Anti hes1

λ-Phosphatase treatment,^{19 (link)} GST pull down,^{19 (link)} far western,^{19 (link)} protein extracts preparation,^{12 (link)} immunoprecipitation,^{12 (link)} immunoblotting assays^{12 (link)} and biotinylation assay^{12 (link), 62 (link)} were performed as previously described. Immunoblot analysis was performed using the following antibodies: anti-Flag (Sigma, Cat#F3165), anti-Flag-HRP (Sigma; Cat#A8592), anti-MPM2 (05-368, Millipore), anti-Notch1_Val1744 (Cell Signaling, Danvers, MA, USA, Cat#2421), anti-Notch3 (Cell Signaling; Cat#2889); anti-Notch3 M20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-7424), anti-Pin1 (Santa Cruz Biotechnology; Cat#sc-46660), anti-β-actin (Santa Cruz Biotechnology; Cat#sc-47778), anti-Lck (Santa Cruz Biotechnology; Cat#sc-433), anti-α-tubulin (Santa Cruz Biotechnology; Cat#sc-803), anti-LaminB M20 (Santa Cruz Biotechnology; Cat#sc-6217) and anti-Hes1 (Santa Cruz Biotechnology; Cat#sc-25392). The antibody against the activated Notch3-IC protein (N3_IC-act) was kindly provided by Genentech (South San Francisco, CA, USA). The anti-N3_EC (5E1) antibody was kindly provided by Professor A Joutel.^{62 (link)} The anti-pTα antibody was kindly provided by H von Bohemer.^{63 (link)}

Each sample was lysed in buffer containing 0.1% sodium dodecyl sulfate, 1.0% Nonidet-P40, 0.5% sodium deoxycholate, 50 mM Tris, pH 8.0, 150 mM NaCl, and a protease inhibitor (Roche Applied Science, Vienna, Austria, pH 7.4). Tissue samples were minced with scissors, and then the total protein was extracted. Electrophoresis was performed as described previously [45 (link)]. The primary antibodies involved included anti-SHMT2, anti-OCT4, anti-SOX2, anti-NANOG, anti-non-phospho-β-catenin, anti-β-catenin, anti-CYCLIN D1, anti-Slug, anti-Snail, anti-β-actin (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA), anti-GAPDH, anti-E-cadherin, anti-N-cadherin, anti-NICD1, and anti-HES1 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). After incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology), immune reactive bands were visualized by enhanced chemiluminescence detection (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Human TNBC cell lines MDAMB468, HCC1806, HCC1569, Hs578t, BT549, MDAMB231, and HCC1937 (Wu et al., 2015 (link)) were procured from American Type Culture Collection (ATCC, Manassas, VA), revived from early passage liquid nitrogen vapor stocks as required and maintained at 37°C in 5% CO₂ and 95% humidity. All cells were authenticated via short tandem repeat testing. No mycoplasma contamination was noted. DAPT and GANT61 were procured from Sigma-Aldrich, St. Louis, MO, and Selleck Chemicals, Houston, TX. For Western blot, immunoprecipitation, immunofluorescence and immunohistochemistry, anti-ALDH1A, anti-Nanog, anti-Oct4, anti-KLF4, anti-c-MYC, anti-NICD, anti-JAGGED, and anti-Ki67 antibodies were purchased from Cell Signaling Technology, Beverly, MA. Antibodies anti-GLI1, anti-Sox2, anti-SHH, anti-FOXM1, and anti-HES1 were procured from Santa Cruz Biotechnology Inc Anti-HEY1 was purchased from ABclonal Technology, Woburn, MA. Mouse monoclonal β-Actin was procured from Sigma-Aldrich, St. Louis, MO. Horseradish peroxidase conjugated goat anti-rabbit IgG, goat anti-mouse IgG, and donkey anti-goat IgG were purchased from Sigma-Aldrich, St. Louis, MO. 3-(4,5-Dimethylthiazol-2-yl)–2,5-diphenyltetrazolium bromide (MTT) were procured from Sigma-Aldrich, St Louis, MO. Chemiluminescent peroxidase substrate was procured from GE Healthcare, UK.