Fig. 2

Detection of ER in breast cancer cells in culture by immunofluorescent technique, Western blot, and Northern blot analysis. (A, B) Immunofluorescent technique. Cells were grown in tissue culture chamber slides in complete medium (DMEM supplemented with 10% FBS, 2.8 µM hydrocortisone, 1 µg/ml insulin, and 12.5 ng/ml of EGF). A standard immunofluorescent technique was used with a monoclonal antibody (TE1-11) as described in Materials and Methods. 70N, cultured cells from normal breast tissue; MCF-7, ER+; 21PT, ER−; T47D, ER+; and MDA-231, ER−, as measured by ligand-binding assay (38,39). (A) Staining of nuclei by Hoechst. (B) Immunostaining by ER Mab shows positive signals in MCF-7, 21PT, and T47D and no visible immunofluorescent signals in 70N and MDA 231. The low-level immunofluorescent signals in 21PT cells were consistently observed in three separate experiments; thus 21PT can be reclassified as ER+. (C) ER protein level as determined by Western blot analysis and enhanced chemiluminescence (ECL) immunodetection system by using a monoclonal human anti-ER-antibody as primary antibody (SRA 1010, Stress Gen, Victoria, Canada). The mobility of 70 and 60 kD prestained markers (Gibco-BRL) analyzed simultaneously with the extracts from the indicated breast cancer cells is shown by arrowheads. This analysis also classified 21PT cells as ER+, confirming the immunofluorescent assay. A control blot incubated with equivalent amounts of mouse IgG as primary antibody and goat anti-mouse IgG-HRP conjugate as secondary antibody did not generate any visible signals with extracts from either of these cell lines. (D) ER-specific mRNA as determined by Northern blot analysis. Here 10 µg of total cellular RNA from T47D, MCF-7, and 21PT cells was probed with ³²P-labeled cDNA-ER. Arrows on the right show the mobility of 28S and 18S RNA. One major and one minor cDNA-hybridizable mRNA species are detected as indicated by the arrows on the left. These mRNA species have a size range similar to that of the previously reported mRNA ER species of 6.3 kb and 3.7 kb, and they further characterized 21PT cells as ER+. A photograph of the RNA samples applied in each lane is shown on the right of panel D. These samples served as loading control for the amount of RNA in these lanes. This experiment was repeated twice.