Diverse vertebrate RIPK3 proteins activate NF-κB. (A) Percent similarity of RIPK3 from the indicated species compared to humans. (B) RIPK3 proteins were transfected into WT or RIPK1 KO HEK293T cells, along with NF-κB firefly luciferase and control renilla luciferase reporter plasmids (see Materials and Methods), and NF-κB activity was measured at 18h post-transfection. (C) RIPK3 proteins were transfected into HEK293T cells with and without human ZBP1 and MLKL. At 18h post-transfection, cells were stained using the ReadyProbe Cell Viability kit and fluorescence was measured using a plate reader. Species shown are mouse (Mus musculus), cat (Felis catus), pig (Sus scrofa), lizard (Anolis carolinensis), turtle (Chelonia mydas), and lamprey (Petromyzon marinus). Data are representative of 3-5 independent experiments with n=3-6 replicates per group. Data were analyzed using two-way ANOVA with Šidák’s multiple comparisons test. ns = not significant, **** = p<0.0001.