Recombinant human wnt3a 5036 wn

CCD-18Co (ATCC CRL-1459) human colon myofibroblasts were cultured in Minimum Essential Medium plus 10% foetal bovine serum (FBS) (both from Life Technologies). IMR-90 (ATCC CCL-186) human lung fibroblasts and BJ-hTERT (ATCC CRL-4001) human foreskin fibroblasts immortalized with hTERT were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) plus 10% FBS. Cell lines were periodically authenticated using the GenePrint 10 System (Promega) and the results were sent for comparison against the ATCC cell line database. Lyophilised recombinant human Wnt3A (#5036-WN, R&D Systems) was reconstituted at a concentration of 200 μg/ml in PBS containing 0.1% BSA following manufacturer’s indications. Cells were treated with a final concentration of 100 nM 1,25(OH)₂D₃ (Sigma-Aldrich), 100 ng/ml recombinant human Wnt3A, and/or with the corresponding volume of vehicles (ethanol for 1,25(OH)₂D₃; 0.1% BSA in PBS for Wnt3A) for the indicated times (in long experiments cells were retreated every 48 h).

Tamoxifen (T5648) was purchased from Sigma-Aldrich. Recombinant human NRG1-β1/HRG1-β1 (396-HB) and recombinant human Wnt3a (5036-WN) were from R&D Systems. Mouse monoclonal antibody against β-catenin (610154) and rat monoclonal antibody against CD31 (PECAM1) (553370) were from BD Biosciences. Rabbit polyclonal antibody against GFP (ab6556) was from Abcam. TO-PRO^®-3 Iodide (642/661) (T3605) was from Invitrogen. Rabbit polyclonal antibody against ErbB2 (Neu) (C-18) (sc-284), rabbit polyclonal antibody against ErbB4 (C-18) (sc-283) and mouse monoclonal antibody against phosphorylated tyrosine (PY99) (sc-7020) were from Santa Cruz Biotechnology. Rabbit monoclonal antibody against GAPDH (14C10) (#2118) was from Cell Signaling Technology. Immunoprecipitation beads (Protein A Sepharose 4 Fast Flow (17-5280)) was from GE-Healthcare. Magnetic-activated cell sorting (MACS) beads, anti-rat IgG microbeads (130-048), were from Miltenyi Biotec. Secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 were from Molecular Probes. Hypoxyprobe^TM-1 Omni Kit (HP3-100) was from Hypoxyprobe Inc.

Luciferase activity in cell extracts was determined using a dual‐luciferase reporter assay kit (Promega). The reporter activity was normalized to co‐express β‐galactosidase activity. All luciferase plasmids were purchased from Addgene. The transcriptional activity of YAP/TEAD in the Hippo pathway was determined using the pGL3b_8xGTIIC‐luciferase plasmid (plasmid #34615). The transcriptional activity of β‐catenin/HTCF‐4 in the Wnt pathway was detected using the luciferase plasmid M50 Super8x TOPFlash located upstream of the minimal c‐fos promoter driving luciferase expression. Recombinant human Wnt3a (#5036‐WN; R&D Systems, Minneapolis, MN, USA) was added to PBS containing 0.1% bovine serum albumin at a concentration of 10 μg/ml and used in experiments at 50 ng/ml.