Expression of Skp2 is required for anchorage independence in breast epithelial cancer cells. (a) Proliferating normal HBECs (lanes 1–3), three different breast cell lines (lanes 4–12), (b) normal human fibroblasts (lanes 1–3), and other transformed cell lines (lanes 4–12) were trypsinized and replated in suspension. Cells were collected at the indicated times, and protein extracts were analyzed by immunoblotting with Ab’s to the indicated proteins. (c) Comparative analysis of the number of colonies larger than 200 μM in diameter obtained growing breast cancer cells infected with various retroviruses in soft-agar. A total of 105 MCF-7 cells or 5 × 103 MDM-MB-435 cells were seeded in 0.3% agar supplemented in 5% calf serum. Colonies were scored after 3 weeks of growth. The number of colonies scored represented at least three independent experiments ± the SD of the mean.