Mef2c

Cells were lysed with Laemmli sample buffer. Samples were boiled for 5 min in sample buffer containing bromophenol blue and 1×β-ME, and the proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic separation was carried out on 15% polyacrylamide gels (Bio-Rad, Hercules, CA, USA), and proteins were subsequently transferred to an Immobilon-P PVDF transfer membrane (Millipore, Billerica, MA, USA). Membranes were blocked in PBS-Tween 20 (PBS-T) with 5% non-fat dry milk powder, and incubated with the primary antibodies β-actin (1:10000, Sigma-Aldrich) and MEF-2C (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were then washed with PBS-T and incubated with anti-mouse or anti-goat secondary antibody (1:5000, Santa Cruz Biotechnology).

We collected 10 μm-thick cryosections in accordance with standard procedure for Immunohistochemistry and EdU staining. When combined with EdU detection, we carried out firstly EdU staining using Click-iT EdU kit (Invitrogen, C10340) in accordance with the manufacturer’s instructions, then followed by a standard immunohistochemistry protocol as previously described (Fei et al., 2017 (link)). In brief, after PBS wash and permeabilization with PBST, slides were blocked in 5% serum prepared in PBST, then incubated with primary antibodies overnight at 4°C, followed by sequential PBST washes and secondary antibody (with DAPI, Sigma-Aldrich, D9542) incubation, and finally mounted using Mowiol 4-88 (Sigma-Aldrich, 9002-89-5) mounting medium after several PBST washes. Fluorescence images were acquired with an Olympus IX83 microscope (Olympus, Tokyo, Japan), using ×20 objectives and analyzed using FIJI. The following antibodies were used in this study, MBP (Genetex, GTX761141), PRRX1 (Gerber et al., 2018 (link)), βIII-TUBULIN (R&D, MAB1195), SOX9 (Chemicon, Ab5535), PAX7 (DSHB, AB528428), MEF2C (Santa Cruz, sc-365862), phospho-Histone H3 (Abcam, 10543), MHC (DSHB, A4.1025). Alexa 488-donkey-anti-mouse IgG (Jackson, 711-547-003), Alexa 555-donkey-anti-rat IgG (Invitrogen, SA5-10027), Alexa 647-donkey-anti-mouse IgG (Jackson, 715-607-003), CY3-donkey-anti-mouse IgG (Jackson, 715-165-151).

Western blot was performed using 30 μg of total protein. The primary antibodies Mef2c (sc-13268; Santa Cruz Biotechnology, Dallas, TX, USA) and Tubulin (sc-8035; Santa Cruz Biotechnology, Dallas, TX, USA) were used at a concentration of 1:100 and 1:5000, respectively, and incubated overnight at 4 °C and the secondary antibody-HRP conjugate (#170-6516, Biorad, Hercules, CA, USA) at 1/5000 for 2 h at room temperature. Blocking was carried out with 5% milk and washed with PBST according to the antibody manufacturer’s recommendations.

For immunocytochemical analysis, the primary cells (first passage) grown in chamber slides were fixed with 1% paraformaldehyde and permeabilized with 0.25% Triton for 30 minutes. After 1 hour of blocking in 5% normal goat serum (NGS), cells were incubated for 1 hour with primary antibodies, including polyclonal anti-cTnT (DAKO, Germany) 1:100 and polyclonal anti-GATA-4, isl-1, Nkx-2.5, and MEF-2c 1:50 (Santa Cruz, USA). After washing in 1% NGS-PBS buffer, we added secondary TRTIC-conjugate antibody (goat IgG, Santa Cruz) and incubated for 60 minutes. Nuclei were counterstained with DAPI (DAKO), and the chamber slides were sealed with Prolong Gold (Life Science, USA). All visualization was digitalized by using phase-contrast microscopy (Olympus BX61, Japan) with software (SIS F-View).