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. 2017 Dec 1;7:16812. doi: 10.1038/s41598-017-16782-8

Figure 2.

Figure 2

Effect of Crmp4 or Crmp4 S540Y gene expression on the dendritic arborisation of cultured hippocampal cells. S540Y mutation in mouse Crmp4 gene is homologous to S541Y in human CRMP4 gene found in the ASD patient. Representative images of cultured hippocampal cells from wild-type (WT) mice transfected with control (pEGFP) vector, those from Crmp4-KO mice transfected with pEGFP vector, those from Crmp4-KO mice transfected with pEGFP-Crmp4 vector, and those from Crmp4-KO mice transfected with pEGFP-Crmp4 S540Y (a). Cultured cells were fixed at DIV-3 (3 days in vitro) with 4% paraformaldehyde and then immunocytochemically stained with antibodies against MAP2 (red) and EGFP (green). Nuclei were stained with Höechst 33258 (blue). The total length of MAP2- and EGFP-double labelled dendrites and the number of the dendritic branching (arrow heads) were measured for each double labelled neuron. Scale bar: 50 µm. Bar graphs showing the average of total dendritic length (b) and the average of the number of dendritic branches (c). n = 81, 71, 82, and 61 for WT neurons transfected with pEGFP, Crmp4-KO neurons transfected with pEGFP, Crmp4-KO neurons transfected with pEGFP-Crmp4, and Crmp4-KO neurons transfected with pEGFP-Crmp4 S540Y, respectively, from at least three independent experiments. Bars indicate mean ± SEM. Asterisks indicate statistical significance (*p < 0.05, ** p < 0.01, *** p < 0.001; one-way ANOVA followed by Fisher’s PLSD post-hoc tests).