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Fig. 1 | BMC Biology

Fig. 1

From: Structural basis for potency differences between GDF8 and GDF11

Fig. 1

GDF11 is a more potent ligand than GDF8. a Overview of the well-established canonical activin A, activin B, GDF8, GDF11, and TGFβ receptor utilization and downstream SMAD pathway. b, c, d Potency differences between GDF8 and GDF11. Luciferase reporter gene assay ((CAGA)12 promoter) following titration of GDF8 (blue) and GDF11 (orange) ligands in HEK293 (b) and HepG2 (c) cells. Luciferase activity was assessed 18–24 h post ligand treatment. The black arrows in (b) indicate the ligand concentrations utilized in panels e and f. In d, mouse gonadotrope (LβT2) cells were treated with increasing doses of GDF8 (blue) or GDF11 (orange). Follicle-stimulating hormone (FSH) release was measured 24 h later, as previously described [92]. Refer to Additional file 1: Table S1 for a corresponding analysis of the activation curves. e Short exposure to GDF11 results in a significantly enhanced SMAD3-dependent response compared to GDF8. The experimental design (left) is such that the ligand was added to HEK293 cells stably transfected with the (CAGA)12 promoter driving the luciferase gene for the indicated time followed by replacement of media without ligand. Activity was measured 24 h after initial treatment. Cells were treated with GDF8 or GDF11 at a ligand concentration of 25 pM (middle) and 125 pM (right). f Time-dependent differences in the SMAD3 activation by GDF8 and GDF11. Similar experimental design (left) as in e, but instead cells were lysed and assessed for luciferase activity at the indicated time of ligand treatment. Cells were treated with GDF8 or GDF11 at a ligand concentration of 25 pM (middle) and 125 pM (right). Data information: In b, c and e, f, data are presented as fold activation above background (0 nM ligand concentration). Each concentration was performed in triplicate and shown as the mean ± standard error of the mean (SEM) of three independent experiments. In d, data are presented as fold FSH release above background (0 nM ligand concentrations). Each concentration was performed in triplicate and shown as the mean ± SEM of one experiment; data shown are representative of 10 independent experiments. In e, f, curves were compared using two-way ANOVA with Bonferroni correction (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001). Ligand sources: for b, e, f, gift from Acceleron Pharma; for c, d, purchased from R&D Systems; Cat. no. 788-G8-CF and Cat. no. 1958-GD-010-CF)

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