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. 2022 Oct 6;11(19):3139. doi: 10.3390/cells11193139

Figure 4.

Figure 4

Necrostatin-1 reduced CI/R-induced cell death through the inhibition of RIPK1-dependent apoptosis and necroptosis in BEAS-2B cells. BEAS-2B cells were incubated in a 4 °C chamber filled with 50% oxygen for 18 h with ice-cold lung preservation Perfadex® solution to simulate the procedure of clinical donor lung preservation and then subjected to a warm culture medium to mimic reperfusion. Necrostatin-1 (Nec-1, 30 μM) was added to the medium at the beginning of cold-ischemia and sustained during reperfusion. (A) Propidium iodide (PI) and Hoechst staining in BEAS-2B cells after CI/R. (B,C) Quantification of necrotic and apoptotic cells in panel A. (D) Western blotting of necroptosis- and apoptosis-related proteins (RIPK1, phosphor-RIPK1, RIPK3, phosphor-RIPK3, MLKL, phosphor-MLKL, cleaved RIPK-1, and Cleaved caspase 3) in BEAS-2B cells. (E–L) Quantification of proteins from Western blots in panel D. (M) The colocalization of RIPK1 and RIPK3 determined by immunofluorescence staining. (N) Cell counting of the RIPK1 and RIPK3 double-positive cells. Three independent experiments were performed. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with Control; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with CI/R. One-way ANOVA.