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. 2019 Feb 22;12:47. doi: 10.3389/fnmol.2019.00047

FIGURE 3.

FIGURE 3

Neurite outgrowth induced by ARTN (A), artefin-dendrimer (B), and artefin-monomer (C) in CGNs. Cells were stimulated with ARTN (0.013, 0.042, 0.13, 0.42, and 2.1 nM) or artefin (0.16, 0.47, 1.4, 4.2, 12.6, and 37.9 μM) for 24 h. For the unstimulated control, the absolute length of neurites was 11.47 ± 1.5 μm. Results from four independent experiments are expressed as percentage of untreated control set to 100% and presented as mean ± SEM. The mean neurite outgrowth lengths were compared using one-way ANOVA for repeated measurements. The effect of individual concentrations of ARTN and artefin was compared to control using one-way ANOVA with Dunnett’s post-hoc test, ∗p < 0.05; ∗∗p < 0.01. (D) Neuritogenic effect of artefin, scrambled and reverse peptides (tetramers). CGN neurons were treated with peptides in concentration of 4.2 μM for 24 h. The results are expressed as percentage of untreated control set to 100% and presented as mean ± SEM. The neuritogenic effect was compared using Student’s paired t-test where + indicates p-values for comparison to the negative control of untreated neurons, and ∗ indicates p-values for comparison to the positive control of neurons treated with artefin. ++p < 0.01; ∗p < 0.05, ∗∗p < 0.01. (E) Inhibition effect of ARTN on neurite outgrowth, by competing with artefin. CGNs were grown for 24 h in the presence of none-neuritogenic concentration of ARTN (2.1 nM), artefin (4.2 μM), or co-incubated with ARTN together with artefin in the same concentrations. Results are expressed as percentage of untreated control set at 100% and presented as mean ± SEM. The neuritogenic effect was compared using Student’s paired t-test, where + indicates p-values for comparison to the negative control and ∗ indicates p-values for comparison to neurons stimulated with artefin alone. +++p < 0.001; ∗∗∗p < 0.001. (F) Representative images of CGNs stimulated with ARTN (0.042 nM), artefin-dendrimeric (atrefin-d; 4.2 μM) and artefin-monomer (artefin-m; 4.2 μM) double-stained for GAP-43 (red) and Hoechst (blue). Scale bar: 100 μm.