Expression of Myogenic Regulatory Factors

For this assay C2C12 cells were seeded in black Advanced TC 96-well microplates (Greiner Bio-One, Kremsmünster, Austria). Cell culture and treatments with recombinant proteins were performed in both proliferation and differentiation stages according to the above-described protocols (Figures 1A,B). Immunofluorescence staining and image analysis were performed as described before (Alvarez et al., 2020). At 24 h after treatment with the recombinant proteins, cells were washed, fixed for 1 h with cold 4% paraformaldehyde, permeabilized for 5 min and blocked with 1% bovine serum albumin for 30 min. The primary antibodies rabbit anti-mouse Pax7, Myf5, MyoD (1:1000; Santa Cruz Biotechnology, Dallas, TX, United States), and myogenin (1:2000; Sigma-Aldrich) were added and incubated overnight at 4°C. After washing (three times), the cells were incubated with a Hoechst DNA-specific stain (1:3000; Thermo Fisher Scientific, Waltham, MA, United States) and anti-rabbit secondary antibodies conjugated to Alexa Fluor 488, 568, 647, and 594 respectively (1:1000; Thermo Fisher Scientific) at room temperature for 1 h. Finally, the plates were subjected to high content imaging analysis on MetaXpress High-Content Image Acquisition & Analysis Software (Molecular Devices). The system was used to acquire 16 images per well at 20× magnifications, and the quantitative data shown represent the mean fluorescence intensity of each protein, corrected with the secondary antibody control.