Fig. 4

miR-6240 inhibits myoblast fusion through regulating Igf2bp3. A, B After transfection with miRNA-6240 mimics or a negative control, the levels of fusion markers were detected by RT-qPCR (A) and western blot (B). C C2C12 myoblasts were transfected with miRNA-6240 mimics or a negative control; their fusion was tracked by measuring the diameter of the myotube, the fusion index, and the MyHC (green) levels using immunofluorescence. With the use of DAPI (blue), cell nuclei were seen. D, E After co-transfection with miR-6240 mimics and the pcDNA3.1(+) vector or a negative control, the levels of fusion markers were detected by RT-qPCR (D) and western blot (E). F MyHC-stained (green) C2C12 myoblasts that were induced to fuse for five days following transfection were examined using immunofluorescence. DAPI (blue) was used to visualize the cell nuclei. For C and F, micrographs of MyHC and merge were taken using 200× magnification (scale bar, 100 µm) and magnifying the partial part of the merge image by 4× (scale bar, 25 µm). Quantification was performed on the fusion index and myotube diameter. Gapdh and α-tubulin were used for standardization. N = 3 in each group. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t-test)