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. 2022 Mar 3;11:e74307. doi: 10.7554/eLife.74307

Figure 7. Inhibition of Rho-kinase attenuates periocular neural crest (pNC) migration on nephronectin (Npnt).

(A–D) Comparisons of pNC migration from explants cultured for 24 hr on either (A) Npnt substrate alone or in the presence of (B) the ROCK inhibitor Y27632. (C, D) Quantification of cell area taken from N = 8 Npnt and N = 8 Npnt plus ROCK inhibitor showing significant reduction in (C) cell area and (D) cell migration in the presence of ROCK inhibitor. (E) Quantification of cell orientation from N = 6 Npnt and N = 6 Npnt plus ROCK inhibitor explants showing significant difference in the fraction aligned between 90° ± 30°. (F, G) Cells stained for actin (phalloidin) and focal adhesion (pY118 paxillin) showing (F) formation of actin stress fibers and focal adhesions by pNC migrating on Npnt. (G) Substantial decrease in actin stress fibers in the presence of the ROCK inhibitor. (H) Quantification taken from N = 4 Npnt and N = 4 ROCK inhibitor explants, showing significant increase in the number of pY118 paxillin-positive puncta. *p<0.05; **p<0.01. Scale bars: (A, B) 100 μm; (F, G) 50 μm.

Figure 7.

Figure 7—figure supplement 1. Analysis of periocular neural crest (pNC) migration on nephronectin (Npnt) substrate in the presence of inhibitors of components of the migratory signaling pathway.

Figure 7—figure supplement 1.

pNC explants were examined for cell migration after 24 hr culture on Npnt substrate with or without inhibitors. (A, B) Control showing robust migration of pNC on Npnt substrate and strong phalloidin staining. (C, D) Migration of pNC is attenuated in the presence of α8β1 inhibitor. (E, F) Inhibition of the focal adhesion kinase (FAK) also attenuated pNC migration and in addition; only a few cells remained attached to the Npnt substrate. (G, H) The ROCK inhibitor substantially decreased the formation of actin stress fibers as indicated by the low level of phalloidin staining compared to (B). Scale bar, 100 μm.