G2B-10A benign mammary epithelial cells enhance the anchorage-independent growth of R2-T1AS breast cancer cells in soft agar. (A) Soft agar co-culture of R2-T1AS cells and G2B-10A cells. G2B-10A cells and R2-T1AS were mixed at the indicated ratios and plated in soft agar cultures in low-glucose, 5% serum media. Colony numbers were scored after 21-day incubation. R2-T1AS cells were plated at a density of 1 × 104 cells/well in each culture (denoted as 1 in the graph labels), and G2B-10A cells were admixed at 1 × 104 to 8 × 104 cells/well, as indicated (denoted as 1 to 8 in the graph labels). Red bar - R2-T1AS plated alone; red stippled bars - R2-T1AS plated with G2B-10A cells. Graph shows mean number of R2-T1AS colonies (± standard error) obtained in 4 independent experiments, with each experiment performed in 4 replicate wells. ** p < 0.001 (B) Soft agar colonies are composed exclusively of R2-T1AS cells. Brightfield (panels i & ii) and fluorescent images (panels iii & iv) of colonies formed after 21-day incubation. Panels i & iii: The R2-T1AS-only culture; Panels ii & iv: R2-T1AS/G2B-10A co-culture containing exclusively colonies formed by red-marked R2-T1AS cells (red).