Fig. 2: Hypoxia induces necroptosis in vascular endothelial cells.

A CCK8 assay was performed to determine changes in cell survival after hypoxia, n = 6. B Changes in HUVEC cell survival after hypoxia treatment in the presence of 50 μM Nec-1, 100 μM z-VAD-fmk, 10 μM Fer-1, and 1 mM NAC, n = 9. C Changes in cytotoxicity after hypoxia as determined by LDH release, n = 5. D Representative transmission electron microscopy images of HUVEC after hypoxia treatment. HUVEC in the hypoxia group exhibited translucent cytoplasm, loss of cytoplasmic contents, and membrane damage. Scale bar = 2 μm (merged image) and 500 nm (magnified image). E Representative immunoblot bands and statistical analyses of HIF-1α, RIPK3, p-RIPK3, MLKL, p-MLKL in Ctrl, hypoxia, and hypoxia+Nec-1 groups, Actin was used as the reference protein, n = 3. F Representative confocal images and statistical analysis results of p-RIPK3 (red) after 12 h of hypoxia treatment. scale bar = 100 μm, n = 5. G HUVEC from normoxic and hypoxic groups were lysed and immunoprecipitated with anti-RIPK3 antibodies, followed by immunoblotting with anti-RIPK3, anti-MLKL, and anti-RIPK1 antibodies. H Representative images of CD31 (green) and p-MLKL (red) staining of aortic vascular sections from mice after 4 weeks of hypoxic treatment. Arrows indicate CD31 and p-MLKL co-localized fractions. scale bar = 50 μm. I Representative immunoblot bands and statistical analyses of aortic vascular tissues for IL-1β, TNFα, MLKL, and p-MLKL in mice after 4 weeks of hypoxia treatment, Actin was used as the reference protein, n = 5. Data are expressed as mean ± SEM. Relevant experiments in this section were performed independently at least three times. *p < 0.05, **p < 0.01, ***p < 0.001.