Fig. 5: AMG-47a inhibits necroptosis downstream of MLKL activation.

A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL^(1-180)-gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL^(1-180)-gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above (A). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).