Fig. 3: Activation of necrosome components and deactivation/loss of executioner caspases is driven by ROS in high-glucose conditions.

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 antioxidant, N-acetylcysteine (NAC), for 2.5 h followed by nonreducing SDS–PAGE and immunoblotting. A high MW, oxidized form of RIP1 is exhibited in 50 mM glucose, but is prevented by NAC. B U937 cells were treated as in A followed by reducing SDS–PAGE and immunoblotting. Total levels of RIP3 and MLKL were normalized. RIP3 and MLKL phosphorylation (p-RIP3 and p-MLKL, respectively) occurs in 50 mM glucose and is prevented by NAC. C U937 cells were treated as in A followed by immunoprecipitation of RIP1, reducing SDS–PAGE, and immunoblotting. RIP3 and MLKL co-precipitate with RIP1 in 50 mM glucose, but this is prevented by NAC. D U937 cells were treated as in B. Caspases-3, -6, and, -7 exhibit a decrease in activation and abundance in 50 mM that is prevented by NAC. All western blot images are representative of three independent experiments.