Figure 4.

AR decreased angiogenesis via angioinhibitory TSP1. (a) RT-PCR detection of TSP1 mRNA in PC3-V, PC3-AR^WT and PC3-AR.⁸⁷⁷ Note decreased TSP1 mRNA by Fl in PC3-AR^WT but not AR.⁸⁷⁷ (b) Western blot of Conditioned Media from PC3-V, PC3-AR^WT and PC3-AR⁸⁷⁷ ± Dox. All cells were DHT-stimulated. (c) The induction of TSP1-Luc reporter in AR(+) and control cells. (d) ChIP of the putative ARE in the 1st intron of TSP1 gene. IC: isotype control. PCR control: cloned TSP1 promoter amplified with the same primers. DNA input is shown. (e) Endothelial cell chemotaxis with the CM from PC3-V, PC3-AR^WT and PC3-AR⁸⁷⁷. The cells were treated with Dox and stimulated with DHT, where indicated and the CM collected and tested at increasing concentrations with the TSP1 neutralizing antibody (•) or with isotype control antibody (IgA) (○). Note the that CM from AR(+) cells are less potent at inducing endothelial cell chemotaxis, and that TSP1 neutralizing antibody, but not isotype control improves migration (arrowheads). (f) Immunostaining of the PC3-V, PC3-AR^WT and PC3-AR⁸⁷⁷ tumors in control and Dox-treated male mice. Cryosections were stained for CD31 (red) and TSP1 (green).