Tcf reporter plasmid kit

The dual luciferase assay was performed with the dual-luciferase®reporter assay system (Promega Corp., Madison, WI). All reagents were prepared as described by the manufacturer of the TCF Reporter Plasmid Kit (Millipore Corp., MA, USA). After the transfection complex was formed with FOP DNA, pRL-TK Vector renilla (Promega Corp., Madison, WI) and 50 μl of DNA Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA), the cells were seeded into 96-well plates with 100 μl/well. U2OS cells were divided into four groups: oleandrin (time): 50 nM oleandrin for 0, 24 and 48 h; oleandrin (concentration): 0, 25 and 50 nM oleandrin for 24 h, LiCl + oleandrin (time) and LiCl + oleandrin (concentration). For the LiCl groups, the cells were pretreated with 20 mM LiCl for 6 h to activate the Wnt signaling pathway. After the cells were lysed with PLB for 15 min, firefly luciferase reagent LARII (100 μl) and Stop & Glo Reagent (100 μl) (Promega Corp., Madison, WI) were added. The OD values of the TOP flash and the FOP flash were detected and the TOP/FOP ratio reflected the activity of the Wnt/β-catenin signaling pathway.

RPMI-1640 medium, fetal calf serum (FCS) and trypan blue were purchased from Seromed (Berlin); anti-human GLO1 monoclonal antibody (mAb, #02–14) was from BioMac (Leipzig, Germany); cell proliferation WST-1 reagent from Roche; anti-human β-actin mAb was from Abgent (Hamburg); HRP-labeled goat anti-mouse Ab and Real Detection System Peroxidase/3,3'-diaminobenzidine (DAB) Rabbit/Mouse Kit from Dako (Hamburg); anti-human GAPDH (cat.no. 5174), anti-human phospho(Ser9)-glycogensynthasekinase-3β (anti-phospho GSK3β (Ser9) (cat.no. 9322), anti-human GSK-3β (cat.no. 9315), pan-phospho-β-catenin (Ser33/37/Thr41) (cat.no. 9561) antibodies from Cell Signaling; protease inhibitor cocktail, RNAse, EP, EL¸ annexin-V-fluoresceine isothiocyanate (FITC), propidium iodine (PI) and LDH-1 were obtained from SigmaAldrich (Taufkirchen); chemiluminescence detection kit from Boehringer (Mannheim); RT² Profiler™ PCR Array: Human WNT Signalling Pathway(Cat. No. PAHS-043F-2) from SA Bioscience (Hilden); plasmid pCMV-GLUC was obtained from Prolume Nanolight Inc. (Pinetop, AZ); TCF-Reporter Plasmid Kit from Millipore (Schwallbach); TransIT®-LT1 from Mirus Corporation (Madison) and Gaussia luciferase transfection kit and coelenterazine from PJK (Kleinbittersdorf).

Neochlorogenic acid (99.3%), cryptochlorogenic acid (99.8%), and caffeic acid (98.5%) used in quantification analysis were purchased from Biopurify, Chengdu, China, and chlorogenic acid (99%) from was purchased from Extrasinthese, Genay, France. Chlorogenic acid ≥ 95% (titration) product reference (Ref.) C3878-1G, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Wnt CHIR 99021 inductor, and iCRT14 inhibitor were purchased from Sigma-Aldrich, Burlington, MA, USA. Acidified isopropyl alcohol, PBS, fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin, and streptomycin were purchased from GIBCO, Grand Island, NY, USA. The TCF Reporter Plasmid Kit Ref. # 17-285 was purchased from Merck Millipore, Burlington, MA, USA.

The TCF Reporter Plasmid Kit (Merck KGaA, Darmstadt, Germany) was used to measure the response of breast cancer cells to conditioned medium from macrophages. A total of 1 μg TOPFlash or FOPFlash and Renilla luciferase reporters were co-transfected into BT549 or HCC1937 cells using Lipofectamine 3000 and incubated for 24 h. Then, the cells were treated with conditioned medium from macrophages for 24 h. Luciferase reporter activity was measured by the dual-luciferase assay (Promega) according to the manufacturer's protocol.

Full‐length SLC35C1 cDNA with a flag tag amplified from a human foetal brain library was cloned into the pcDNA3.1 vector (#v70920, Invitrogen). The LentiCRISPR V2 system was from Addgene (#52961). The TCF reporter plasmid kit containing TOPFlash reporter plasmid and activator plasmid was from EMD Millipore (#17‐285). The Renilla Luciferase control plasmid pRL was from Promega (#E2261). The integrity of all constructs was confirmed by gene sequencing. The shRNA‐SLC35C1 plasmids and control plasmids were from Santa Cruz Biotechnology (#sc‐96300‐SH).