Fig. 4: Glucose-independent induction of ROS causes the activation of necrosome components and deactivation/loss of executioner caspases.

A U937 cells were grown in 10 or 50 mM glucose followed by the treatment with TNF-α/CHX in the presence or absence of superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), for 4 h. Cells were then stained with MitoSOX Red and analyzed by flow cytometry. Glucose-independent induction of ROS with DDC produces a fold increase in MitoSOX staining similar to that exhibited in 50 mM glucose. Results are from three independent experiments. Graphed values represent mean ± standard deviation. Two-way ANOVA, ***p < 0.001. B U937 cells were grown in 10 or 50 mM glucose followed by the treatment with TNF-α/CHX in the presence or absence of DDC for 2.5 h followed by nonreducing SDS–PAGE and immunoblotting. RIP1 exists as a high MW, oxidized species in 50 mM glucose as well as in 10 or 50 mM glucose + DDC. C U937 cells were treated as in B followed by reducing SDS–PAGE and immunoblotting. Total levels of RIP3 and MLKL were normalized. RIP3 and MLKL are phosphorylated (p-RIP3 and p-MLKL, respectively) in 50 mM glucose and 10 or 50 mM glucose + DDC. D U937 cells were treated as in B followed by immunoprecipitation of RIP1, reducing SDS–PAGE, and immunoblotting. Both RIP3 and MLKL co-precipitate with RIP1 in 50 mM glucose and 10 or 50 mM glucose + DDC. E U937 cells were treated as in C. Caspases-3, -6, and -7 decrease in activation and abundance in 50 mM glucose and 10 or 50 mM glucose + DDC. All western blot images are representative of three independent experiments.