Characterization of the LNCaP-S14 screening cell line. (a) Basal levels of p27 and SKP2 were examined by immunoblotting in LNCaP and HeLa cells. (b) Parental LNCaP cells as well as cells stably transfected with empty vector (P10) or Myc-SKP2 (LNCaP-S14) were examined for the expression of p27 and SKP2 by immunoblotting. The numbers under each lane represent the tubulin normalized levels of p27 and SKP2 relative to untransfected LNCaP cells. (c) Basal levels of p27 and SKP2 in LNCaP and LNCaP-S14 were visualized by immunofluorescence. Images were taken using a Nikon Eclipse E600 microscope at 60× magnification. (d) The levels of p27 and SKP2 were analysed in total cell lysate from LNCaP-S14 cells treated with the proteasome inhibitors MG132 (20 μM), epoxomycin (1 μM) and bortezomib (100 nM) or the CDK inhibitor roscovitine (20 μM) for 18 h. The numbers under each lane represent actin normalized levels of p27 and SKP2 relative to DMSO treated cells. (e) p27 and SKP2 staining in cells treated with the indicated compounds for 18 h. (f) Cells seeded in a 384 well plate and treated with vehicle (dimethyl sulfoxide; DMSO) or roscovitine (20 μM) for 18 h were fixed and stained for p27. Images were obtained using the ImageXpressMicro automated microscope (Molecular Devices) at 20× magnification. (g) The graph represents the percentage of p27 positive cells +/- standard deviations in LNCaP-S14 cells treated with DMSO or roscovitine and analysed with two imaging platforms (MetaXpress and CytoShop) as described in the Methods section. Data are representative of three independent experiments.