Figure 3.

Identification of COL6 as the extracellular signal initiating axonal fasciculation.

(A) Total protein bands in the different groups. Protein bands were detected using Tris-Glycine eXtended Stain-Free technology. Yellow brackets indicate the specific protein bands in the co-immunoprecipitation group. (B) Set analysis of liquid chromatography-mass spectrometry/mass spectrometry results identified 17 protein candidates, and subsequent GO annotation further identified 5 ECM candidates out of 17. (C) Representative images of axons growing on substrates of rat collagen 1 (R-COL1), human laminin 521 (H-LAM521), and human collagen 6 (H-COL6). Distinct axonal fasciculation occured only in the H-COL6 group. (D) Representative images showing axonal trajectories in different groups. Axonal fasciculations were observed in the COL6-rich substrates (COL6 + Vehicle and Matrigel + COL6 groups) but not in the Matrigel + Vehicle substrate. (E) Axon bundle diameter distribution (n = 5 cultures, *P < 0.05, ****P < 0.0001, one-way analysis of variance followed by Tukey's multiple comparison tests). (F) Immunoblotting of NCAM1 in DRGs ex vivo preparations growing on different substrates. Histogram of densitometry showing increased NCAM1 levels in the COL6 + vehicle (1) and Matrigel + COL6 (3) groups, compared with that in the Matrigel + vehicle (2) group (n = 3 cultures, ****P < 0.0001, Kruskal-Wallis test followed by Dunn-Bonferroni post hoc test). (G) Immunoblotting of COL6 and β-actin in the components of DNM-G and Matrigel. The histogram of densitometry shows a lower COL6 level and a higher β-actin level in Matrigel, compared with that in DNM-G (normalized by total protein, n = 4 independent experiments, ****P < 0.0001, Student's t-test). Scale bars: 5 μm in C and 20 μm in D. COL6: Collagen VI; DNM-G: decellularized nerve matrix-gel; GO: gene ontology; H-COL6: human collagen 6; H-LAM521: human laminin 521; R-COL1: rat collagen 1.