Fig. 2

Effect of CaM-antagonist, W7, on ERE-ER complex formation in ER(+) human breast cancer cell lines. MDA-MB231, 21PT, and MCF-7 cells (10⁵ cells/ml) were plated in 25 ml of rich medium in 150 mm tissue culture dishes. At about 70% confluency the medium was changed to rich (R) or basal (B) medium [Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% dextran coated charcoal-treated fetal bovine serum]. 48 hr later the cells were treated with W7, and 1 hr following W7 addition, the cells were treated with estradiol (E2) at the indicated concentrations for an additional 4 hr. These cells were harvested and nuclear extracts were made as described (23–25). ³²P-labeled E2 response element-specific receptor (³²P-ERE-ER) interaction in 10 µg of nuclear extracts was measured in duplicate samples by (A) Nitrocellulose filter binding assay (NMBA) and (B) electrophoretic mobility shift assay (EMSA) followed by autoradiography. Control for nonspecific DNA protein interaction was determined by carrying out a reaction with the same amount (10 µg) of nuclear extract from ER-deficient breast cancer cell line MDA-MB-231, designated by 231 (panel A, lane 10 and panel B, lane 1). ER+, ER-positive; ER−, ER-negative.