Generating Breast Cancer Cell Lines Resistant to FOXM1 Inhibitors

All breast cancer cell lines were obtained from the ATCC and were maintained and cultured as described [7 (link),38 (link),39 (link)]. Cells resistant to the growth suppressive effects of inhibitors were developed by a selection of surviving cells by continuous exposure to increasing concentrations of NB73 or NB115 compounds over a period of 6 to 9 months, starting at 0.1 µM and increasing until a maintenance concentration of 10 µM was reached. Aliquots of pooled resistant cells were frozen and stored in liquid nitrogen. Aliquots were thawed when cells were in culture for more than 10 passages. The cell aliquots were faithful to the original phenotype based on the experiments described herein. All cells were tested for mycoplasma using MycoSensor PCR Assay Kit from Agilent Technologies (Santa Clara, CA, USA). FOXM1 inhibitors were synthesized as described previously [11 (link)]. Fulvestrant and lapatinib were obtained from Sigma Aldrich (Burlington, MA, USA). Antibodies against ERα, FOXM1, E2F1, CCND1, CCNE2, CDK4, STAT1, pSTAT1, STAT3, and pSTAT3 were from Cell Signaling Technology (Danvers, MA, USA); the antibody to HER2 was from Abcam; the antibody to EGFR was from Santa Cruz Biotechnology (Dallas, TX, USA); and the antibody to β-actin was from Sigma Aldrich. Further antibody information is provided in Supplementary Table S1.