Fig. 5: DLK1 inhibits Myostatin-mediated ACVR2B activation in reporter cells and myoblasts.

A Illustration of Myostatin-ACVR2B signaling in the presence or absence of DLK1. Binding of the canonical ligand Myostatin to ACVR2B and ACVR1B leads to the formation of a 2:2:2 complex and subsequent activation of SMAD2/3 (left). The EGF5 domain of DLK1 binds to ACVR2B to inhibit ligand signaling (right), and the DLK1(EGF5-6) region used for co-crystallization is indicated with a dashed circle. Created in BioRender. Antfolk, D. (2025) https://BioRender.com/p73c456B DLK1 inhibits Myostatin-ACVR2B signaling in a HEK293-(CAGA)₁₂ reporter assay. Myostatin treatment at 2 nM is represented as 100% activation, and a decrease in activation was observed upon treatment with increasing concentrations (125 nM-16 µM) of soluble DLK1. Data is represented as normalized relative luciferase units (RLU) represented as the mean of triplicate wells from one representative experiment. The experiment was independently repeated three times. C DLK1 transfected into HEK293-(CAGA)₁₂ reporter cells inhibit Myostatin signaling. Data is represented as RLU based on quadruplicate wells from one representative experiment. The experiment was independently repeated two times. D Representative microscopy images showing C2C12 myoblast differentiation in the presence of Myostatin, Myostatin + DLK1, or Myostatin + DLK1^R193D (loss-of-ACVR2B-binding mutant). Control cells were allowed to differentiate for 72 h. Myostatin treatment (4 ug/ml) inhibits C2C12 myoblast differentiation into myotubes as determined by MyoHC staining. C2C12 cells were fixed with 4% PFA, immunostained with an anti-MyoHC antibody and an anti-mouse IgG Alexa Fluor 488 secondary antibody. Nuclei were counterstained with Hoechst 33342. Nuclei represented with pseudo color (magenta) in zoom in panels. Scale bar, 100 μm. The experiment was independently repeated four times. Source data are provided as a Source Data file.