Engrailed-2 reduces synaptic cluster area.

<p>(A-D) Frequency distribution of the area of vGlut1 (green) and PSD95 (red) (A, B) and vGlut1/PSD95 overlap (i.e. synaptic overlap, brown) (C, D) clusters analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181350#pone.0181350.g004" target="_blank">Fig 4</a>. Kolmogorov-Smirnov test on two samples (<a href="http://www.physics.csbsju.edu/stats/KS-test.html" target="_blank">http://www.physics.csbsju.edu/stats/KS-test.html</a>): A, cont-PSD vs En1-PSD (D = 0.072, p = 0.178), cont-Vglut vs En1-Vglut (D = 0.084, p = 0.055); B, cont-PSD vs En2-PSD (D = 0.183, ***p<0.0001), cont-Vglut vs En2-Vglut (D = 0.055, p = 0.568); C, cont-overlap vs En1-overlap (D = 0.074, p = 0.208); D, cont-overlap vs En2-overlap, (D = 0.112, *, p = 0.047).</p

Engrailed increases immature spine density in vitro.

<p>(A-E) Hippocampal cells were transfected with pEGFP, daily treated with 150ng/ml of Engrailed or En1SR, a membrane non-permeant mutated construct, from div15 to div18 and imaged at div19. (B) Quantification of dendritic spine density after treatment with either En1 or En2, or (C) after treatment with either En1 or En1SR. Significance of the differences were assessed with a Kruskal-Wallis test in B (****, p< 0.0001), and C (***, p = 0.0001), followed by a Dunn's Multiple Comparison Test. *, p<0.05; **, p<0.01; ***, p<0.001. Values are the mean +/- s.e.m. of measures from 3 independent experiments with an average of 39 pyramidal cells per condition. (D) Quantification of branched spines after treatment with En1. ***, p = 0.0004 (t-test). (E,F) Morphometric analysis of dendritic spines under control conditions or after En1 treatments. (E) Spines were categorized using Imaris software as described in Methods. En1 increased the density of “stubby”, ****, p<0.0001 and “thin” spines, ***, p = 0.0002 (t-test) but not the one of “mushroom” spines, p = 0.1874 (Mann-Whitney test). (F) En1 increases the maximum diameter of stubby spines (KS test, D = 0.159, p<0.0001), does not increase thin spine length (KS test, D = 0.06, p = 0.054), and slightly increases mushroom spine volume (KS test, D = 0.119, p = 0.01). Values in D-F are the mean +/- s.e.m. of measures from 2 independent experiments each cumulating an average of 30 pyramidal cells per condition. Bar in A, 5μm.</p

Engrailed increases mTORC1 activity and protein synthesis in hippocampal cells.

<p><b>(</b>A) De novo protein synthesis in hippocampal neurons visualized with SUnSET. DIV18-20 neurons were analyzed without treatment (cont) or after a 1-hr treatment with either En1 or En2. Shown are inverted grayscale images. (B) Phosphorylation of S6 (Ser240/244) assayed by immunofluorescence in untreated hippocampal neurons (cont) or in neurons treated as in A. (C) The SunSET signal was strictly dependent on the presence of puromycin and strongly suppressed by anisomycin indicating that it was reporting de novo protein synthesis. (D) Quantification of SUnSET images. (E) Quantification of phospho-S6 images. Values in D and E are the mean (+/- s.e.m.) fluorescence intensity in neurons from N = 19–22 images from 2 independent experiments. D and E, One-way ANOVA (****, p< 0.0001) followed by a Tukey’s Multiple Comparison Test. C, Kruskal-Wallis test (**** p< 0.0001) followed by Dunn's Multiple Comparison Test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Detailed P-values are given in supplementary information. Bar in A and B, 20μm.</p

Exogenous Engrailed increases dendritic complexity.

<p>(A) Div9 hippocampal cells not-treated (A, left) or daily treated with 150ng/ml of purified En for 6 days (div4-div9, A, right) and immunolabelled with MAP-2. Gabaergic cells were identified by immunolabelling with an anti-GAD67 antibody. (B-G) quantification of dendritic nodes (B, E) dendritic tips (C, F), and dendritic length (D, G) per non-treated pyramidal cells (cont) and pyramidal cells treated with either En1 or En2 (B-D), and per non-treated GABA cells (cont) and GABA cells treated with either En1 or En2 (E-G). Values are the mean +/- s.e.m. of measures from a total of 40–45 pyramidal cells and from a total of 30–40 GABA cells/condition from 3 (En1) and 2 (En2) independent experiments. Significance of the differences were assessed with a Kruskal-Wallis test, in B, D, F, G (****, p< 0.0001), C (***, p = 0.0006), and in E (***, p = 0.0002), followed by a Dunn's Multiple Comparison Test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Detailed P-values are given in supplementary information. Bar in A, 35μm.</p