Suppressive effects of Nec-1 on necroptosis in a mouse model of LPS-treated ALI. (A) Western blotting of lysates from mouse lung tissues was used to estimate the expression levels of necroptosis-associated proteins (RIP1 and RIP3). (B) Semi-quantification of the protein expression levels of RIP1 and RIP3. Representative images of at least three independent experiments are shown. (C and D) Estimation of the degree of apoptosis in primary lung cells, as determined by flow cytometry after staining with Annexin V and PI. (E) Ultrastructure of alveolar epithelial cells in the indicated mouse models (green arrow, lamellar body; red arrow, microvilli). Data are presented as the mean ± SEM from at least three independent experiments (n=5 mice/group). *P<0.05, as indicated. ALI, acute lung injury; LPS, lipopolysaccharide; Nec-1, necrostatin-1; PI, propidium iodide; RIP, receptor-interacting serine/threonine-protein kinase.