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. 2016 Dec 23;108(1):42–52. doi: 10.1111/cas.13109

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© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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Development of reporter assay to measure Wnt5a and Wnt5b signaling. (a) Schema of the reporter system used to measure Wnt5a/b signaling using CHO cells stably expressing a Ror2– low‐density lipoprotein receptor‐related protein 6 (LRP6) chimera and TOP‐flash (CHO/Ror2‐LRP6‐TOP cells). GSK3‐β, glycogen synthase kinase 3β; TCF/LEF, T‐cell factor/lymphoid enhancer factor. (b) CHO/Ror2‐LRP6‐TOP cells were stimulated with 1 mL control, Wnt3a, or Wnt5a conditioned medium (CM) for 3 h, and cell lysates were probed with anti‐phospho‐LRP6 and anti‐LRP6 antibodies. (c) CHO/Ror2‐LRP6‐TOP cells and parental CHO cells stably expressing only TOP‐flash were stimulated with control or Wnt5a CM for 8 h. A luciferase assay was carried out and the activity was expressed as a fold increase compared with samples without Wnt5a and Ror2‐LRP6. Results are shown as means ± SD of three independent experiments. *P < 0.05. (d) CHO/Ror2‐LRP6‐TOP cells, which had been transfected with control or Frizzled‐2 (Fz2) siRNAs, were stimulated with control or Wnt5a CM for 8 h. (e) CHO/Ror2‐LRP6‐TOP cells were stimulated with Wnt5a CM for 8 h in the presence of the indicated concentrations of secreted Frizzled‐related protein‐2 sFRP2. (f) CHO/Ror2‐LRP6‐TOP cells were stimulated with the indicated concentrations of purified Wnt5b for 8 h.