Figure 4

High-throughput screening: targeting pure luminobasal (pLB) cells with drugs from an NCI-approved 89-drug oncology library. (A) 1:1 mixtures of pLUM:ZsG-pLB were co-cultured in triplicate 96-well plates in 1 nM 17β-Estradiol (E; not shown, see panel B) or E-free (shown) conditions, then treated 48 h with a panel of 89 NCI-approved oncology drugs (1 μM) or 0.05% dimethyl sulfoxide (DMSO) controls. Media were aspirated, cells were fixed, immunostained for CLD3 (red) and counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Total (blue), pure luminal (pLUM) (red) or pLB (ZsG, green) subpopulations were imaged and quantified at 20×. There were 9 fields/well captured, averaging 8,000 cells/field in controls (Additional file 3: Figure S1A). Mean numbers of pLB (red bars) and pLUM (green bars) cells, and % pLB (black bars) are shown as the average of triplicates ± standard error of the mean (SEM). Erlotinib, P ≤0.0001, gefitinib, P = 0.0005. (B) Details of vehicle, erlotinib or gefitinib data from panel A in both +E and –E conditions; ***P ≤0.001; ****P ≤0.0001 for pLB cell numbers in gefitinib or erlotinib versus vehicle control.