Figure 6: TβRI promotes expression of Snail and invasion of prostate cancer cells in a TGFβ-dependent manner.

(a) PC-3U cells were treated with or without TGFβ. Endogenous TβRI and p300 are visualized by immunofluorescence using V22 (TRITC) and p300 (FITC) antibodies. Note the TGFβ-induced nuclear accumulation of endogenous TβRI and colocalization with p300 (shown in merge; yellow). (b) PC-3U cells were treated as indicated. Endogenous TβRI and PML are shown by immunofluorescence using V22 (TRITC) and PML (FITC) antisera. (c) TGFβ induces association between endogenous TβRI and p300. Cell lysates from PC-3U cells treated with TGFβ were immunoprecipitated with the V22 antibody against TβRI and subjected to immunoblotting with p300 antibody. (d) Cell lysates from PC-3U cells transiently transfected and treated as indicated, were immunoprecipitated with an antibody against p300 and subjected to immunoblotting with HA antibody. (e) Cell lysates from PC-3U cells, transiently transfected and treated as indicated, were immunoprecipitated with an antibody against HA and subjected to immunoblotting with acetyl-Lys (AcK) antibody. (f, g) qRT-PCR analysis for expression of p300, Snail-1, MMP2, PAI1 and Smad7 was performed on mRNA extracted from PC-3U cells transiently transfected with wt HA-TβRI (filled bars) or the E161A mutant (open bars) and treated as indicated. (h) Chromatin immunoprecipitation assay for the Snail promoter using V22 antibody against the endogenous TβRI in PC-3U cells treated or not with TGFβ. (i) Invasion assay for PC-3U cells, transiently transfected and treated as indicated. Cells were visualized by staining with crystal violet cell stain solution. Right panel presents mean values for optical density (OD) of invasive cells. (j) Immunofluorescence stainings of cytoskeletal reorganization of actin and subcellular localization of TβRI in TGFβ-treated primary prostate epithelial cells (PREC) and PC-3U cells for comparison. (k) Quantification of the number of cells in (j) showing endogenous TβRI in the nucleus, where N=200 cells were counted in each group. (a, b, j) Staining with DAPI was used to visualize cell nuclei. Scale bar 20 μm. Data in (f, g, h, i, k) are representative of three independent experiments (mean and s.d). *P<0.05 and **P<0.005 (ANOVA except for relative Smad7 expression, where Students t-test were used).