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. 2011 Mar;13(3):230–243. doi: 10.1593/neo.101340

Figure 6.

Figure 6

The additive up-regulation of CXCL8 induced by EGF + estrogen: Involvement of NF-κB. (A) Activation of NF-κB determined by expression levels of IκBα. MCF-7 cells were grown in the presence of estrogen (10-8 M, 54 hours), EGF (10 ng/ml, last 6 hours), EGF + estrogen (estrogen: 10-8 M, 54 hours; EGF: 10 ng/ml, last 6 hours), or ethanol as control. The expression levels of IκBα were determined by Western blot analysis. The experiment represents similar stimulations, preformed under varying exposure times of EGF, estrogen and both together (see Materials and Methods). (B) Inhibition of NF-κB activities by PDTC. MCF-7 cells were grown as detailed in Figure 1A. Two hours before addition of EGF, the cells were pretreated with conventional concentrations of the specific NF-κB inhibitor PDTC (60 εM; diluted in water; the drug was active in other experimental designs—data not shown). Then, the growth of the cells was continued in the absence or presence of the drug. CXCL8 extracellular expression was determined in the supernatants of the cells by ELISA and was analyzed in the linear range of absorbance. Δ, The net amount of CXCL8 added to cell supernatant on stimulation. These values were used for statistical evaluations of differences between the control group and the inhibitor-treated group. A representative experiment of n = 3 is presented. *P < .05, **P < .01 for the difference between cells stimulated by EGF/estrogen/EGF + estrogen and untreated cells.