Skip to main content
. 2020 Jan 27;10:3087. doi: 10.3389/fimmu.2019.03087

Figure 6.

Figure 6

miR-221-3p is directly interfering with JAK3/STAT3 signaling in TLR4-stimulated M2-macrophages. (A) Location of two predicted miR-221-3p targets, Site-A and Site-B, within the 3'UTR region of the human JAK3 mRNA. Sequence of Site-A contains seed match region identical to already established miR-221-3p target on CDKN1B gene (gray shading). (B) Luciferase reporter assay of constructs carrying JAK3 Site-A and JAK3 Site-B target sites, co-transfected with increasing concentration of miR-221-3p (221 mimic) or RNA duplex carrying randomized base pairs (negative ctrl). Luciferase activity represents Renilla/firefly values normalized 0 nM and is expressed as mean ± S.D. N = 3–8, *p < 0.05. Differentiated M2-macrophages derived from CD14+ cells of HD blood were transfected with miR-221-3p mimic (221 mimic) or a respective control miR (ctrl mimic) and stimulated with 100 ng/ml LPS for (C) 16 h and (D) 1 h or left untreated. Protein levels of (C) JAK3 and phosphorylated STAT3 and nuclear shuttling of (D) p65 (NF-κB) and IRF3 were analyzed by SDS-PAGE and visualized by immunoblotting. TBP and β-Tubulin were used as loading controls for nuclear protein extract or whole cell lysates, respectively. Shown are representative images and quantification of N = 3–5. Quantification of protein expression was measured using ImageJ software and represented as a relative density ratio (protein of interest to loading control). Values are expressed as mean ± S.D. N = 3–5, *p < 0.05.