Goat serum

Sperm slides were permeabilized and blocked with 5% goat serum (Beyotime, China) for 45 min. Primary antibodies were treated overnight at 4 °C. After three washes with PBS, Alexa Fluor 484- or 555-labeled donkey anti-rabbit IgG (Beyotime, China) was incubated for 45 min. The nuclei were counterstained with DAPI dye (Beyotime, China). The antibodies used in immunofluorescence staining are provided in Figure 7—source data 1.

Cells were seeded on round coverslips (Corning Inc.) and cultured for 4 days. After various treatments, the cells were carefully washed three times with PBS, fixed with 4% paraformaldehyde for 1 h, and permeabilized with 0.5% Triton X-100 for 10 min. Then, they were blocked with 5% goat serum (Beyotime) for 1 h and incubated for 1 day at 4 °C with antibodies against RUNX2, OPN, DNMT1, DNMT3a, DNMT3b, 5-MC (28692S), β-catenin, or LEF1. The next day, the samples were incubated with a fluorescent dye-conjugated secondary antibody (Beyotime) for 1 h. Nuclei were counterstained with 4ʹ6-diamidino-2-phenylindole (Beyotime) for 10 min and phalloidin (Beyotime) was used to stain microfilaments for 10 min. Cells were imaged under a laser scanning confocal microscope (Olympus, Japan).

Cultured cells were fixed in 4% PFA (Sigma-Aldrich) for 15 min, and then blocked in 5% goat serum (Beyotime) with 0.1% Triton X-100 (Beyotime) in PBS for 30 min at room temperature. The cells were then incubated overnight with anti-GALNS antibody (1:200; Proteintech) at 4 °C, followed by Alexa Fluor-488 donkey anti-rabbit IgG (1:500) (Invitrogen) for 1 h at room temperature. After counterstaining with DAPI (Beyotime), the cells were viewed by confocal microscopy.

After 72 h of transfection, the DM of SMSCs was removed, and the cells were washed with PBS (Gibco) 3 times before SMSCs were fixed with 4% paraformaldehyde for 30 min. Subsequently, 0.1% Triton X-100 was used to permeate the SMSCs. The 5% goat serum (Beyotime) was used to block the SMSCs so that the cells could better bind to the primary antibody of Myosin (Santa Cruz, United States; 1: 200). SMSCs were incubated with Rhodamine (TRITC) AffiniPure Goat Anti-Mouse Immunoglobulin G (IgG) (ZenBio; 1: 1000) at 37°C for 1 h. Then, DAPI (Beyotime; 1: 50) was used to stain the cell nuclei before the required images were captured. The results were determined using Image-Pro Plus software.

Chicken primary myoblasts were seeded in 48-well plates, and the cells were induced to differentiation for 72 h after transfected by treatments. The cells were fixed in 4% formaldehyde for 30 min and permeabilized by 0.1% Triton X-100 for 20 min and then blocked by 5% goat serum (Beyotime) at room temperature for 30 min. Thereafter, the cells were incubated with primary antibody anti-MyHC (Santa Cruz; 1:250) at 4°C for 12 h and then incubated with the Rhodamine (TRITC) AffiniPure Goat Anti-Mouse IgG (ZenBio; 1:1,000) at 37°C for 1 h. Finally, cell nucleus was stained with DAPI (Beyotime; 1:50) for 5 min. Three images were randomly captured by a fluorescence microscope (Olympus, Japan). The MyHC labeled myotube area and DAPI-labeled nuclear area (Internal control) were measured by Image-Pro Plus software, and the ratio of myotube area to nuclear area is taken as the relative myotube area.

Cells were cultured in confocal dishes for the stipulated duration, fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS (Beyotime) for 15 min, and then blocked with 5% goat serum (Beyotime) in PBS for 30 min at room temperature. The cells were incubated overnight with anti-TOM40 antibody (1:200) (Proteintech) at 4 ℃, followed by incubation with Alexa Fluor-488 donkey anti-rabbit IgG (1:500) (Invitrogen) for 1 h at room temperature. After counterstaining with DAPI (Beyotime), the cells were imaged with a Leica TCS SP5 confocal microscope.