Figure 6.

Down-regulation of β1D subunit by siRNA inhibits myoblast fusion. (A) siRNA was used to down-regulate the level of β1D integrins expression during myogenic differentiation to study the effect on cell fusion. Myoblasts were transfected with different amounts of siRNA oligonucleotides (100, 250, or 500 pmol/well) and then cultured in DM. Analysis by Western blots demonstrates an effective inhibition of β1D integrin up-regulation upon differentiation at all doses tested. The α5 integrin subunit, used here as a control, remained unchanged. (B) Immunofluorescence of β1A and β1D integrin subunits in cells treated with siRNA and cultured for 48 h in DM. In control myotubes, both isoforms were localized at focal adhesions. In cultures treated with β1D subunit siRNA, only the β1A isoform was detected. Bar, 20 μm. (C) Myoblasts treated by siRNA were induced to differentiate to determine the effect of β1D subunit inhibition on morphological differentiation. Cells treated with β1D subunit siRNA formed myotubes of a smaller size. Bar, 100 μm. (D) Determination of fusion index in siRNA-treated cultures. Fusion was delayed in β1D siRNA-treated cultures but ultimately reached a similar plateau to that seen in control cultures, suggesting that primary fusion was relatively normal in the absence of the β1D isoform (*p < 0.05). (E) Determination of myonuclear content in siRNA-treated cultures. Cells transfected with siRNA were cultured in DM for 72 h. The proportion of myotubes containing a large number of nuclei was decreased in β1D siRNA-treated cultures suggesting a selective inhibition of secondary fusion (*p < 0.05).