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. 2013 Jan 30;8(1):e55237. doi: 10.1371/journal.pone.0055237

Figure 5. Mutation of the NES2 (L135A) counteracts the effects of Ngn3 overexpression on neuronal morphology and synaptic inputs.

Figure 5

(A–C) Hippocampal neuronal cultures were co-transfected with constructs encoding EGFP and full-length myc-tagged wild-type Ngn3 (myc-Ngn3), Ngn3 with leucine 135 mutated to alanine (myc-Ngn3-L135A) or empty vector expressing myc-tag as control. After 16 h, double immunostaining was performed using an anti-GFP antibody to visualize transfected neurons and an anti-synaptophysin I antibody to determine the morphology and the total number of synapses of the transfected neurons. (D–F) Lower panels show the boxed regions at higher magnification (G) Number of primary dendrites of the transfected neurons. (H) Counts of synaptophysin I immunoreactive terminals in contact with a neuron within a circular region of interest (ROI) with a diameter of 100 µm and centered in the neuronal soma. Data are mean+s.e.m. and significance levels were determined using ANOVA followed by the Bonferroni post hoc test; *** p<0.001 versus control neuron values and ### p<0.001 versus myc-Ngn3 expressing neuron values.