Fig. 5

Exosomal miR-30d-5p activates NF-κB in macrophage via targeting SOCS-1 and SIRT1. a Sequence alignment between miR-30d-5p and its putative binding sites (in red letters) in the SOCS-1/SIRT1 3′-UTR. Mutation of the miR-30d-5p target sites (in blue letters) is also shown. b Detection of the relative luciferase activities of WT and Mut SOCS-1/SIRT1 reporters by luciferase reporter assay, using Renilla luciferase vector as the internal control. RT-qPCR analysis of relative SOCS-1/SIRT1 mRNA level (c) and Western blot (d) of SOCS-1 and SIRT1 in Raw264.7 macrophages transfected with miR-30d-5p mimics as indicated. e, f Treatment of Raw264.7 macrophages with PBS/PBS-Exo/TNF-Exo for 24 h. e Detection of mRNA levels of SOCS-1 and SIRT1 by RT-qPCR. f Western blot analysis of SOCS-1, SIRT1 and p65-Acetyl 310. g Prior to co-culturing with TNF-Exo for 24 h, Raw264.7 macrophages were transfected with control or miR-30d-5p inhibitors for 24 h. The expression levels of SOCS-1, SIRT1 and p65-Acetyl 310 in Raw264.7 cells were measured by Western blot. Student’s t test (b, c) or one-way analysis of variance with Tukey's multiple comparisons test (e) was used for the analysis. Graphs represent means ± SEM, n ≥ 3; *P < 0.05, **P < 0.01 compared within two groups