Figure 4. Pharmacologic inhibition of Signaling Pathways Predicted to influence RPC migration.

RPCs were treated with selected inhibitors of signaling pathways identified in IPA bioinformatics analysis and evaluated for migration over 24hrs using n=6 Boyden Chamber assays per inhibitor. A) Analysis of RPC migration following pre-treatment with selected inhibitors reveal that significant inhibition of migration was observed in low 20ng EGF condition using anti-EGFR (0.4499 ± 0.10 SEM (p=<.0001*) to antagonize EGFR cell-surface binding, AG1478 0.3427 ± 0.06 SEM (<.0001*) to inhibit phosphorylation of the EGFR cytoplasmic domain, AG490 (0.5913 ± 0.02 SEM (p=0.0012*) to inhibit STAT3 activity and Wortmanin (0.4699 ± 0.04 SEM (p=<.0001*) to inhibit PI3K activity. The use of PD98059 to inhibit ERK1/2 activity did not result in significant inhibition of migration. In the presence of a higher 40ng/ml concentration of EGF, significant inhibition of migration was observed with anti-EGFR (0.4370 ± 0.12 SEM (p=0.0002*), while PD98059 1.5810 ± 0.12 SEM (p=0.0001*) increased migration.

Statistical significance was determined using Dunetts analysis and data are presented as normalized mean. B) Robust activation of identified signaling pathways was observed in 20ng/ml EGF concentrations. Western Blot analysis demonstrated the presence of both non-phosphorylated (inactivated) and phosphorylated (activated) STAT3, ERK1/2 and P13K proteins RPCs. Inhibition with significant p-values are denoted with an asterisk.