Figures & data
Figure 1 Study design. After four days of differentiation, cells were treated for two days (three times per day) with differentiation media (DM) and vehicle (10 mM Tris-NaCl), 10 ng/ml recombinant GDF-8 MSTN, 10 ng/ml MSTN and 10 μM leucine, 10 ng/ml MSTN and 10 μM of HMB, or 10 ng/ml MSTN and 10 μM creatine monohydrate.
Figure 2 Efects of MSTN in the absence or presence of leucine, HMB, or creatine on markers of skeletal muscfle hypertrophy. Legend: Data are presented as means ± standard error. Data in A show that MSTN treatment decreases myotube diameter, while Leu, HMB, and Crea rescue MSTN-induced atrophy. Data in B & C are n = 5-6 plates per treatment. Muscle protein synthesis data in E are n = 4 treatments per plate; of note, MSTN+Leu, MSTN+HMB and MSTN+Crea samples were separately analyzed for MPS (data not shown). One-way ANOVA with LSD post-hoc comparisons performed where applicable; difference superscript letters = p < 0.05 and NS = no significant differences. In subfigure D of 10x light micrographs, white bar is 100 μm.
Figure 3 Effects of MSTN in the absence or presence of leucine, HMB, or creatine on the expression of MSTN-related mRNAs. Legend: Data are presented as means ± standard error (n = 6–9 plates per treatment). Effects of MSTN with or without each ingredient on the mRNA expression of explicit atrogenes (A), MSTN signaling repressors (B), and MSTN transcriptional targets (C). Sub-figures D and E demonstrate that Akirin-1/Mighty mRNA expression was modestly-to-strongly correlated with measured hypertrophy variables. Sub-figure F shows that Akirin-1/Mighty mRNA expression was modestly correlated with the early differentiation marker MyoD mRNA. One-way ANOVA with LSD post-hoc comparisons performed in sub-figures A/B/C; difference superscript letters = p < 0.05. In sub-figure C, ** indicates that MSTN down-regulated MyoD mRNA compared to all other groups (p < 0.01).
Figure 4 Experiment demonstrating Akirin-1/Mighty mRNA knockdown affects myotube size. Legend: Data are presented as means ± standard error (n = 4–5 plates per treatment). Effects of shRNA-mediated Akirin-1/Mighty mRNA knockdown on myotube size versus a scrambled shRNA control condition (A), and confirmation that Akirin-1 mRNA was reduced in shRNA-transfected myotubes (B). Photographs in sub-figure C are 10x representative images of GFP-positive cells (arrows) that were transfected with either a scrambled (CTL)-shRNA or Akirin-1/Mighty-shRNA.
