Fig. 4. Exosome-derived miR-499 inhibits NF-κB signaling pathway activation.

a NF‐κB p65 immunofluorescence staining (red) of the uterine horn of non-pregnant (G.D.0, n = 3) and early pregnant cows (G.D.60, n = 3), scale bar, 50 μm. b Translocation of the p65 subunit from the cytoplasm into the nucleus was evaluated by immunofluorescence. Blue spots represent cell nuclei, and red spots represent p-p65 staining. c Western blotting analysis showing the effect of placental exosome (p-EXO) and non-pregnant cow exosomes (n-EXO) on IκBα and phosphorylated p65 levels. β-actin/total protein (NF-κB p65) was used as a control. d The effect of overexpression of miR-499 on the phosphorylation level of NF-κB p65 induced by LPS; total protein (NF-κB p65) was used as a control. e Western blotting analysis of the phosphorylation levels of NF-κB p65 using a specific NF-κB inhibitor, BAY-117082 (20 μM); (NF-κB p65) was used as a control. f qPCR analysis showing the effect of a specific NF-κB inhibitor on the expression of proinflammatory cytokines TNF-α and IL-6. GAPDH was used as the internal control. G_D.0 and G_D.60 represent gestational day 0 and 60, respectively. n-EXO and p-EXO represent exosomes isolated from cow plasma at G.D.0 and G_D.60, respectively. NC represents the negative control (agomiR-NC). Data represent three independent experiments and are presented as the mean ± S.E.M (error bars). Two-tailed Student’s t-test, *P < 0.05; **P < 0.01