Fig. 8

TWEAK strongly amplifies TNF-α cytotoxic activity within the range of concentration secreted by VS tumors. (A) Experimental paradigm for testing the contribution of TWEAK in TNF-α mediated cytotoxicity. Co-treatment of L929 cells with 50 ng/mL TWEAK and 0.01–100 ng/mL TNF-α dramatically reduced cell viability compared to the cells treated separately with TWEAK or TNF-α. (B) Cytokine co-treatment results in significantly reduced metabolic activity in L929 cells. (C) TWEAK and TNF-α co-treatment in the presence of ATA, an Fn14 signaling pathway inhibitor, abrogates the cytotoxic effect of the co-treatment. (D) Cells co-treated with TWEAK and TNF-α released significantly higher levels of LDH than cells treated with either factor alone, indicating an increased rate of cell death in the co-treated cultures. (E) Cells co-treated with TWEAK and TNF-α exhibited increased CellTox Green Dye staining, confirming compromised membrane integrity and indicating necroptosis as the underlying form of cell death associated with TWEAK and TNF-α cytotoxicity. The cytotoxic effect of TWEAK and TNF-α was manifested 4–5 h after the co-treatment and reached a maximum 18 h later. Metabolic activity and LDH levels are presented relative to untreated control L929 cells. CellTox Green Dye staining is expressed as a mean of fluorescence intensity (mean). Each data point represents technical replicates; data are shown as mean ± s.e.m. MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; LDH, lactate dehydrogenase; ATA, aurintricarboxylic acid