Figure 4.

Effect of Lf on myoblast differentiation. (A) Myoblasts were cultured in differentiation medium containing Lf at various concentrations for six days. (B) Myoblasts were cultured in differentiation medium in the presence of Lf for six days (6 days) or in differentiation medium in the presence of Lf for one day and in the absence of Lf for the next 5 days (1 day). (A and B) The expression of MyHC and β-actin was analyzed by western blots. (C) Myoblasts were cultured in differentiation medium containing Lf (50 µg/ml). cDNA was synthesized and genes were amplified by polymerase chain reaction. (D) Myoblasts were transfected with control siRNA (siControl) or LRP1 siRNA (siLRP1) and were differentiated in the presence of Lf (50 µg/ml) for three days. The expression of MyHC, LRP1, and GAPDH was analyzed by western blotting. (E) (Upper panel) Fixed cells were reacted with anti-MyHC antibody and a fluorescence-labeled secondary antibody (green). The nuclei were stained with DAPI (blue). (Lower panel) The fusion index was calculated. Statistically significant differences were determined by one-way ANOVA and Tukey's post-hoc test. *P<0.05, **P<0.001 vs. Lf (0 µg/ml) group. ^#P<0.05 vs. Lf (1 µg/ml) group. Each result is representative of three (A, B, C, and E) or two independent experiments (D). Scale bar, 100 µM for all images. Lf, lactoferrin; LRP1, low-density lipoprotein receptor-related protein 1; MyHC, myosin heavy chain; siRNA, small interfering RNA.