Sema3c fc

Serum starved PC3 or DU145 cells plated on coverslips were treated with PBS, Sema4D-Fc, Sema3C-Fc (R&D, Abingdon, UK, 2 μg/mL), or dexamethasone (Sigma, 10 nM) for 60 min. Cells were then fixed (4% paraformaldehyde), permeabilized (0.2% triton), stained by immunofluorescence with mouse anti-GR(CST) followed by anti-mouse Alexa Fluor488 (Life Technologies, Carlsbad, CA, USA), phalloidin-TRITC (Sigma) and DAPI. Serum starved 22Rv1 cells were treated with PBS, Sema4D (2 μg/mL) or dihydrotestosterone (DHT, Sigma, 1 nM) for 60 min. Cells were then fixed (4% paraformaldehyde), permeabilized (0.2% triton), stained by immunofluorescence with mouse anti-AR followed by anti-rabbit Alexa Fluor488 (Life Technologies), phalloidin-TRITC (Sigma) and DAPI. Images were taken at x63 magnification using a Zeiss LSM510 confocal microscope and the intensity of staining of Alexa488 in the cytoplasm and nucleus was measured using ImageJ using DAPI staining to outline the nucleus and actin staining to identify the cytoplasm. The ratio of nuclear to cytoplasmic staining for each cell was calculated. (n = 3, A minimum of 44 cells were scored per treatment).