Fig. 1: Necroptosis stimulation induces an inflammatory storm.

WT BMDMs were treated with IFNβ (100 U/mL), zVAD-fmk (50 µM), and Nec-1 (10 µM). At 6 h, differential gene expression and GSEA were performed on microarray data comparing WT BMDMs treated with IFNβ, IFNβ + zVAD, and IFNβ + zVAD+Nec1 (A–D). Expression of TNFα was measured in the supernatants collected at 7 h after treating the WT BMDMs with EMR (10 µM) and different LPS concentrations (E). Expression of IFNβ, and IL-12p70 was measured in the supernatants collected at 7 h after treating the WT BMDMs with LPS (1 ng/mL) and EMR (10 µM) at 7 h (F, G). H, I WT BMDMs were stimulated with LPS (1 ng/mL), EMR (10 µM), Nec1-s (10 µM) and the impact on cell death was evaluated at 24 h by MTT (H). Secretion of TNFα was measured in supernatants collected at 7 h (I), and the activation of various proteins was evaluated by performing western blotting of cell extracts collected at various time intervals (J). Each experiment was repeated at least three times. (**P < 0.01, ***P < 0.001, ****P < 0.0001).