Fig. 4

M2 macrophages promote myofibroblast differentiation of LR-MSCs through the Wnt/β-catenin signaling pathway. a RAW 264.7 cells were treated with LPS (10 ng/ml) or IL-4 (10 ng/ml) for 24 h to induce M1 and M2 macrophage differentiation, respectively. The mRNA expression levels of inducible nitric oxide synthase (iNOS, M1 macrophage marker), arginase (Arg-1, M2 macrophage marker), and Wnt7a in differentiated macrophage subtypes were determined by q-PCR. Results are expressed as means ± SD (n = 5; *p < 0.05 vs. M1 macrophage). b Wnt7a levels in the culture supernatant of differentiated macrophage subtypes were determined by ELISA. Data were expressed as means ± SD (n = 5; *p < 0.05 vs. control). c LR-MSC lysates were subjected to coimmunoprecipitation (Co-IP) with anti-Frizzled-1 antibody, and the blot was probed with anti-Wnt7a antibody. Moreover, blots were re-probed with anti-Frizzled-1 antibody to confirm equal protein loading. Co-IP with mouse IgG served as a negative control. The presence of Wnt7a in the cell lysate was detected by western blotting, serving as a positive control. d The ratios of Wnt7a/Frizzled-1 were determined by densitometry and were expressed as means ± SD (n = 3; *p < 0.05 vs. control). e The nuclear translocation of β-catenin was evaluated by measuring protein levels in the cytosolic and nuclear extracts. Histone H3 and GAPDH were used as loading controls for nuclear and cytoplasmic proteins, respectively, and also used as a control for the purity of the preparation. f M2 macrophages were transfected with control or Wnt7a siRNA and then cocultured with LR-MSCs in a transwell system. The expression of α-smooth muscle actin (α-SMA) and collagen I in LR-MSCs was measured by western blotting. g and h Purified primary LR-MSCs were cocultured with M2 macrophages or control medium in a transwell system. In some of the cocultured LR-MSCs as indicated 1 μM salinomycin (a specific Wnt/FZD/LRP5 complex inhibitor) or the solvent DMSO was added to the medium of the cocultured LR-MSCs to block Wnt/β-catenin signaling. Expression of α-smooth muscle actin (α-SMA) and collagen I on LR-MSCs was measured by immunofluorescence assay (g) and western blotting (h)