Fig. 3: GDF11 modulates neuronal activity and enhances autophagy in hippocampal neurons in vitro.
a, Schematic representation of the primary hippocampal neuronal cultures in vitro. Neurons were transfected with a GFP plasmid on DIV11 (only for the spine density experiment) and treated on DIV18. Scale bar: 20 μm. b, Representative confocal images of primary hippocampal neurons in vitro treated with vehicle (Ctrl), cLTP (positive control) or rGDF11 (40 ng ml−1) for 2 h on DIV18 and immunostained with cFos (red) and Hoechst (blue). Scale bar: 100 μm. c, Representative confocal images of GFP+ hippocampal neurons transfected with GFP (green) on DIV11 and treated with either vehicle (Ctrl) or cLTP (positive control) or rGDF11 (40 ng ml−1) on DIV18. Scale bar: 4 μm. d, Quantification of % cFos+ neurons per field (nCtrl = 12 fields, ncLTP = 10 fields, nGDF11 = 13 fields; F (2, 32) = 8.3). e, Quantification of dendritic spine density after a 2-h stimulation with either GDF11 or cLTP or vehicle (nCtrl = 18, ncLTP = 19, nGDF11 = 15 neurons examined over three independent experiments; F (2, 49) = 14.4). Numbers represent the number of spines for every 10 μm of primary dendrite. f, Western blots images of lysates from hippocampal neurons treated with GDF11 or control (vehicle). g–j, Quantification of western blots by optical density for phospho-SMAD2/3 (g) (n = 4 biologically independent samples per condition), Beclin 1 (h) (n = 8 biologically independent samples per condition), LC3 (i) (n = 4 biologically independent samples per condition) and p62 (j) (n = 4 biologically independent samples per condition). One-way one-sided ANOVA and Tukey’s post hoc test for multiple comparisons; two-sided Mann–Whitney test for two-group comparisons; F (DFn, DFd) values presented for each ANOVA statistical analysis; P values <0.05 are represented on the graph; mean ± s.e.m.
