Fig. 2: RIP1 and mitochondrial ROS act on one another during the hyperglycemic shift to necroptosis.

A rip1 CRISPR KO U937 cells or those transfected with nontargeting control guide RNA (NTC) were grown in 10 or 50 mM glucose, treated with TNF-α/CHX for 4 h followed by staining with MitoSOX Red and flow cytometry. The robust increase in MitoSOX Red staining in NTC cells in 50 mM glucose is blunted in rip1 CRISPR KO cells. Results are from three independent experiments. Graphed values represent mean ± standard deviation. Two-way ANOVA, ***p < 0.001. B rip1 CRISPR KO or NTC cells were grown in 10 or 50 mM glucose, treated with TNF-α/CHX for 2.5 h followed by lysate preparation and immunoblotting. Caspases-3, -6, and -7 decrease in NTC cells in 50 mM glucose, however, this decrease is not exhibited in rip1 CRISPR KO cells. C rip1 CRISPR KO or NTC cells were treated as in B followed by immunoprecipitation of RIP3 and immunoblotting. RIP3 is phosphorylated (p-RIP3) and MLKL co-precipitates in NTC cells in 50 mM glucose. Phosphorylation of RIP3 and co-precipitation of MLKL with RIP3 was not exhibited in rip1 CRISPR KO cells. WT U937 cells were treated as in B in the presence or absence of D antioxidant, N-acetylcysteine (NAC) or E superoxide dismutase inhibitor, diethyldithiocarbamate (DDC). Total RIP1 levels were normalized. Phosphorylation of RIP1 (p-RIP1) is exhibited in 50 mM glucose and prevented by NAC. Treatment with DDC led to p-RIP1 in 10 or 50 mM glucose. All western blot images are representative of three independent experiments.