2.4. Immunofluorescence Assay

First, the cells in 12-well plate were fixed in 4% paraformaldehyde for 15 minutes and rinsed 3 times for 5 minutes each in PBS. Then, the cells were permeabilized with 0.1% Triton X-100 for 10 minutes, followed by blocking with 1% BSA (Beyotime, Shanghai, China) for 30 minutes. Next, the cells were incubated with anti-MyHC antibody (1:100; Developmental Studies Hybridoma Bank [DSHB], Iowa City, IA, USA) at 4 °C overnight, and then with FITC-labeled Goat Anti-Mouse IgG (H + L; 1:1000; Beyotime) at room temperature for 1 hour. After DAPI (Sigma-Aldrich) staining for 5 minutes, the cell nuclei were examined using a TCS SP8 confocal microscope (Leica, Wetzlar, Germany).