Alexa488 conjugated secondary antibodies

Cells (1 × 10⁵) were seeded in six-well plates overnight, washed thrice with PBS, and then fixed for 10 min at room temperature 18 °C with 4% paraformaldehyde in PBS. Cells were permeabilized with 0.3% (w/v) Triton X-100 in PBS, incubated with blocking buffer (PBST containing 5% bovine serum albumin), and sequentially probed with anti-CFP1, anti-SETD1B, anti-H3K4me3, anti-Ki-67, anti-p21, and anti-cleaved caspase 3 primary antibodies (Supplementary Table S1, 1:200) and Alexa488-conjugated secondary antibodies (Cell Signaling Technology). Slides were mounted using VectaShield containing 4′,6-diamidino-2-phenylindole (Vector Laboratories). Digital images were acquired with a confocal laser scanning microscope FV3000 (Omnicon,) at ×6–100 magnification.

The antibodies used for blast cell identification by Flow Cytometry (FACSCanto II, Becton Dickinson, Rutherford, NJ, USA) were: anti-CD45-VioBlue, anti-CD45-APC-Vio770, anti-CD34-PerCP and anti-CD117-APC all from Miltenyi Biotech (Bergsch gradbach, Germany). For Western blot analysis, anti-β-catenin, anti- Ser675-phospho-β-catenin, anti-Ser33/37/Thr41-phospho-β-catenin, anti-non-phopho-β-catenin, anti-GSK-3β, anti-Ser9-phospho-GSK-3β, anti-GSK-3α, anti-Ser21-phospho-GSK-3α anti-Histone H3 antibodies and Alexa 488-conjugated secondary antibodies were from Cell Signaling (Danvers, MA, USA); anti-GAPDH, and HRP-conjugated secondary antibodies against mouse or rabbit were from Sigma-Aldrich (Darmstadt, Germany). Wnt modulators used for proliferation and vitality assays, i.e., Wnt-3a, PNU-74654, Niclosamide, IWP-2, Lithium Chloride (LiCl), and AR-A014418, were all purchased from Sigma-Aldrich. For the analysis of cell death, Propidium iodide (PI) and FITC-conjugated Annexin V were from Miltenyi Biotechnology. CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (MTS, Eden Prairie, MN, USA) was from Promega (Promega, Milano, Italy). Cytarabine (Ara-C) and Idarubicin (Ida) were provided by the Pharmacy Unit of the University Hospital of Verona.

MDA-MB-231 cell lines grown on coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.2% Triton X-100 for 10 min at room temperature, blocked in 1% BSA for 1 hour at room temperature and incubated with primary antibodies overnight at 4°C. The slides were then incubated with Alexa 555–conjugated or Alexa 488–conjugated secondary antibodies (Cell Signaling Technology) for 2 hours at room temperature. DNA staining was performed using Fluoroshield Mounting Medium with DAPI (Abcam). Images were captured with a Leica SP5 confocal laser microscope (Leica Microsystems, Buffalo Grove, USA).