Luciferase levels were measured using the Dual-Luciferase Reporter Assay System (Promega) 24 hrs

Luciferase levels were measured using the Dual-Luciferase Reporter Assay System (Promega) 24 hrs. MDA-MB-231, MDA-MB-436, HEK293T and MCF7 cells were obtained from ATCC and maintained in Dulbeccos Modified Eagle Medium (DMEM) (Life Technologies, Waltham, MA) with 10% fetal bovine serum (FBS). HMLE cells were provided by Dr. Jing Yang (University of California, San Diego) and maintained in F12 media (Life Technologies) supplanted with 10% FBS, 0.1% insulin, 2 g/ml hydrocortisone and 10 ng/ml epithelial growth factor. H146, Orexin 2 Receptor Agonist obtained from ATCC, and 67NR, 168FARN and 4TO7 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% FBS. Human colon epithelial cells were obtained from Dr. Jerry Shay (University of Texas Southwestern) and cultured under DMEM with 10% FBS. Human mammary epithelial cell line (AG11132) was obtained from Coriell Institute for Medical Research (Camden, NJ), cultured using MEGM complete medium (Lonza, Basel, Switzerland). MCF7R cells [43] were from Dr. Marc Lippman at the National Cancer Institute using Dulbeccos Modified Eagle Medium (DMEM) (Life Technologies, Waltham, MA) with 10% fetal bovine serum (FBS). For non-adherent 3-D culture of 67NR and H146 cells, plates were coated with 12 mg poly 2-hydroxyethyl methacrylate (polyHEMA; Sigma Aldrich, St Louis MO)/ml of 95% ethanol and allowed to evaporate. 2105 cells per ml were plated and cultured for 48 hr. ATCC cells were used within 5C6 generations and other cells were tested for mycoplasma using PlasmoTest-Mycoplasma Detection (InvivoGen, San Diego, CA). For CD177 shRNA, Lentivirus containing shRNA sequences were packaged in HEK293T cells and media containing packaged virus was collected. 67NR cells were incubated with media containing the packaged shRNA lentivirus for 24 hr and stable cells lines expressing the CD177 shRNA were generated by selection of transduced cells with 4 g/ml puromycin (Thermo Fisher Scientific). The mouse CD177 shRNAs targeting sequences: Sh1 5-GCCAAGACTTGATAATGCTCC ?3; Sh2 5-ACCCAGGCGATTGGGACCTTG-3 were used to silence CD177 in 67NR cells. For soft agar colony assay, 5104 cells were suspended in 0.4% agarose/media mixture and plated on top of solidified Orexin 2 Receptor Agonist 0.8% agarose/media mixture. IEGF Colonies were cultured for two weeks and counted. For monolayer growth curves, 1105 cells were plated and counted at 24, 72, and 120 h. Cell lysates, immunoprecipitation and immunoblots For membrane and cytosolic fractionation, we followed our previously described protocol Orexin 2 Receptor Agonist [44]. For immunoprecipitation, 1 mg of cell lysate was incubated with 1 g/mL of antibodies at 4 C overnight. Immunocomplex was precipitated using protein A or G sepharose beads (Thermo Fisher Scientific). Sepharose beads were resuspended in SDS loading buffer and separated by SDS-PAGE and visualized by Western blotting. For in vitro pull-down assay, 1 g of FC-fusion CD177 (14501-H02H, SinoBiological, Beijing, China) and His-Tag full-length -Catenin (11279-H20B, SinoBiological), both purified from HEK293T cells, were incubated using RIPA buffer, with or without the presence of 1 mg of cell lysates from MCF-7 or MDA-MB-231 cells. Ni-NTA agarose was used to pull down His–Catenin complex, following with SDS-PAGE and Western Blotting. Mammary gland whole mount Mammary glands were removed from mice and fixed in Carnoys fix (6 parts ethanol, 3 parts chloroform, and 1-part glacial acetic acid) overnight. They were then rehydrated with ethanol washes, stained with carmine alum stain, cleared, and mounted. Whole mount slides of mammary glands were marked an inch above the inguinal lymph node and all branch points within this inch were counted. Immunohistochemistry Tissues were processed with standard IHC protocols. High pH 9 (Vector Labs) was used for antigen retrieval and blocked with background punisher (BioCare Medical, Concord CA). Slides were incubated with primary antibody, anti Ki67 antibody (D2H10; Cell signaling), anti-KRT5 antibody (Poly 19055; Biolegend, San Diego, CA), anti-active -catenin (D13A1; Cell Signaling), anti-ER (C-311; Santa Cruz Biotechnology, Dallas, Orexin 2 Receptor Agonist Texas), Orexin 2 Receptor Agonist or anti-PR (D8Q2J; Cell Signaling) for 2 h. Next, rabbit or mouse-on-rodent polymers (BioCare Medical) for 30 min, developed with 3,3-diaminobenzadine (DAB and 0.3% H2O2.