Fig. 2

MTAs activate membrane TNF signaling to induce bystander cell death. a, b Effect of Tnfr1 (a) and Tnf (b) knockout on MTA-induced necroptosis in L929 cells. c Pretreatment (2 h) of neutralizing antibody against TNF rescued cells from MTA-induced necroptosis. d MTA-treated L929 cells were tested for the presence of soluble TNF (solTNF) in the cell culture media. Samples were harvested for ELISA analysis to determine the concentration of solTNF, as described in the “Methods” section. LPS-primed Raw264.7 cell medium was used as a positive control for measuring the autocrined soluble TNF. e MTA-treated L929 conditioned medium (CM) was applied to naïve cells. Left panel, a schematic representation of the experimental design. Right panel, conditioned medium-fed L929 cell viability was determined by ATP levels at 12 h post treatment. f Influence of TACE inhibitors on MTA-induced cell death in L929 cells. TACE inhibitors were pretreated for 2 h followed by MTAs treatment. g Immunoblotting analysis of membrane-bound TNF in crude membrane fraction of NCZ-treated WT, Tnfr1 KO, and Tnf KO L929 cells. Integrin β1 was used as the loading control of plasma membrane fraction. h, i MTA-induced bystander cell death analysis in L929 Tnf KO and Tnfr1 KO co-culture system, as described in the “Methods” section. Representative images show NCZ-induced necroptotic cells by PI staining (h). Scale bar, 100 µm. The number of necroptotic cells per well was quantified by IncuCyte (i). D, DMSO; NCZ, nocodazole; VCR, vincristine; PTX, paclitaxel. Cell viability was determined by measuring ATP levels. The data are represented as mean ± SEM of duplicate wells (a–f and i). Results are reported from one representative experiment. Experiments were repeated independently for four (a, b, and g) or three (c–f, h, and i) times