Fig. 4

TNF-Exo activates NF-κB signaling pathway in macrophage via miR-30d-5p. a–e Prior to co-culturing with TNF-Exo for 24 h, Raw264.7 macrophages were transfected with negative control (NC) or miR-30d-5p inhibitors (anti-miR-30d-5p) for 24 h. a, b Flow cytometry detection of CD11c and CD86 expression. c Detection of inflammatory cytokine mRNA (IL-6, IL-1β, TNF-α) and iNOS mRNA expression by RT-qPCR. d Western blot of NF-κB p-p65 and p65 in Raw264.7 macrophages. e Detection of NLRP3 and caspase-1 mRNA expression by RT-qPCR. f–h Transfection of Raw264.7 macrophages with negative control (NC) or miR-30d-5p inhibitors for 24 h, followed by stimulation with TNF-Exo plus ATP. f Flow cytometry detection of Alexa Fluor 488-labeled caspase-1 FLICA expression. g Western blots of pro-caspase-1 (Pro-casp-1) and activated/cleaved caspase-1 (Casp-1 p20) in whole-cell lysates of Raw264.7. h Cleavage of GSDMD by immunoblotting. Student’s t test was used for analysis. Graphs represent means ± SEM, n ≥ 3; *P < 0.05, **P < 0.01 compared within two groups