Figure 3.

Four modes of necroptosis execution exhibit variability in temporal dynamics and differ in rate constant values and responses to CYLD and A20 knockouts. (A) Clustering analysis of simulated time courses (100 ng/mL TNF) from 10,628 parameter sets reveals four distinct modes of execution (M1, …, M4). Dissimilarity (“distance”) between dynamical signatures (digitized time courses) was quantified using the longest common subsequence (see materials and methods). (B) Simulated time courses (100 ng/mL TNF) of the necroptosis marker, pMLKL, show significantly more variability in TTD (defined as the time at which pMLKL reaches its half-maximal value) in modes 1 and 2 than in modes 3 and 4. Time courses for all parameter sets associated with each mode are shown. Experimental western blot data (black circles; quantified from Fig. 2A) are included to illustrate the model fit for each mode. (C) Variations in the values of 10 rate constants distinguish the four modes of execution. Parameter indices (PN) match reaction indices (RN) in Fig. 1 and Table 2. Note that second-order rate constants (P4, P13, P15, and P38; see Table 2) were converted from units of μM⁻¹ min⁻¹ to min⁻¹ assuming a spherical cell of diameter 5 μm (see materials and methods). (D) Knockouts of CYLD and A20 (100 ng/mL TNF) differentially affect TTD, relative to wild-type (WT), across the four modes of execution (each dot corresponds to a parameter set). Note that CYLD;A20 double knockout inhibits cell death in all cases (TTD = ∞). The number of parameter sets that do not result in cell death (n_∞) are included for all modes under all conditions. KO, knockout; DKO, double knockout.