Figure 4

TNFα + WT-Ras up-regulate CXCL8 expression via the activation of NF-κB and induce AP-1 stimulation. MCF-7 cells were transfected with WT-Ras vector or with control vector, and were not-stimulated or stimulated by TNFα (50 ng/ml). (A) p65 phosphorylation was determined by WB. Control vector-transfected non-stimulated cells were given the value of 1. (B) NF-κB activation was determined in cells transfected to express the luciferase gene under the control of 3 conserved repeats of NF-κB binding sites, using dual luciferase assay. Control vector-transfected non-stimulated cells were given the value of 1. The results obtained in each of the 3 repeats are presented in Table 1. (C) WT-Ras-expressing cells were transfected with a pool of 4 siRNAs targeting p65 (25-35 nM), or with appropriate control siRNA. CXCL8 protein expression levels were determined in cell CM by ELISA. (D) c-Jun phosphorylation was determined by WB, following c-Jun immunoprecipitation. GAPDH was used for determination of protein amounts in original cell lysates, prior to immunoprecipitation. Control vector-transfected non-stimulated cells were given the value of 1. The direct roles of AP-1 in mediating the TNFα + WT-Ras stimulation of CXCL8 are presented in Table 2. In panels A and D a representative experiment of n≥3 is presented. Each of the results presented in Panels B and C show the average ± SD of n=3. *p < 0.05, ***p < 0.001 compared to non-stimulated cells. Please see “Methods” for additional details on the experimental procedures and statistical analyses performed in this part of the study.