Fig. 4

GDF11 acts via STAT3, SOCS3, and iNOS to induce proteolysis in muscle wasting in vitro. A–D NF-κB, ERK, Smad, or STAT3 dependent luciferase reporters in C2C12 myotubes treated with rGDF11 with the dose ranging from 0 to 100 ng/ml. E Representative Western blots of target proteins (iNOS, phosphorylation and total STAT3, phosphorylation and total Smad2/3, socs3) and loading control (GAPDH) from myotubes treated with rGDF11 with the dose ranging from 0 to 100 ng/ml for 48 h. F NO levels were measured in supernatant from the myotubes described in the panel. G Total protein content of rGDF11-treated myotubes. H Representative western blotting images of ubiquitin from myotubes. I Protein expression of trim63, fbx32, and foxo1 in myotubes treated with rGDF11 with the dose ranging from 0 to 100 ng/ml. GAPDH was used as an internal control. J Bright-field images of myotubes treated with rGDF11, with or without the 26S ribosome inhibitor MG-132 (10 μM) for 48 h; diameter of myotubes for conditions represented in the panel. Scale bar is 50μm. Data presented as mean ± SEM. Versus vehicle control, *P < 0.05, **P < 0.01, ***P < 0.001; versus rGDF11 control, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001; n=3