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Fig. 4. β-catenin is involved in axon growth downstream of BDNF- and HGF-signalling. (A) Western blot from hippocampal neurons infected with lentiviral-driven scrambled shRNA or β-catenin shRNA#1 or #2 shows the decrease in β-catenin expression following expression of β-catenin shRNAs compared with scrambled shRNA (scr) or non-infected (ni) neurons. Actin was used as a loading control. Plot represents quantification of β-catenin corrected against the loading control (n4 experiments). Panels show confocal images for anti-β-catenin and anti-GFP (indicating transduced neurons) immunostainings in neurons expressing scrambled shRNA or β-catenin shRNA#1. Note the lower levels of β-catenin especially along the axon in β-catenin-shRNA#1-expressing neurons. Arrows indicate colocalization between GFP and β-catenin immunostainings in neurons expressing scrambled shRNA that is reduced in neurons expressing β-catenin shRNA#1. Scale bar: 20 µm. (B) Hippocampal neurons expressing scrambled shRNA or β-catenin shRNA#1 fixed and immunostained against GFP (driven by the lentiviral vector) at 3 DIV show the decrease in axon length obtained by expressing β-catenin shRNA#1. Scale bar: 40 µm. (C) Quantification of axon length of neurons expressing scrambled or β-catenin shRNA, untreated or treated with BDNF or HGF (normalized to untreated neurons expressing scrambled shRNA). Neurons expressing β-catenin shRNA#1 or #2 display axons that are shorter than controls. Stimulation with BDNF or HGF (50 ng/ml) promotes axon growth in neurons expressing scrambled shRNA, but not in neurons expressing β-catenin shRNA#1 or #2 (n=4-8 experiments). ^**P0.01, ^***P0.001 when compared with the untreated neurons expressing scrambled shRNA; #, P0.05; ##, P0.01 when compared with the BDNF- or HGF-treated neurons expressing scrambled shRNA. (D) Quantification of axon length in neurons expressing intracellular N-cadherin or β-catenin, either untreated or stimulated with BDNF or HGF (normalized to the respective untreated neurons). Neurons expressing β-catenin display axons longer than controls upon BDNF or HGF (50 ng/ml) stimulation. By contrast, in neurons expressing intracellular N-cadherin, BDNF or HGF cannot stimulate axon growth (n=4 experiments). ^*P0.05, ^***P0.001 when compared with the corresponding untreated control; #, P0.05, ##, P0.01 when compared with the BDNF- or HGF-stimulated β-catenin-expressing neurons.

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