Mandra1

Protein extracts from tissues and cultured cells were prepared using a RIPA buffer (Cell Signaling). Protein concentration was determined using the BCA assay (Pierce). Fifty micrograms of total protein per lane were used for differentiated MPCs and muscles from injected adult mice. Samples were denatured at 99°C for 5 minutes before being loaded on to 3%–8% TA precast gels (Invitrogen). Dystrophin and vinculin (loading control) were detected by primary antibodies Mandra1 (1:100, Abcam) and V4505 (1:1,000, Sigma), respectively, followed by horse anti‐mouse IgG HRP‐conjugated secondary antibody (1:1,000, Cell Signaling Technology). A ChemiDoc imaging system (Bio‐Rad) was used to detect chemiluminescence after using a Supersignal West Dura ECL kit (Thermo Fisher). Intensities of dystrophin and vinculin bands were quantified using ImageJ (NIH) and the gel analysis function.

EHHTs with mold were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Alphen aan den Rijn, the Netherlands), snap frozen in cold isopentane, and stored at −80°C. Frozen cryosections (7 μm), hiPSCs, or iCMs on glass coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with phosphate-buffered saline (PBS), the slides and glass coverslips were blocked with 4% BSA with 0.15% Triton X-100 at room temperature for 1 h. The slides and glass coverslips were incubated with primary antibodies including dystrophin (MANDRA1, 1:200), cardiac troponin T (cTnT; ab209813, 1:200, Abcam), OCT4 (PA5-27438, 1:300, Invitrogen), or NANOG (PA1-097, 1:200, Invitrogen) at 4°C overnight. After that, the slides and glass coverslips were warmed up at room temperature for 30 min, washed with PBS and incubated with secondary antibodies Alexa Fluor 555 goat anti-mouse IgG (1:400, Invitrogen) or Alexa Fluor 488 donkey anti-rabbit IgG (1:400, Invitrogen) for 1 h at room temperature. The slides and glass coverslips were sealed with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratory, Burlingame, CA, USA). All images were taken under a Nikon Ti-E fluorescence microscope (magnification 200×).