Fig. 2.

MyD88 mediates myoblast fusion in vitro. a Primary myoblasts were prepared from hind limb muscles of WT mice. The cells were incubated in DM and samples were collected at indicated time points and processed by Western blot to detect MyD88, MyHC as a differentiation marker, and an unrelated protein GAPDH. b Primary myoblasts prepared from hind limb muscle of WT and MyD88-KO mice were plated at equal densities and incubated in DM for 24 h or 48 h followed by staining for MyHC and DAPI. Representative photomicrographs are presented here. Scale bar: 50 µm. c Quantification of the percentage of myotubes containing indicated number of nuclei per myotube in WT and MyD88-KO myotubes cultures after 24 h of addition of DM. d Quantification of the percentage of myotubes containing indicated number of nuclei per myotube in WT and MyD88-KO cultures after 48 h of addition of DM. e Average diameter of myotubes in WT and MyD88-KO cultures after 48 h of addition of DM. f Quantification of number of MyHC⁺ myotubes containing 2 or more nuclei and g number of mononucleated MyHC⁺ cells per unit area (~0.15 mm²) in WT and MyD88-KO cultures after 48 h of incubation in DM. h Primary myoblasts prepared from WT and MyD88-KO mice were incubated in DM and samples were collected at indicated time points. Protein lysates were prepared and blotted with antibodies against MyHC, myogenin and unrelated protein GAPDH. i In a separate experiment, WT and MyD88-KO cells were collected at 0 and 48 h following addition of DM and mRNA levels of MyHC and myogenin were measured after normalizing to the levels of β-actin. Results are from four to five independent experiments. Error bars represent s.d. *p < 0.05 from corresponding WT cultures for all experiments by unpaired t-test