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Conditioned media were prepared by culturing 3 ��106 Leydig cells for 24 hours as described above. After 16 hours of culture, MLECs were washed twice http://www.selleck.cn/products/XL184.html with phosphate-buffered saline and lysed in 60 ��L of lysis buffer (Promega, Madison, Wisconsin). Cell extracts were assayed for luciferase activity with an assay kit from Promega and a Netzschalter 090003 luminometer (GSG Nuclear, Milan, Italy). Parallel MLECs were incubated with known increasing concentrations of TGF-�� (T1654; range, 0.0625�C4.0 ng/mL; Sigma-Aldrich) to obtain a standard curve. For zymography of PA, 20 ��L of the same conditioned medium used for the TGF-�� bioassay was separated by 10% SDS-PAGE under nonreducing conditions according to the procedure of Laemmli (1970). Molecular weights were calculated from the position of prestained molecular weight markers (Sigma-Aldrich) subjected to electrophoresis in parallel lanes. PA was then visualized by placing the Triton X-100�Cwashed gel on a casein-agar-plasminogen underlay, as previously described (Granelli-Piperno http://www.selleckchem.com/products/z-vad-fmk.html and Reich, 1978; Vassalli et al, 1984). Densitometric analysis of the band was performed. Gelatinolytic activity was assayed using the Heussen and Dowdle (1980) method, adapted for a minigel format. Twenty-microliter aliquots of conditioned media were applied directly, without prior heating or reduction, to 10% (wt/vol) acrylamide gels containing 2 mg/mL gelatin (Sigma-Aldrich). After removal http://www.selleckchem.com/products/ly2157299.html of SDS from the gel by incubation in 2.5% (vol/vol) Triton X-100 for 30 minutes, the gels were incubated at 37��C for 18 hours under continuous stirring in 50 mM Tris-HCl (pH 7.6) containing 0.2 M NaCl, 5 mM CaCl2, and 0.02% (wt/vol) Brij-35. Gels were stained for 30 minutes in Coomassie brilliant blue diluted in 30% methanol in 10% glacial acetic acid 0.5% (vol/vol) and then destained in the same solution without the dye. The gelatinolytic activity of each gelatinase, identified by the molecular weight, was evident as a clear band against the blue background of stained gel. The intensity of the bands was quantified with specific densitometry (Molecular Analyst Software; Bio-Rad, Hercules, California). All experimental data are expressed as means �� SE of at least 3 separate experiments. Statistical analysis was performed by Student's t test. Differences were considered significant at P