Fig. 1: Few MLKL orthologues reconstitute necroptotic signaling in mouse and human cells. | Nature Communications

Fig. 1: Few MLKL orthologues reconstitute necroptotic signaling in mouse and human cells.

From: Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues

Fig. 1

Genes encoding human, mouse, rat, horse, pig, chicken, tuatara, frog and stickleback MLKL were stably introduced into Mlkl−/− mouse dermal fibroblast (MDF) (a) and MLKL−/− human U937 (b) cells and expressed upon doxycycline treatment (induced). Cells were either untreated (UT) or treated with a necroptotic stimulus (TNF, Smc mimetic, IDN-6556; TSI) to examine the capacity of each orthologue to reconstitute necroptotic signaling. Cell death was measured by propidium iodide (PI) uptake by flow cytometry. Data shown are mean ± SEM of independent experiments on one U937 cell line (n = 3 for mouse, rat, horse and chicken MLKL; n = 4 for human and frog MLKL; n = 5 for pig MLKL) or two biological replicate MDF lines (n = 6, except for n = 8 for mMLKL-FLAG). * represents statistical significance of p < 0.05 using a paired, two-tailed t-test: a mouse-FLAG p = 0.000015, horse p = 0.0017, pig p = 0.0290; and b human p = 0.0104, pig p = 0.0003. Source data are provided in a Source Data file. An example of the flow cytometry gating strategy used throughout this study is shown in Supplementary Fig. 2. Expression of introduced genes was verified by western blot (Supplementary Fig. 3).

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