Fig. 8: Synergetic effect of Rb-EGF and Rb-Sema3A through EGFR/ERK signaling regulated by NRP1.

A NHEKs were serum starved and subsequently stimulated with Rb-Sema3A for 5 min to 1 h and subjected to IF analyses. B Combined treatment with Rb-EGF and Rb-Sema3A was utilized in keratinocytes at the indicated time points. EGFR and NRP1 localization/expression was shown by IF staining. Wound healing assays (C) and Transwell assays (D) were performed in Rb-Sema3A-,Rb-EGF and EGFR plasmids incubated in Hacat cells after transfected with si-NRP1 or negative control. The percentage of wound closure is displayed as the mean ± SEM; n = 3. Bars indicate the mean fold changes ± SEM relative to the corresponding control; n = 3. E Western blot analysis of EGFR-ERK pathway after treatment at the indicated time points. F Immunofluorescence in Hacat cells. si-NRP1 or si-NC plasmids were transfected in EGFR-overexpressing cells. Recombinant EGF and Sema3A cytokines were added in cells to test the process of NRP1 and EGFR activation and degradation. Scale bar = 20 µm. G A schematic of the proposed mechanism. **P < 0.01; ***P < 0.001.