Figure 3: miR-142-5p and miR-130a-3p regulate the profibrogenesis of macrophages.

(a–g) Human macrophages were transduced with control, miR-142-5p ASO or miR-130a-3p mimics or both. After 24 h, the macrophages were stimulated with IL-4 for 12 h and co-cultured with primary human fibroblasts for another 48 h. (a) The schematics of the approach. (b) Representative images of fluorescent FAP/DAPI staining in fibroblasts. Scale bar, 100μm. Fluorescence intensity was quantitated.(mean±s.e.m., n=3 independent experiments; *P<0.05; **P<0.01. P values were obtained using two-tailed Student’s t-test). (c) Fibroblast contractility in three-dimensional collagen matrices (mean±s.e.m., n=4 independent experiments; *P<0.05; **P<0.01. P values were obtained using two-tailed Student’s t-test). (d) Representative images of western blot analysis of α-SMA, collagen I and collagen III in fibroblasts (n=3). The numbers above the blots present the intensity ratio of indicated protein/GAPDH analysed by ImageJ. (e) Extracellular acid-soluble collagen production of fibroblasts was measured by the Sircol assay. (mean±s.e.m., n=4 independent experiments; *P<0.05; **P<0.01; ***P<0.001. P values were obtained using two-tailed Student’s t-test). (f) The proliferation of fibroblasts was determined by BrdU incorporation assay (mean±s.e.m., n=4 independent experiments; *P<0.05; **P<0.01. P values were obtained using two-tailed Student’s t-test). (g) Total TGF-β1 (acid-treated) and active TGF-β1 (not acid-treated) in the media of the macrophage/fibroblast co-culture system (mean±s.e.m., n=4 independent experiments; *P<0.05; **P<0.01; ***P<0.001. P values were obtained using two-tailed Student’s t-test).