Fig. 3: The effect of Resolvin-D2 on the myogenesis capacity of myogenic cells is mediated by Gpr18.

a Representative Western blot and b quantification of Gpr18 expression in proliferating myoblasts and differentiated myoblasts (1 day in differentiating medium) isolated from mdx mice (data are presented as mean ± SEM, n = 5 biologically independent samples, two-tailed unpaired Student’s t-test; p = 0.0018). c siRNA knockdown of Gpr18 on primary myoblasts isolated from mdx mice. d–f Dystrophin-deficient myoblasts were treated with siRNA-scrambled (Ctrl) or siRNA-Gpr18 (siRNA) and cultured in differentiating media containing Resolvin-D2 (RvD2, 200 nM) or not for 4 days. d Representative images of myotubes stained for MyHC. Scale bars = 75 μm. Quantification of (e) the fusion index (siRNA vs. Ctrl, p = 0.5967; RvD2 vs. Ctrl, p = 0.0038; siRNA+RvD2 vs. Ctrl, p = 0.8042; RvD2 vs. siRNA, p = 0.0018; siRNA+RvD2 vs. siRNA, p = 0.4428; siRNA+RvD2 vs. RvD2, p = 0.0055), and f the myotube diameter (siRNA vs. Ctrl, p = 0.9937; RvD2 vs. Ctrl, p = 0.0003; siRNA+RvD2 vs. Ctrl, p = 0.5762; RvD2 vs. siRNA, p = 0.0003; siRNA+RvD2 vs. siRNA, p = 0.5710; siRNA+RvD2 vs. RvD2, p = 0.0005). g–j Representative Western blot and quantification of the p-Akt (phosphorylation on Ser473)/total Akt-1 ratio in primary myoblasts isolated from g, h mdx mice (0 vs. 30 min, p = 0.0329; 0 vs. 60 min, p = 0.0062; 5 vs. 30 min, p = 0.0181; 5 vs. 60 min, p = 0.0034; 15 vs. 60 min, p = 0.0128) or i, j Gpr18-knockout mice cultured for 0, 5, 15, 30, 60, or 120 min with RvD2. For Western blot experiments, the samples derive from the same experiment and the gels/blots were processed in parallel. Data are presented as mean ± SEM, n = 3 biologically independent samples performed in technical duplicates and analyzed with one-way ANOVA uncorrected Fisher’s LSD test (e, f, h, and j). All data were analyzed with a 95% confidence interval. *p < 0.05, **p < 0.01, ***p < 0.001.