Stem cell content population is increased in tamoxifen-resistant (TAM-R) cells and is affected by anti-Nicastrin (NCST) monoclonal antibodies (mAbs) and gamma secretase inhibitor (GSI) treatment. (A) Percentage of CD44+/CD24- cells assessed by FACS in TAM-R compared to MCF7 cells. The graph shows CD44+/CD24- percentage assessed by FACS in TAM-R cells pre-treated with mAbs or GSIPF as in 2B. All treatments reduce the stem content population. (B) TAM-R cells form more primary and secondary mammospheres. Sphere formation efficacy (SFE) was calculated as the number of spheres formed in 10 days from the original number of single cells seeded and expressed as percentage. Bars represent mean percentage of mammospheres ± standard deviation (SD) from five separate replicates and three separate experiments. A representative image is shown at 10X magnification (*P <0.05, **P <0.01). (C) SFE in secondary mammospheres is greatly reduced in mAb1 and GSIPF-treated cells. A representative image of mammospheres is shown as in B (bars represent SD) *P <0.05, **P <0.01) (D) MCF7 and TAM-R stem cells were fractionated into cytoplasm, membrane and nucleoplasm. Lysates were immunoblotted and probed for NCST, Notch1, and Notch4. Tubulin was used as cytoplasmic marker. ActinB was used to ensure each fraction was loaded equally.