pGL3-(CAGA)12-luciferase reporter assay

As described in detail previously [19], capacities of truncated, MSTNpro proteins to inhibit MSTN or GDF11 activities in vitro were measured using the pGL3-(CAGA)₁₂-firefly luciferase reporter assay in HEK293 cells stably expressing (CAGA) ₁₂-luciferase gene construct [25]. Cells (40,000 cells/well) were seeded on a 96-well culture plate at in DMEM with 10% fetal calf serum, penicillin-streptomycin plus fungizone, and grown for 24 hr at 37°C with 5% CO₂. 1 nM of MSTN (R&D Systems) or GDF11 (R&D Systems) plus various concentrations of commercial mouse MSTNpro (R&D Systems) or pig MSTNpro proteins in DMEM without serum were added to each well after removing the medium, and incubated for 24 hrLuminescence was measured using Veritas microplate luminometer (Turner Biosystems Inc., CA, USA) after adding Bright-Glo luminescence substrate (Promega, Madison, WI, USA). The % inhibition of MSTN activity was calculated by the following formula: % inhibition = (luminescence at 1 nM MSTN—luminescence at each ligand concentration)*100/(luminescence at 1 nM MSTN–luminescence at 0 nM MSTN). The MSTN-inhibitory activities were analyzed by regression analysis using Prism5 program (Graphpad, San Diego, CA). To examine the differences in MSTN-inhibitory capacity of these proteins and peptides, IC₅₀ (ligand concentration inhibiting 50% of MSTN activity) values were estimated using a non-linear regression model defining dose response curve. The equation for the model was as follows: Y = Bottom + (Top–Bottom)/(1 + 10^(X–LogIC₅₀)), where Y is % inhibition, Bottom is the lowest value of % inhibition, Top is the highest value of % inhibition, and X is Log ligand concentration. IC₅₀ values were analyzed by ANOVA (Analysis of Variance) using the same program.