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Fig. 2 | Critical Care

Fig. 2

From: Exosomal miR-30d-5p of neutrophils induces M1 macrophage polarization and primes macrophage pyroptosis in sepsis-related acute lung injury

Fig. 2

TNF-Exo promotes M1 macrophage activation and primes macrophage pyroptosis through NF-κB pathway in an in vitro co-culture model. ac Treatment of Raw264.7 macrophages with PBS-Exo/TNF-Exo for 24 h. An equal volume of PBS was used as negative control. a Flow cytometry detection of CD11c and CD206 expression on Raw264.7 macrophages. b Detection of expression levels of iNOS, IL-1β, TNF-α, Mrc1, Arg1, Fizz1 and Ym1 mRNA by RT-qPCR. c Detection of the concentration of inflammatory cytokines (IL-6, TNF-α) in the supernatant of Raw264.7 macrophages by ELISA. d–g Stimulation of Raw264.7 macrophages with PBS/PBS-Exo/TNF-Exo for 24 h and treatment with PBS, 5 mM ATP or 20 mM nigericin for 2 h. d Flow cytometry evaluation of macrophage death by Annexin-V and PI double-staining. e Flow cytometry evaluation of macrophage pyroptosis by Caspase-1 and TUNEL double-staining. f Cleavage of GSDMD by western blot. GSDMD-FL: full-length GSDMD, GSDMD-N: N-terminal cleavage products of GSDMD. g Analysis of culture supernatants for IL-1β secretion by ELISA. h–i Treatment of Raw264.7 macrophages with PBS/PBS-Exo/TNF-Exo for 24 h, and detection of NLRP3 and caspase-1 mRNA expression by RT-qPCR. One-way analysis of variance with Tukey's multiple comparisons test was used for the analysis. Graphs represent means ± SEM, n ≥ 3; *P < 0.05, **P < 0.01 compared within two groups

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