L929 cells were pretreated for 1 h in the presence or absence of zVAD (10 μM) followed by stimulation with TNF (10 ng/ml). At the indicated time points, cell death was analysed by AnnexinV/PI staining. (f) L929 cells were pretreated for 1 h in the presence or absence of zVAD (10 μM) followed by stimulation with TNF (10 ng/ml). After 24 h, cell death was analysed by PI staining. (g) L929 cells were pretreated for 1 h with the indicated concentrations of Nec1 and zVAD (10 μM), followed by addition of TNF (10 ng/ml). After 24 h, cell death was scored by morphology. (h) L929 cells were electroporated with either non-silencing siRNA or siRNA directed against RIPK1, RIPK3 or RIPK1 and RIPK3, as indicated. Forty-eight hours later, cells were pretreated in the presence or absence of zVAD (20 μM) for 1 h, followed by addition of TNF (10 ng/ml). After 5 h, cell death was scored by morphological assessment, and cell lysates were analysed by immunoblotting for levels of endogenous proteins. Error bars represent the mean±S.E.M. of triplicate determinations from a representative experiment. For cell death analyses, triplicate counts were performed on a minimum of 300 cells per treatment. All data are representative of at least three independent experiments