Fig 2: EGFR and AREG expression in pancreatic cancer cells. (A) The mRNA expression of EGFR was examined by RT-qPCR. (B) Western blotting was performed to determine the protein levels of EGFR in a panel of pancreatic cancer cell lines. Equal amounts of protein were subjected to immunoblotting using the EGFR and β-actin antibodies. (C) The mRNA expression of AREG was examined by RT-qPCR. (D) AREG ELISA was performed using conditioned medium collected from PANC-1, AsPC-1, Bxpc-3, and Mia PaCa-2 cells. The data represent the mean ± SD of at least three independent experiments. *P<0.05, **P<0.01. EGFR, epidermal growth factor receptor; AREG, amphiregulin; ELISA, enzyme-linked immunosorbent assay.

Fig 3: The NF-κB pathway is involved in AREG-induced migration and invasion of pancreatic cancer cells. (A) Representative images and analysis of western blots for IκBα, p-IκBα, nuclear p65 in AsPC-1 cells transfected with AREG siRNA (siAR-390) or control siRNA (NC). (B) Representative images and analysis of western blots for IκBα, p-IκBα, nuclear p65 in PANC-1 cells treated with AREG (100 ng/ml), U0126 (20 µM), PD153035 (5 µM), and normal IgG (100 ng/ml, NC). (C) Wound-healing assay. QNZ inhibited the migratory ability of PANC-1 cells induced by AREG. (D) Transwell assay. QNZ abolished the invasive properties of PANC-1 cells induced by AREG. PANC-1 cells treated with AREG (100 ng/ml) alone (AREG), AREG combined with QNZ (20 µM, AREG+QNZ), or normal IgG (100 ng/ml, NC). The data represent the mean ± SD of at least three independent experiments, *P<0.05, **P<0.01. AREG, amphiregulin.

Fig 4: AREG promotes EMT in pancreatic cancer cells. (A) Western blot results of the EMT-related proteins from AsPC-1 cells transfected with AREG siRNA (siAR-390) or control siRNA (NC). (B) Western blot results of the EMT-related proteins derived for PANC-1 cells treated with AREG (100 ng/ml) alone (AREG), AREG combined with QNZ (the selective NF-κB inhibitor, 20 µM, AREG+QNZ), or normal IgG (100 ng/ml, NC). (C) Immunofluorescence staining results of E-cadherin and vimentin in AsPC-1 and PANC-1 cells. Magnification, ×400. AREG, amphiregulin; EMT, epithelial-mesenchymal transition. The bars represent the mean ± SD of triplicate analyses. *P<0.05, **P<0.01.

Fig 5: Effects of AREG on the growth of DPSCs. A The CCK-8 assay was used to detect cell proliferation in different AREG-treated groups (0.01–1 mg/mL) and untreated DPSCs at 1, 3, 5 and 7 days (n = 5, *P < 0.05). B The cell cycle proliferation index (PI = G2/M + S) in AREG-treated and control groups analyzed by flow cytometry. C Flow cytometry analysis of AREG-treated and control groups