Fig. 1. Rheb C180S expression induced axonal elongation. (A) Primary rat hippocampal neurons were transfected with plasmids for EGFP fusion proteins of Rheb WT, Rheb C180S or control vector constructs at E18 and stained and photographed on 3 d.i.v. for GFP (green), tau (red, axonal marker) and MAP2 (blue, dendrite marker) along with a Hoechst staining (white, nucleus). Arrowheads indicate terminal ends of the axon. The scale bar is 50 µm. (B) The length of GFP-positive axons (mean±S.E.; *and #, *p<0.05 vs. control, ***p<0.0001, #p<0.05 vs. Rheb WT, ###p<0.0001 with one-way ANOVA and Tukey post hoc test, compared with control; n>35). (C) The percentage of neurons without an axon (0), with a single axon (1), and with multiple axons (≥2). (D) Confocal images of coronal sections of postnatal 5 days (P5) mice transfected by in utero electroporation with pCAGIG-Control, pCAGIG-Rheb WT, and pCAGIG-Rheb C180S and at E15.5 and immunostained for GFP (green) and Cux1 (red). The scale bar is 500 µm. (E) Distribution of GFP positive cells in contralateral Layer II (mean±S.E.; *, *p<0.05 vs. control with one-way ANOVA and Tukey post hoc test, compared with control; n>5 animals). (F) Primary rat hippocampal neurons were transfected with plasmids for EGFP fusion proteins of Rheb WT, Rheb C180S constructs at E18 and stained and photographed on 3 d.i.v. for GFP (green), GM130 (red, Cis-Golgi marker), LAMP1 (red, Lysosome marker) along with a Hoechst staining (white, nucleus). The scale bar is 5 µm.
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