Pro-apoptotic effects of testosterone-HSA in Caco2 cells. Caco2 cells stimulated with 10-7 M androgen conjugate for the indicated time periods. (A) Cells were stained with Annexin V and visualized by confocal laser scanning microscopy. Magnification, ×400. (B) Quantitative APO-Percentage apoptosis assay of testosterone-HSA stimulated Caco2 cells according to the manufacturer's instructions. Bars present the mean OD measured at 550 nm (** P < 0.01, N = 4). (C) Non-transformed intestinal IEC 06 cells were exposed to 10-7 M testosterone-HSA for 24 and 48 hours. No apoptoric response was evident by the APOPercentage apoptosis assay. Cells serum starved for 24 hours served as positive control for apoptosis. Bars present the mean OD measured at 550 nm, N = 3. (D) Caspase-3 activity was measured at 405 nm in lysates derived from testosterone-HSA treated cells in the presence, or absence of caspase-3 inhibitor DEVD-fmk for the indicated time periods and then incubated with the caspase-3 substrate DEVD conjugated to the chromophore pNA according to the manufacturer's instructions (** P < 0.01, * P < 0.05, N = 4).