Myogenin

Samples were lysed in ice-cold radioimmunoprecipitation assay buffer with a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). A bicinchoninic acid assay kit (Pierce Biotechnology, Waltham, MA, USA) was used to determine the concentrations of proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to separate the protein samples (30 μg), and the samples were then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Subsequently, the membrane was blocked at room temperature for one hour in Tris-buffered saline with Tween 20 (TBS-T), containing 10% skim milk, and probed with primary antibodies (1:1000 dilutions), including calsarcin-2, myogenin (MYOG), myoblast determination protein 1 (MYOD) (Abcam, Cambridge, UK), and pan-actin (Millipore) at 4 °C overnight. The blots were then washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution) at room temperature for one hour. The protein bands were visualized with Immobilon™ (Millipore). Actin was used as an internal control. The relative signal intensity was determined using ImageJ software.

The lysates of muscle tissue samples on 1 w post injury were loaded on SDS-PAGE gel and transferred to the PVDF membrane. Then CD68 (1 : 1000, Abcam), iNOS (1 : 1000, Abcam), ArgI (1 : 1000, Abcam), TNF-α (1 : 1000, Abcam), α-SMA (1 : 1000, Cell Signaling Technology), desmin (1 : 50,000, Abcam), fibromodulin (FMOD, 1 : 1000, Abcam), myogenin (1 : 1000, Abcam), MyoD (1 : 100, Santa Cruz Biotech), collagen I (1 : 1000, BOSTER), HGF (1 : 100, Santa Cruz Biotech), AKT (1 : 1000, Cell Signaling Technology), pAKT (1 : 1000, Cell Signaling Technology), and GAPDH (1 : 20,000, Santa Cruz Biotech) were probed with the membrane as the primary antibodies at 4°C overnight. The membranes were rinsed and incubated with anti-mouse or anti-rabbit second antibodies at room temperature for 50 min. The blots were visualized by exposure to chemiluminescence ECL reagents (Beyotime). The gray values of the target bands were calculated by Image J software. Each result was obtained by at least three independent experiments.

Cells were grown on glass coverslips 1 cm in diameter and fixed with 4% paraformaldehyde for 15 min. Then cells were washed with PBS and incubated in PBS with 0.2% Triton X-100 for 3 min to permeabilize membranes. Cells were incubated with primary antibodies against desmin, myogenin (Abcam, Cambridge, UK), Cx43 (Transduction Laboratories, Lexington, KY, USA), or Cx45 (Invitrogen, CA, USA) for 1 h or with Alexa Fluor 594 phalloidin (Invitrogen, CA, USA) for 30 min at 37°C. Then cells were rinsed in PBS with 1% BSA and incubated with secondary goat anti-mouse IgG H&L antibodies conjugated with Cy5 or with donkey anti-rabbit IgG H&L Alexa Fluor 488 (Abcam, Cambridge, UK). Coverslips were attached with a Vectashield Mounting Medium with DAPI (Vector Laboratories, CA, USA). The analysis was performed with an inverted fluorescence microscope Olympus IX81 (Olympus Europa holding Gmbh, Hamburg, Germany) equipped with an Orca-R² cooled digital camera (Hamamatsu Photonics K.K., Japan), the fluorescence excitation system MT10 (Olympus Life Science Europa Gmbh, Hamburg, Germany), and the fluorescence imaging system XCELLENCE (Olympus Soft Imaging Solutions Gmbh, München, Germany).

Induced myogenic cells (iMCs) grown on Matrigel-coated 8-well IbiTreat slides (Ibidi) were fixed with 3.7% Formaldehyde (10 min). Cells were permeabilized with 0.1% Tween 20 in TBS and blocked in 0.1% Triton X-100 + 1% FBS in TBS for 30 min. Primary antibodies were diluted in blocking solution and incubated overnight at 4 °C on a shaker (Desmin (1:1000, Abcam ab15200), fast MyHC (1:200, Sigma-Aldrich M4276), MyHC3 (1:200, Santa-Cruz sc-2064), Myogenin (1:800, Abcam ab1835), PAX7 (1:200, Santa-Cruz sc-81648), TUJ1 (1:1000, Sigma-Aldrich T8578)). Next day, secondary antibodies were also diluted in blocking solution and again incubated overnight at 4 °C on a shaker (AlexaFluor 568 goat anti-mouse (1:1000, Thermo Fisher A11031), AlexaFluor 568 donkey anti-rabbit (1:1000, Thermo Fisher A10042), AlexaFluor 488 goat anti-mouse (1:1000, Thermo Fisher A11001)). Hoechst (1:10,000 in DPBS) was incubated for 5 min at RT. Confocal immunofluorescence imaging was performed using the Laser Scan Microscope LSM 700 (Carl Zeiss, Jena, Germany) and for mosaic image acquisition a Leica DMI 6000 B microscope equipped with a XY scanning stage (Leica Microsystems) was used.

Cells were lysed by RIPA buffer and added the bromophenol blue loading buffer, and then the samples were boiled for 10 min and centrifuged at 12,000 rpm for 5 min. The whole-cell lysate was separated into 8% SDS-acrylamide gels and transferred to PVDF membranes. After that, the membranes was blocks by 5% milk in TBST and probed with primary antibodies including mouse SETD3 (3B3, generated by Wuhan Dia-An Company), rabbit polyclonal SETD3 (Abclonal, A8071), MyoD1 (Proteintech, 18943-l-AP), HDAC1 (Abclonal, A2238), Cyclin E1 (Cell Signaling Technology, 20808 S), Myogenin (Abcam, ab124800; or Santa Cruz, D-10, sc-13137), MHC (Developmental Studies Hybridoma Bank, MF-20), β-Actin (Proteintech, 6008-I-Ig), and α-Tubulin (Sigma, T9026). For generation of mouse monoclonal SETD3 antibody, His-tagged full-length human SETD3 protein was expressed in E. Coli and purified as described previously^{14 (link)}. Purified His-SETD3 proteins were immunizated into 5-8 weeks old Balb/C mice and boosted additional 4 times. After several steps including hybridoma production, screening, cloning, and expanding the hybridomas, a subclone named 3B3 was validated and amplified followed the procedure described as before^{39 (link)}. Membranes were further probed with horseradish peroxidase (HRP)-conjugated secondary antibodies and the protein bands were visualized using chemiluminescence detection reagents.

Mouse brains were processed, immunostained, and quantified as previously described [67 (link)–69 (link)]. In brief, mice were placed under isoflurane anesthesia and decapitated. Harvested brains were fixed by immersion in 4% formaldehyde for 24 h and then transferred to a graded ethanol series and embedded in paraffin and sectioned along the sagittal midline. Samples were stained and imaged using an Aperio Scanscope and quantified via automated cell counting using Tissue Studio (Definiens).
Primary antibodies used were: H3K27me3 diluted 1:200 (Cell Signaling, #9733), pRB diluted 1:3000 (Cell Signaling, #8516), cC3 diluted 1:400 (Biocare Medical, #CP229C), NeuN diluted 1:10,000 (Millipore, MAB377), Myogenin (MYOG) diluted 1:500 (Abcam, ab124800), SMYD1 diluted 1:100 (ThermoFisher, PA5-84544), and CDKN2A diluted 1:500 (Abcam, ab241543). Stained images were counterstained with DAPI.