Fig. 4.

The endocrine-resistant breast cancer cell line SKBR3 overexpresses EGFR/HER2 and shows increased expression and activity of cPLA₂. A and C, Total mRNA was extracted from untreated MCF-7 and SKBR3 cells, reverse transcribed into cDNA, and either subjected to semiquantitative PCR using specific primers for EGFR, HER2, cPLA₂α and COX-2 (expression levels were normalized for GAPDH (a representative agarose gel is shown along with densitometric analysis of six experiments) (A) or subjected to real-time quantitative PCR with specific primers for HER2 and cPLA₂ (C). mRNA expression levels were normalized to 18S and expressed as fold difference in relative quantity relative to MCF-7. Data are mean values ± se. *, P < 0.001; **, P < 0.01 compared with MCF-7 values. B, Western blot analysis of total EGFR, HER2, cPLA₂, and COX-2 was performed on unstimulated MCF-7 and SKBR3 cells. β-Actin was used for protein level normalization. Densitometric analysis of three different experiments is shown with a representative blot. Data are mean values ± se. *, P < 0.001; **, P < 0.01 compared with MCF-7 values. D, cPLA₂ enzymatic activity was measured in total lysates from MCF-7 and SKBR3 cells. *, P < 0.01 compared with MCF-7 values.