CXCL1 enhances NF-κB activation and CXCR2 increases CXCL1 promoter activity via NF-κB site.

<p>(A) Comparative effects of CXCL1 on NF-κB activation in SKA and SKCXCR2 cells. Cells were treated with CXCL1 (100 ng/ml) and results examined in a time-dependent manner. Whole cell lysates were prepared and Western blots carried out using antibodies specific to IκB, phosphorylated IκB (pIκB) and CXCR2. β-actin was used as a loading control. (B) Effect of CXCL1/2/3 antibody on NF-κB luciferase activities in SKA and SKCXCR2 cells. After transfection using the NF-κB luciferase vector overnight, cells were incubated with normal IgG (C) and the CXCL1/2/3 antibody (Ab, 1∶100 dilution)) for 4 h. (C) Effect of Bay11-7082 on cell proliferation in SKA and SKCXCR2 cells. Cells in A-B were incubated with vehicle (C) or Bay11-7082 (Bay, 2 µM) for 48 h. The cell proliferation assay was performed using MTT and values were normalized to untreated controls. * and # in 5B and 5C (p ≤ 0.05) when compared to Control (C) and SKA cells, respectively, by the paired Student’s <i>t</i>-test. (D) Inhibitory effects of Bay11-7082 (2 µM) on CXCL1-induced cell invasion in SKA and SKCXCR2 cells. * (p≤0.05) when compared to Control (C) and SKA cells, respectively, by the paired Student’s <i>t</i>-test. (E) Effect of CXCR2 on luciferase activity of mouse CXCL1 promoter in parental SKOV-3 cells. Site-directed mutants were generated from the KC701LUC using primers with mutant κB sites (termed m1, m2 and m3 indicates mutation site): −615/−585 mutant κB site (KC701LUC<u>m3</u>), −102/−71 mutant κB site (KC701LUC<u>m2</u>) and −83/−52 mutant κB site (KC701LUC<u>m1</u>). Additional mutants of κB sites were generated as KC701LUC<u>m3m2</u>, KC701LUC<u>m3m1</u>, KC701LUC<u>m2m1</u>, and KC701LUC<u>m3m2m1</u>. After transfection with CXCL1 luciferase vector overnight, a luciferase assay was performed. Results were normalized to the activity of internal control (pRL-TK vector) and expressed as a fold increase compared to empty vector (pA) controls. * (p≤0.05) when compared to its own control by Student’s <i>t</i>-test. Cross circles indicate κB site mutants. pA = empty vector transfection; pCXCR2 = CXCR2 vector transfection. (F) Effect of Akt1 siRNA on CXCL1 promoter activity in parental SKOV-3 cells with empty vector or CXCR2 transfection. After transfection with CXCL1 luciferase vector and siRNAs for control and Akt1 (final concentration: 10 nM) overnight, a luciferase assay was performed. Results were normalized to total protein concentrations and expressed as a fold increase compared to each control. All experiments were performed at least in triplicate and all data are shown as mean ± S.E. * and # (p ≤ 0.05) when compared to Control siRNA and pA transfected cells, respectively, by Student’s <i>t</i>-test. We confirmed knockdown of Akt1 (main Akt isoform in parental SKOV-3 cells) by using Akt1 siRNA. Whole cell lysates were prepared and a western blot was carried out using Akt1 specific antibody. β-actin was used as a loading control.