Figure 6. Confirmation of CXCR2-potentiated NF-κB signaling in OVCAR-3 cells.

(A) CXCR2 protein expression in OVA versus OVCXCR2 cells. Western blot and immunofluorescent staining were carried out using antibodies specific to CXCR2 and β-actin as a loading control. (B) Comparison of growth rates in OVA and OVCXCR2 cells. Cells were incubated for 0, 24, 48 and 72 h and growth rates normalized to 0 h densities in each cell line. Experiments were performed in triplicate and all data are shown as mean ± S.E. * and ** (p≤0.05) in each group by ANOVA and Tukey’s pairwise comparisons. # (p≤0.05) between OVA and OVCXCR2 cells by Student’s t-test. (C) Effect of TNF (10 ng/ml) effects over time (0-120 min) on NF-κB activation in OVA and OVCXCR2 cells. Whole cell lysates were prepared and Western blots carried out using antibodies specific to IκB, IKK and their phosphorylated forms (pIκB and pIKK). β-actin was used as a loading control. (D) Comparison of EGFR activation in OVA and OVCXCR2 cells. Whole cell lysates were prepared and Western blots carried out using antibodies specific to EGFR, Akt, Erk and the phosphorylated forms (pEGFR, pAkt and pErk). The non-phosphorylated forms were used as loading controls. (E) Effects of EGFR downstream inhibitors on NF-κB luciferase activity in OVA and OVCXCR2 cells. After transfection with NF-κB luciferase vector overnight, cells were treated with vehicle (C), AG-1478 (AG, 2 µM), LY294002 (LY, 2 µM) or PD98059 (PD, 20 µM) for 4 h. (F) Effect of CXCL1/2/3 pan specific antibody for neutralization on cell proliferation in OVA and OVCXCR2 cells. Cells were incubated with normal IgG (C) and antibody (1∶100 dilution) for 48 h. The cell proliferation assay was performed using MTT and values were normalized to untreated controls. (G) Inhibitory effects of Bay11-7082 (2 µM) on CXCL1-induced cell invasion in OVA and OVCXCR2 cells. All experiments were performed at least in triplicate and data are shown as mean ± S.E. * and # (p≤0.05) as calculated by Student’s t-test.