Fig 2: Full immunoblots from AAV8.Mstn- and AAV8.GDF11-treated mice A, BQuadriceps (A) and heart (B) lysates from mice described in Figs 2 and EV2 were immunoblotted for phosphorylated and total content of SMAD3, SMAD1/5/8, Akt, p38 MAPK, ERK1/2, total SMAD4, and total NOX4. GAPDH content was used as a loading control and normalization standard. Note that the cropped version of many of these images is found in Fig 5A and B.CPhosphorylation of TAK1 is not different between control and AAV8.GDF11-treated hearts or quadriceps.

Fig 3: GDF11PRO-Fc blocks GDF11/MSTN-induced myotube atrophy in C2C12 cells. a Schematic detailing experimental timeline in C2C12 myotubes. AAV6-EGFP or AAV6-GDF11PRO-Fc was added to C2C12 myotubes at a MOI of 105 on day 5 post-differentiation and 100 ng/ml rGDF11 or rMSTN was added on day 7. Myotubes were stained and analyzed on day 10. b EGFP expression was evident at 48–72 h in C2C12 myotubes treated with AAV6-EGFP (MOI 10 [5]). Scale bars represents 50 µm. c Vector genome copy number per diploid genome in C2C12 myotubes 72 h after addition of AAV6-EGFP or AAV6-GDF11PRO-Fc (MOI 10 [5]). d Representative immunofluorescence images of C2C12 myotubes. C2C12 myotube membranes were visualized by staining with an anti-dystrophin antibody (red). Nuclei were stained with DAPI (blue). Inset shows a zoomed-in region. Scale bars represent 50 µm (main panel) and 25 µm (panel inset). e The fraction of nuclei incorporated into myotubes (differentiation index) was calculated and presented as a percentage of control. f Average myotube diameter relative to control and (g) distribution of diameter measurements. For myotube diameter measurements, each myotube was measured at three points along the length of the myotube and averaged. h Number of nuclei incorporated per myotube. A minimum of 50 myotubes were analyzed per experimental condition. i pSMAD2/3 relative to tSMAD2/3 was assessed by western blot. Equal protein loading was verified by Ponceau S staining and GAPDH was used as a loading control. Data represents results from three separate experiments. All error bars represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant; compared to AAV6-EGFP-treated control. †p < 0.05; ††p < 0.01; †††p < 0.001; compared to AAV6-EGFP + ligand-treated. pSMAD2/3: phosphorylated SMAD2/3; tSMAD2/3: total SMAD2/3

Fig 4: GDF11PRO-Fc associates with GDF11 and MSTN. Protein-protein interactions between GDF11PRO-Fc or MPRO-Fc and rGDF11, rMSTN, or rActivin A were determined by a pull-down assay. GDF11PRO-Fc or MPRO-Fc was incubated with rGDF11, rMSTN, or rActivin A for 1 h at 4 °C. Fc-fused protein complexes were separated on a protein A/G-coated agarose resin and eluates were run on a 12% SDS-PAGE gel under reducing conditions and probed by western blot. Input control was 5% of the input material. WB western blot

Fig 5: Differential activin receptor levels in skeletal muscle and heart A, BRelative muscle content of the activin type IIB receptor (ActRIIB) in quadriceps (n = 6) and hearts (n = 4) from multiple treatment groups, as determined by immunoblotting (normalized to Ponceau-visualized loading).C–FGene expression of Acvr1b (ALK4 gene; C), Tgfbr1 (ALK5 gene; D and E), and Tgfb1 (F) in quadriceps and heart of untreated 7-week-old C57BL/6 mice (n = 3), as measured by real-time PCR. Relative gene expression values were calculated by the ??Ct method using Gapdh (C, D and F) or Acvr1b (E) as reference genes.GCardiac gene expression of Tgfb1 in control (n = 3), day 3 (n = 4), and day 5 (n = 4) high-dose (1 × 1012 gc) AAV8.GDF11-treated C57BL/6 mice. Relative gene expression values were calculated by the ??Ct method using Gapdh as the reference gene.Data information: Values are displayed as mean ± SEM. In (B–F), statistical analysis performed using two-tailed Student's t-test, with effect size presented as Cohen's d (d). In (G), statistical analysis performed using one-way ANOVA with Tukey's HSD post hoc test (non-connecting letters indicate P < 0.05 between groups) and effect size presented as eta-squared (?2).