FIGURE 4:
Expression of TNAP control axonal growth. (A) Hippocampal neurons transfected at 1 DIV with pEGFP (1), TNAP IRES (2), shRNA TNAP (3), or TNAPmut (4) and fixed at 3 DIV. Scale bar, 50 μm. (B) Effect of overexpression of wild-type or mutant TNAP and silencing of TNAP on axon length and the number of ramifications in hippocampal neurons. The total axonal length (corresponding to the principal axon plus ramifications), length of the principal axon, and length and number of the axonal ramifications were analyzed. The 100% values in control neurons for total axonal length of EGFP-transfected neurons, principal axon length of EGFP-transfected neurons, and length of ramifications correspond to 310.8 ± 28.0 μm, 242.4 ± 28.7 μm, and 126.9 ± 24.2 μm, respectively. The number of axonal ramifications in EGFP neurons was 2.6 ± 0.9. Values represent mean ± SEM (n = 5) with at least 20 neurons analyzed in each experiment. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA). (C) Representative images of electroporated neurons with shRNA TNAP and its neighboring nontransfected neurons (immunostained with βIII-tubulin [red]). Scale bar, 50 μm.