Materials and methods Cell cultures The Caco2 and HCT116 human colon cancer cell lines and the non transformed intestinal IEC06 cells were obtained from the American Type Culture Collection and were studied between passages 55 and 70. Preparation of steroid solution Before www.selleckchem.com/products/CP-690550.html each experiment testosterone 3 oxime human serum albumin, was dissolved in serum free culture medium at a final concentration of 10 5 M. This stock solution was incubated for 30 min at room temperature with 0. 3% charcoal and 0. 03% dextran, centrifuged at 3,000 g and passed through a 0. 45m filter to remove any potential contamination with free steroid. This is highly important for the interpretation of the results to disconnect any possible intracellular testosterone and or iAR interference with the effects mainly induced by the mAR activation.

Testosterone HSA, estradiol and dihy drotestosterone solutions were used at a final concentration of 10 7 M throughout the study unless otherwise mentioned. All treatments and incubations with steroids including apoptosis assays were performed Inhibitors,Modulators,Libraries in serum containing medium. Testosterone HSA FITC or control HSA FITC constructs were generated by conjugat ing Testosterone HSA or HSA with FITC using standard techniques. Preparation of paraffin blocks from HCT116 colon cancer xenografts HCT116 cells or p53 deficient HCT116 were injected sub cutaneously at the axillary region of 7 9 weeks old male SCID mice according to the British practice of bilateral Inhibitors,Modulators,Libraries trocar implants as described previously. Each inoculum contained 106 cells exponentially growing at the time of harvesting.

When tumors reached about 1000 mm3 in size, animals were sacrificed and tumors were excised and fixed in buff ered formalin embedded in paraffin. 4m sections were prepared with the aid of a Leica microtome. Subsequently, sections were stained with hematoxylin eosin and examined under a microscope to assess the histological phenotype of the tumor, the type and degree of differentiation, Inhibitors,Modulators,Libraries and the presence of regres sive changes. Other sections were de paraffinized and sub jected to standard immunofluorescence analysis using fluorescent Testo HSA FITC or HSA FITC control conju gates as described in the following section. All animals were Inhibitors,Modulators,Libraries treated according to the Greek law and the instruc tions of the European council governing the use and handling of animals in experiments.

Immunofluorescence analysis and confocal laser scanning microscopy Cells were cultured on glass cover slips with testosterone Inhibitors,Modulators,Libraries HSA FITC or Y-27632 129830-38-2 control HSA FITC using the concentrations and the incubation periods indicated in the figure leg ends. For testosterone HSA FITC staining, cells or speci mens were washed twice with PBS containing 1. 5% FBS for 1. 5 min and incubated for 1 h with 1% BSA in PBS at room temperature. After two washes with PBS 1.