Fig 1: Heregulin1 is the major mitogenic factor for cell proliferation: (A-D) EGFR-ErbB3–expressing FDC-P1 cells were seeded in 96-well plates and treated with different EGFR and ErbB3 ligands or IL-3 at 20 ng/ml at different times. (E-F) NSCLC cells were seeded in 96-well plates and were treated with EGFR and ErbB3 ligands at 20 ng/ml for 72 hours. The relative cell number was determined with CCCK8. The OD value at 450 nm was measured with an iMark microplate reader. Results were shown as cell growth curves for EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing FDC-P1 cells in response to ligand stimulation, respectively (A-D). The difference above the nontreatment control was compared in order to determine which ligand was the major mitogenic factor for NSCLC cells with EGFRmut (E-F). Data are plotted as mean +/− SD of tetraplicates. The data are representative of three independent experiments.
Fig 2: Comparison of expression level of EGFR and ErbB3 on EGFR-ErbB3 expressing FDC-P1 cells and NSCLC cell lines. Real-time PCR was performed with 200 ng of RNA with an iTaq Universal SYBR One-Step Kit. Fold expression was calculated as relative fold expression = 2 cq value of GAPDH-cq value of receptors. Data are plotted as mean ± SD of triplicates.
Fig 3: EGF and TGF-α inhibit HRG1-induced ErbB3 phosphorylation in EGFRwt-ErbB3 cells but not in EGFRmut-ErbB3 cells: Cells at 50% confluence were serum starved for 24 hours. The cells were untreated or treated with different ligands at 20 ng/ml for 15 minutes. The cell lyses were subjected to SDS-PAGE. Membranes were first probed with anti-pErbB3 antibody. The membranes were stripped and reprobed with anti-ErbB3 antibody as loading control and anti-pErk1/2 antibody. The data are representative of three independent experiments.
Fig 4: Expression of EGFR, ErbB3, and their ligands on patient samples: 16 tumor tissues were fixed in 4% polyoxymethylene, embedded in paraffin, sectioned to 3 μm, and mounted on adhesion microscope slides. IHC was performed by following standard procedures of hospital. (A) EGFR; (B) ErbB3; (C) HRG1; (D) AREG; (E) EREG.
Fig 5: (A) High expression of EGFR, ErbB3, and their ligands: IHC was performed by the following standard procedures of hospital: Pictures were taken at junction area between tumor cells at right side and normal alveoli or stroma tissue at left side with 10×10 amplification from the same one patient with NSCLC harboring EGFR mutant of E746-A750del.(B). Different response to binding of EGFR and ErbB3 ligands: When EGF binds to wild-type or mutant EGFR-ErbB3 heterodimer, the EGFR configuration change allows it to activate MAP kinase pathway and a phosphatase that dephosphorylates ErbB3, which results in a weak proliferation signal (A and E). When HRG1 binds to EGFR-ErbB3 heterodimer, the EGFR configuration change does not allow it to activate MAP kinase pathway but allows it to phosphorylate ErbB3 and generates a strong proliferation signal (B and F). When EGF and HRG1 bind to their receptors simultaneously, the configuration change of wild-type EGFR allows it to activate MAP kinase pathway and activates a phosphatase that dephosphorylates ErbB3, which results in a weak proliferation signal (C), whereas the same event allows mutant EGFR to activate MAP kinase pathway and phosphorylate ErbB3, which results in a stronger proliferation signal (G). When AREG and HRG1 bind to their receptors simultaneously, the configuration change of either wild-type or mutant EGFR allows EGFR to activate MAP kinase pathway and phosphorylate ErbB3, which also results in a stronger proliferation signal (D and H).
Supplier Page from Sino Biological, Inc. for Human ERBB3/HER3 transcript variant 1 Gene ORF cDNA clone in cloning vector