Fig. 4

MTAs induce memTNF-mediated apoptosis in RIP3-deficient human carcinoma cell lines. a Immunoblotting analysis of apoptosis markers using whole cell lysates from recombinant TNF (soluble TNF, solTNF)-treated HeLa cells in the presence or absence of pan-caspase inhibitor z-VAD (Z). Cells were treated as indicated for 24 h. b Immunoblotting analysis of apoptosis markers using whole cell lysates from 1 µM MTA-treated HeLa cells in the presence or absence of 20 µM TACE inhibitor TAPI-1 (TACEi) or 20 µM pan-caspase inhibitor z-VAD (Z) for 36 h. c Immunoblotting analysis of apoptosis markers using whole cell lysates from 1 µM MTA-treated WT and TNFR1 KO HeLa cells for 36 h. d Effect of TNFR1 knockout on MTA-induced cell death in HeLa cells. Cells were treated as indicated for 48 h. e–g Immunoblotting analysis of apoptosis markers using whole cell lysates of 1 µM MTA-treated HCT116 (colon cancer, e), MDA-MB-468 (breast cancer, f), and BT549 (breast cancer, g) cells for 36 h. h, i qRT-PCR analysis of JUN mRNA level (h) and in flow cytometric analysis of memTNF (i) in MTA-treated HeLa, HCT116, MDA-MB-468, and BT549 cells for 12 and 20 h respectively. D, DMSO; NCZ, nocodazole; PTX, paclitaxel; Z, z-VAD. Cell viability was determined by measuring ATP levels. The data are represented as mean ± SEM of duplicate wells (d). Results are reported from one representative experiment. Experiments were repeated independently three (c, d, and h) or two (a, b, e–g, and i) times