NOTCH1 regulates Nanog expression in mouse mammary tumor cells. (A) Nanog expression is suppressed on NOTCH1 inhibition. RNA was harvested from tumor cell lines left untreated or treated with doxycycline (2 μg/ml) for 24 hours and c-Myc, Hes1, Deltex1, Hey1, and Nanog expression levels determined with quantitative real-time PCR. Expression is normalized to the untreated control. (B) Doxycycline treatment reduces Nanog protein levels. The mouse mammary tumor lines (8542, 8526) were left untreated or treated with doxycycline (2 μg/ml) for the time periods indicated. Lysates were analyzed for ICN1 and Nanog expression with immunoblotting, and relative band intensities were quantified. (C, D) Nanog is expressed in the nuclei of NOTCH1-transformed mouse mammary tumor cell lines and primary tumorspheres. The mouse mammary tumor cell line 8542 was grown on coverslips, and primary tumorspheres were dissociated and centrifuged onto slides, followed by fixation in 4% paraformaldehyde. The cells were then permeabilized with Triton X-100 and immunostained by using antibodies against cytokeratin 8/18 (red) or Nanog (green). Cells were photographed at 400 × magnification. Scale bars, 100 μm. (E) NOTCH1 regulates NANOG expression in human breast cancer cells. The human basal-like breast cancer cell line MDA-MB-231 was left untreated or treated with the γ-secretase inhibitor Compound E (10 μM) for the time periods indicated, and intracellular NOTCH1 and NANOG expression levels examined with immunoblotting.