Fig 1: The SVF-induced proliferation of L6 myoblasts is reduced by Norleual (Nor), an HGF inhibitor. a Immunofluorescence staining of the HGF receptor c-Met (green) in L6 myoblasts, counterstained with DAPI (blue). Bar = 10 µm. b The effect of different concentrations of HGF on the proliferation rate of L6 myoblasts. c 100 pM Norleual effectively inhibits the proliferation induced by 10 ng/ml HGF. d Indirect co-culture of L6 myoblasts and SVF cells for 3 days in the presence of 100 pM Norleual. Norleual was tested on cells alone and no toxicity could be observed. e The expression of p-ERK1/2 and total ERK1/2 protein in L6 myoblasts after co-culture with SVF cells. Norleual had no effect on baseline levels of p-ERK1/2. Statistical analysis was performed using one-way ANOVA with post hoc test (Bonferroni correction). Results are presented as mean ± standard deviation. n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant
Fig 2: Differentiation of myoblasts into myotubes is inhibited by SVF cells and HGF. a Immunofluorescence staining of fast type myosin heavy chain (green) in C2C12 mouse myoblasts after 4 days in growth medium (GM), differentiation medium (DM), indirect co-culture with SVF cells in DM (SVF), and DM supplemented with 10 ng/ml HGF (HGF). Cells were counterstained with DAPI (blue). Bar = 200 µm. The expression of myogenic regulatory factor 5 (Myf5), slow type myosin heavy chain (Myhc1), and fast type myosin heavy chain (Myhc2) mRNA in C2C12 myoblasts after b 7 days or c 14 days of differentiation in the various culture conditions. Western blot of fast type myosin heavy chain (MyHC2) protein is also shown. ß-actin was used as a loading control. Statistical analysis was performed using one-way ANOVA with post hoc test (Bonferroni correction). Results are presented as mean ± standard deviation. n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant
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