Immunocytochemistry.

Myofilament proteins were visualized by immunocytochemistry. Cells were grown on Matrigel-coated (60 μg/cm²), 35-mm, glass bottom imaging dishes (MatTek; Ashland, MA) or plastic, as detailed above, with the modification that the media were changed daily. Cells were fixed with 4% paraformaldehyde (Fisher Scientific, Atlanta, GA), permeabilized with 0.2% Triton X-100 (Fisher), and blocked with 5% BSA in PBS for 1 h at room temperature. Cells were incubated overnight at 4°C in fast-twitch skeletal muscle myosin antibody (1:500, MY-32; Sigma) followed by secondary antibody (1:100, anti-mouse IgG; Molecular Probes) to visualize myofilaments or 1 μM tetramethylrhodamine isothiocyanate-labeled phalloidin (Sigma) to stain actin to visualize the entire cell. Cells were imaged using a Nikon Ti-E inverted microscope with C2 confocal at ×40 for myofilament measures or an Olympus BX51 with QImaging Retiga R6 at ×10.