Tamoxifen-resistant cells (TAM-R) are resistant to tamoxifen (4-OH-TAM), phenotypically distinct, more invasive and migratory compared to wild-type MCF7. (A) MCF7 and TAM-R cells treated with vehicle (EtOH) or 10-7 M 4-OH-TAM were plated (3 × 103/well) in 96-well plates and allowed to adhere. One plate was fixed and annotated as Day 0. A sulforhodamine B (SRB) assay was performed every two days until Day 6. The experiment was repeated three times and each time six technical replicates were used. (B) Cells were stained with Crystal Violet and 10X images were taken with a bright-field microscope when cells were 50% confluent (bar represents 400 μm). (C) Boyden chamber-based assay was used to determine the cells invasive or migratory capacity. Cells were allowed to invade or migrate for 72 or 18 hrs respectively before the insert was fixed, cut, and mounted in Mowiol infused with DAPI. 4X images were taken with a fluorescent microscope (bar represents 1,000 μm). The results are representative of three biological and two technical replicates. (D) Quantification of microRNA (mRNA) levels of epithelial to mesenchymal transition (EMT) markers or Notch genes (G) analysed by qRT-PCR. Fold change is shown in TAM-R compared to MCF7 cells, normalised to GAPDH. Results represent three biological as well as three technical replicates of each. (Bars represent standard deviation (SD) *P <0.05 **P <0.01, ***P <0.0001, t Student, two-tails). (E) Western blot validation for representative EMT markers and Notch proteins (H). ActinB was used as loading control. (F) Representative images showing E-cadherin expression in MCF7 and TAM-R cells at cell-cell contact.