Fig. 3: Phosphorylation of kinase domain regulates RIPK3 functions.

a Necroptotic cell death mediated by wild-type or phosphorylation site mutants of human RIPK3 in RIPK3^−/− HT29 cells. Wild-type (WT) or mutant human RIPK3 expression was induced with 2.5 ng/mL doxycycline (dox) for 16–24 h, and cell death after 24 h of necroptotic stimulation, dTSI (d, doxycycline; T, TNF; S, Smac mimetic Compound A; I, pan-caspase inhibitor IDN-6556), was measured. Cell death (quantified by SYTOX Green uptake) was measured as a percentage of total number of cells (quantified by DRAQ5 uptake) using IncuCyte imaging. The means of three independent experiments (n = 3) are displayed as bars. Values of each independent experiments are shown as points. Asterisks (*) indicate RIPK3 mutants that mediated significantly less cell death than the wild-type RIPK3 at 24 h post-necroptotic stimulation (p < 0.05 unpaired t-test). b Evaluation of the abilities of RIPK3 phosphorylation site mutants to mediate MLKL phosphorylation and RIPK3 autophosphorylation in RIPK3^−/− HT29 cells. After being induced with 2.5 ng/mL doxycycline (dox) for 16–24 h, cells expressing wild-type or mutant human RIPK3 were treated with necroptotic stimuli, dTSI, for 7.5 h, before lysis and analysis by immunoblotting with antibodies indicated. Data are representative of three independent experiments (n = 3). c RIPK3^−/− HT29 cells expressing S227D RIPK3 displayed necroptotic morphology upon dTSI treatment for 24 h, despite failing to uptake SYTOX Green. In contrast, cells with wild-type RIPK3 underwent necroptosis upon dTSI treatment, and cells with T224D/S227D RIPK3 underwent attenuated necroptosis, whereas S227A RIPK3 failed to mediate necroptosis. IncuCyte micrographs taken with the phase channel (greyscale) and green fluorescence channel (green, detect SYTOX Green signal) are overlaid. Data are representative of three independent experiments (n = 3).