Fluorescence microscope

The cells grown on coverslips were fixed in 4% (v/v) paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature or overnight at 4°C. After rinsing in PBS, the fixed cells were permeabilized and nonspecific epitopes were blocked using 2% bovine serum albumin (Bovogen Biologicals, Keilor East, Vic, Australia) in 0.1% Tween-20/PBS, followed by incubation in the diluted primary antibody for 1 h at room temperature or overnight at 4°C. After three washes in PBS, samples were incubated for 1 h at room temperature with secondary antibodies diluted in PBS. Prepared samples were then mounted using Vectashield medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) and photographed using a fluorescence microscope (Nikon Corporation, Tokyo, Japan). The manufacturers and catalog numbers of the antibodies employed were as follows: mouse anti-myosin heavy chain (MHC; cat. no. MAB4470; R&D Systems) mouse anti-myogenin (cat. no. ab-1835; Abcam, Cambridge, MA, USA), mouse anti-desmin (cat. no. D1033; Sigma-Aldrich, St. Louis, MO, USA), Alexa-568 goat anti-mouse IgG (cat. no. A-11004), Alexa-488 goat anti-rabbit IgG (cat. no. A-11008) (both from Life Technologies). Quantification of immunofluorescence staining confirmed that four slides were used for each condition. Graphs represent the average of multiple tests from three independent experiments.