Figure 2. Stat3 is a key mediator of Fzd2-mediated downstream signaling, EMT program and cellular migration.

A. Comparison of 45 different signal transduction pathways in FOCUS cells transfected with Fzd2 or control shRNA using a 45-transcription factor reporter array. Signaling pathways which showed significant change in Fzd2 knockdown samples are indicated. Neg and Pos denotes negative and positive luciferase controls. B. Bar graph showing increase in transcription activity of Stat3 upon Wnt5a stimulation in Fzd2-expressing Huh7 cells. C. Bar graph showing decrease in phosphorylation of Stat3, Erk1 and Mek1 upon Fzd2 knockdown in FOCUS cells. The relative phosphorlation of Akt (Ser473) is unchanged in Fzd2-shRNA expressing cells. D. Wnt5a stimulation increases phosphorylation of Stat3, Erk and Mek in a Fzd2-dependent manner. E. Treatment with Stat3 inhibitor reduces FOCUS cell migration. Dose response curves showing EC₅₀ (50% reduction in cell migration compared with DMSO control) in FOCUS, and SNU449 liver cancer cell lines treated with Stat3 or Mek inhibitors. F. Western blots showing Fzd2 and Stat3 associate in a co-immunoprecipitation assay. Lysates immunoblotted with anti-Stat3, and anti-Fzd2 are also shown. G. Perturbing Stat3 expression reverses EMT in FOCUS cells. Bar graph showing expression of epithelial and mesenchymal marker genes in FOCUS cells with knockdown of Stat3. H. Stat3 activity regulates cell migration. Knocking down expression of Stat3 decreases Fzd2-mediated cell migration in FOCUS cells (left) while expression of constitutively active Stat3 (Stat3C) increased migration of Dld1 epithelial cells (middle). Treatment with Stattic (Stat3 inhibitor) decreased migration of Fzd2 over-expressing Huh7 cells (right) in a dose dependent manner.

See also Figures S3, S4, S5 and Table S1