Figure 4.

Functional analysis of the p.M64T, WNT4 variant in HFK-PC shows significant loss of function and diminished canonical WNT/b-catenin signaling. (A) Wild-type WNT4, but neither WNT3A nor mutant WNT4, activates the canonical WNT pathway in HFK-PC. HFK-PC cells are cotransfected on a 6-well plate, with TOPFlash reporter plasmid (4 µg) and wild-type WNT4, mutant WNT4, or WNT3A expression plasmid (4 µg). pCMV-Renilla plasmid (0.4 µg) is used as internal control. *P<0.05 (t test). Data are presented as the mean ± SEM from three separate experiments. (B) Illustration of the conditioned media experiments. HEK293 cells are transfected separately with wild-type WNT4, mutant WNT4, or empty vector (control). Conditioned media containing WNT proteins were applied on HFK-PC 24 hours after transfection. Cells are harvested for total RNA and total protein 6–24 hours later. (C) Results of a representative Western blot analysis of the HFK-PC cells after they are treated with wild-type WNT4, mutant WNT4, and control conditioned media, with the use of anti-active β-catenin antibody. Cbl (95 kD) is used as a loading control. Active β-catenin protein levels are adjusted to Cbl and are quantified compared with the control (empty vector). Three separate experiments yield a similar result in which wild-type WNT4 induces a significant increase in active β-catenin as opposed to the p.M64T Wnt4 variant, which results in a nonsignificant change. (D) Quantification by real-time RT-PCR of the expression of mRNA axin2 and ccnd1 in HFK-PC after treatment with wild-type WNT4, mutant WNT4, and control conditioned media.