Fig. 2

Overexpression of miRNA-203 promote the nuclear translocation of NF‐κB. A The cultured BV2 cells were transfected with miRNA-203 mimics or miRNA-203 inhibitors. Mock transfections were performed in the blank controls and LPS-induced NF‐κB translocation was used as the positive control. Subcellular localization of endogenous NF-κB was visualized by immunofluorescent using monoclonal antibody of the p65 subunit of NF‐κB and a Alexa Fluor 647-labeled secondary antibody (red). B The fluorescent intensities of NF‐κB in the nuclei, defined by Hoechst 33342 staining (blue), were quantified and the results of five independent experiments were presented, with the height of columns representing the mean and the error bars showing the S.D. The statistical significance between samples were indicated by asterisks (ANOVA, Turkey’s post hoc). The scale bars were 50 μm. C Western blots showed increased expression of IL-1 β and NF-κB in BV2 cells transfected with miR-203 mimics, as well as in LPS-induced cells. Untransfected cells were used as the negative controls. Transfection of miR-203 inhibitors reversed the LPS-induced elevation of IL-1β and NF-κB. D and E Luminescence-based relative quantification of protein (IL-1 β:β-actin; NF-κB:β-actin) were expressed as mean ± S.D. of three biological replicates (ANOVA, Dunnett’s post hoc). F HEK293T cells were transfected with miRNA-203 mimic with or without and pmirGLO vector containing wild type Akirin2 3ʹUTR. The intrinsic levels of 14-3-3θ were detected using western blots. Mock transfection was performed in blank controls. G Luminescence-based relative quantification of protein (14-3-3θ: β-actin) was performed in three individual biological replicates, with error bars representing the standard deviation (ANOVA, Turkey’s post hoc). Downregulated expression of 14-3-3θ in cells transfected with miR-203 mimics was reversed by overexpression of Akirin2 3′UTR. H Akirin2 3ʹUTR and 14-3-3θ 3ʹUTR Luciferase activity in 293T cells transfected with miR-203 mimics and pSilencer 4.1 containing wild type or mutant form of Akirin2 3ʹUTR and 14-3-3θ 3ʹUTR. Asterisks indicate statistical significance between samples (*P < 0.05; **P < 0.01; ***P < 0.001)