Fig. 8

Generation of GDF8/GDF11 chimeric ligand confers potency to GDF8. a Luciferase reporter gene assay ((CAGA)₁₂ promoter) following transient co-transfection of HEK293 cells with GDF8/GDF11 chimera constructs (25 ng, 50 ng, or 100 ng), furin (50 ng), and TLL2 (50 ng). All constructs contain human wild-type GDF8 prodomain followed by human wild-type or mutated mature GDF8. 18–24 h post transfection, the culture media were replaced with serum-free media and allowed to incubate for an additional 24 h, at which point the cells were lysed and measured for luciferase activity. wt = GDF8; point mutations are indicated by number. For chimera number 12 (GDF8 pro/GDF11 mature), each non-identical residue between GDF8 and GDF11 was mutated to generate the equivalent wt GDF11 ligand. A surface representation of GDF8 dimer is shown. Each monomer is colored independently (monomer A = blue; monomer B = gray). Non-identical residues are shown in orange. b, c Luciferase reporter gene assay ((CAGA)₁₂ promoter) following exogenous addition of purified empty vector, wt, or GDF8/GDF11 chimeric latent protein complexes (see Additional file 6: Figure S5). The latent complexes (~1.5 ng mature ligand) were activated by treatment with acid (b; adjusted to pH 2.5 for 1 h and then neutralized), or the HEK293 cells were transiently transfected with TLL2 prior to protein addition (c). AA acid activated, Not AA no acid activation, Not AA + GASP1 no acid activation but complexes added to cells in the presence of 100 nM GASP1, AA + GASP1 acid activation in the presence of 100 nM GASP1, EV empty vector transfected, EV + GASP1 empty vector transfected, but complexes added in the presence of 100 nM GASP1, TLL2 transfected with TLL2, TLL2 + GASP1 cells transfected with TLL2, complexes added in the presence of 100 nM GASP1. Data information: In a, b, c, data are presented as a ratio of the fold activation (mutant/wt GDF8) where each was normalized to the response of empty vector control. Each concentration was performed in duplicate or triplicate, and each bar is shown as the mean ± SEM from two to three independent experiments. ^a P ≤ 0.05, ^b P ≤ 0.01, ^c P ≤ 0.001, and ns = not significant (Student’s t test). Ligand sources: produced and purified as described in “Methods”