Identification of the small molecule C-6. (A) PE1007070 and hTERT-HMEC cells were treated with 20 μM of the compound library in duplicate. After four days, a luciferase-based ATP assay was performed and data were normalized to DMSO vehicle control wells to determine viability (%). The difference in viability between the hTERT-HMEC and PE1007070 cells is plotted. The hit limit was 2.7 times the standard deviation. (B) The structure of C-6, which had a 62% difference in viability between the hTERT-HMEC and PE1007070 cells. (C) Dose response curves of C-6 and EC50 values of hTERT-HMEC, PE1007070, PE1008032, and PE904557a cells and (D) MCF-10A, MCF-7, T47D and MDA-MB-231 cells after four days of treatment. N/A indicates data could not be fitted. (E) MCF-10A, MCF-7, MDA-MB-231 and T47D cells were treated with DMSO or 15 μM C-6 for 24 or 48 hours followed by addition of 10 μM BrdU for 30 minutes. The cells were stained for BrdU and propidium iodide and analyzed by flow cytometry. The average ± standard deviation of the percent BrdU positive cells (S phase) of three replicates was calculated. Asterisks (*) denote P-value < 0.05. BrdU, 5-bromo-2-deoxyuridine; DMSO, dimethyl sulfoxide; HMEC, human mammary epithelial cells; hTERT, human telomerase; PE pleural effusion.