Figure 6. FOG2 is a mediator of ZEB1.

(A) Coimmunoprecipitation of FOG2 and GATA3 in 344SQ cells. A GFP-tagged GATA3 expression vector was generated to discriminate between exogenous GATA3 and IgG heavy chain on the basis of size. Extracts were subjected to immunoprecipitation with IgG, anti-FOG2, or anti-GATA3 antibodies. Total extracts and immunoprecipitates were then blotted with anti-FOG2 or -GATA3. (B) qPCR analysis (bar graph) and Western blot analysis (gels) of FOG2 expression. qPCR values, normalized on the basis of ribosomal protein L32 mRNA levels, represented the mean ± SD from triplicate samples and were expressed relative to 393P_vector cells (Vec), which were set at 1.0. (C) 3′-UTR reporter assays. 344SQ cells were transiently cotransfected with Fog2 3′-UTR reporters, in which the predicted miR binding sites were wild-type or mutated (mut) and control, miR-183, or miR-200c precursors. Mean ± SD from triplicate samples. (D) Western blot analysis of FOG2 expression in 344SQ cells that had been transiently transfected with indicated miRNA precursors. (E and F) Western blot analysis of FOG2 (E) and p110α (F) in 344SQ_scr shRNA cells (Scr) and 344SQ_Fog2 shRNA cells. (G) Invasion assays. Invasive 344SQ_Fog2 shRNA (#3 and #4) and 344SQ_scr shRNA cells were photographed (images) and quantified (bar graph). Mean ± SD from triplicate samples (bar graph). Scale bars: 100 εm. (H) Scatter plots of primary tumor weight (left) and numbers of visible lung metastases (right) in syngeneic mice injected with 344SQ_Fog2 shRNA (#3 and #4) or 344SQ_scr cells. Mean ± SD for each cohort. Actin was included as a loading control for Western blots.