Figure 4.

Functional Assessment of Mutant WNT10B in Wnt Signaling

(A) Effects of mutant WNT10B on luciferase activity. The Wnt signaling in HEPG2 cells was assessed with TCF reporter plasmids. HEPG2 cells were transiently transfected with either pTOPFLASH or pFOPFLASH and then cultured in no supernatant (Mock), wild-type WNT10B supernatant, or each of the four indicated mutant WNT10B supernatants. Error bars depict SDs; ^∗∗p < 0.01, ^∗∗∗p < 0.001. The results are from three independent experiments performed in triplicate.

(B) Impact of WNT10B on endothelial differentiation of DPSCs. DPSCs were isolated and cultured as described in our previous study.²⁵ Cells were treated with medium conditioned with wild-type or mutant WNT10B (containing 50 ng/ml of each of the recombinant WNT10B proteins) for the purpose of inducing endothelial cell differentiation. Recombinant human VEGF-C (50 ng/ml, Sigma) served as a positive control. After 7 days, cells were lysed and prepared²⁶ for western blots (ECL system, GE Healthcare Life Sciences) with anti-VEGFR2 polyclonal antibody (5 μg/mL, Invitrogen). The amount of tubulin, used as a loading control, was detected by monoclonal anti-α-tubulin antibody (1:500, Sigma).