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. 2017 Oct 23;13(10):e1007048. doi: 10.1371/journal.pgen.1007048

Fig 8. Nrp 1 mediates the semaphorin-3A-induced inhibition of neurite outgrowth.

Fig 8

SGN explants were transfected with either 50nM Nrp1 siRNA or scrambled siRNA, and treated with semaphorin-3A (250 ng/ml). Neurons were stained with β-tubulin III monoclonal antibodies. Control (A); Semaphorin-3A alone (B); Nrp1 siRNA alone (C); D. Nrp1 siRNA and semaphorin-3A (D); Scrambled siRNA alone (E); Scrambled siRNA and semaphorin-3A (F). Scale bar = 200 μm. (G,H, J) Recombinant Sema3a inhibits SGN outgrowth in whole cochlear cultures. (G, H) Three-dimensional confocal z-stacks from E17.5 cochlear cultures treated with 20nM of either control IgG-Fc or Sema3a-Fc. Tissue samples were stained with anti TUJ1 to mark SGNs (white) and Myo6 for HCs (blue). Note the dramatically diminished presence of fibers in the sample treated with Sema3a-Fc (arrowheads). Scale bar = 10um. (I) Average length of neurites grown from the SGN explants showing statistically significant decreased neurite outgrowth for Sama3a, Nrp1 siRNA, and scrambled siRNA/Sema3a when compared to the control group. * p<0.001. (J) Panel J showing average percent neurite coverage of sensory epithelium. Error bars, SEM. * p<0.0001. (K) Western immunoblots of Nrp1 protein from SGN explants after transfection with either Nrp1 siRNA or scrambled siRNA control as indicated. β- Actin was used as a loading control.