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Fig. 1. Cytochalasin D disrupts microfilament networks in primary mammary epithelial cells. (A) First-passage mammary epithelial cells were plated on basement-membrane-coated coverslips in DMEM-F12 supplemented with insulin and hydrocortisone for 24 hours. Under these conditions, the cells form aggregates and become completely surrounded by basement membrane, developing into hollow structures resembling alveoli (Aggeler et al., 1991). Most epithelial cells interact directly with basement membrane (Streuli et al., 1991). The micrographs depict sections at the edges of hollow `alveoli', where more cells are available for inspection, but the lumens are not visible. Confocal micrographs of control cultures that were fixed and stained for the presence of actin using TRITC-conjugated phalloidin revealed a cortical actin network (arrows) that was not disrupted after 30 minutes or 6 hours of treatment with colchicine (col). By contrast, the cortical actin network was completely disrupted within 30 minutes of cytochalasin-D treatment (CD), and the staining became punctate (arrowheads). Scale bar, 10 µm. (B) Mammary cells were plated on collagen-I-coated coverslips and were either left untreated as controls or treated with 2 µM cytochalasin D (CD) for 30 minutes, then rinsed and harvested at 4 hours following cytochalasin-D removal (washout). Cells were stained for microfilaments using FITC-conjugated phalloidin. Similar experiments using cells plated on basement membrane indicate that the cortical actin organization can be restored after washing out cytochalasin D for several hours (data not shown).
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