Figure 3.

Distinct patterns of EGFR activation by EGF and AREG. (A): Western analysis of phospho-EGFR (Tyr1173), total EGFR and GAPDH protein levels in airway epithelial cells derived from basal cells (BC) treated with EGF or AREG (both 10 ng/ml) versus unstimulated BC at indicated time-points; representative of >3 independent experiments (see Supporting Information Fig. S3A for quantification data). (B): Top seven categories enriched in the AREG-coexpressed airway epithelial gene set (r ≥ 0.5, p < .05) annotated using Gene Ontology (G) and protein family classifications (P, Protein Information Resource; I, InterPro; S, The Human Spliceosome Protein-Protein Interaction Resource) using GATHER (for G, additional criteria: Beyes factor >6) and DAVID (for G, P, I, S); ranked by false discovery rate-corrected p value (top axis; bars); green dots represent enrichment score (% input genes; bottom axis). (C): Venn diagram: overlap of the top AREG-coexpressed airway epithelial genes (r ≥ 0.6, p < .05) with the EGFR feedback signature. (D): Fold-change in expression of the EGFR feedback genes (see text for full gene names) in the epithelium derived from BC during 28 days of air-liquid interface (ALI) ± basolaterally added EGF or AREG versus control (mean ± SD; n= 5 independent experiments). (E): FACS analysis: histogram plots of the cell surface EGFR expression in control basal cells (BC) (blue) and BC treated with AREG or EGF; at 1 hours and 12 hours after stimulation; representative of three independent experiments. (F): IF images of cytospins of epithelial cells derived from BC after 14 days of ALI culture ± AREG or EGF for expression of EGFR and BC marker KRT5. Nuclei are stained with DAPI (blue). Scale bars-10 µm. See Supporting Information Figure S3C for more examples. Abbreviations: AREG, amphiregulin; DUSP, dual specificity protein phosphatase; EGFR, epidermal growth factor receptor.