Pro-apoptotic effects of testosterone-HSA, DHT and estradiol in the absence or presence of inhibitors in Caco2 cells. (A) Quantitative APOPercentage apoptosis assay of testosterone-HSA, DHT or estradiol stimulated Caco2 cells and in the presence/absence of cytochalasin B (Cyto B) or flutamide. Cells were exposed to 10-7 M testosterone-HSA, DHT or estradiol for 24 hours and pro-apoptoric responses were assessed by the APOPercentage apoptosis assay. Equally, cells pre-treated or not with 10-7 M Cyto B or flutamide, were exposed to testosterone-HSA for 24 hours. Cells serum starved for comparable periods of time served as a positive control for apoptosis. Bars present the mean OD measured at 550 nm (** P < 0.01, N = 4). (B) Cells were pre-treated or not with Cyto B or flutamide for 1 h and then exposed or not to 10-7 M testosterone-HSA for 4 h, lysed and incubated with the caspase-3 substrate DEVD conjugated to the chromophore pNA according to the manufacturer's instructions. Caspase-3 activity was measured at 405 nm (** P < 0.01, N = 4). (C) Immunoblot analysis for iAR expression in membrane preparations. Membranes were isolated as described in Materials and Methods and subjected to immunoblot analysis using a specific iAR antibody, and subsequently a Na+/K+ ATPase or actin antibody as a positive/negative control respectively.