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

Figure 2.

Figure 2

The additive up-regulation of CXCL8 induced by EGF + estrogen: Involvement of ErbB2 and ErbB1. (A) ErbB2 activation. MCF-7 cells were stimulated by EGF (10 ng/ml), estrogen (10-8 M), or EGF + estrogen (EGF: 10 ng/ml; estrogen: 10-8 M) or ethanol as control for 10 minutes. Time points were selected based on kinetics analyses as described in Materials and Methods. ErbB2 phosphorylation was determined by Western blot analysis. A representative experiment of n > 3 (with estrogen used for 1.5 or 10 minutes) is presented. (B) The involvement of ErbB2 (B1) and ErbB1 (B2) in EGF-, estrogen-, and EGF + estrogen-induced up-regulation of CXCL8 expression. MCF-7 cells were grown as detailed in Figure 1A. Two hours before addition of estrogen, the cells were pretreated with conventional concentrations of the ErbB2 inhibitor AG825 (10 εM; B1), the ErbB1 inhibitor AG1478 (0.5 εM; B2), or DMSO (control = the drug's solubilizer). Then, the growth of the cells was continued in absence or presence of the drug (or DMSO as control) as required. 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, ***P < .001 for the difference between cells stimulated by EGF/estrogen/EGF + estrogen and untreated cells.