Figure 1

Inhibition of serine proteases, but not metalloproteases, cathepsin or calpain/cysteine proteases protects from TNF-induced necroptosis. A. Cells were stimulated or not with 100 ng/ml TNF for 5 (L929Ts), 16 (NIH3T3, HT-29), or 20 h (Jurkat) with optional addition of 20 (L929Ts, NIH3T3, HT-29) or 50 μM (Jurkat) of the broad-spectrum caspase inhibitor zVAD-fmk to prevent apoptosis, 2 (Jurkat) or 5 μg/ml (HT-29) cycloheximide (CHX) to sensitize for necroptosis[14] and 50 (L929Ts, NIH3T3) and 25 μM (Jurkat, HT-29) TPCK, or 50 μM of the necroptosis inhibitor necrostatin-1 (Nec-1, to confirm necroptosis). Subsequently, the cells were analyzed for loss of membrane integrity as a marker for cell death by PI staining and flow cytometry. Asterisks indicate statistical significance (t-test), *p < 0.05, **p < 0.01, ***p < 0.001. Micrographs show the morphology of untreated vs. necroptotic vs. L929Ts cells protected by TPCK. Scale bar: 100 μm. B. L929Ts and NIH3T3 cells were preincubated for 2 h with TAPI-1, GM 6001 and marimastat and subsequent addition of TNF/zVAD as in A before cell death was analyzed. C. L929Ts cells were incubated with TNF/zVAD as in A with optional addition of 20 μM zFA-fmk, CA-074 Me, E-64 or (in a separate experiment) zFF-fmk before cell death was analyzed. For all flow cytometric analyses of membrane integrity, we measured the percentage out of a total of 10,000 analyzed cells that show loss of membrane integrity (this is calculated as 100% minus the percentage of intact, large PI-negative cells to account for disintegrated dead cells that have lost their PI staining again due to diffusion). For all figures, representative data from one out of at least two or more experiments are shown and error bars indicate the standard deviations (SD) from at least triplicate determinations.