Figure 1

Dvl1, Dvl2, and Dvl3 expression are each required for Wnt5a/Fz2/NF-AT signaling. A, mouse F9 stable clones expressing rat Frizzled1 (Fz1), rat Frizzled2 (Fz2) or empty vector (EV) [30] were treated with vehicle (-), Wnt3a or Wnt5a for 6 hours and the activity of NF-AT-dependent luciferase was measured. ***p < 0.001, compared with corresponding vehicle-treated group; ###, p < 0,001, compared with Wnt5a-treated "EV" group. B, Myc-Dapper1 (Myc-Dpr) was expressed (assayed by immunoblotting, bottom panel) in F9 clones expressing Fz2. The activity of NF-AT reporter in response to Wnt5a was determined as described. C, Suppression of Dvl1, Dvl2, or Dvl3 using siRNA targeting a single Dvl isoform was conducted in F9 clones expressing Fz2. The activation of NF-AT-sensitive transcription in the presence (+) or absence (-) of Wnt5a was assayed by using a luciferase reporter. **, p ≤ 0.01, compared with basal (-Wnt5a) in control group; ##, p ≤ 0,01, compared with Wnt5a-treated (+Wnt5a) control group. Suppression of Dvl1, 2 or 3 by using siRNA was examined by immunoblotting. The blots of actin were shown as loading controls. D, F9 cells expressing Fz2 were treated with variable amounts of siRNAs targeting a single Dvl isoform. The activation of the NF-AT-dependent luciferase in response to Wnt5a was measured. The data are plotted as "% of maximal" of the Wnt5a-sensitive luciferase response versus the extent of knock-down (KD) in individual Dvl isoforms produced by treatment of increasing amounts of siRNA. The level of Knock-down for each Dvl was verified by immunoblotting (data not shown). E, Increased amounts of Dvl were expressed in F9 clones expressing Fz2. The activation of NF-AT-sensitive transcription in the presence (left panel) or absence (right panel) of Wnt5a was assayed by using a luciferase reporter. The luciferase activities were plotted versus the amount of immunoreactive staining for the HA-tag quantified in the blots (data not shown).