Fig 1: Effects of AM9928 on cell survival, adhesion, and trans-endothelial migration of TNBC cells across HBMEC cultures. (a) Effect of AM9928 on MDA-MB-231 and MDA-MB-BrM2 Cell Viability. AM9928 at different concentrations as noted (250 nM, 500 nM, or 1 µ?) were incubated with 5 × 104 cells/well for 72 h and treated as detailed in the manufacturer’s protocol (Abcam Cat# ab211091, MTT assay) to determine cell viability. Absorbance was measured at OD 590 nm using Biotek reader. Error bars represents the mean ± S.D. (b) Adhesion: After maintaining HBMECs at confluence for 5 days, approximately 40,000 labeled MDA-MB-BrM2 cells were seeded over the monolayer in the presence or absence of AM9928 and were allowed to adhere to HBMEC monolayer for either 0, 10 min or 30 min, as indicated. The TGL (a triple-modality reporter gene of thymidine kinase (T), GFP (G), and luciferase (L)TGL-tagged cells were then counted and compared to control (untreated) cells. Data shown are representative of at least three independent assays performed on duplicate or triplicate wells. The error bars indicate standard deviations. (c) Transmigration: After maintaining HBMECs at confluence for 5 days, approximately 40,000 labeled MDA-MB-231 or MDA-MB-BrM2 cells were seeded over the monolayer in the presence or absence of AM9928 for 5 h. Green labeled, transmigrated cells across the HBMEC were then counted and expressed as the percentage of cells transmigrated relative to control. Data shown are representative of at least three independent assays performed on triplicate wells. The error bars indicate standard deviations. n.s not significant.