Mouse recombinant wnt3a

L-cells which express recombinant Wnt3a (Wnt3a L-cells) and control L-cells which do not express Wnt3a were purchased from ATCC (cat#CRL-2647 and cat#CRL-2648, respectively). Cells were grown in ATCC-Formulated Dulbecco's Modified Eagle's Medium (ATCC cat#302002) supplemented as described above and with 0.4 mg/ml G-418 for Wnt3a L-cells. Wnt3a L-cells and control L-cells were grown to full confluence, re-fed with fresh media without G-418, and media were conditioned for 2 days, and repeated once. Media were pooled, filtered, and aliquots stored at −20°C. Wnt3a- or control-conditioned media at a concentration of 20% in growth medium were used in all experiments. Selected experiments were performed with 150 ng/ml mouse recombinant Wnt3a purchased from Preprotech (cat#315-20).

The WNT/β-catenin pathway was stimulated with mouse Recombinant WNT3a (Peprotech, NJ, USA) at a final concentration of 100 ng/ml. β-catenin transcriptional activity was inhibited by treating cells with iCRT14 (Santa Cruz, CA, USA) at a final concentration of 10 μM. c-Met signalling was stimulated with recombinant human HGF (R&D Systems) at a final concentration of 75 nM, whilst it was inhibited with Capmatinib (Biozol Diagnostica, Eching, Germany) at a final concentration of 2 nM. c-Met and EGFR signalling were concomitantly stimulated with recombinant human EGF (Corning, NY, USA) at a final concentration of 20 nM. Downstream signalling was inhibited with Erlotinib at a final concentration of 5 μM. Recombinant WNT3a, HGF, and EGF were diluted in 0.1% BSA in PBS, which was therefore used as a control in all experiments and is indicated with the “unstimulated” label in respective figures. iCRT-14 and Capmatinib were diluted in DMSO, whilst Erlotinib was diluted in PBS. Thus, the respective vehicle controls (veh. ctrl) were used in the experiments.