Fig. 4: High glucose-induced shift to RIP1-dependent cell death is concurrent with increased markers of necroptosis and necrosis.

a Levels of total RIP3 from U937 cells treated with TNF-α/CHX in 10 or 50 mM glucose were normalized to one another and phosphorylation of RIP3 was measured. RIP3 phosphorylation increases in high glucose conditions. b MLKL migrates to the membrane/organelle fraction (M) of U937 cells following TNF/CHX-induced death in high glucose conditions but not in normal glucose conditions. MLKL also increases in abundance in the cytoplasmic fraction (C) following TNF-induced death in high glucose conditions. CD71 = membrane-specific protein, GAPDH = cytoplasm-specific protein. c Flow cytometry of U937 cells following activation of TNF/CHX-induced apoptosis. Annexin V staining (apoptosis marker) decreases in high glucose following TNF/CHX treatment. Annexin/PI staining (necroptosis marker) increases in high glucose and is prevented by the RIP1 inhibitor, nec-1s. Two-way ANOVA, ***p < 0.001. d U937 cells shrink in diameter following TNF/CHX treatment in 10 mM glucose. At 50 mM glucose cellular diameter increases prior to cellular shrinkage. e Immunoprecipitation (IP) of HMGB1 from supernatants of U937 cells treated with TNF-α/CHX at low (10 mM) and high (50 mM) glucose. High levels of HMGB1 are released into the supernatant high glucose conditions