Fig 2: Treatment with MAPK and PI3K inhibitors following Fgf-2 treatment. (A) Inhibition of Npnt expression by Fgf-2 is regulated by JNK. Following pretreatment with MAPK inhibitors (+PD: 10 µm PD98059, +SB: 10 µm SB203580, +SP: 10 µm SP600125) for 1 h, and MC3T3-E1 cells were treated for 24 h with 1 ng·mL-1 of Fgf-2 and each MAPK inhibitor. Real-time PCR was performed using cDNA derived from total cellular RNA obtained from each sample to determine the expression levels of Npnt and Gapdh mRNAs. Values are shown as the mean ± SD of three samples as compared to the group without treatment. **P < 0.01 (ANOVA). (B) Inhibition of Npnt expression by Fgf-2 is regulated by PI3K. Following pretreatment with 10 µm of the PI3K inhibitor (LY294002) for 1 h, MC3T3-E1 cells were treated for 24 h with 1 ng·mL-1 of Fgf-2 and the PI3K inhibitor. Real-time PCR was performed using cDNA derived from total cellular RNA from each sample to determine the expression levels of Npnt and Gapdh mRNAs. Values are shown as the mean ± SD of three samples as compared to the group without treatment. **P < 0.01 (ANOVA).

Fig 3: 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.

Fig 4: Nephronectin (Npnt) expression during early ocular development.Expression of Npnt mRNA and protein was examined via section in situ hybridization (A–C) or immunohistochemistry (D–L). (A, B) Npnt expression in the retina pigment epithelium layer of the optic cup and region of the presumptive lens fiber cells at embryonic day (E)4 and E5. (C) Initial expression of Npnt by periocular neural crest (pNC) is observed during the second wave of migration into the stroma (black arrows). (D–F) At E4, Npnt protein was detected in the optic cup, lens epithelium, in the periocular mesenchyme proximal to the presumptive cornea region (arrow), and in the matrix of the primary stroma (asterisk). (G–I) At E5, vivid expression of Npnt protein is localized in the primary stroma adjacent to the corneal epithelium and diffusely throughout the primary stroma (asterisk), and persists at low levels in the optic cup and lens epithelium. At E6, vivid expression of Npnt protein persists in the primary stroma adjacent to the corneal epithelium and it remains diffusely expressed throughout the primary stroma (asterisk). At this time, low expression of Npnt protein is also observed in the migratory periocular neural crest cells invading the primary stroma (arrows in L). ec, ectoderm; oc, optic cup; pom, periocular mesenchyme; rpe, retinal pigment epithelium; st, stroma; en, corneal endothelium; ep, corneal epithelium. Scale bars: 100 µm.

Fig 5: Inhibitory effects of Fgf-2 occur in dose- and time-dependent manners. (A) Dose-dependent effects of Fgf-2 on Npnt mRNA expression. MC3T3-E1 cells were treated with 0, 0.01, 0.1, 1, 10, or 100 ng·mL-1 of Fgf-2 for 24 h. Values are shown as the mean ± SD of three samples as compared to the level in cells without treatment. **P < 0.01, relative to level in untreated cells (ANOVA). (B) Time-dependent effects of Fgf-2 on Npnt mRNA expression in MC3T3-E1 cells. Cells were treated with 1 ng·mL-1 of Fgf-2 for 6, 12, 24, or 48 h. Total cellular RNA was extracted, and then, mRNA levels for Npnt and Gapdh were examined using real-time PCR. Values are shown as the mean ± SD of three samples as compared to the value at 0 h. *P < 0.05, **P < 0.01; relative to level in cells without treatment at each time point (Student's t-test).